001 by wulinqing

VIEWS: 2,834 PAGES: 369

									  EHRLICH II –2nd World Conference
         on Magic Bullets
            Celebrating the 100thAnniversary of the

Celebrating the 100th Anniversary of the
   Nobel Prize Award to Paul Ehrlich

          Nürnberg, Germany

           October 3-5, 2008




              Abstract Book
                                                              EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                   Celebrating the 100th Anni versar y of the
                                                                     Nobel Prize Award to Paul Ehrlich
                                                                        Nürnberg, October 3-5, 2008

                                                                                           Introduction of supercritical fluid extraction as a new s ample-prep aration
Combined targeting of gro wth-promoting genes with antisense
                                                                                           procedure for isolation and identification of a ph armaceutical from biolog ical
oligonucleotides in human color ectal cancer cells: Chemo-sensitization
                                                                                           fluids: Application to dispo sition kin etics
potential
                                                                                                                1 ,2           3         3           3                2            1
                                                                                           ABD EL-ATY AM               , CHOI JH , KO MW , KHAY S , GOUDAH A , SHIN HC , SHIM
ABAZ A M-S I AND BAHMAN A M
                                                                                           JH 3
Department of Bi ological Scienc es, F aculty of Scienc e, Kuwait U niversity, P. Box      1
                                                                                               Konkuk U niversity, Seoul, Republic of Korea; 2 C airo Uni versity, Giza, Egypt;
5969, 13060 Safat, State of Kuwait.                                                        3
                                                                                               Chonnam N ational Uni versity, Gwangju, Republic of Korea
Background: Aberrant expression of growth-promoti ng genes c ontributes to the
                                                                                           Background: Since its commercial development in the earl y 1990s, supercritical
growth advantage of tumor cells, targeting suc h genes with phosphorothioated
                                                                                           fluid extrac tion (SFE) has attr acted consi derable attention as a sampl e-preparation
antisense oligonucl eoti des, AS[S]ODNs, might therefore be useful in c ontrolling
                                                                                           procedure. However, other different s ample preparation procedures , incl uding
the abnormal proliferation of c anc er cells. To explore the potenti al of combi nation
antigene therapy in human c olorectal canc er cells, we have examined the in vitro         precipitati on, liquid- and/or solid-phase extraction in biological fluids, als o remain in
                                                                                           use. Aims : In this investigati on, SFE was introduc ed to isolate and identify
effec ts of AS[S]ODNs targeting c- myb, c-myc and c dc2 in human colorec tal
                                                                                           orbifloxacin (OBFX) from plas ma and milk of lac tati ng does.
cancer c ell lines.
                                                                                           Methods: Four parameters, including the temperature and the press ure of
Method: Cancer cells were treated with c-myb-, c- myc- or cdc2-AS[S]ODNs
                                                                                           supercritical fluid, modifier ratios, and dynamic extracti on time, were evaluated and
indivi dually or in c ombination. The effects of growth promoti ng -gene AS[S]ODNs
                                                                                           opti mized to obtain the best yield of the anal yte from the bi ological fluids.
on mRNA and protei n expressi on were determined by RT-PCR and blot anal ysis.
                                                                                           Determi nations of the OBFX in the extrac ts were c arried out using high-performanc e
The effects of thes e combinati ons on c ell growth, chemo-sensitization, apoptosis
                                                                                           liquid chromatography c oupled with fluoresc ence detec tor (HPLC -FLD). The
and genes controlling cell growth and apoptosis were monitored by MTT ass ay,
                                                                                           linearity of the calibration cur ves as well as the instrumen t limit of detec tion /limit of
DNA fragmentati on and Real-time RT-PCR.
                                                                                           quantitation (LOD/LOQ) were evaluated.
Results: mRNA and pr otein expression wer e dr amaticall y reduc ed after treatment
                                                                                           Results: The optimum conditions of the extraction process that yielded the
with c-myb-, c-myc-, or cdc 2-AS[S]ODNs. C ombined targeting of c-myb/c-myc and
                                                                                           maxi mum anal yte extracti on efficiencies were 150°C vs. 60°C, 250 kg/cm2 , 30% vs.
c-myb/cdc 2 had much higher dose and ti me dependent synergistic growth
                                                                                           35% methanol, and 40 min vs. 2 0 min, for plas ma and mil k, res pecti vel y. Good
inhibitor y effects ; 5-100% and 5-95% , respecti vel y; compared to single antigen        linearity (at least r 2 ≥ 0.999) of the c alibration cur ves was obtained over the range
therapy (5-55%). Combined targeting of c-myc / cdc2 als o produced greater dos e
                                                                                           from 0.2 to 0.01 µg/mL. The method s howed good a rec over y rate (74.2–127.73%)
and time dependent additi ve or s ynergistic growth inhi bitor y effects (10-100%)
                                                                                           and precision (expressed as relati ve standard deviations (RSDs) 1.64 –20%). The
compared to single antigene ther apy (5-60%). The combined targeting of c-myb /
                                                                                           instrumental LOD and LOQ val ues wer e 0.004 µg/mL vs. 0.01 µg/mL or 0.006
c-myc / cdc 2 exhibited much higher growth inhi bitor y effects ( 10-90%) compared
                                                                                           µg/mL vs . 0.02 µg/ L, for plasma and mil k, respecti vel y. The method was
to single antigene therapy (20-50%). Combined targeting of c-myb / c-myc / cdc 2
                                                                                           successfully applied to es timate the pharmacokinetic variables of orbifl oxacin in
produc ed marked inducing effects on both apoptosis and c hemo-sensitization to
                                                                                           lactating does .
taxol, 5FU, doxorubicin and vinbl astine. Real-time RT-PCR indic ated down-
                                                                                           Conclusions: To the best of our knowledge, this is the first time that SFE has been
regulation of mRNAs of cdk1, c yclin B1, cdk2, c yclin E1, cdk4, c yclin D 1, Bc L2 and
                                                                                           applied to isolate an antimicrobial agent from biological fluids. This method is
BclxL and up-regulati on of mRNAs of p21, Bax and c aspase.
                                                                                           promising for clinical applications and for pharmacokinetic studies of various
Conclusion: Our study suggests that c ombination antigene ther apy targeting c-
                                                                                           phar mac euticals in bi ological fluids.
myb, c-myc and cdc 2 can inhibit human color ectal c anc er cell prolifer ation more
effec tivel y than monogene therapy by bloc ki ng the c ell c ycle and inducing
apoptosis. Combinati on antigene ther apy is thus a promising approach for cancer
therapy.




Misoprostol and Po stpartum Hemorrh age                                                    Magic bullets in pro state cancer

ABDEL-ALEEM H                                                                              ABDELBAKY A, HUSSAIN M, KAISARY AV

Department of Obstetrics and Gynecol ogy, F aculty of M edicine ,Assiut Uni versity,       Department of Urolog y, The Royal Free NHS Trus t, London, U.K.
Assiut , Egypt.
                                                                                           It has been 100 years since Paul Ehrlich popularized the c oncept of a " magic bullet"
Abstract:                                                                                  during his 1908 Nobel prize lecture. Scientists have sinc e keenl y embar ked on the
Every year mor e than half a million women los e their lives because of                    search for thes e magic bullets. In the UK prostate cancer is the most c ommon
complications of pr egnanc y and childbirth. Pos tpartum hemorrhage is the leading         cancer diagnos ed in men and the s econd leading cause of c anc er death in men (1).
cause of death es peciall y in developing countries. Strategies have been                  Consequentl y a magic bullet is highl y desired by patients and urol ogists alike.
devel oped to prevent and treat postpartum hemorrhage. One of these is the use of          The first bullet c omprises hormonal therapy res ulting in androgen abl ation, which
uterotonic drugs to pr event and treat atonic pos tpartum hemorrhage. Misopros tol         represent the mainstay of treatment of metastatic pros tate c ancer. LHRH analogues
(prostaglandi n E1 analogue) has been emerged as an uter otonic drug with special          can be impl anted into patients where they c ause a negative feedbac k effec t,
advantages ; being heat stable, can be us ed orall y and rather cheap. So, it can be       resulting in a decr ease i n LHRH production from the anterior pituitary and a
considered as a potential "Magic Bullet" in the management of postpartum                   subsequent reduction in testic ular testos terone production. D egarelix, an LHRH
hemorrhage. Bec aus e of this potentiality, World Health Organizati on has been            antagonist has an immediate onset of ac tion s uppressing LHRH and testosterone,
conducting big multicentre r andomized clinical trials to evaluate effic ac y and safety   thus avoi ding tumour flare. Studi es suggest a PSA decrease by 97-98% after 1 year
of misoprostol in the pr evention and tr eatment of postpartum hemorrhage. The             and the medi an time to 90% reduction in PSA was 8 weeks (2).
findings and i mplications of thes e trials will be pres ented and discussed.              Brachytherapy is ultras ound guided implanation of radi oacti ve seeds i nto the
                                                                                           prostate. Its popularity has increased as the us e of transrec tal ultrasound (TRUS)
                                                                                           makes it easier to accur atel y direct the magic bullet. Patient s election is a key. In
                                                                                           this scenario brachytherapy achieves a 12- year progression free s ur vival of 6 6%
                                                                                           (3).
                                                                                           Heat effec ts are attrac tive. HIFU is a bullet where prostate tissue is heated to the
                                                                                           point of coagul ati ve necrosis using high-energy ultr asound allowi ng the selec tive
                                                                                           destructi on of tissues. Long-term results from clinical trials are awaited. Freezi ng or
                                                                                           cryotherapy of the prostate cancer has increased over the past few years,
                                                                                           especi ally with the impr ovement in cryotec hnology. Multipl e hollow core pr obes are
                                                                                                                                                                              -    -
                                                                                           placed percutaneously under TRUS guidance. This causes cell destructi on at 20 -
                                                                                           40ºc, us uall y achi eved by appl ying two cycl es of freeze thaw. 96% of men with
                                                                                           localised disease achieve a PSA of <0.2ng/ml within 6 months. Again long term
                                                                                           results are awaited.
                                                                                           Finally, Doc etaxel, chemother apy agent, is a magic bullet that may be used to treat
                                                                                           androgen-independent pros tate cancer, with promising results (4).

                                                                                           1) http://info.cancerresearchuk.org/cancerstats/types/prostate/
                                                                                           2) Ragde H, Korb LJ, Elgamal AA et al.Modern prostate brachytherapy. Prostate specific antigen
                                                                                           results in 219 patients with up to 12 years of observed follow-up.Cancer.2000: 89;135-41.
                                                                                           3) T annock If Ronald de Wit, William R. Berry,et al Docetaxel plus Prednisone or Mitoxantrone plus
                                                                                           Prednisone for Advanced Prostate Cancer. N Engl J Med. 2004 Oct 7;351(15):1502-12.
                                                                                           4) Van Poppel H, T ombal B, de la Rosette JJ,et al Degarelix: A Novel Gonadotropin-Releasing
                                                                                           Hormone (GnRH) Receptor Blocker-Results from a 1-yr, Multicentre, Randomized, Phase 2 Dosage-
                                                                                           Finding Study in the T reatment of Prostate Cancer. Eur Urol. 2008 May 8 [Epub ahead of print]




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                      Page A-1
                                                         EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                     Nürnberg, October 3-5, 2008
Non-Syst emic Deliver y of Ocu lar Brimonidine to the Br ain: Ext ending the          A Role of L ysosom al Phospholipase A2 in C ationic Amphiphilic Drug-
Therapeutic Benefits of Brimonid ine to the CNS                                       induced Phospholipidosis

ABDULRAZIK M 1,2, TAMILVAN AN S1, BENITA S1                                                       ABE A1, KELLY R 1, KOLLMEYER J1, HIRAOKA M 2, SHAYMAN J A1
1                                                                                                 1                                               2
 Phar maceutics Department, School of Phar mac y, H ebrew Uni versity of                           University of Michigan, Ann Arbor, MI, USA, Nippon Medical Sc hool, Sendagi,
Jerusalem, J erusal em, Israel, and 2D epartment of Ophthal mol ogy, Shaare Zedek                 Bunkyo-ku, T okyo, Japan
Medical Center, J erusal em, Israel.
                                                                                                  Background: Many of c ationic amphiphilic drugs (CADs) devel oped as
Background. Des pite rec ent advanc ements in neurosci enc e, and ever-emerging                   therapeutic drugs, including those with anti-inflammator y, antineoplastic and
novelti es in the field of drug deli ver y, the blood brain barrier is still considered a         antiangiogenic properties, and have been known to induce phospholipidosis in
major obstacl e that critically limits the delivery to the brain of hydrophilic drugs or          human and other mammalian tissues . Generally, CADs tend to acc umulate in
thos e with modest or weak hydr ophobic character. Brimonidi ne, a sel ecti ve alpha-             lysos omes and to inhibit lys osomal enz yme acti vities . Recentl y, it was found that
2 agonist, is a widel y used oc ular hypotensi ve agent with promising merits as                  the al veolar macrophages (AMs) prepared fr om l ysos omal phospholipase A2
neuroprotectant.                                                                                  (LPLA2) deficient mic e suffer from c ellular phos pholipid accumulation and
Aim. To demons trate the efficient delivery to the brain of Brimonidi ne following                phos pholipidosis. This phenotype suggests a potenti al connection between CAD -
ocular administration and to eluci date the route of non-s ystemic drug flow along                induc ed phos pholipidosis and LPLA2. The study on the reaction properties of
the eye-brai n axis.                                                                              LPLA2 could find a clue to understand the molec ular mec hanism of CAD-induced
Methods. Brimonidi ne was us ed as a probe to study the eye and br ain                            phos pholipidosis.     LPLA2 shows an increas ed acti vity towards z witterionic
phar mac okinetics followi ng topical oc ular administrati on of one droplet of the               phos phatidylcholine lipos omes contai ning negati vel y charged lipids under acidic
radio-labeled drug. Ani mals were s acrificed at different time points following the              conditi ons. In the present study, the effect of negativel y charged lipid on LPLA2
administration of single 0.2% 3H-Brimonidine droplet to the rabbit eye. Brain and                 activity was investigated.
eyes were dissec ted and s elected brain and eye tiss ues, as well as s ystemic blood             Methods: Purified recombinant mous e LPLA2 was used as the source of LPLA2.
samples, were proc essed for detec ting the concentration of radio-labeled                        Sulfati de, a lipid that is resistant to LPLA2, was chosen as a negati vel y charged
Brimoni dine. Fluorescent-labeled Dextr an with molec ular weight of 40 kDa was                   lipid.
used as a probe to eluci date the r oute of the non-sys temically medi ated eye-to-               Results: Sulfatide incorporated into 1, 2-dioleoyl-glycero-3-phos phoc holine
brain dr ug flow following periocul ar administration.                                            (DOPC) liposomes enhanc ed LPLA2 acti vity under acidic conditions . The
Results. Brimoni dine accumulation in s tudied intracrani al tissues was significant              enhanc ement by sulfatide was linear until 10% molar ratio of sulfatide to DOPC
already at 5 minutes followi ng single oc ular admi nistration while the detected                 and weakened by the addition of NaCl in the reaction mi xtur e. Amiodarone
Brimoni dine levels i n the s ys temic bl ood were ver y low at all time points.                  (AMD), a cationic amphiphilic drug that interac ts with negati vel y charged lipids,
Evaluation by fluorescent microscopy s howed that 40 kD a fl uoresc ent-labeled                   reduced the LPLA2 acti vity in a conc entr ation dependent manner. In addition, the
Dextr an that was administered to the perioc ular spac e didn‘t per meate the oc ular             co-sedimentati on of LPLA2 with negati vel y c harged liposomes occurred in
barriers and was flowing through veins that drain the eye and orbital tissues                     centrifuge at 150,000 g and was mar kedl y reduc ed by the addition of NaCl or AMD
towards the intracranial cavernous sinus. Tissue secti ons that included the eye                  in the reaction mixture. T hese results indic ate that LPLA2 adsorption to the
and its major vessels s howed extensive acc umulation i n vei ns but with no signal of            negati vel y charged lipid membr ane s urface through an electr ostatic attraction
the fl uoresc ented probe inside the ciliar y arter y, which corres ponds to non-                 enhanc es the rate of phospholipid hydrol ysis by LPLA2 at lipid- water interfaces .
significant flow of the probe through the s ystemic circulation.                                  Conclusions: CAD-induc ed c ellular phos pholipidosis is linked to the i mpairment
Conclusion. Brimonidine, a non-toxic agent to the eye with well appreci ated                      of phospholipid catabolism by inhibition of the binding of LPLA2 to the membrane
therapeutic merits, can accumulate in intr acranial tissues at significant                        treated with CDA.
concentrati ons already at few minutes after simple oc ular administrati on with ver y
low s ystemic accumulation. Our results s uggest an efficient method for the
extension of Brimonidine therapeutic merits to the brai n while avoi ding s ystemic
side effects .




Effect Of Flunixin On Enrofloxacin Clearance And Stead y-State Serum                              Selen ium Derivatives as Cancer Preventive Agents
Concentrations In Calves
                                                                                                  ABOUL-FADL T
ABO-EL-SOOUD K 1, AL-ANATI L2
                                                                                                  Departments of Phar maceutical C hemistry, King Saud Uni versity, Riyadh, Saudi
1
 Phar macol ogy D epartment, Faculty of Veterinar y Medicine, Cairo Uni versity, Giza             Arabia.
12211, Egypt; 2Dept. of Veterinary Basic Scienc es, F aculty of Veterinar y Medicine,
Jordan Uni versity of Sci ence and T echnolog y, J ordan.                                         Background: The role of seleni um in the prevention of cancer has been recentl y
                                                                                                  established by laborator y experiments, clinical trials, and epidemiological data.
Background: To evaluate whether the conc omitant administration of flunixi n may                  Most of the effects are related to the function of s elenium in antioxi dant enz yme
alter enrofloxacin pharmac okinetic parameters and the hepato-renal acti vities.                  systems . Ani mal data, epidemiological data, and intervention trials have s hown a
Methods:: Ten clinically healthy, Freisian calves weighting 200-250 kg and 5-7                    clear role for s eleni um derivati ves i n both pr evention of specific cancers and
months ol d were used. They were divi ded up into two groups. First group was                     antitumorigenic effects i n postinitiation phases of c anc er.
injected a single dose of enrofloxacin 2.5 mg/kg of body weight (BW) intravenousl y               Methods: Selenazolidine prodrugs (SCA) of selenoc ys teine were s ynthesized by
(IV). Sec ond group was injec ted the s ame dose intramusc ularl y (IM). After 1 month            the reaction of sel enoc ys teine with the appropriate c arbonyl deri vati ve. Male CF1
was hout period, each of the 10 animals was given flunixi n (IM) at a dose 2.2 mg/kg              mice were treated daily for 7 days with equi-seleni um ( 1.25 mg Se/kg) dos es of
of BW one-hour prior to with the injecti on of enrofloxacin i n a dos e of 2.5 mg/kg              each agent, by either the i ntraperitoneal (ip) or intr agastric (ig). route and the
BW in cal ves of the first group IV. or the IM i njection in the sec ond group.                   effec ts compared with thos e of selenoc ysti ne. H epatic parameters were
Results: Co-administration of flunixin with IV injection of enrofloxacin reduced the              determined 24 hours after the last dose.T he efficac y of SCA in reducing NNK [4-
volume of distribution at steady state Vd(SS) and total body clearance (Cltot ) by                (methyl nitrosamino)-1-(3-pyridyl)-1-butanone]-induced lung adenomas in female
33.9% and 30% respectively. After IM injection of enrofloxacin, the elimination half-             A/J mic e, a model for tobacc o-related lung tumorigenesis, has been investigated
life (t1/2el) and mean residence time (MRT) were shorter in the flunixin-medicated                with sel enazolidines in the diet for 1 month prior to c arcinogen admi nistration and
calves.                                                                                           during the subsequent 4 months of tumor development.
                                                                                                  Results: In general, few significant (p <0.05) changes were s een with ig as
                                           Enrofloxacin alone     Enrofloxacin + flunixin         compared to ip admi nistration. 2-butyl SCA, 2-cycl ohexylSCA, 2-phenylSC A and 2-
                              Unit                                                                oxoSCA were chemopreventi ve, significantly reduci ng mean lung tumor numbers
          Parameters
                                                                                                  from the 10.9 of unsupplemented c ontrols to 4.7, 5.3, 2.8 and 4.7, res pec tivel y.
    I/V
                                                                                                  When sel enazolidine supplementation began three days after carcinogen
            Vd(SS)           L kg-1             2.24  0.17             1.48 ± 0.04**             administration (i.e., post-initiation), 2-butylSCA, 2-c yclohexylSCA, and 2-oxoSCA
             ClB            ml/min/kg           1.73  0.10             1.21 ± 0.04**             were c hemopreventi ve. In both regimens, s elenoc ystine was also
           AUC0-?           g.h/mL             1.58  0.13             2.17 ± 0.06**             chemopr eventi ve. Both 2-butyl SCA and 2-phenylSCA retained their
    I/M                                                                                           chemopr eventi ve acti vity (44% and 40% tumor number reduction, respecti vel y),
             Cmax            g/mL              0.31 ± 0.01             0.26 ± 0.01**             when the s upplementati on was shortened and restricted to a pre-initi ation period
             Tmax              h                0.94 ± 0.02             0.69 ± 0.05**             (days −9 to−2).
            MRT                h                3.62 ± 0.24             2.23 ± 0.29**             Conclusions: Although this study has not identified the mechanism, it firmly
            (t1/2el)           h                2.24 ± 0.14             1.45 ± 0.11**             establishes that 2-substituted s elenazolidi ne- 4(R)-carboxylic acids poss ess
           AUC0-?           g.h/mL             1.42 ± 0.11             0.83 ± 0.03***            chemopr eventi ve ac tivity against NNK-induced lung tumors in a murine model.
                                                                                                  Dependent on the natur e of the 2-substituent, the chemopreventi ve ac tivity can
Cmax: maximum plasma or milk concentration; T max: time to peak concentration; AUC0-∞:areaunder   arise from changes elicited in the post-initiati on period, similar to selenoc ysti ne, or
the curve from zero to infinity by the trapezoidal integral; * * P<0.01; * * * P<0.001            in the pre-initiation period.
Conclusions: Conc omitant administrati on of flunixi n with enrofloxacin induced
significant alterations in pharmacokinetic parameters and hepatic-renal functi ons i n
calves. Therefore, concurrent administrati on of flunixi n with enrofloxacin should be
avoided.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                       Page A-2
                                                                     EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                           Celebrating the 100th Anni versar y of the
                                                                             Nobel Prize Award to Paul Ehrlich
                                                                                Nürnberg, October 3-5, 2008
Xenon: a m agic bullet for the treatment of acute brain ischemia?                                Central Nervous Systems involvement in dog s naturally infected b y
                                                                                                 Leishman ia chagasi/infantum
ABRAINI JH,1,2 DAVID HN,2 HAELEWYN B,2 RISSO JJ.3
                                                                                                 ABREU-SILVA AL1, DE CARVALHO DA SILVA AP1 , D E MACEDO AA1, DE
1
  ERT 1083 ―Phar mac ologie et Formulation de Gaz-Médicaments‖, UMR 6232,                        CARVALHO NET A AV2, AND DA SILVA CALABRESE K3
                                                                              2
Université de Caen, CNRS, CEA, Centr e CYCERON, Caen, Franc e; NNOXe
                                           3                                                     1
Phar maceuticals, Quebec, Canada ; IMNSSA, Toulon, France.                                         Departamento de Patologia da Uni versidade Estadual do Maranhão, São Luís –
                                                                                                 Maranhão Brazil.,
Background: As a general consensus, the noble and remar kabl y safe anesthetic                   2Departamento de Qui mica e Biol ogia da Uni versidade Estadual do Mar anhão,
gas xenon, which possess es low-affinity antagonistic properties at the NMDA                     São Luís – M aranhão Brazil.,
                                                                                                 3
receptor, has been clearly demonstrated as a promising preclinic al neuroprotec tive               Laboratório de Imunomodulaç ão, Departamento de Protozoologia do Instituto
agent for the treatment of acute cerebral ischemi a (and other hypoxic-ischemic                  Oswal do Cruz, Rio de J aneiro, Brazil.
insults), with no proven adverse side effects when us ed at subanesthetic
concentrati ons. However, surprisingly, possible interacti ons between xenon and                 Blood br ain barrier (BBB) was described by Paul Ehrlich when he injec ted dyes in
tissue-type plas minogen acti vator (t-PA), a s erine protease with thrombol ytic                the blood str eam and obs er ved that all tissues stained, except the organs of
activity that is the only drug approved for the tr eatment of acute br ain ischemi a,            central ner vous s ystem (CNS). This barrier is very efficient in c ontr olling
have never been investigated.                                                                    substances that could c aus e damage to the brain. However, s everal pathogens
Methods: We review in vitro and in vivo unpublished and published structural,                    are abl e to cross it. In order to demonstrate that Leishmania chagasi/ infantum is
electrophysiological, elec trochemic al, biochemical, and pharmacological data on                also capable to produc e lesions in CNS, the brain of thirty dogs naturall y infected
the mec hanis ms of action and the neuroprotec tive effects of xenon, and its                    by the protoz oan were studied. The s amples were submitted to histopathological,
interactions with t-PA.                                                                          imunohistochemic al exams and Poli meras e Chain R eaction (PCR).
Results: We show that xenon woul d produce its antagonistic action at the NMDA                   Histopathological anal ysis revealed that the amastigotes forms were associ ated to
receptor by binding on a specific hydr ophobic poc ket that may be loc ated on the               an inflammatory reac tion. It was obser ved mai nly in the cerebellum and thalamus.
NR1 subunit. In addition, we also s how that xenon has exc eptional neuroprotec tive             Both PCR and i mmunohistochemistr y c onfirmed the presence of the parasite in
properti es that take place withi n a limited but interesti ng therapeutic window                20% of the exami ned brains. Previ ous studies in our laborator y evidenced that
followi ng ischemia onset. Finall y, we show de nov o that xenon at concentrations               mice experimentally infected by L. amaz onensis also developed infl ammator y
higher than 25 vol% reduces the thrombol ytic and pr oteol ytic pr operti es of t-PA             infiltrates c ompos ed by eosinophils and parasitized macrophages. Mas t cells were
both in vitro and in vi vo in rats s ubjec ted to a thrombo-embolic model of middle              frequentl y obs er ved. Both studies demonstrated that Leis hmani a is able to change
cerebral arter y by inter acting directl y with the catal ytic site of t-PA.                     the permeability of BBB leading to lesions . Although, th e brain is consi dered a
Conclusions: Overall, these data i ndicate that xenon should be used for the                     healthy plac e immunologicall y privileged several pathogens can cross this barrier
treatment of ac ute ischemic stroke acc ording to a well-defi ned therapeutic                    and caus e inj ury. Different hypotheses have been postulated to explai n how the
sequenc e. We pr opos e that xenon at concentrati ons higher than 25 vol% s hould                efficient BBB is i mpaired. In acc ordance to Persids ky et al. (2006), it occ urs during
not be admi nistered in patients suffering ischemi a due to the ris k of inhibiting the          inflammator y proc ess due to disrupti on of junction compl exes between br ain
benefits of t-PA-induced thrombol ysis or possible endogenous fibrinolysis in                    microvascul ar endothelial cells with s ubs equent for mation of a paracellul ar route
patients who c annot be treated with t-PA. T hen, onc e blood flow would be r estored            that facilitates entr y of leukoc ytes into cer ebral parenc hyma. We s uppose that
spontaneousl y or by t-PA, xenon c ould be used at higher subanes thetic dos es, if              Leishmania reached brain of mice and canine thr ough i mmature monoc ytes .
necess ary, to increase neur oprotection and to reduc e the risk of adverse
proteolytic side- effec t of t-PA therapy that is hemorrhaging.




New Silver Compounds as W ound Healing Material Problems and                                      PKC-ι inhibition b y IC A-1 redu ces cell pro liferation in Neuroblastoma
Opportunities
                                                                                                  PILLAI P, DESAI S, WIN H, OSTROV D * AND ACEVEDO-DUNCAN M
ABU-YOUSSEF M A , GOHAR Y B, ÖHRSTRÖM LC,D , LANGER VC , MASSOUD
AA,C                                                                                              Department of Chemistr y, U niversity of South Florida, Tampa, Fl
                                                                                                  James A. Haley Veteran‘s H ospital, Tampa, Fl
a
 Department of Chemistry, F aculty of Science, Alexandria Uni versity, P.O. Box                   *University of Florida, Gaines ville, Fl
426 Ibr ahimi a, 21321 Al exandria, EGYPT, bDepartment of Microbi ology, Fac ulty of
Science, Al exandria Uni versity, 21321 Alexandria, EGYPT, c Department of
Chemic al and Biol ogical Engineering, Chalmers University of Tec hnology, SE-412                 Abstract
96 Gothenburg, SWEDEN, dRebact, Stena C enter 1C, 412 92 Göteborg,
SWEDEN.                                                                                           Neuroblas tomas are highl y lethal tumors and 85% of cerebr al neuroblastoma
Wounds are a maj or medic al problem affecti ng about 10% of all hos pitalized                    occurs in children and 15% in adults. Neurobl astoma is the fourth most c ommon
patients and c osting more than $5 billion US$ per year in the USA al one. 1 The                  type of c anc er in c hildren. Accor ding to the American Cancer Society, ther e are
major problem, especiall y with large wounds, s uch as burns, and chronical                       approxi matel y 650 new cas es of neuroblastoma each year in the U nited States.
wounds, is the risk of infecti on. And that this not a mere inc onvenience, event                 Despite significant educational efforts, improved diagnostic tec hniques , and
today infec ted wounds may be lethal to hospitalized patients. The antibac terial                 rigorous ther apies, neuroblastoma c ontrol remains s tatic. To address this health
properti es of sil ver and its low toxicity, combined with the ease of topical                    issue, the obj ecti ves of this researc h was to inves tigate the us e protein ki nas e C-
applications and surface coatings using the metal and its compounds has led to a                  iota (PKC- ι ) inhibitors on the proliferati on of neuroblas toma c ells. Previ ous wor k
revi val of silver use in medici ne.2 Sil ver contai ning wound dressings are today               has shown that inhibition of PKC- ι is a promising means by which to prevent and
based on mai nly silver or simple sil ver s alts suc h as the nitrate or the s ulphate.           treat certain cancers. Here, we report the i dentification of a PKC- ι inhibitor (1H-
The only sil ver coordination c ompound us ed to any extent is the                                imidaz ole-4-carboxamide, 5-ami no-1-[2,3-di hydroxy-4-[(phos phonooxy) methyl]
[Ag(sulfadiazine)]n c oordination pol ymer patented in 1973, a prescription drug in               cyclopentyl]-,[1R-(1α, 2β, 3 β, 4 α)] (ICA-1) that targets a unique s equence (amino
the U S and sold under trade names suc h as Sil vazine in preparations as a 1%                    acid residues 469-475) in the c atal ytic domain of PKC- i nhibits PKC- ι acti vity
cream.                                                                                            and is effec tive in bl oc king the proliferati on of BE(2)-C neurobl astoma c ells. Our
In this contribution we will discuss some problems affecti ng different types of sil ver          data support significant proof of concept that ICA-1 i nhibits the proliferati on of
therapy, i.e. silver resistant bacteria (contrar y to popular belief this does exist and          neuroblastoma c ells and may be a novel c hemother apeutic for the treatment of
may be a growing problem), coupling of silver and antibiotic resistance (remains to               patients with neuroblastoma.
be proven, but a link exists), in vitro and in vivo studies (difficult to extrapolate and
documented effect of existi ng preparations not always good).
We will also examine the opportunities that new c oordination c ompounds may give
in this respect and what grounds there are to expect that other species other than
just the ―naked‖ Ag + ion c ould be the acti ve antimicrobial agents. New sil ver
complexes were s ynthesized by direct mi xing of aqueous s olutions of sil ver nitrate
and ethanolic sol utions of the c orresponding ligand. Als o solid state techniques
were empl oyed to s ynthesis [Ag(2-aminopyridine) 3](NO3) and [Ag 3(2-
aminopyridine) 4(NO3) 2](NO3). Further s ynthesis and structural details are
described elsewhere.3 UV- as well as 1H-NMR titrations were carried out to
investigate the structural behavior and stability at different ratios . Antibac terial
activity of the compl exes was determi ned acc ording to the rec ommendations of
NCCLS (1999) for the use of a broth microdiluti on method. The acti vity (1/MIC) of
some Ag(I) nicotinate and iso-nicotinate compounds c ompar ed to
silversulfadiazine against s ome clinicall y isolated multi-resistant bacteria.3
1
  J. E. Williamson, Dressing for success: New wound care products aid healing, efficiencies,
Healthcare Purchasing News, Jan., 2005. 2 S. Silver, L. T . Phung, G. Silver, J Ind. Microbiol.
Biotechnol., 2006, 33, 627–634. 3 M. A. M. Abu-Youssef, R. Dey, A. A. Massoud, Y. Gohar, V.
Langer, L. Öhrström, Swedish Patent Application, 0701314-7, 2007; M. A. M. Abu-Youssef, R.
Dey, A. A. Massoud, Y. Gohar, V. Langer, L. Öhrström, Inorg. Chem., 2007,5893; Morsy A. M.
Abu-Youssef, Vratislav Langer and Lars Ohrstrom, Dalton T rans.,2006, 2542.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                       Page A-3
                                                          EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                               Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                     Nürnberg, October 3-5, 2008
Gener ation of Human Gen e Knockout Cell Lines: Application to                     Magic Bullet s ag ainst the Autoimmune Diseases
Assessm ent of Genotoxic Anticancer Drugs
                                                                                   ADAMS DD
ADACHI N
                                                                                   University of Otago Medical Sc hool, Dunedin, N ew Zeal and
International Graduate Sc hool of Arts and Sciences, Yokohama City Uni versity,
Yokohama, Japan.                                                                   Abstract

Background: DNA damage causes c ancer, is used to treat canc er, and is                   In 1908 Paul Ehrlich was awarded the Nobel Prize for discovering Sal varsan, a
responsi ble for many of the side effec ts of cancer therapies. It is thus important to   ―magic bullet‖ that was more toxic for the spiroc haete of s yphilis than for man, and
understand how chemotherapeutic agents c aus e DNA damage and how c ells                  for showing that i mmune reacti vity against host c omponents did not nor mall y occ ur
cope with suc h damage, particularl y in the context of human somatic cells. Many         but, as an abnormality, could be the basis of many dis eas e proc esses . Today,
of chemotherapeutic agents c urrentl y us ed in the clinic target DNA                     knowledge of the autoi mmune diseases has reached the stage of showing how they
topoisomerases; for example, camptothecin is a topois omerase I inhibi tor, while         can be cured by ―magic bullets‖ against the forbidden clones of B and T l ymphoc ytes
etoposide a topoisomeras e II inhibitor. These agents, li ke ionizing radiati on, have    that cause them. The magic bullets can be made by isolating the target autoantigen
been shown to induce DNA double-strand breaks (DSBs), but clearl y by distinct            and coupling it to a c ytotoxic moi ety, suc h as bungarotoxi n or a mol ecule containing
mec hanis ms. In human somatic cells, the rol es of two maj or DSB repair                 a radioac tive element such as 131 Iodine emitting short-range radi oacti vity in the form
pathways, homologous recombi nation (HR) and nonhomologous end-joini ng                   of beta particles (el ectrons).          Such therapeutic molec ules, administered
(NHEJ), in the repair of topoisomeras e-induc ed DNA damage ar e not compl etel y         intravenousl y, would home c ytotoxicall y on the l ymphoc ytes of the pathogenic
understood.                                                                               forbidden clones , selecti vely destr oying them. The autoantigen for rheumatoid
Methods: We have recentl y developed a s ystem using the human pr e-B cell line           arthritis, calpastatin, is already available for us e in this way, and so is that for the
Nalm-6 that enables rapid production of knoc kout cell lines . Using this s yste m,       probabl y autoimmune lethal stage of HIV infec tion, AIDS, in the for m of the CD4
we generated human cell lines deficient for Rad54, a HR protein, and/or DNA               structure on helper T lymphoc ytes. Additionall y, dangerousl y severe autoimmune
ligase IV (Lig4), a critical NHEJ ligase. Cells l ac king Artemis, a nuclease invol ved   diseas es can be c ured by chemical or radi ological generalised immune ablation,
in NHEJ, were also created by gene targeting using Nalm- 6. Sensiti vity assays           which will destr oy the forbidden clones, followed by i mmune restitution with
were perfor med by clonogenic survi val assays or by using the CellTiter-Glo              autologous s tem c ells, taken previously from the patient‘s own bone marrow.
Luminesc ent Viability Assay kit (Promega).                                               Because unluc ky semi-random s omatic mutati ons in immunoglobulin variable region
Results: We find that NHEJ is critical for repairing topois omerase II- as well as        (V) genes are res ponsible for occ urrenc e of the pathogenic forbidden clones, they
low-dose irradiation-induced DNA damage, while HR is important for repairing              are unlikely to r ecur in the regenerated immune s ystem (Burnet‘s F orbidden Clone
topoisomerase I-induc ed DNA damage. Intriguingly, NHEJ negativel y affects               Theor y) and this has been demonstrated therapeuticall y in cas es of s evere
survi val of cells treated with the topois omerase I poison c amptothecin. We also        scleroder ma.
find that loss of Artemis not onl y leads to increas ed camptotheci n resistanc e,
independentl y of Lig4, but also alleviates hypers ensiti vity of Lig4-null cells to
topoisomerase II i nhibitors.
Conclusions: 1) HR and NHEJ have different rol es in the repair of
topoisomerase- mediated DNA damage. 2) Our data have significant implicati ons
for chemotherapy invol ving topois omerase inhibitors. 3) A series of human gene
knoc kout c ell lines are us eful in assessing c ellular DNA damage and repair
induc ed by chemotherapeutic agents.




Nicotinamide M echan ism s of Neuroprotection in Stroke                                   Prevention of Mucositis and Improvem ent in Complian ce of Head & Neck
                                                                                          Cancer Patients undergoing Radio-Chemother ap y b y Curcumin
ADAMS JD
                                                                                          ADHVAR YU MR 1 , VAKH ARIA BC 2, REDDY MN 1, SRIVASTAV SP3
University of Southern Califor nia, School of Pharmac y, Los Angeles , CA, USA
                                                                                          1                                                         2
                                                                                           Veer Nar mad South Gujarat U niversity, Surat, India; Shree Gurudev Sarvajani k
Background: Stroke is a major caus e of death in the USA. Although stro ke can            Charitable trust, Surat, Indi a; 3 Lions C anc er Research Center, Surat, India.
be treated by removing intracerebral clots or by administration of clot diss olvi ng
agents, therapy for stroke is inadequate. Pati ents frequentl y do not r ecover           Background: Oral mucositis (OM) is a common complicati on and a dose limiting
normal speech or motion following strokes. The current work explores the                  toxicity i n up to 90% of head & nec k cancer patients (HNCP) undergoing radio-
devel opment of a new therapeutic agent, nic otinamide, for recovery from stroke.         chemotherapy (RCT). Its advers e effects on sc hedule of therapy, quality of life and
Methods: The foc al ischemia and reperfusion model of stroke in rats was used,            economic burden to the patients and s ystem warrant an urgent need for a potent c ell
as well as the global ischemia and reperfusion model in mice. Mechanistic                 specific radioprotector. Sever al adj uvant agents li ke folic acid, Vit-E, anti biotic mouth
experiments were perfor med with intrac erebroventricular t-butylhydroperoxi de           rinse etc have been tried to prevent OM without remar kable succ ess. Curcumin has
treated mice. Nicotinami de was adminstered at various doses and various ti mes           shown radioprotecti ve as well as radios ensitizing potential in in vitro studies with its
before or after ischemia and reperfusi on. Brai n cell damage was exami ned by            antioxi dant, free radical scavenging acti vity and ability to arrest cell cycl e in G2 and
light and el ectron microsc opy. T he brain levels of pyridine dinucl eotides,            M phas e in malignant cells. Objecti ve of this pilot s tudy was to evaluate effects of
glutathione and ATP were meas ured. DNA fragmentati on and pol y(ADP-ribose)              curcumin on OM in HNCP under going RCT.
pol ymerase acti vity were ass essed.                                                     Methods: 95 HNCPs were given conventional RCT and 109 HNCP wer e g iven 2 gm
Results: Mechanistic studies show that glutathione is oxi dized, NAD and ATP              of curcumin/day orall y in 3 di vided doses in addition to RCT for two months starting
levels decrease during oxidati ve stress in the brain .              Pol y(ADP-ribose)    from 3 days before planned radiation. OM gradati on as per WHO oral toxicity scale
pol ymerase is acti vated and DNA fragmentation occurrs during oxi dati ve stress.        was done weekl y for the whol e RCT period and follow-up was c onti nued for 6
Ischemia and reperfusion res ult in a central necrotic c ore surrounded by a limit        months in both the groups. Incidenc e, onset and duration of OM in eac h grade, and
that includes apoptotic cells. Nicotinamide used as a pr etreatment or post-              patient compliance in term of uninterrupted completion of scheduled RCT were
treatment is able to prevent both necrosis and apoptosis in the brain suffering           compared in both controlled and curc umin treated group by Chi-square test (P≤
from ischemi a and reperfusi on. Nicoti namide is partiall y effecti ve even when         0.05).
given 6 hours after reperfusion. Nicotinamide increas es brai n NAD and ATP               Results: There was a significant decreas e in incidenc e of grade III and IV OM (P≤
levels and regul ates pol y(ADP-ribos e) pol ymeras e acti vity.                          0.001) among curcumin treated group. Patient compliance in terms of c ompleti on of
Conclusions: Nicotinamide is an effec tive and safe neuroprotecti ve agent. The           scheduled RCT increas ed from 53% to 89.0% (P ≤ 0.001).
mec hanis m of nicotinamide neuropr otecti on i nvol ves inhi bition of poly(ADP-
ribose) polymerase and sti mul ation of NAD s ynthesis.              Pol y(ADP-ribose)                    Mucositis characteristics              Control (n = 95)      Trial (n = 109)
pol ymerase r egulates the acti vities of a number of enz ymes and genes. These                         Total Patients with OM (%)                   88 (92)               56 (51)
results will be discussed in ter ms of cas pase acti vation and the induction of IAPs.                      Gr-III and Gr-IV (%)                     49 (51)               14 (13)
                                                                                                        Mean onset of any OM (Gy)               19.1 (17.5-20.6 )     26.9 (24.5-29.3)
Author‘s disclo sure statement: T he author is part owner of the US patent for                        Mean duration of any OM (days)            33.8 (28.9 - 38.7)   13.3 (11.2 - 15.3)
the use of nicoti namide as a neuroprotecti ve agent in stroke and other c onditi ons.              Duration of OM in Gr-III & IV(days)          39.7 (30.4-49.3)      17.1 (13-21.3)
                                                                                                  Patients completing scheduled RCT (%)              50 (53)               97 (89)

                                                                                          Conclusion: 1. Curcumin showed a significant adju vant protecti ve acti vity against
                                                                                          RCT induced OM in HNCP 2. Long term follow-up required for its effec ts on
                                                                                          recurrence and s urvi val i n HNCP.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                  Page A-4
                                                     EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                            Celebrating the 100th Anni versar y of the
                                                               Nobel Prize Award to Paul Ehrlich
                                                                 Nürnberg, October 3-5, 2008
Prepar ation and Evaluation of Mucinated Sodium Alginat e Microparticles for      Trends in Co-Occuring Diseases in Patients Treat ed for Alcohol and Drug
Oral Deliver y of Insulin                                                         Problems in Can ada.

ADIKW U MU 1 , BUILDERS FP2                                                                ADRIAN M
1
  Department of Pharmaceutics, U niversity of Nigeria, 410001 Ns ukka, Nigeria;            Nova Southeaster n Uni versity, Ft. Lauderdale, F L, USA
2
  Department of Pharmaceutical T echnolog y and R aw Material Development,
National Institute for Pharmac eutical Research and Development, Abuja, Nigeria            Background: Understanding the diseas e experience of alc ohol and drug patients
                                                                                           (AD) is needed for rational decision-making in health care and diseas e prevention.
Background: Effecti ve oral ins ulin delivery remains a challenge to the                   Aim: 1) To improve measurement of co-occ urring disease (co-morbidity) in AD
phar mac eutical industr y. A novel formulation of ins ulin usi ng new biomaterials is     patients using population-based data 2) to easil y identify reduc ed c o-mor bidity
necess ary to formul ate and administer insulin via the oral r oute. Aims: 1) To           using a new more s ensiti ve meas ure.
devel op new bioc ompati ble deli ver y materials through cross-linking for the delivery   Method: Co-morbi dity was measur ed using the standardized morbi dity rati o (MR),
of insulin. 2) To study the level of protection of ins ulin against degradation when       comparing 1985-86 Hospital Medic al Rec ords Institute data for all inpatient
formul ated using the new bi omaterial. 3) To study the in vitro and in vivo rel ease of   cases(n=52,200) with primary (P) or sec ondar y (S) alcohol or drug diagnoses i n all
the i nsulin and its effec t on bl ood glucose level i n diabetic rabbits.                 Ontario hos pitals to data for all patients in all Canadi an hos pitals, using indirect
Methods: Cross-linking of mucin-sodi um alginate of rati os 0:1 to 1:3 was effec ted       age-sex standardization and adjusti ng for multiple diagnoses. The modified MR
in liquid paraffi n using ac etone at -30 oC. The microparticles wer e diffusion-filled    (mMR) was us ed to improve meas urement and visual display (graphing) of
and further c oated with 1.5 % w/v c ellulose ac etate phthal ate in acetone at - 30 oC.   reduced c o-mor bidities.
Diabetes was induc ed in 32 rabbits weighing between 1.8-2.5 kg using 120 mg/kg            Results:       Compared to all patients, AD patients had more co- morbi dity.
of alloxan and thes e used for the anti-diabetic ass essment. Ins ulin-loaded              Generally, MR was higher in pati ents with primary (P) rather than s econdar y (S)
microparticles contai ning 50 IU/kg was gi ven to the rabbits in 2 ml of distilled water   AD diagnoses Patients treated for the use or misus e of prescription drugs had the
using gastric intubation using distilled water as the negati ve control and 50 IU/kg       highest MR (MR P=13.3 and MR S=3.5). MR was intermediate for patients who
subcutaneousl y as the positi ve c ontrol.                                                 used illegal drugs (MR P=8.9 and MR S=4.7), and lower for alcohol patients
Results: The microparticles for med were generally multi-particulate, discrete and         (MR P=6.7 and MR S =4.1). Co-morbidity in alcohol patients s temmed from
free fl owi ng. Before ins ulin loading, microparticles were round and s mooth,            practicall y all diagnostic categories, but c o-morbidities in drug patients were due to
becoming fluffier, less s pherical and larger with rough and pitted s urface after         fewer diagnostic categories. AD patients had partic ularly high MR for mental
insulin loading. The mean dissol ution time of ins ulin from the microparticles            disorders, i nfec tions and parasitic c onditi ons, and inj uries and poisonings.
prepar ed with sodi um alginate, mucin, sodium alginate: mucin rati os of 1:1, 3:1         Cocaine cas es had high MR for i nfec tious and parasitic dis eas es, and diseas es of
and 1:3 were 11.21±0.75, 3.3±0.42, 6.69±023, 8.52±0.95 and 3.48±0.65 ( min)                the s kin and subcutaneous tiss ue. Amphetamine cas es had high MR for diseas es
respecti vel y. T he percentage bl ood glucose reduction for the subcutaneously            of the digesti ve s ys tem, and of the musculos keletal s ystem and c onnecti ve tissue.
administered insulin was significantl y (p < 0.001) higher than for the for mulations.     Graphing results using the mMR clearly show AD patients had reduced co-
The blood glucose reduction effect produc ed by the orall y administered ins ulin-         morbidity from complications of pregnanc y, childbirth and the puerperium, and
loaded microparticles prepared with 3 parts of sodi um alginate and 1 part of mucin        from c onditi ons originating in the perinatal period, but a higher rate of c ongenital
after 5 h was, however, equal to that produced by the subcutaneously                       anomalies .
administered ins ulin sol ution.                                                           Conclusion: 1) AD patients had more c o-morbidity than all patients combi ned,
Conclusion: This study shows that the or al route could be an effecti ve alter nati ve     with c o-morbidity invol ving more diagnostic categories for alcohol than drug
for the delivery of insulin using this pol ymer cross-link.                                patients, but they had less c o-morbidity related to reproduc tion than all patients.
                                                                                           2) mMR is a s ensiti ve meas ure of reduced c o-morbi dity.




TOPICAL IMMUNOTHERAPY W ITH DIPHENYLCYCLOPOPENONE IN                                       Studies on Inter action s of W ater Soluble Vitamins with Zinc Through cell,
VITILIGO: A PR ELIMINARY EXPERIENCE                                                        Protein and Anim al Models in H ealth and Disease

AGH AEI S, MD 1, SAFAEE-ARDEKANI GR, MD 2                                                  TUPE RS and AGT E VV

1- Jahrom Uni versity of Medical Scienc es, School of Medicine, J ahrom, Iran              Agharkar Research Institute, G.G. Agar kar Road, Pune 411004, India
2- Shiraz Uni versity of Medical Scienc es, School of Medicine, Shiraz, Iran
                                                                                           Background: Zinc (Zn) is required as a catal ytic, struc tural and regulatory ion for
Background: Des pite rec ent significant therapeutic advances, vitiligo remains as         enz ymes, pr otei ns and transcripti on factors and is thus a key trac e element in
a clinical conundrum. T opical i mmunotherapy has been e xtensivel y tested in the         many homeostatic mechanis ms of the body. Vitami ns li ke ribofl avin, nicoti nic aci d,
treatment of various der matologic disorders especiall y those believed to have an         thiamine, folic acid and asc orbic aci d have functional groups capable of forming
immunol ogic basis. The aim of this study was to evaluate the role of topical DPCP         complexes with Z n. H owever, the interacti on of water-soluble vitamins with Zn has
in the treatment of vitiligo.                                                              not rec ei ved much attention.
Methods: Nineteen pati ents with limited vitiligo lesions were enrolled in this study.     Methods: We have examined the Zn- vitamin i nter actions at a variety of conditions
After s ensitization with 2% DPCP, progressivel y higher conc entr ations beginning        like differ ent Zn c onc entrations, different cell and pr otein models and under nor mal
at 0.001% were applied weekl y for 6 months to depigmented s kin. The maximum              and oxidati ve stress (OS) conditions . The interactions were studied in vitro, by
concentrati on of DPCP in acetone was 2%.                                                  using erythroc ytes under deficient, normal and excess Zn states .
Results: Thirteen of 19 patients were eval uated at the end of 6 months. Four              Results: Under Zn-deficient state, thiamine significantl y enhanc ed the erythroc yte
patients with foc al vitiligo, 3 pati ents with vitiligo vulgaris, and 3 patients with     Zn uptakes (p<0.05), whereas asc orbic acid and riboflavin inhi bited it (p<0.05). In
segmental vitiligo showed marked (grade 3) repigmentati on.                                another s tudy, an i n vitro erythroc yte Z n uptake was compared among healthy and
Conclusion: Marginal and central repigmentation with hyperpigmented borders                diabetic subjects and it was found that Zn uptakes of healthy subjects were 17-
was seen in the majority of lesions. Further controlled trials should be undertaken        52% higher than those for diabetic subjects. Further more, er ythroc yte s uper oxide
to evaluate the us e of topical DPCP in vitiligo, as this preliminar y study is            dismutase, pl asma ascorbic acid and status of ribofl avin were negati vel y
encouraging.                                                                               correlated with Zn uptakes in healthy s ubjects (p<0.01). These i nterac tions were
                                                                                           also studied i n precision c ut rat li ver slices, where it was found that folic acid
                                                                                           showed inhibitor y effect on Zn uptake under both normal and OS conditions as
                                                                                           seen by dos e res ponse c ur ves. Ascor bic acid showed marked enhancing effec t on
                                                                                           Zn uptake under OS. T hese in vitro interacti ons were c onfirmed in vi vo usi ng male
                                                                                           Wistar rats . The 21 days old rats were used to exami ne the effec t of ni acin
                                                                                           suppl ementation on Zn abs orption under c hronic OS gener ated by tert-butyl hydro
                                                                                           peroxide at a dos e of 0.2 mM/Kg body weight. Niaci n supplementation increased
                                                                                           the Z n absor ption and i mproved antioxidant enz yme pr ofile. T he albumin being the
                                                                                           major Z n carrier protein i n plas ma and the albumi n bound Z n (ABZn) c omprises
                                                                                           80% plas ma Zn. Folic acid and thiamine significantl y enhanc ed the ABZ n
                                                                                           (p<0.010), while nicoti nic acid i nhibited Z n bi nding to al bumin.
                                                                                           Conclusions: These res ults collecti vel y suggest that vitamins are pl aying an
                                                                                           important role in distribution of circulating Zn among albumi n, blood cells and liver
                                                                                           and gi ving a new di mension to their functi onality i n Zn metabolism in health and
                                                                                           diseas e conditions.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                               Page A-5
                                                        EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                             Celebrating the 100th Anni versar y of the
                                                                Nobel Prize Award to Paul Ehrlich
                                                                  Nürnberg, October 3-5, 2008
Association of Fluoroquinolone and ESBL-Resistance in Gr am-Negative               Clinical Applications of Ghrelin
Organism s from On colog y Patients of Lagos Universit y Teaching Ho spital
(LUTH), Nigeria                                                                    AKAMIZU T1, IWAKURA H 1, ARIYASU H 1, KANGAWA K1 ,2
                                                                                          1
AIBINU, IE, ADENIPEKUN, EO, NWAKA, DUC, AD ELOWOTAN, AO, AJEKIGBE,                         Kyoto Uni versity, Kyoto, J APAN; 2 National C ardiovasc ular Center, Suita,
AT. ADEYEMI, OF AND ODUGBEMI, TOLU                                                        JAPAn.

College Of Medicine, Uni versity Of Lagos . Nigeria                                       Background: Ghrelin is a 28-amino-aci d peptide hormone that was discovered in
                                                                                          1999 as an endogenous ligand for the growth hor mone (GH) -secretagogue
Background: This study determi ned the gram- negati ve bacilli associated with            receptor (GHS-R). Ghrelin plays a critical rol e in a variety of physiological
various c anc er infections and defined fluor oquinolone susc epti bility and Extended-   processes including stimulati on of GH secretion and food. T hes e ac tions of ghrelin
Spectr um Beta-Lactamas e pr oduc tion in isolated strains .                              shoul d be invaluable for the development of novel treatments and diseas e
Methods: Materials for research wer e bl ood c ulture, urine, aspirates, fluids and       diagnostic techniques.
swabs from canc er wounds. Samples were cultured and organisms isol ated were             Methods: Study 1: we attempted to eval uate the clinical response to repeated
determined usi ng API s ystem (Bio-Merieux). Anti microbial resistanc e was               ghrelin administration in patients with anorexia c aused by functional disorders.
estimated by the disc diffusi on method accordi ng to NCCLS/CLSI                          Subjec ts were incl uded in this study, who 1) are diagnos ed with functional
recommendations and ESBL detection was c arried out using the Double Dis k                anorexi a, 2) ar e lean, and 3) exhi bit decreas ed FI. Subjects rec ei ved an
Synergy Test method                                                                       intravenous infusion of 3 microg/kg ghrelin for thirty minutes t wice a day for two
Result: Of the 103 str ains isolated 22 (21.4%) were found to be resistant to onl y       weeks. We inves tigated the effects of ghrelin admi nistration on food i ntake (FI),
ciprofloxacin. Only 1 of thes e resistanc es to ciprofl oxacin was obs erved to have an   appetite, hor mones, and metabolic parameters. Study 2: we evaluated the effects
accompanying producti on of ESBL . Of the 7 isolates that had resistanc e to a            of ghrelin admi nistration on physical perfor mance and body compositi on in
combinati on of two fl uoroquinolones, 2 (28.6%) were found to be ESBL-pr oduc ers.       patients undergoing electi ve total hip replacement (THR) as treatment for
Cross resistance to the 3 quinolones tested, occurred in 40 (38.8%) of the strains        osteoarthritis (OA). Thirty-two patients were assigned to two groups of si xteen
isolated. The strains in this group were obs erved to be associ ated in most of the       subjects each; the ghrelin group recei ved intravenous injections of 2 microg/kg
cases with MDR [35 (37.5%)] and produc tion of ESBL [16(41%)]. This group was             ghrelin, while the plac ebo group r ecei ved vehicle alone. Subj ects rec eived twice
obser ved to be predominant amongst strains of E.c oli, Pseudomonas spp and               daily injecti ons for three weeks beginning one week before surgery.
Klebsiella s pp.                                                                          Results: Study 1: Ghr elin administration tended to increase dail y F I in c omparison
Conclusion: Cross-resistance to fl uoroquinolones has emerged amongst thes e              to levels before and after completion of treatment, although this differenc e that
clinical isolates and more worrisome is its ass ociation with ESBL-Pr oduction and        was the primar y endpoint of this study did not reac h statistical significance. Hunger
Multidrug Resistance. Multi-drug resistance may be one of the contributing factors        sensation was significantly el evated at the end of drip infusion. Study 2: While
to the high mortality rate amongst these group of patients reported in devel oping        ghrelin significantly i ncreas ed lean body mass after the three- week i njection
countries. Antibi otic resistanc e s urveillance is thus of utmost i mportance in         period, it di d not affec t muscl e strength or wal king ability. Significant decreas es in
contributing to the reduc tion of the high morbidity and mortality rate reported          fat mass and GH res ponses to ghrelin injection wer e also obser ved.
amongst cancer pati ents i n this part of the world.                                      Conclusions: Ghrelin treatment has sti mulator y effects on appetite in patients
                                                                                          with functional anorexi a. On the other hand, ghrelin admi nistration did not provide
                                                                                          any favor able effect on physical perfor manc e in patients with OA undergoing THR,
                                                                                          despite increased l ean tissue res er ves. F urther studies remain necess ary to
                                                                                          confirm the efficac y of ghrelin treatment for anorexia- and catabolism/aging-related
                                                                                          disorders.




Effect of Diabetes Mellitus on Ph armacokin etics (PK) and                                Chemotherap y of advan ced colorectal car cinoma und er hemodialysis
Pharmacod ynamics (PD) of Immunosuppressive agents: Ciclosporin,
Tacrolimu s and M ycophenolic Acid                                                        AKIYAMA S, T ANAKA M
                 1            1                  1              1            2
AKHLAGHI F , CHITNIS SD , MENDONZA AE , PAT EL CG , GOHH RY                               Mitsukaido Sakura Hos pital, Mits ukaido, IB, J apan.
1
 Clinical Phar macoki netics R esearch Laborator y, Uni. of Rhode Island, Kingston,       Background: Recent progress of hemodial ysis therapy (HD) has prolonged the
RI, USA; 2Di vision of Transpl antation, Br own Uni. M edical School, Providence, RI,     life span of patients sufferring from end stage renal dis ease (ESRD) and it
USA.                                                                                      increased the complication of malignant tumors. It is reported that the prevalenc e
                                                                                          of c olorectal cancer in HD patients is higher than that in a c ontrol popul ation. Saltz
Background: A large percentage of kidney trans plant recipi ents are diabetics but        regimen; combination therapy of irinotec an, 5-fluorouracil, and l euc ovorin is wi del y
little is known on the effect of diabetes on the disposition or effect of                 used for the treatment of metastatic color ectal cancer after the large randomized
immunos uppressi ve agents .                                                              phas e III trials. Chemotherapy of HD patients is difficult bec aus e ther e are few
Methods: The PK and PD studi es included 42 and 32 stable kidney trans plants,            phar mac okinetic data of most c ytotoxic agents muc h less combination
sampled for 12 or 2 hours post dose, respecti vely. Approximatel y 50% of patients        chemotherapy. We enc ountered a patient of c olorectal carcinoma on HD. It was
were diabetic (D) that wer e demographicall y matched with non-diabetic (ND)              decided to give combination chemotherapy with monitoring the pharmacokinetics
controls.      Immunos uppr essive agents‘ total, unbound and metabolite                  of the pati ent.
concentrati ons were measur ed by LC-MS/MS. Mar kers of T-cell acti vity (ATP             Methods: CPT-11 was administered j ust after HD at a dose of 50 mg/m2 by 90-
concentrati on in CD4 c ells, intracellul ar cytokines IL-2, IFN-          -              min intravenous infusion; followed by l-LV 10 mg/m2, administer ed over the course
cells) and B-cell activity (co-sti mulator y protei ns CD54 [ICAM-1], CD86 [B7.2] and     of 15mi n; and 5-FU (400 mg/m2), gi ven by bolus i ntravenous injecti on after l-LV.
CD95 [Fas antigen] in stimul ated CD19) were determined. Inosine                          Three drugs were given on days 1, 8 and 15 of a 35-day c ycl e. Blood samples
monophos phate dehydrogenas e (IMPDH) acti vity was als o meas ured in s ome              were c ollected before administrati on and at 0.5, 4, 12, 24 hours after
patients.                                                                                 administration. Plas ma concentration of CPT-11, SN-38 and 5-FU were compared
Results: Phar macoki netics: As summarized in the Tabl e, both ciclosporin (CsA)          to those in referenc e c ontrol values without ESRD. The pl asma concentration of
Cmax and AUC was reduc ed in Ds and CsA fr ee frac tion was higher. Tacrolimus            CPT-11, SN-38 and            5-FU were anal yzed using high performance-liquid
absorption was delayed with no effect on the overall AUC. Diabetes delayed                chromatography (HPLC).
mycophenol ate mofetil (MMF) absorption rate but not the enteric coated Na-MPA.           Results: The pharmacokinetic results for each post administration val ue were not
The concentrati on of s ome metabolites of ciclos porin or MPA was l ower in Ds.          statisticall y different compared with the data of normal renal func tion. As for side
                  ics
Phar macodynam : The mean ± SD of ATP concentrati on was 298 ± 78 ng/mL in                effec ts, he experienced grade III hematol ogical toxicity, whic h was easil y
D and 359 ± 86 in ND patients (P=0.021). Expression of intracellular IL-2 in CD3          controlled with G-CSF.
cells was l ower in D (mean floresc ence intensity: D 104 ± 42, ND 145 ± 74;
P=0.03). Also CD95 c ell surfac e expression on B-cells was lower in Ds (D: 73 ± 28       Time after administrati on (hrs) 0       0.5                 4              12       24
ND: 114 ± 68; P=0.01) however despite a trend, expr ession of CD54 or CD86 did            CPT-11 ( ng/ml)                    0     820-845             610-790        340      95
not differ. IMPDH acti vity was 17.5  2.8 vs. 46.6  2.5 nmol XMP/h/µgprotein, in D
and ND, respecti vel y (P<0.0001).                                                        SN-38    ( ng/ml)                  0     8.4-12.0            7.5-8.2        5.2      4.4
Conclusions: Diabetes variabl y affect the PK/PD of i mmunosuppressi ve agents.           5-FU     ( ng/ml)                  0     7,200-12,300        0              0        0
Adjusting i mmunosuppressi ve dose guided by the PD mar kers may prove us eful in
dose indi vidualizati on of i mmunosuppressi ve agents in diabetics.                      Conclusions: Thes e data suggest that Saltz regimen can be feasibl e for
                     Absorption rate   Exposure       Free fraction    Metabolites        colorectal c anc er patients rec eivi ng HD without dose reducing
                                        (AUC)              (fu)
    Cyclosporine        Delayed           ?             Increased     ? AM1, ? AM19

    Tacrolimus          Delayed            ?               ??         ? Clearance,
                                                                      metabolites??
    Mycophenolic     Delayed as MMF        ?               ?             ? MPAG
    Acid              ? EC-Na-MPA                                     ? Acyl-MPAG




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                 Page A-6
                                                            EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                 Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                      Nürnberg, October 3-5, 2008
In vitro Activities of Extr acts from N aturals Plants on the Human F ilar ia Loa      The Role of Ancient Iraqi People (Sumerian, Assyrian , Bab ylonian and
loa                                                                                    Arabian) in the Development of Medicin e as Viewed b y W estern W riters

MENGOME LE1, FEUYA TCHOUYA GR 1, SOUZA A1 , NSI EMVO E1, AKUE JP2                           AL MOSAW I AJ
1
  Institut de Pharmac opée et de M édeci ne Traditionnelle (IPHAMETRA), Libreville,         University H ospital i n Al Kadhimi yia ,Baghdad,Iraq.
Gabon;
2
  Centre International de Rec herches Médicales de Franceville (CIRMF),                     Background: The region of Iraq was historicall y known as Mesopotamia and was
Franceville , Gabon.                                                                        home to the world's first known civilizati ons These ci vilizations produced some of
                                                                                            the earliest writing and s ome of the first scienc es and medicine of the world; henc e
Background: Loa loa is a human filaria endemic to Wes t Africa. Current drugs               its common epithet, the "Cradle of Ci vilization". T he ai m of this paper is to study
such as Diethylcar bamazine (DEC) and Iver mecti n induce s evere adverse                   the views of the distinguished western writers regarding the role of anci ent people
reactions li ke enc ephalitis and pati ent death in high microfilaremic indi viduals.       of Iraq in the development of Medicine.
Therefore there is a need for new drugs. Plants are known as potential                      Methods: the views of the disti nguished western writers regarding the role of
nematocids, but no study has been c arried out to examine their effect on Loa loa.          ancient people of Ir aq in the devel opment of Medicine wer e reviewed and studied.
Aim: to screen nematocidal plants us ed in traditional medici ne for their filaricidal      Results: Samuel Noah Kr amer considered the consider ed the Sumerian
activity.                                                                                   phar mac ological tablets as the first pharmac opoeia. In summarizing the effects of
Methods: Plants wer e collected acc ording to their traditi onal usage an identified        Arabian in the development of medicine Stubb and Bleight stated in 1931‖With
using botanic criteria. Extracts were obtai ned by using different organic s ol vents       Arabs began the real cr aft of apothec aries‖. Me yer hof i n 1944 wrote in his book
(Dichloromethane, Methanol, Water). T he resulting products wer e dissol ved at             ―Pharmacolog y during the golden age of Arabian Medicine‖ ―The establishment of
1mg/ml in Dimethylsulfoxi d (DMSO). Co cultures of extract and filaria were made            hospitals was originated by Arabians‖
in duplicate using a 96 well cell culture plaque containing LLCMK2 cells, MEM               Conclusions: The anci ent Iraqi peopl e played an important r ole in the earl y
medium with 10% F etal calf s erum and anti biotics. 5 Loa loa microfilaria were            devel opment of medicine.
added i n each well and plaques were inc ubated at 37°C in 5% CO 2 atmos phere
for 120 hours. The effects of plant extracts were measured by obser ving the
motility and mobility of filaria in wells containing different concentrations of extr act
compared to negati ve controls (no extract or drug in the well) and positi ve controls
(DEC or Ivermectin in the well ) at different c onc entrations .
Results: Extracts from s even pl ants s peci es of the Euphorbiac eae,
Caesalpi niacae, Compositeae, Mi mosac eae, Ochnac eae, Rutac eae, Annonac eae
famil y had microfilaricidal acti vity at conc entrations of 95.6µg and 47.8µg, while
plants from Lecytidaceae had no effects on filaria at thes e concentrations.
Conclusion: Crude extracts of s ome plants us ed i n traditional medicine have in
vitro effect on the human filaria Loa loa.




Retargeting anticancer drugs to drug r esistant cancers b y using polymer                   Novel reporter probes for HSV1-tk gene expr ession
Biotransport technolog y. Clinical proof of the concept
                                                                                            ALAUDDIN MM
ALAKHOV VY
                                                                                            Center for Advanced Biomedical Imaging Research (CABIR), The U niversity of
Suptarek Pharma Inc.,Montr eal, Canada                                                      Texas M D Anderson C anc er center, Houston, T X, USA.

Background: Anthrac yclines are amongst the most wi del y us ed anticanc er agents          Herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene is being used as a
in man, but are limited by toxicity c onsiderations, as well as by inherent or induced      suicide gene for gene therapy of cancer. Cancer cells are transduced with a retroviral
drug resistanc e. We have recentl y disc overed that nonionic bloc k c opol ymeric          vector carrying HSV1-tk gene. The gene expressed in the proliferating cells produces
surfactants, i.e. hydrophobic pol yethylene oxide pol ypropylene oxide bloc k               the HSV1-Tk enz yme that selectivel y phosphor ylates a guanosine derivati ve,
copol ymers (Pl uronics) can c onsiderabl y reduce drug resistance of various tumour        ganciclovir, to its monophosphate. The ganciclovir monophosphate is then converted to
cells to anthrac yclines and other c ytotoxic drugs. Following this fi nding, we have       its di-phosphate form by the c ellular kinases and/or HSV1-TK, and finally to the
devel oped a Pluronic-based formulation of doxorubicin (SP1049C) that is                    triphosphate form by cellular kinases. The ganciclovir tri-phosphate becomes a pro-
thermodynamicall y stable, safe and provi des doxorubicin with high effic ac y agai nst     drug and inhibits DNA polymerase, thus kills the tumor cells.
both drug resistant and drug sensiti ve tumours.                                            Malignant tumors have been successfully treated in animal models with suicide gene
Methods: Safety and pl asma pharmacokinetics of SP1049C were evaluated in                   therapy using HSV1-tk gene and ganciclovir. However, clinical results with this method
phas e I clinical trial that was c urried out in 26 advanc ed c anc er pati ents ; and      showed that gene delivery to the tumor cell in human was not sufficient. Therefore, an
efficac y of the pr oduc t was tested in phas e II clinical trial, i n 21 patients with     in vivo method to assess the HSV1-TK enzyme activity after gene transfer is required
metastatic adenocarcinoma of the esophagus.                                                 for the optimization of gene delivery and establishment of treatment efficacy. Positron
Results: The r esults of phase I trial demonstrated comparable s afety and PK               emission tomography (PET) is a non-invasive modality for in vivo imaging of HSV1-tk
profiles of SP1049C to that of doxor ubicin. The phas e II trial reveal ed that the         gene expression using reporter gene and reporter probe, and can provide repeated
produc t has a high anticanc er activity. The over all responc e rate to SP1049C was        and quantitative assessment of the expression of genes in tissues and organs.
47%, (95% CI: 24 - 71), and medial sur vi val was 10 months, whic h compares                Non-invasive imaging of transgene expression invol ves the appropriate combination of
favourabl y with other most acti ve single agents tasted in this i ndicati on.              a reporter gene and a reporter probe. Model systems have been established and
Conclusions: The performed clinical studies have ass uared a further clinical               validated by HSV1-tk gene as a reporter gene, and radiolabeled pyrimidine nucleoside
devel opment of SP1049C. At pres ent it is entering an expanded international               and ac ycloguanosine analogues as reporter probes. Reporter probes can be us ed (i) to
clinical phase II/III efficac y program including a single 300 patient pivotal phas e III   image vector targeting and level of HSV1-tk expression, (ii) to image the regulation of
trial in patients with upper gastrointesti nal trac t.                                      endogenous genes and signal-transduction pathways; and (iii) to monitor and
                                                                                            quantitatively assess the expression of a second transgene that is cis-linked to the
                                                                                            reporter gene by an internal ribosome entry site sequence.
                                                                                            Various radiolableld reporter probes have been developed during the past decade, and
                                                                                            these are primarily purine and pyrimidine nucleoside analogues, such as 18F-FHPG,
                                                                                            18
                                                                                               F-FHBG, 18F-FMAU, 18F-FIAU, 18F-FEAU, 18 F-FFAU etc., and also radioiodinated
                                                                                                                                          18        18             18
                                                                                            analogues of FIAU. Among these probes, F-FHBG, F-FIAU and                 F-FEAU have
                                                                                            been studies extensively. 18F-FHBG and 18F-FEAU have been recognized as ideal
                                                                                            probes for PET imaging of HSV1-tk gene expression. We have studies these probes in
                                                                                            the animal models and demonstrated that both 18 F-FHBG and 18 F-FEAU have efficacy
                                                                                            for imaging of HSV1-tk gene expression, and they can be used selectivel y in PET
                                                                                            imaging of native and mutated HSV1-tk gene expression. Thus 18 F-FEAU is suitable for
                                                                                            PET imaging of the native HSV1-tk gene expression; and 18F-FHBG is suitable for PET
                                                                                            imaging of mutated HSV1-tk gene expression. This presentation will focus on these
                                                                                            novel probes used for PET imaging of HSV1-tk gene expression with an emphasis on
                                                                                            their application in pre-clinical animal models and human studies.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                              Page A-7
                                                   EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                         Celebrating the 100th Anni versar y of the
                                                            Nobel Prize Award to Paul Ehrlich
                                                              Nürnberg, October 3-5, 2008
Drug-Drug and Drug-Biomolecule Photoinduced R eactions in Preparations         Protective Effect of Dim ebon on the TNF-alpha–Induced Lipid Disord ers in
for Topical Use                                                                Mice Brain

ALBINI A1, FASANI E1, DONDI D 2, SERPONE N,1 FAGNONI M 1                                       ALESSENKO AV1, KARAT ASSO YO1., SHEVTSOVA EF 2, *SHINGAROVA LN 1,
                                                                                               BACHURIN SO2
1                                      2
    Dep. Org. Chem., Uni v. PAVIA; D ep. Gen. Chem., Uni v. PAVIA, Pavi a, Ital y.
                                                                                               1
                                                                                                Institute of Bi ochemical Physics of the Russian Academy of Scienc es, Mosc ow,
Background: There is a growing concern for the advers e effec ts c aused by                    Russia, 2 Institute of Physiologicall y Acti ve Substanc es, Russi an Academy of
expos ure to indoor/outdoor light after assumpti on of dr ugs. The first concern is            Sciences , Chernogolovka, Russi a.
obviously for topical preparations. The rather sparse literature on phototoxicity
rarely examines the chemical reacti ons underlying to the biol ogical effect. In               Background: Dimebon (Dimebolin) is an antihistamine drug whic h has been used
general, there is a feeling that oxygen acti vati on is the main mechanism, but the            clinically in Russia since 1983. Recently Dimebolin has attr acted renewed inter est
example bel ow show that this is not always the c ase.                                         after being shown to have positi ve effects on persons suffering from Alzhei mer‘s
Methods: In vitro studies wer e carried out by irradiating aqueous s olution of the            diseas e. Ani mal studies have shown that di mebon operates through multi ple
drugs under indoor/outdoor light at a meas ured flux. T he photoproducts were                  mec hanis ms of action, both bloc ki ng the acti on of neur otoxic beta-amyloid proteins
isolated and the s tructur e deter mine d. The explorati on was repeated i n the               and inhi biting L-t ype c alcium channels, modulating the acti on of AMPA and NMDA
presenc e of heteroc ycles mi micki ng the behavi our of nucleotides. Time-resol ved           glutamate receptors, and may exert a neur oprotecti ve effec t by bloc king c ytotoxic
studies for the detection of chemical intermediates wer e also performed. Thes e               signals induced by proinfl ammator y citokines suc h as TNF-
studies and the structure of the products allowed to propos e viabl e mechanis ms,             believed to play a central r ole in Alzheimer's diseas e. Inflammator y respons e
here conc erning some UV filters used in sunscreens as well as some bactericides               induc ed by TNF-alpha suggests that this c ytokine affects the phos pholipid
with eye-toxicity.                                                                             metabolism and subsequent production of eicosanoids , ceramide, and ROS that
Results: Dibenzoyl methanes (DBM, in the tautomeric form under the applicative                 may potentiate brai n injur y.
conditi ons) are a virtuall y constant c omponent of commercial sunscr eens in view            Methods: This study included 65 male mice (weight: 20  2 g, aerage  SD). TNF-
of the high absorpti on in the UV-A. T hes e products are quite photostable but are            alpha (10mkg per mous e), dimebon (0,2 mg/kg ) and their combinati on were
often associ ated with c ynnamates in the preparations, because the latters abs orb            injected to mice i nterperitoneal y. Changes in level of phos pholipi ds molec ular
the UV-B. However, if the combinati on of the two filters is expos ed to light, both           species (phosphatidylcholine, l ysophosphatidylc holine, phosphatidylethanolami ne,
are rapidl y cons umed via a photoc ycloaddition reaction. Furthermor e, irradiated            sphingomyelin) and galactos ylceramide in hippoc ampus , cer ebellum and cerebral
DMBs photo-add to a variety of molecul es pres ent in the s kin, e.g. to fats.                 cortex within 30 mi n, 2, 4 and 24 hours after injection were detected by chromato-
As for antibacterials we refer here to fluoroquinolones used in eye drops, suc h as            mass-spectrometr y.
lomefloxacin (LOM), and to oxaz olidinones suc h as linez olid, known to c aus e               Results: Maximal c hanges in phospholipids and galactos ylc erami des contents of
damage to the eye. In both cases a fas t photoinduc ed decomposition takes plac e,             different mol ecular s pecies after single TNF-alpha administrati on wer e found in the
with liberation of fluoride and formation of an aggressive i nter mediate (an ar yl            hippocampus, and were less expressed in the cerebral cortex and c erebellum after
cation) that adds to a various c ompounds, including elec tron-rich heter oc ycles             24 hours. Dimebon itself did not induce changes in lipids spec trum in br ain
considered as a model of nucleoti des, but not to water.                                       sections , but protec ted lipids against disorders induced by TNF-alpha i n mice
This negati ve effect support that exposure to light should be minimiz ed (e.g. LOM            brain.
has been found to be genotoxic), but also suggest that fl uorina ted heter oc ycles            Conclusions: Modern strategies in the s earch of new therapeutic approaches are
may be consi dered for a novel type of photodynamic therapy bas ed on the                      based on the multitarget properties of new drugs. According to our results TNF-
selecti ve additi on to biomol ecules by photoproduc ed ar yl cati ons, veritabl e magic       alpha may ser ve as a new target for di mebon. Dimebon preventing lipids
bullets.                                                                                       disorders in brain i nduced by TNF-alpha might have a positi ve anti-inflammator y
                                                                                               effec ts, pr eventing the negati ve r esponse of ner ve cells to the pathological
                                                                                               process.




The Role of cell membr ane lipid environment in antigenic peptide structure-                   Protective effects of Nigella sativa extract and its components against
function                                                                                       chromium VI-induced toxicit y in Nile tilapia (Oreo chromis niloticus) and
                                                                                               zebrafish (Danio rerio)
                1, 2
                       , YU, H.F. 1, 2 and BOMSEL, M.   1, 2
ALFSEN, A.
                                                                                               KHALIL WKB1**, AL I Fagr K 2, BELATTAR N 3, SENATOR A2, ABDEL-WAHHAB
1                                                                                                4**
 Entr ée muqueus e du VIH et Immunité muqueuse; D épartement de Bi ologie                      MA
Cellulaire, Ins titut Cochi n, Uni versité Paris Descartes, CNRS (UMR 8104), Paris,
France; 2 Inser m, U567, Paris.                                                                1
                                                                                                 Cell Biology D epartment, 2Water Polluti on Department, 4F ood Toxicol ogy &
                                                                                               Contaminants Department, Nati onal R esearch Centre, 12622 Dokki, C airo, Eg ypt
                                                                                               2
Background : The cell membranes c ontrol the extra- and intr acellular exc hange.                Laboratory of Applied Biochemistr y, Biolog y Department, F aculty of
The lipid struc ture, their organization in the membrane and around membrane                   Sciences , Ferhat Abbes Uni versity, 19000 Sétif, Algeria.
proteins but also their dynamics i n the two dimensions of the membrane are all
essential characteristics of eac h cell type, for their selecti ve role in the cell traffic.   Background: Chromium is an incr easing h ealth c onc ern for aquatic
The aim of our s tudy was to address the role of the lipids on an HIV-1 vaccine                environments, however, the mechanis m of c hromi um toxicity i n aquatic s pecies is
epitope structure and on the inter action with target cell membranes : either CD4+             yet unknown. Hexavalent chr omium VI (CrVI) is the dominant toxicant at some
T cells or epitheli al cells.                                                                  Superfund sites within Egypt.
Methods: Biophysical, bi ochemical and biol ogical methods have been used.                     Methods: The aim of the current study was t o evaluate the genotoxicty potenti al
Pepti des deri ved from HIV-1 glyc opr otein envelope gp41 were c hemicall y                                                                      is
                                                                                               of chromi um VI (CrVI) in Nile tilapia (Oreochrom nilotic us) and zebrafish (Danio
synthesized or produced as recombinant pepti des. To study lipid dynamics and                  rerio) using semi-quantitati ve revers e-transcription polymerase chain reaction
domain organization due to protein interacti on, liposomes of different lipid                  (RT-PCR), and possible protecti ve effects of N. s ativa. The test fis h within the two
composition, mimic king either the viral membrane or target cell membranes were                species were divi ded into eight groups and treated with Cr(VI) alone or in
chemically obtained and fl uoresc enc e resonance energy transfer (FRET) has been              combinati on with the crude extract of N. sativa, N. sativa oil, or its deri vati ve
used. Nuclear Magnetic Resonance (NMR) allowed defining peptide s olution                      thymoquinone for 30 days.
structure in presence of micelles . Bindi ng of known HIV-1 antibodies to the                  Results: The semi-quantitative RT-PCR results indicated that treatment with
epitopes inserted i nto lipos omes wer e meas ured by enz yme linked i mmunoass ays            Cr(VI) at 4.37 and 1.75 mg/l for tilapi a and z ebrafish, res pec tivel y resulted i n a
(ELISA).                                                                                       significant increas e in hepatic and br ain mRNA level of cytoc hrome 450 gene
Results: Different pepti des from cons erved regions of the HIV-1 envelope                     famil y including CYP1A2, CYP3A and CYP2E1 in both fish speci es compar ed to
glycoprotein gp41 have been studied, includi ng: 1) P1, the minimal Membrane                   control group. Moreover, Cr(VI) was found to induce s evere histological changes
Proxi mal Region (MPR) that permits interaction with muc osal galactos yl ceramide             in liver, brai n and gills of the tested fish. On the other hand, the combi ned
HIV-receptor and c ontains epitopes recogniz ed by gp41-specific, broadl y                     treatment showed that mRNA level of CYP1A2, CYP3A and CYP2E1 decreased
neutralizing IgGs, 2F5 and 4E10, and 2) P5, P1 extended at its N-terminal by a                 significantly i n the groups treated with Cr(VI) plus N. sativa oil or thymoquinone
calcium binding site. The alpha-helical struc ture of the peptides on DPC micelles             compared to the groups treated with the crude extrac t or Cr(VI) al one
appeared pH-dependent. P1 was deri vatiz ed with phos phatidylethanol amine (PE)               accompanied with a significant i mprovement in the histological picture of the liver,
at its C-ter minal and ins erted into lipos omes of varied lipi d composition, thereby         brain and gills. However, N. s ati va was found to be more effecti ve.
enabling P1 to move laterally within liposome bilayer. The Kds of both 2F5 and                 Conclusion: It could be concluded that N. sati va is a promise c andidate against
4E10 IgGs meas ured on viral lipos ome and infectious virus are si milar in the nM             DNA damage res ulted from the exposure to different environmental pollutants.
range and much lower than for the binding of the free P1 pepti de. For P5, the role
of cell calcium on its s tructure and its antiviral pr operties when in interaction with       ** Corresponding author: Fax: +202-3337-0931, E-mail: wagdykh@yahoo.com
lipids was shown essential.                                                                    ** Corresponding author: Fax: +202-3337-0931, E-mail: mosaad_attia@yahoo.com
Conclusions/Significance: Thus, the defined lipid environment of MPER-deri ved
peptides and environmental c onditions (pH, c alcium, ...) appear as essential for
their structure and therefore for the design of an i mmunogen inducing broadl y
neutralizing anti bodi es to HIV-1, and also microbicides.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                     Page A-8
                                                         EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                     Nürnberg, October 3-5, 2008
Synthesis and effect of silver nanoparticles on the antibact erial activity of        Effect of gentamicin on serum digoxin level in patients with congestive h eart
different antibiotics against Staphylococcus aureu s and Escherichia coli             failure.

ALI F AKHIMI AND AHMAD REZA SHAHVERDI                                                        ALKADI HO , MAJED AN, YAHIA AR

Department of Phar maceutical Biotechnolog y and M edical Nanotechnol ogy                    Univ. Sana'a , Sana'a, Yemen
Research C enter, Fac ulty of Pharmac y, T ehran Uni versity of Medical Sci ences ,
P.O. Box 14155/6451, Tehran, Iran                                                            Background: Gentamicin is frequently us ed to tr eat i nfec tious diseases i n patients
                                                                                             receivi ng digitalis therapy. The ai m of this study is to evaluate the effec t of
Abstract                                                                                     gentamicin on s erum digoxin level.
               Silver nanoparticles have been known to ha ve inhibitory and                  Methods: Twenty-four diabetic patients and patients with congesti ve heart failure
bactericidal effec ts. Resistanc e to antimicrobi al agents by pathogenic bacteria has       and twelve normal healthy volunteers were enrolled in this study. T he patients
emerged in rec ent years and is a major health probl em. The combi nati on effects of        received digoxin treatment 0.25 mg/day. Gentamicin in a dose of 80 mg i.m. twice
silver nanoparticles with the antibac terial acti vity of antibiotics have not been          a day for 7 days was prescribed for thes e patients to treat ches t infec tion. Serum
studied. H ere, we report on the s ynthesis of metallic nanoparticles of silver usi ng a     digoxin and creatinine levels were determi ned before and after gentamicin
reduction of aqueous Ag+ ion with the c ulture s upernatants of Klebsiella                   administration.
pneumoni a. Also in this investigation, these nanoparticles were evaluated for their         Results: Gentamicin induc ed a significant increase in serum digoxi n level of
part in increasing the anti microbial acti vities of different antibiotics agai nst          diabetic pati ents and patients with congesti ve heart failure. Serum creatinine level
Staphyloc occus aureus and Escherichia coli. The antibacterial acti vities of                increased significantly before and after i.m. i njection of gentamicin.
penicillin G, amoxicillin, er ythromycin, clindamyci n and vancomycin were                   Conclusions: The pres ent study indicated that increase s erum digoxin level when
increased in the presence of silver nanoparticles agai nst both test strains. The            combined with gentamicin s hould be c onsidered a ris k factor for digitalis toxicity.
highest enhanci ng effects were obs erved for vanc omycin, amoxicillin and penicillin
G against Staphyl ococc us aureus.




Controlling of Systemic Absorption of Gliclazide through Incorpor ation into                 Pharmaceutical An alysis using Sequential Injection Analysis (SIA): A review
Alginat e Bead s.                                                                            of present Applications and future possib ilities

AL-KASSAS RS1, AL-GOHAR Y OM 2, AL-FAADHEL MM 2                                              Al-KINDY SMZ, SU LIMAN FO
1                                       2
    Univ. AUK, Auc kland, New Zealand; U niv. KS, Riyadh, Saudi Arabia                       Sultan Qaboos Uni versity, Muscat, Oman

Background: Gliclazide is an oral hypoglyc aemic sec ond generation s ulfonyl urea           Background: T he need for automation in pharmaceutical analysis has lead to the development of
                                                                                             rapid and sensitive detection methods. Our aims are to develop robust and simple sequential
drug whic h is useful for a long term treatment of non ins ulin dependent diabetes           injection analysis (SI) methods for the essay of pharmaceutical samples and to investigate various
mellitus. However, the abs orption rate of gliclazide from gastrointes tinal tract is        luminescence detection modes in combination with SIA system. The approaches compared will be
slow and varied among the subj ects. Several studies on healthy volunteers and               spectrophotomety, micellar enhanced fluorescence, and lanthanide enhanced luminescence and
diabetic patients reveal ed that the ti me to reac h plas ma c onc entration (t max )        chemiluminescence (CL).
ranged from 2 hours to 8 hours following a single oral administration of 80 mg of            Method: T he development of analytical protocols for monitoring of various drugs is presented. T he
                                                                                             chemical system is developed based on the structural properties of the given drug. When
gliclazide tablet. Aims : (1) to develop controlled releas e for mulations of gliclazide
                                                                                                                                                                                       ing
                                                                                             compounds lack efficient chromophores the use of a metal ion or organic derivatiz agents that
using alginate beads. (2) to study the effect of proc essing conditions on the               can form highly absorbing or fluorescent products is necessary. On the other hand lanthanides
physical characteristics of the prepared beads and the in vitro rel eas e rate of the        such as Eu(III), and T b(III) are used to sensitize the luminescence of drug samples offering
drug (3) to investigate the hypoglycaemic effec t of the prepared gliclazide loaded          excellent analytical characteristics.
alginate beads on the diabetic rabbits and to compare the developed s ystems with            CL is also emerging as an efficient tool when coupled to flow techniq ues. Ru(III) complexes were
                                                                                             used to develop simple and robust assay for group of common drugs such as chlorpheniramine
mar keted conventional gliclazide tablets .                                                  (CPA) and ephedrine(EP) and some fluoroquinolone antibiotics. Several types of SI designs were
Methods: Gliclazide loaded Ca- alginate beads wer e prepared usi ng ionotr opic              employed.
gelation method. The variati on in pol ymers concentrati ons, stirring speed, inter nal      T he development of the analytical system thereafter hinges on the selection of the most suitable
phas e volume and the type of surfactants in external phase were examined                    environmental factors that result in an enhanced signal -to-noise ratio. Therefore a sy          stematic
systemic ally for their effec ts on particle siz e, incor porati on efficienc y, flow        optimization protocol must be used for this purpose. T hen the methods developed are validated
                                                                                             and compared to standard and official methods.
properti es of the beads and in vitro drug release rates . The in vi vo studies i nvol ved   Results: using spectrophotometric techniques penicillamine (PA) was complexed with Fe(II) ions in
measuring blood glucose l evel of 18 diabetic male New Z ealand white rabbits                acidic media forming blue complex that absorbs strongly at 600 nm. A linear dynamic range for
treated (6 rabbits per treatment) with the following formul ations (1) nor mal saline        the determination of PA of 25-300 ppm was obtained with sampling frequency of 50 h -1. PA and
solution (2) mar keted conventional gliclazide tablet (Gliclazide ®) (3) gliclazide          ephedrine were determined using tris(bipyridyl)ruthenium(II) as a CL reagent and potassium
loaded alginate beads .                                                                      peroxydisulfate as an oxidant in the presence of light.. Derivatization of PA and EP with aldehydes
                                                                                             produced a significant enhancement of the CL emission, leading to detection limits (LOD) of
Results: The aver age mean diameters of gliclazide beads decreas ed with                     0.1ppm for PA and 0.03 ppm for EP.
decreasi ng polymer concentration, increasi ng speed of stirring, and increasing the         Optimum conditions for the determination of BRZ in pharmaceutical formulation were 0.6 % tergitol
internal phas e volume. All prepared beads possess ed excellent flowability. The             surfactant in the presence of 0.1 M lactose.
swelling behavi our was s trongly dependent on pol ymer c oncentrati on i n the              Piroxicam (PX) and ibuprofen (IB) were assayed using lanthanide sensitized luminescence. Eu(III)
formul ation and the pH of the medium. The in vitro releas e experiments revealed            when complexed to PX resulted in a huge enhancement in the emission of the EU(III) bands..
                                                                                             which allow the determination of 100–1000 ppb of PX with LOD of 29 ppb. Recoveries of PX in
that the s welling is the mai n parameter c ontrolling the releas e r ate of gliclazide      pharmaceutical formulations and in urine samples were 100.87±1.7% and 97.57±2.0%. IB was
from the beads. In vi vo studies on diabetic rabbits s howed that the hypoglyc aemic         determined after complexation with T b (III) ions giving a detection limit of 1.0 × 10 −7 mol/L.
effec t induc ed by gliclazide loaded alginate beads was significantl y greater and          Fluoroquoinolone antibiotics eg levefloxacin ( LV), were determined using Ce(IV) ions as oxidant
more prolonged than that i nduced by the marketed conventional gliclazide tabl et            and tris(1,10-phenanthroline)ruthenium(III) as the CL reagent giving characteristic orange emission
(gliclazide ®).                                                                              with LOD of 0.02 ppm.
                                                                                             Conclusions: SIA is a powerful tool when coupled to s ensitive luminescence methods for the
Conclusions: Alginate beads can control, improve and prolong the s ystemic                   determination of drugs in the pharmaceutical formulations and in biological fluid. T his combination
absorption of the gliclazide through their muc oadhesi ve properties . This effect           results in an increased sampling frequency and an enhanced sensitivity. T he use of micelles has
results in mai ntai ning tight blood glucose l evel and improved patie nt complianc e.       lead to an increase in the solubilisation of BRZ and an improvement in the sensitivity of the
                                                                                             method. Lanthanide sensitized luminescence lead to a sensitize detection of the drugs with
                                                                                             emission in the longer wavelength of the lanthanide ion that achieve a better selectivity of the
                                                                                             methods. Future work include the development of methodology for the determination of drugs in
                                                                                             various env  ironmental samples such as round water and sewage, in order to cope with the
                                                                                             increase problem of pollution brought about by the continuous charging of drugs into the
                                                                                             environment.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                              Page A-9
                                                           EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                Celebrating the 100th Anni versar y of the
                                                                  Nobel Prize Award to Paul Ehrlich
                                                                     Nürnberg, October 3-5, 2008
Clinical Phar macokinetics of Gentamicin: Estim ation of Initial Dosing               Complexation of Itraconazole with C yclodextrins for Enh anced Solubilit y,
Param eter s in Hospit alized Patients at Ku waiti Hospital.                          Dissolution and Bio availab ilit y

AL-LANQAW I Y1, CAPPS P2 , ABUDLMALEK K3, AL-ANEZI K4, THUSU A5, AND                      AL-MARZOUQI AH 1, JOBE B1, SHEH ATTA I2 , HASSAN HA1 , HAMZA AA1
SHARMA P
                                                                                          1                                                  2
                                                                                           UAE Uni versity, Al-Ain, United Arab Emirates; Mans oura U niversity, Mans our a,
1, 3, 4, 5
     AL-Amiri Hospital, Ministry of Health, Kuwait, and 2,6F aculty of Phar mac y -       Egypt.
Kuwait Uni versity, Kuwait.
                                                                                          Background: Itrac onazole is an antifungal agent whose poor aqueous s olubility
Background: Gentamicin has a narrow range between its therapeutic and toxic               restricts its us e for the treatment of or ophar yngeal candidiasis, which is the first
blood levels. This has prompted the development and wide use of                           symptom of HIV infection. Therefore, the ai m of this study was to prepare
pharmacokinetic dosing equations in order to maximize drug safety and efficacy.           itraconaz ole-c yclodextrin i nclusion compl exes i n solid state by differ ent methods in
Dosing equations commonly rely on estimating gentamicin clearance (Cl gent) and           order to enhanc e the s olubility, dissoluti on and bi oavailability of itraconaz ole.
volume of distribution (Vd). These parameters are subject to considerable                 Methods: The formation of inclusion complexes between itraconaz ole and α-, β-,
variability‘s. The objective of this study was to: (a) develop equations for              γ-, and hydroxylpropyl-β-cyclodextrin (HP-β-CD) were assess ed using phas e
estimating Cl gent and Vd based on Kuwaiti population and (b) evaluate these              solubility tec hniques. Solid state inclusion complexes between itraconazol e and
equations by comparison with other methods in their predictive ability to estimate        cyclodextrins were pr epar ed using supercritical (SC) CO 2 and conventional
Clgent and Vd.                                                                            methods. The physico-chemical pr operti es of the products were c harac terized by
Methods: Clgent and Vd were calculated in 47 patients (group 1) using the                 UV, DSC, FTIR, PXRD and SEM. Dissol ution amounts of the products obtained by
Sawchuk-Zaske method. Regression analysis was used to derive a correlation                different methods were measured in gastric fluid. Finally, pharmacokinetic studies
between creatinine clearance (Cl cr ) and Cl gent, Vd and actual body weight (ABW).       of the inclusi on complexes wer e conduc ted in blood, liver and kidney of m ale
Based on actual Cl gent and Vd values, the predictive ability of the estimated            Wistar rats to assess the bi oavailability of the prepar ed c omplexes .
parameters from the regression equations was validated and compared with 4                Results: The aqueous sol ubility of itraconazol e increased linearl y as a function of
methods, using mean error (ME) (bias), mean squared error and root mean                   cyclodextrin concentration accordi ng to the rank order: HP-β-CD > β-CD > γ-CD >
squared error (MSE and RMSE, respectively) (precision). All equations were also           α-CD. Inclusion for mati on was i nfluenc ed by the preparation technique. Products
evaluated in an independent second group (group 2) of 23 patients.                        obtained by the SC CO2 method were among the ones showi ng the highest
Results: (a) The mean (±SD) for Cl gent and Vd, was 4.0 (±1.8) L.h-1 and 16.8 (±6.7)      interaction between itraconazole and the CDs, leading to about three ti mes higher
L, respectively. (b) The derived equations were: Cl gent = (0.760) (Cl c r )+1.117 (r =   dissolution amounts than pure itraconaz ole. Temperature, pressure and dr ug:CD
0.701) and Vd = (0.165)(ABW)+5.604 (r = 0.532). In comparison to the four                 ratio had significant effects on the inclusion yield. In vivo drug pharmacokinetic
published methods, the derived equations were found to be less biased                     studies showed that the itraconaz ole-β-CD product prepared using SC CO2 res ults
(ME=0.00), and more precise (MSE=1.68, RMSE=1.02) in predicting Clgent                    in higher bioavailability of itraconaz ole than those obtained by physic al mi xing or
(p<0.05), and less biased (ME=-0.01) with no difference in precision (MSE=36.22,          coprecipitation methods.
RMSE=4.59) in predicting Vd, (p>0.05). This precision was confirmed in the                Conclusions: Cyclodextrins significantl y improved the sol ubility of itraconaz ole in
second group of 23 patients, where the derived equations were less biased (ME=-           aqueous solutions, which should improve the therapeutic effects of itraconaz ole
0.1) and more precise (MSE=3.22, RMSE=1.48) in predicting Cl gent (p<0.05) and            against antifungal infections. SC CO2 method proved to be an effecti ve technique
no differences were found for prediction of Vd (p>0.05).                                  for preparing solid c omplexes between c yclodextrins and itrac onaz ole. Sinc e SC
Conclusion: The equations developed in this study provided a reliable estimation          CO2 method has no toxic sol vent residue, products obtai ned by this method
of Cl gent and Vd. It is planned to use them at Kuwait Hospitals help provide more        shoul d provi de mini mal side effec ts in humans.
individualized patient dosing information to physicians.




Pathogenesis of Osteoarthritis: possible targ et molecules for new                        Molecular D ynamics Simulations of human membr ane tran sport proteins
therapeutic strategies.                                                                   from the Major Facilitator Superfamily.

ALMONTE-BECERRIL M, CRUZ R, GONZALEZ EC, MIRANDA M, ROJ AS-                               ALMQVIST J (A), HUANG Y (B), HOVMÖLLER S (A), LAAKSONEN A (C) AND
ORTEGA M, KOURI JB                                                                        WANG D-N (D).

Investigation c enter and advanced studi es of National Pol ytec hnic Institute           (a) Division of Structural Chemistr y, Arrhenius Laborator y, Stoc khol m Uni versity,
(CINVESTAV-IPN); Méxic o, DF.                                                             Sweden.
                                                                                          (b) Department of Molec ular Biol ogy, Upps ala Bi omedic al Center, Swedish
Background: Changes in the morpholog y and function of the chondr oc ytes during          University of Agricultural Sciences, Sweden.
the Osteoarthritis (OA) progression can be rel ated with the expr ession of               (c) Division of Physic al Chemistr y, Arrhenius Labor ator y, Stoc khol m Uni versity,
molec ules invol ved in the i nflammatory process . Some of these molecul es could        Sweden.
be an important target to avoid, retard or even stop the progression of OA. Ai ms:        (d) Kimmel Center for Biol ogy and Medicine at the Skirball Ins titute of
1) To describe the chondroc ytes phenotype during early OA. 2) To i dentify               Biomolecul ar Medicine and D epartment of Cell Biolog y, New Yor k Uni versity
possible target molecul es invol ved in phenotype c hanges in OA chondroc ytes. 3)        School of Medicine, USA.
To identify programmed cell death of OA chondroc ytes.
Methods: The experimentall y OA-induc ed model was acc omplished by unilateral            The Major F acilitator Superfamily (MFS) constitutes the largest group of sec ondar y
knee menisectomy and post-surger y training; normal rats were us ed as a control.         active tr ansporter protei ns, with members ubiquitous in all kingdoms of life. R ecent
Right femoral condyles were removed and processed for either electron microscopy (EM)     crystal struc tures of bac terial MFS proteins - including the Oxalate transporter,
or Immunohistochemistr y (IHQ). Samples from rats with 1 to 5 tr aining days (td)         Glycer ol-3-phos phate transporter,
were used to evaluate ultrastr uctural changes by EM. Moreover, cartilage from            Lactose permeas e and the multidrug resistance protei n D - has revealed a
rats with 3, 6 and 10 td were anal yzed by IHQ for IL-1, IL-10, TGF-1 and TNF-          common struc tural architec ture. This MFS fold featur es two transmembrane
. Finall y, programmed cell death was identified with TUNEL, c aspasa 3 and LC3II        domains composed of six helices eac h, connected with a cytos olic linker region of
at 20 and 45 td. Fluorescent signals were anal yz ed by confocal microscopy.              approxi matel y 30 amino acids. Human MFS trans porter structures, however, have
Results: EM reveal ed c hanges in the s uperficial zone c hondroc ytes, where cell        not yet been obtained.
phenotype changed from elongated to a rounded shape, in addition, the cells
devel oped prominent endoplas mic reticulum (ER) with dilated cisterns and                From a pharmaceutical standpoint, the human MFS transporters are of significant
enhanc ed Golgi membranes. Expression of the proinflammator y c ytokines IL-1β            interest. This talk will present our studies on two human MFS proteins ; (1) the
and TNF-α and began at 6 td, in the s uperficial zone and reached their highest           glucose-6-phosphate trans porter (G6PT), directl y invol ved in the glycogen storage
levels at 10 td in all the c artilage z ones . TGF-1 s howed diminis hed expression at   diseas e type 1b and (2) the vesicular glutamate tr ansporter (VGLUT1) fr om the
6 and 10 td. IL-10 was constant in all samples. Finall y the cell death anal ysis,        membranes of s ynaptic vesicles l ocated in excitator y neural cells in the
showed a c o-expression of autophagic and apoptotic mec hanisms.                          mammalian central ner vous s ystem. In the absenc e of structural data for thes e
                                                                                          important human proteins, we have modelled atomic s tructur es of G6PT and
Conclusions: Our res ults suggest that i n earl y OA, chondroc ytes c hanges its
                                                                                          VGLUT1 from high-resolution cr ystal structures of bacterial homologues , and
phenotype i n order to s ynthesize protei ns required for extrac ellular matri x (ECM)
                                                                                          carried out extensi ve Molec ular Dynamics simulations to better understand
repairment. However, when its capacity is over whelmed, c hondroc ytes begin th e
synthesis of catabolic molec ules li ke IL- 1β and TNF-α that s timulate an               fundamental questions related t o substrate bi nding, inhibition and c onformational
inflammator y pr ocess. F urthermore, the decrease of anti-inflammator y molec ules       change of human MFS protei ns.
could be involved in the beginning of the OA. Finally, the c hondroc ytes exec ute its
own proc ess of c ell death, that i nclude both autophagy and apoptosis.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                             Page A-10
                                                        EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                             Celebrating the 100th Anni versar y of the
                                                               Nobel Prize Award to Paul Ehrlich
                                                                   Nürnberg, October 3-5, 2008
Verap amil R everts Acute Renal Functional Impairm ent Induced b y                 Anti-tumour Vaccination in Advanced Malignancy with Class I & II hTERT
Angiotensin II Converting Enz ym e Inhibitor s                                     Peptide-pulsed Dendritic Cells (DCs) Generates Suboptimal Antigen Specific
                                                                                   CD8+Cytotoxic T C ell (CTL) Responses and Induces R egulator y T C ells in
                        1 ,2                     2                     2
ALVAREZ GREGORI JA , BOUCHOUTROUCH Y , MACIAS NUÑEZ JF                             the Circulation
1                                                                                                                                                     1             2             2              3                    3
    CS. Casto Prieto. UDMFYC. SACYL. Sal amanc a;                                                                                     ALOYSIUS MM , VERMA C , ROBINS RA , EREMIN JM , SREENIVASAN T ,
2
    Faculty of Medicine. Uni versity of Salamanca                                                                                     FARZANEH F4, HARDWICK N 4 , HABIB N 5, KECHNIE AMC 1, HARRISON PR 6,
                                                                                                                                      EL-SHEEMY M 4 , AND EREMIN O1 , 4
Background: Antihypertensive agents have been found ef fective in arresting glo merulosclerosis. Init ially, it was though t
                                                        y
that the healthy ef fect of these drugs was exclusivel due to their hemodynamic effects. However, it has become clear that            1                         2
nonhemodynamic actions of these agents are an important component of their beneficial e ffects. Among thepharmac                        Section of Surgery and Institute of Infecti on and immunity, Sc hool of Molec ular
ological agents that may have a favorable                                                                                             Medical Sciences , Notti ngham Uni versity H ospitals, Uni versity of Nottingham, NG7
influence in the course of renal failure, angiotensin converting enzyme (ACE) inhibitors, and calcium channel blockers
(CCB) have generated the most interest .                                                                                              2RD, UK.
                                                                                                                                      3
Angiotensin-converting enzyme inhibitors (ACE I) have proven to be effective drugs for the treatmen t of hypertension a nd              Lincoln Onc ology Centr e and 4 R esearch and D evelopment Department, Lincoln
represent a major therapeutic breakthrough in the management of hypertension and renal function preservation in diabetic
and nondiabetic nephropathies. In some patients, AC EI may induce a rapid deterioration of renal function, assessed as an             County Hospital, Li ncoln, UK.
                                                                                                                                      4
increase in serum creatinine (SCr), which can be reversed by withdrawing the drug.In these cases, maintain ing the rena l               Department of Haematological & Molecular Medicine, Rayne Institute, Ki ng‘s
protection due to ACE I, instead o f wi thdrawing these drugs could be desirable.
Calcium antagonists are a heterogeneous group of agents with diverse effects in terms of nephroprotection. Some of these              College, 123 Col dharbour Lane, London, SE5 9NU.
                                                                                                                                      5
differences relate to their effects on renal microcirculation. Dihydropyridine agents appear to act only on the afferent                Surgery Sec tion, Department of Surgical Oncolog y and Technolog y, Imperial
arteriole, increasing intraglomerular pressure, and albumin excretion rate. In con trast , nondihydropyridine agents like
verapamil, may dilate efferent arterioles in addition to af ferent arterioles and with normalization of the systemic blood            College London, Du Cane R oad, London, W12 0NN, UK.
                                                                                                                                      6
pressure, verapamil may reduce intraglomerular pressure, and proteinuria. However, some other no n hemodynamic                          The Li ver Unit, King's College Hospital, Denmark Hill, London SE5 9RS, UK.
protective effects of CCB could be explained by its capacity to inhibit the extracellular calcium influx, an important signal for
the proliferative e ffect o f mesangial cells mitogens, its in fluence in the decrease in mesangial en trapment o f
macromolecules and, possibly, its e ffect as free radical scavenger                                                                   Purpose: Human tel omerase reverse transcriptas e (hTERT) is expr essed in
Münter et al. describe that nephroprotective effects of ACEI/CCB combina tion can occur at doses, which do not significantly
alter systemic blood pressure in the stroke -prone SHR. We have des cribed that the combined therapy with these agents                >85% of human cancers and is bec oming an increasi ngly popular mol ecular target
provide in the remnant kidney model a synergistic effect in preventing renal injury, independently of their e ffects on blood         for anticancer immunotherapy. We have previousl y shown that autologous DCs
pressure.In a preliminary study, we have demonstrated in a small group of patien ts that nondihydropiridin (nonDHP) CCB
are able to revert renal function reduction associated to AC EI treat ment .                                                          pulsed with class I epitopes of hTERT are capable of generating hTERT
The main purpose of this study was to assess the efficacy and safety of low doses of verapamil (180 mg/day) added to the              tetramer+, CD8+ CTLs in circulation of advanced c anc er pati ents, albeit s hort-
previous ACEI treat ment for reverting decreased glomerular filtrat ion rate observed in patients treated with ACE I. A
secondary purpose was to test the ability of the fixed combination Verapamil- SR 180 mg plus Trandolapril 2 mg in attaining           lived. By including CD4+cognate T cell help in the vaccination, through pulsing
BP control and main taining there renal function throughout the study .                                                               autologous monoc yte-deri ved DCs with class II promiscuous hTERT peptides, we
Methods: This was a multicenter, nonrandomized, prospective, open study developed in five Spanish Hospitals. All patients
were referred from the outpatients Departments of Internal Medicine and Nephrology. The Institutional Review Boards                   aimed to optimiz e the hTERT+, CD8+ CTL res pons e in magnitude and duration.
approved the study protocol and each patien t en tering the study signed a writ ten in formed consent.                                Experimental Design: A Phase I clinical trial in advanced canc er patients was
Eligible pa tients presented a previous diagnosis of hypertension and an increase in SCr o f 20 % or 45 m mol/L from las t
values, in the course of ACEI treatment for more than four weeks. Exclusion criteria were renal insufficiency, defined as SCr         perfor med to evaluate the immunol ogical and clinical impac t of vacci nati ng (6
>354 mmol/L (4 mg/dL), stroke, AM I in the last three months, unstable angina, cardiac failure, other causes of renal hypo -          occasions, 2 to 3 weekly) advanced canc er patients with the HLA-A2-restricted
perfusion, or low volume syndrome such as dehydration, vomiting , diarrhea, laxative, and known hypersensitivity to
verapamil.                                                                                                                            class I hTERT epitopes I540 & I865 (n=10), with or without cl ass II epitopes 766 &
In the selected group, a clinical evaluation was carried out, including clinical history, physical examination, B P                   675 (n=5), pulsed on autol ogous monoc yte-derived ex vivo generated DCs.
measurement, ECG , and biochemistry (Visit 1). Pa tien ts fulfi lling the inclusion/exclusion criteria were enrolled in the study.
Three to ten days later a new BP measurement, serum, and urine creatinine and 24 h proteinuria were also measured (Visit              Results: Peptide/MHC tetramer-CD8+ CTLs and CD4+CD25+ foxp3+ T cells
0 or Baseline) and creatinine clearance (CrCl) calculate d. If the SCr was not higher than 10% respect to Visit 1, the patients       were tr ac ked s equentiall y through the vaccinations. Four patients with advanced
were included in the study and Verapamil (180 mg/day) was added. Patien ts returned to the hospita l af ter four weeks for
new measurement of SCr and BP (Visit 1). I f an increase on SCr su perior to 10% respect to Visit 0 or BP was _140/90mm               cancer (pr ostate, renal, head & nec k and c olon) rec ei ved an average (range) of
Hg was detected, AC EI was discontinued , and the pa tient withdrawn from the study. I f not , trea tment was continued for           8.5 (6-12) vaccinations . . hT ERT tetramer+, CD8+ CTLs were induced in 13/15
eight weeks. At th is momen t (Visit 2) pat ients were evaluated again. I f SCr had incre ased more than 20% with respect to
baseline, or BP was 140/90mmHg, the pat ients were withdrawn. If not, the patients were placed on the Trandolapril (2mg) -            patients, with circul ating tumour mar ker level reduc tions i n 4/6 patients with
Verapamil (180 mg) association for eigh t more weeks.                                                                                 prostate cancer. T hes e were onl y short-lived and declined des pite c onti nuing
Patien ts were fol lowed unt il 20 weeks o f
follow-up were completed. All the pat ients                                                                                           vacci nati on. T regulator y c ells (CD4+CD25+foxp3) tended to increased in the
were recommended to lim it sodium in take.                                                                                            circulation during vaccination and showed a negative correlation with tetramer+
At each study visit, blood pressure was
measured three times at 2 min intervals after                                                                                         CD8+ CTL levels.
10 min rest in the sitting position, using                                                                                            Conclusions: These results demons trate that vaccination with autologous
calibrated mercury sphygmomanometers .
Blood press ure values were estimated as the                                                                                          monoc yte-deri ved DCs pulsed with hT ERT class I & II peptides was unable to
mean of the three readings. The mean value                                                                                            induc e an optimal and sustai ned tetramer+, CD8+ CTL respons e but generated T
obtained in Visit 0 was accepted as BP basal
level. Biochemis try and SCr (Jaffe´ reaction)                                                                                        regulator y suppress or cells in the circulation.
were determined in an automa tic analyzer
(Hitachi 747). Creatinine clearance was
calculated as: urinary creatinine (mg/L) urine
volume            (mL)/serum            creatinine
(mg/L)/1.73 m2.
All the basal variables were analyzed descriptively. Da ta are given as Mean ± s tan dard deviation . Adverse events are              Hydrogels for Sustain ed and Selective Release of Diclofenac
codified according to the WHO Adverse Reaction Terminology List (WHO -ARTL) . Repeated measuremen ts ANO VA was
used with an exploratory approach in order to test the following null hypotheses: changes of creatinine, systolic , and
diastolic blood pressure, uric acid and potassium over time. The Wilcoxon test (two tailed) for paired samples was used to            ALVAREZ-LORENZO C
test the following hypotheses: changes of creatinine clearance and albuminuria between inclusion and week 12. Changes in
creatinine between Visi t 1 and inclusion, inclusion and week 12, inclusion and week 20, week 12, and week 20, historic
control, and week 20 . All the p-values are exploratory as type I error has not been adjusted for mult iplicity.                      Univ. Santiago de C ompostela, Santiago de Compos tela, Spain.
Results: Forty -six patients, 23 female and 23 male , with a mean age of 54.9 ±7 .3 years and BMI of 27.3±2 .2 kg/m2, were
included. Five of them (10.9%) had suffered from cerebrovascular disease, eight (17.4%) coronary heart disease, 24 (52%)
had left ventricular hypertrophy on ECG or echocardiography, seven (15%) lower limbs peripheral vascular disease, and 35              Background: The development of hydr ophilic s ystems able to load hydrophobic
(76%) some degree of retinopathy. Fourteen patients had been diagnosed from diabetes 4.5±8.0 years earlier (a median of
2.6 years): one was treated with diet, 12 with oral anti-diabetics drugs, and one with insulin. They were known hypertensive          drugs and to control the site and rate at which they are deli vered is a target issue
for a median of 4.5 years and all of them had been treated with ACEI for at least four weeks previous to SCr deterioration.           in pharmac eutic al technol ogy. T he ai m of our wor k was to prepare functionalized
Previously used ACEI were: enalapril in 12 pa tients , Lisinopril in nine , Cilazapril in seven, Perindopril in five, Qu inapril in
five, Ramipril in four , Trandolapril in two, and Captopril in two. Nine patien ts were withdrawn from the study, three at week       hydrogels based on s emi-s ynthetic cellulos e derivati ves, acr ylic copol ymers or
four and six at week 12. Eight were withdrawn due to no B P control and no trea tmen t compliance in one patien t. In one             cyclodextrins, with high affinity for diclofenac for improving the loading and the
patient , the reason was no BP control plus SCr increase>20% . A total number o f 37 patien ts fina lized the study.
The evolution of the main clin ical and biochem ical parame ters is shown in the following Table.                                     release performance and even able to deliver the drug in res ponse t o certain
                                                                                                                                      stimuli.
                                                                                                                                      Methods: The hydrogels were i) cross-linked cationic c ellulose ethers with
                                                                                                                                      ethyleneglyc ol diglycidylethers (EGDE); ii) cross-linked cellulos e ethers/
                                                                                                                                      cyclodextrins with EGDE; iii) copol ymerized hydroxyethylmethacr ylate (HEMA)
                                                                                                                                      with aminopropyl methacr ylamide (AMPA) or vinylpyridine (VP); and iii)
                                                                                                                                      interpenetrated networ ks of N-isopropylacr ylamide (NIPA) and c hitos an.
                                                                                                                                      Results: Cross-linked cati onic c ellulose networ ks loaded 250 mg diclofenac per
                                                                                                                                      gram and s electi vely releas ed the drug at pH>7, being us eful for s pecialized
                                                                                                                                      intestinal deli ver y. A novel cross-linking method to prepare c yclodextrin hydrogels
All the 46 patien ts were considered for the safety analysis. Throughout the study, 13 patients reported 19 adverse events.
Sympto ms were mi ld/modera te in intensi ty in a ll cases. Any patien t in terrupted the study because of adverse events .           led to s ys tems that hosted the drug in the c yclodextrin cavities (100 mg/g) and
Conclus ions: The major f inding of this study is the possibility o f reverting renal funct ional i mpairment induced by ACE I        sustained the deliver y for 8 hours. Poly(HEMA-co-APMA) and pol y(HEMA-co-VP)
treatmen t in hypertensive patien ts by adding verapamil wi thout further modifica tion in ACE I therapeutical regime . These
patients had normal serum creatinine levels be fore ACEI trea tment .                                                                 loaded 15 mg/g and did not release the drug in water but in the pr esenc e of ions;
ACEI have been demonstrated to reduce morbidity and mortality in patients with heart failure. Renal impairment is the mos t           the rel ease bei ng sustained for several days. Such hydrogels dis play features
important factor associated with prescription of lower -than-recommended doses. A recent review of 12 randomized clinical
trials evaluating renal disease progression and ACEI-based therapy, shows that an acute increase of serum creatinine in               particularl y adequate for the development of medicated s oft c ontact l ens es.
the first two mon ths usually follows the ACE I therapy in patients with preexisting renal fai lure. This is not o ur case, as the    Interpenetrati ng NIPA/chitosan networ ks showed temperature- and pH-sensitive
patients in our study had normal values of plasma creatinine before ACEI trea tment . The review also shows that a limited
elevation, of up to 30%, is strongly associated with long -term preservation of renal function when ACEI therapy is continued.        loading and releas e behavior. The IPNs had a notabl y greater affinity for
In our study, we have observed increases in plasma creatinine up to 50% respect to pretreatment levels. These increases               diclofenac (18 mg/g) than the pur e PNIPA hydrogel and were able to s ustain the
were completely reverted by adding verapamil to the ACE I treatmen t.
Some calciu m antagonis ts seem to o ffer addi tional benefi t in hypertensive patien ts when renal function is i mpaired, and may    drug release for more than 8 h i n 0.9% NaCl solutions or pH 8 phos phate buffer.
reduce proteinuria in hypertensive diabetic patients. The deterioration o f renal function in pat ients receiving ACE-inhibitors      The IPNs with low chitosan postcr oss-linking degree showed high temperature-
seems to be due to a decrease in intraglomerular pressure, and, su bsequently in the filtration net pressure. Thus, as
angiotensin II has a predominant vasoconstrictor effect in the ef ferent arteriole , the decrease in angiotensin II due to AC E       sensiti ve rel eas e patterns.
inhibition leads to an efferent arteriolar vasodilatation, and subsequently to a decrease in glomerular capillary pressure. This      Conclusions: Loading of diclofenac and its releas e pattern c an be tunned
is one of the major beneficial effects of AC E inhibitors, as increased capillary pressure is a major cause of the progression
of renal fai lure, but if the decrease on glomerular pressure is below normal values, glomerular f iltra tion rate wil l be also      designing tailored hydrogels in whic h functional elements performing as high
decreased. However, it is difficult to accept this as the only cause of filtration rate decrease after ACEI trea tment , becaus e     affinity bindi ng points and environment-responsi ve sensors are combined. Thes e
the autoregulation threshold is not always reached. If it is overpassed, an immediate de terioration in renal function mus t
occurs at least in absence of decreased effect ive arterial volu me, by far the com monest cause of acute rise in serum               functi onalized hydrogels may be partic ularly useful for site-specific drug delivery.
creatinine.
The additive effect of verapamil and ACEIs could be owing to that the two drug types pro tect the kidney by differen t
mechanisms. Dworkin et al. have reported that either enalapril or nifedipine reduce renal injury in the remnant kidney model          Authors disclosur e statement:
by different mechanisms: ni fedipine reduces glomerular hypertrophy whereas enalapril reduces glomerula r hypertension.
Verapamil is able to diminish renal vasoconstriction mediated by BK receptor activation, prevent intrarenal vasoconstriction          Some i nfor mation described i n this abstract is the s ubjec t of patent applications
mediated by adenosine, and prevent from mesangial cells contraction and proli feration induced by several agents .                    filed by the Uni versity of Santiago de Compostela (WO 2006/089993; ES
Verapamil is also able to stimu late NO release.
The addition of verapamil to trandolapril treat ment might promote o ther beneficial e ffects on renal structure and function .       200802364).
Thus, CCB are able to inhibi t platele t aggregation, and this inhibition has been repor ted to ame liorate glomerular injury in
rats with reduced renal mass. In addit ion, Harris et a l. have shown tha t verapamil lessened oxygen consumption in the
isolated, perfused remnant kidney and, therefore, it migh t ameliora te glomerular injury by reducing damaging oxygen
radicals production rate.
The results of this study confirm our previous preliminary data suggesting a therapeutical approach, which allows revert the
renal failure induced by ACEI treat ment in hypertensive patients with previous normal re nal function, maintaining this
therapy, and adding verapamil 180 mg/day to the ACE I treatmen t. These f indings are clinically relevant and wil l be o f
paramount interest for the patien ts in which ACE i llicit a doubtless protection of target organs, even in n onhypertensive
patients. The trandolapril-verapamil comb ination a llows mainta in an excellent BP control decreasing further the levels o f
serum creatinine.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                                      Page A-11
                                                                 EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                      Celebrating the 100th Anni versar y of the
                                                                        Nobel Prize Award to Paul Ehrlich
                                                                            Nürnberg, October 3-5, 2008
Clinical importance of drug resistance in antiviral therap y for Hep atitis B               The F1-V Plague Vaccin e Can Activate the Innate Immune Response
Infection                                                                                   Through Toll-Like Receptor2 and 4 but Does Not Need Them for an Antibod y
                                                                                            Response to the Vaccin e
AMAR APURKAR DN
                                                                                            AMEMIYA K
Bombay Hospital, Mumbai
                                                                                            U.S. Army Medical R esearch Institute of Infec tious Diseas es, Frederick, Mar yland,
Background: Hepatitis B Virus (HBV) is a unique DNA virus which replicates                  USA
through an RNA intermediate, lac ks proof readi ng ability, has high viral replication
rate. This leads to random mutations with amino acid substitution in reverse                Background: A new r ecombinant fusion protein F 1-V is i n advanced devel opment
transcriptas e region. In clinical parlanc e anti viral drug resistance is defined as       for preventi ng plague caused by Yersini a pestis. F1 is a c apsul ar protein, and V is
selection of variants bearing amino acid s ubstitution confirming reduced                   a low c alcium respons e (Lcr) protein or V-antigen. V-antigen has been reported to
susceptibility to drug those res ults in primar y or secondary treatment failure. For       activate Toll-like receptor (TLR) 2. We hypothesized that acti vating the innate
the management of HBV i nfec tion in additi on to interferon/Peg                            immune s ystem through T LRs is required for an antibody r esponse to the F 1-V
Interferon/T hymosin alpha. 4 oral anti viral like lamivudi ne, adefovir, entecavir,        vacci ne.
Telbuvidine ar e licensed and T enofovir and emc etar abine are licensed for HIV &          Methods: We evaluated the interac tion of V-antigen and F 1-V with human
HBV coinfection. Succ ess rate of thes e anti viral agents do not exceeded more             embryonic kidney (HEK)-293 cells expressing TLR2 or TLR4 (Invi voGen). Further,
than 30 to 40% in long term treatment and with prolonged therapy, menac e of                we exami ned the antibody response, the proliferati ve respons e, and cytokine
anti viral resistance exists. Clinical consequence of resistanc e are decreased             expression by s plenoc ytes from F 1-V-vaccinated TLR 2, T LR 4, T LR2/4, or
HBeAg clear anc e, reversal of histological improvement, increas ed rate of diseas e        myeloi d differ entiation factor 88 ( MyD88) knoc k-out (KO) mice. Acti vation of TLR
progression, ,clinical dec ompensation or even death in pati ents with cirrhosis, risk      2 and 4 in HEK-293 cells was measured as per manufacturer‘s instructions. Mice
of graft loss and death in liver transplant recipients, transmission of drug                were vaccinated s ubcutaneousl y twice ( 0 and 28 days) with F1-V ( 1 – 2 ug)
resistance strain and vaccine failure mutati on.                                            formul ated with Alhydrogel. Ser um and spleens were removed from anesthetized
Methods: We anal yzed real life data on our patients recei ving long term                   mice 21 days after the boost. T he anti body respons e to the vaccine was measured
Lamivudine tr eatment and devel opment of resistanc e clinical c ons equences and           by end-point ELISA. C ytokine expressi on by antigen-sti mulated splenoc ytes were
their management...                                                                         measured by BD FACSArray anal ysis after 40 h. Proliferation of splenoc ytes was
Results: Our study incl uded 82 patients ( male 66, Age range 5-85 years). Of the           determined by the amount of 3 H-thymidine inc orporated after an additional 18 h
82 patients 50 patients were HBeAg +ve. These patients recei ved mean duration              incubation.
of Lami vudine treatment 32.44 months. 17 out of 50 (34%) developed resistanc e             Results: TLR2 and TLR 4 HEK-293 cell lines were weakl y acti vated by V-antigen
to Lami vudine, 32 patients were HBeAg –ve, Mean duration of treatment with                 but strongly by the F1-V vaccine. Thes e antigens acti vated no other TLR cell
Lamivudine was 28 months. 8 out 32 (25%) developed Lamuvi dine resistanc e.                 lines. Wild- type, TLR2, and TLR4 mutant mice vaccinated with the F1-V vaccine
The pr esentati on of Lami vudi ne resistanc e was clinic al decompens ation 3, sero-       all appeared to respond si milarly to the prime and boost administrati on of the
reversion 7, flar e of liver enz ymes 8, and i ncreas ed viral load 5. All the patients     vacci ne. This included the IgG1 an d IgG2a isotype res ponse to the vaccine.
who devel oped r esistance were tr eated with addition of Adefovir to Lami vudine.          Furthermore, the anti body res ponse i n M yD88 KO mice was si milar to that in wild-
Mean period of 8.5 months of follow up; 2 patients died dues to decompens ation,            type mice. The pr oliferati ve res ponse and c ytoki ne expression was partiall y
remaini ng pati ents are s table with nor malized liver function                            affec ted in the TLR 2 KO but was essentially bac kground le vel in the TLR 4 KO,
Conclusion: Antiviral drug resistanc e is a maj or problem in management of                 TLR2/4 KO, and M yD88 KO mice.
chronic HBV infection. Combining sec ond drug with no cross resistanc e at                  Conclusion: 1. TLR2 and TLR4 medi ated innate i mmune res ponses are not
appropriate time s eem to be best polic y currentl y.                                       required for an antibody r esponse to the plague vaccine. 2. M yD88 is als o not
                                                                                            required for an antibody respons e to the vaccine. 3. Cellular immune respons es
                                                                                            to the vaccine appear to be partiall y dependent on TLR2 but appear to be
                                                                                            completel y dependent on TLR4.




Pharmacokin etic stud y of rivastigmin e in Iranian health y subjects follo wing           Antibiotic r esistance: what can we learn from evolution?
3 and 4.5 mg dosing u sing a simple and sen sitive HPLC-UV method
                                                                                           AMINOV RI
AMINI H 1, AHMADIANI A2
                                                                                           Rowett Inst. of N utrition and Health, Uni v. Aberdeen, Aber deen, UK
1                                                          2
Golestan Uni versity of M edical Scienc es, Gorgan, Iran; Behes hti Uni versity of
Medical Sciences , Tehran, Iran.                                                           Background: The brilliant ―Zauber kugeln‖ (Magic bullets) idea of Paul Ehrlich was
                                                                                           a major breakthrough in treati ng infectious dis eas es. It opened the whole new era
Background: Rivastigmine is relativel y new drug and the evaluation of its                 in medicine and led to the discover y of many antibi otics that saved millions of
phar mac okinetic properties in different ethnic populations is important to opti mize     lives. Despite the consi derable s uccess, however, bac teria i nvented various
the dosage regimens. For the phar mac okinetic study of rivas tigmine, a simple and        shields thus c ompromising the magic power of antibiotics. The number of
rapid but als o a highl y sensiti ve and s electi ve bioanalytical ass ay method s hould   pathogens that learned how to dodge thes e bullets is increasi ng and the question
be available. Ai ms: 1) To develop and validate a sensiti ve and selec tive anal ytical    is how they acquired thes e properties ? Aim: rec onstruc tion of evolutionar y histor y
method for rivastigmine assay in pl asma 2) To perform the pharmacoki netic study          of sel ected antibi otic resistance genes to ans wer this ques tion.
in Iranian healthy subj ects 3) To compare the phar macoki netics of ri vastigmine         Methods: Four sets of genes enc oding resistance to tetrac yclines, macrolides,
followi ng 3 and 4.5 mg dosing.                                                            vancomycin and fluoroquinolones were c hos en for this analysis: 1) tetrac ycline
Methods: A simple and repr oduci ble HPLC method with spectrophotometric                   resistance genes , encodi ng ribosomal protecti on protei ns; 2) the er m genes
detection at 200 nm was developed and validated for the determi nati on of                 encoding enz ymes that methylate the s pecific adenine residue in the 23S rRNA
rivastigmine in human pl asma. The ass ay was used for phar mac okinetic study of          molec ule; 3) the vancomycin r esistance gene cluster repres ented by the
rivastigmine capsul es in healthy Ir anian subjects following 3 and 4.5 mg dosing. 23      concatenated set of vanHAX; and 4) the qnr genes conferring resistance to
healthy and fasted volunteers participated in both groups. Food and drinks were            fluoroquinolones. The sequences were downloaded from GenBank and aligned
not allowed until 3 h after ingestion of the capsul es. Multiple blood samples (5 ml)      using ClustalX ver. 1.83. Maximum-likelihood and Bayesian inferenc e were used
were collected before and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6 and 8 h         to reveal the evolutionary history of thes e genes.
post-dosing. A non-compartmental anal ysis was used in the data processi ng.               Results: Phylogenetic rec onstr uction suggested a long evolutionar y histor y of
Results: In H PLC anal ysis, it was founded that in addition to reversed-phas e            diversification of anti biotic resistance genes that began well before the ―anti biotic
retenti on, other retention mec hanisms s uch as hydrogen- binding or ion- exchange        era‖. Ther e is no indic ation that lateral gene transfer fr om antibiotic-producing
are probabl y invol ved in the c hromatogeraphic behaviour of rivastigmine.                bacteria has played any significant role in shapi ng the pool of anti biotic resistanc e
Triethyl ami ne shoul d not be used in the mobile phas e, while an acidic mobile           genes in pathogenic and commensal bacteria. The primar y anti biotic resistanc e
phas e and chromatography at high temper atures can increase theoritical pl ates for       gene pool originated and diversified within the environmental bac terial
rivastigmine. A selecti ve extraction of rivastigmi ne from plasma was obtained            communities, from which the genes wer e mobilized and penetrated into
using 1-butanol/n- hexane (2:98, v/v) and bac k extraction into diluted ac etic aci d.     taxonomically and ecologicall y distant bacterial populati ons, includi ng pathogens.
The newl y developed HPLC-UV method had an li mit of quantific ation (LOQ) of 0.5          Conclusions: Enormous metabolic di versity of bacteria allows them to c ome up
ng/ml, whic h is c ompar able to LOQ of 0.2 ng /ml obtai ned by current LC/MS              with protection mechanisms even agains t novel antibiotics. To preser ve the magic
methods. The pharmacoki netic studies s howed that ri vas tigmine has a rapid oral         of novel anti biotic bullets we have to pay more attention to the pool of anti biotic
absorption with a large inter-subjects variati ons. The mean val ues of maxi mum           resistance genes in the environment and carefully monitor the possi ble movement
plasma concentration (Cmax), ti me to Cmax (tmax), area under the pl asma                  of suc h genes into c ommensal and pathogenic bacteria.
concentrati on-time cur ve from time 0 to 8 hours (AUC8) and from time 0 to infinity
(AUC∞), and plas ma half-life following admi nistration of the rivas tigmine at 3 mg
dosing were 6.27 ng/ml, 0.98 h, 11.95 ngh/ml, 12.79 ngh/ml and 1.11 h,
respecti vel y, and for 4.5 mg dosing 10.74 ng/ml, 0.91 h, 21.60 ngh/ml, 22.98
ngh/ml and 1.22 h, respecti vel y.
Conclusions: For the first time, a highly sensitive HPLC-UV method was
devel oped and validated for rivas tigmine ass ay in plas ma. Phar mac okinetics of
rivastigmine in Iranian healthy subjects were c omparable with results obtained in
other ethnic populations . As reported by others, the oral bioavailability of
rivastigmine increased with dos e.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                            Page A-12
                                                               EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                      Celebrating the 100th Anni versar y of the
                                                                         Nobel Prize Award to Paul Ehrlich
                                                                            Nürnberg, October 3-5, 2008
Activit y of Antimalarial Constituents of Sp athodea campanulata                            Metalloantibiotics: Synthesis and Antibacterial Activit y of Metal(II)
                                                                                            Complexes Containing Cephalosporin and Sulfathiazo le
AMUSAN OOG
                                                                                            ANACON A JR
University of Swaziland, Private Bag 4, Kwalus eni, Swaziland.
                                                                                            Universidad de Oriente.Departamento de Q uimic a, Cumana. Venez uela
Background: The search for antimal aria drugs is a continuous one bec aus e of the
devas tating effect of the dis eas e. Aim: To determine the anti mal arial properties of    Background: The interaction of anti biotics with main and transition metal ions has
Spathodea campanulata.                                                                      attracted our attention and compelled us to combine their chemistr y in order to
Methods: This study included 165 Swiss albino mic e in Fink and Kretschmar‘s,               improve the stability and efficienc y of anti biotics. T hus, designing a new area of
and Rane in vivo tests. Fi ve mic e were us ed per treatment, weight range: 18-22g.         research in the s ynthesis of stable metalloantibiotic compounds that may be used
In the Fink and Kretsc hmar‘s test, each mouse was inoc ulated with Plas modium             as acti ve drugs wor king effecti vely against antibiotic resistance s pecies .
berghei berghei and treated post-infection subcutaneousl y once daily for 4 days
                                                                                            Methods: The metal(II) complexes wer e prepared by mi xing clear solutions of the
with plant constituents, chl oroquine or blank control. T he % parasitaemia was             appropriate cephalosporin s odium s alt (1 mmol) and NiCl 2.6H 2O or CuCl 2 metal
evaluated on the fifth day post-infection. In the Rane test, the mice were treated          salts (1 mmol) in distilled water (10 mL) and sulfathiaz ole (1 mmol) in EtOH (10
with the drugs onc e daily for 4 days starting 3 days post-infection. The %                 mL). The reacti on mi xture was then stirred at room temperature for 12 h. and
parasitaemia for each mous e was determi ned for 5 days starting fr om the fouth            green preci pitates formed. The pr ecipitated compl exes were filtered off, was hed
day post-infection. The ac tive constituents of the plants wer e isolated by col umn
                                                                                            with water, MeOH and ether and dried under reduced press ure at room
chromatography and characterised.                                                           temperature. Yield 55-65% . No attempts to use different molar ratios to prepare
Results: Anti malarial principl es of stem bark were ursolic acid, tomentos olic acid,      the c omplexes were made. Antibacterial acti vities were tested usi ng the paper disc
20β-hydroxyursolic acid and caffeic acid from l eaves . In Fink and Kr etschmar‘s           diffusion method. The c hos en str ains were Staphyloc occus aureus ATCC 25923,
test ursolic acid at 15-60 mg kg -1day-1 pr oduc ed 34-97% suppresion of                    Escherichia c oli ATCC 11775, Pseudomonas aeruginosa ATCC 27853, Klebsiella
parasitaemia and mean sur vi val period of 13-25 days. Tomentos olic acid at 10-80          pneumoni ae ATCC 23357, Sal monella enteritidis CDC 64 and Bacillus s ubtilis
mgkg -1 day-1 produc ed 35-82% s uppr esion of parasitaemia and mean sur vi val             ATCC 6051.
period of 10-19 days. 20β-hydroxyursolic acid at 20-80mg kg -1day-1 produc ed 11-           Results: Nickel(II) and copper(II) react with cephal osporins pl us sulfathiaz ole
53% s uppresion of parasitaemia and mean sur vival period of 8-13 days. The                 (Hstz) to form the followi ng mi xed ligand complexes: [M(cefazol)(stz)(H 2O)],
aqueous leaf extrac t at 50-400 mg kg -1day-1 pr oduced 0-74% suppresion of                 [M(cephal ot)(stz)(H 2O) 2 ],         [M(cefotax)(stz)],      [M(ceftria)(Hstz)]     and
                                             -1   -1
parasitaemia. Chl oroquine at 10 mgkg day produced 98% suppresi on of                       [M(cefepi me)(stz)]Cl (Hcefazol = c efaz olin, Hcephalot = c ephalothin, Hc efotax =
parasitaemia and mean sur vi val period of 26 days.                                         cefotaxime, H 2ceftria = ceftriaxone) whic h were characterized by physicoc hemical
In Rane test the aqueus leaf extract at 50-400 mg kg -1day-1 produced mean                  and s pectrosc opic methods. Their spectra indic ated that most the cephalosporins
survi val ti me of 11- 15 days. Ursolic acid at 15-60 mg kg -1day-1 produced mean           are ac ting as a monoanionic multidentate chelating agents, the exception being
                                                                                 -1   -1
survi val period of 9-24 days . 20β-hydroxyursolic acid at 20-80 mgkg day                   ceftriaxone which is diani onic. The complexes are ins olubl e in water and common
produc ed mean sur vi val period of 6-16 days. T omentosolic acid at 5-40 mg kg -
1                                                                                           organic solvents and pr obabl y have pol ymeric structur es. They have been
  day-1 produc ed mean sur vi val period of 9-18 days . Blank control gave mean             screened for anti bacterial acti vity in DMSO solutions against several bacteria, and
survi val period of 7 days in both tes ts.                                                  the results are c ompar ed with the acti vity of cephal osporins . The
Conclusions: 1) The anti mal arial principles of Spathodea campanulata                      [M(cefepi me)(stz)]Cl compl exes showed better acti vity than free cefepi me agai nst
demons trated significant sc hizontocidal properties, the acti vity of chloroquine was      all bac teria str ains, i ncludi ng against P. aer uginosa and S. aureus where c efepime
however superior to any of them. 2) The acti vity of the anti malarial princi ples          is inacti ve.
provides the sci entific basis for the us e of Spathodea campanulata in the                 Conclusions: The s ynthesiz ed c ompounds showed antibacterial properties. In
management of malaria i n traditional medicine.                                             comparison, the copper(II) and nic kel(II) complexes containi ng cefepime plus stz
                                                                                            showed better acti vity agai nst several bacterial strains than the c efepime, thus
                                                                                            introduci ng a novel cl ass of metal-based bactericidal agents.




Production, isolation, partial char acter ization and antimicrobial spectrum of             System s-directed targ eted therapies in met astatic tumors: Equitable to
a novel bacteriocin produced b y a Lactobacillu s plantarum str ain in                      reductionist ther ap y approach es?
fermentation
                                                                                            REICHLE A, VOGELHUBER M, VOGT T, BERAND A, BROSS K, WIEST R, KLEBL
ANASTASIADOU S*, PAPAGIANNI M, AMBRO SIADIS I, KOIDIS P                                     F, KULLMANN F, ROGENHOFER R, WALTER B, HAU P, ANDREESEN R

Department of H ygiene and Tec hnology of Food of Animal Origin, School of                  University Hospital of Regensburg, Regensburg, Germany
Veterinary Medicine, Aristotle U niversity of Thess aloniki. Thess aloni ki 54006,
Greece                                                                                      Introduction: As we c onsider the exchange of i nfor mati on between tumor,
                                                                                            adjac ent stroma cells, and cells of the involved organ from a system s
Background: Fer mentation broths (in MRS) of an isolated Lactobacillus plantarum            perspective, we may disregard operati onal prerequisites that a c ombination of
strain exhi bited str ong anti microbial ac tivity against common food spoilage and         activiti es triggered by s pecific acti on s ystems must be intended by single
also food born pathogens. Anti microbial acti vity was assess ed in the agar diffusion      participati ng pathophysi ological mechanis ms, s uch as i nflammation, angiogenesis,
assay using a total of 9 indicator food-grade bacteria. The antimicrobial acti vity         Warburg effect, immune res ponse, extracellular matri x remodeling, proliferation,
appeared to be higher against clos el y related species and Lactobacillus cur vatus         apoptosis, c oagul ation. Acti vities that seem to be operati onally induc ed by the
was chos en as the most s uitable among the tes ted microorganisms to s er ve as            division of function present itself from a systems perspecti ve as an enhancement
indicator for the quantification of the produced bacteriocin.The exhibited                  of complexit y. We hypothesiz ed, that tumor s ystems-directed therapies might
anti microbial acti vity was due to the production of a proteinaceus compound, a            have the capability to use aggreg ated action effects, as adjustable sizes to
bacterocin, mos t of which appears to be cell-associ ated.                                  therapeuticall y modulate the tumor systems‘ stability, homeostasis, and
Methods: A number of mechanical and physicochemic al treatments were applied                robustness.
to was hed c ells in attempting to sol ubilize the bacterioci n. The mos t c on venient     Methods: We performed a retros pec tive anal ysis of recentl y published data on
method for extraction was centrifugati on at 20200 rcf for 10 minutes at 4oC .              278 patients with advanced and heavily pre-treated (10% to 63%) vasc ular
Repeated tricine - SDS - PAGE electrophoresis of samples taken at various                   sarcoma, melanoma, renal clear cell, chol angiocellular, muc oepider moid, and
fermentation ti me-points s howed that bonds as ~ 30kDa were of inter est.The               hepatoc ellular c arcinoma, hormone-refrac tor y prostate c anc er, glioblastoma, and
positon was finall y deter mined by overlying the gel with MRS agar in which L.             multi visceral Langerhans‘ cell histiocytosis enrolled in nine multi-center phas e II
curvatus was embedded.                                                                      trials (13 centers). Eac h patient rec eived a multi-targeted s ys tems-directed therapy
Results: T he M.W. of the bac teriocin was esti mated at 30kDa.T he isol ated               that consisted of metronomic low-dose chemotherapy, a COX-2 inhibitor,
bacteriocin lost its acti vity after treatment with lipas e and ?-chymothr ypsi n, but      combined with one or two transcripti on modul ators, pioglitazone +/- dexamethason
retained ac tivity following proteol ytic treatment. The stability of bacterioci n was      or IFN-alpha.
studied over a range of pHs,T and mec hanical stress es.                                    Results: Thes e treatment schedules may attenuate the metastatic potential,
Conclusions: It appeared to be heat labile, a characteristic indic ati ve, along with       tumor-associated inflammation, may exert site-specific acti vities, and induce long -
M.W. and the s ensibility to lipase and ?-chymothrypsin, of a certai n c ategor y of        term dis eas e stabilization followed by prolonge d objecti ve res ponse (3% to 48%)
anti microbial peptides, the class IV of bacteriocins produc ed by l actic acid             despite poor monoac tivity of the respecti ve drugs. Progression-free survi val (PFS)
bacteria. Fermentation ki netics studies performed in a stirred tank bioreac tor            data are c omparable with thos e of reductionist- designed standard first-line
showed that the production of the bac teriocin was not ass ociated to growth, but it        therapies targeting preferabl y tumor cell-specific pathways .
was r ather for med as a s econdar y metabolite.                                            Conclusions: Differential response patter ns indicate the therapies‘ sys tems
                                                                                            biological acti vity. Struc tured sys tems-directed therapi es in metastatic canc er,
                                                                                            targeting amongst others inflammation and neoangiogenesis, may break through
                                                                                            the barrier of c omplexity of tumor-stroma-inter actions , and get a s ource for
                                                                                            detecting topologies of tumor-associated aggregated action effec ts as adjus table
                                                                                            sizes for targeted biomodulator y therapies . Biomodulati on of sys tems biological
                                                                                            processes facilitates c omparativel y high efficac y at moderate toxicity.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                             Page A-13
                                                                 EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                        Celebrating the 100th Anni versar y of the
                                                                           Nobel Prize Award to Paul Ehrlich
                                                                             Nürnberg, October 3-5, 2008
A diar ylquinoline targ eting the en erg y supply of M. tuber culosis                         The Chemical Interaction of the Anticancer Drugs Irinotecan-HCl and
                                                                                              Epirubicin-HCl in the Sam e Infusion Solution
ANDRIES K, KOUL A, LOUNIS N, GUILLEMONT J*, J ARLIER V**
                                                                                              ÖZDEMIR FA1, ANILANMERT B 2, PEKIN M 1
Tibotec N V, Beerse, Belgium, *Tibotec NV, Val de R euil, Franc e, * *Pitié-
                                                                                              1                                                           2
Salpêtrière Sc hool of Medicine, Paris, Franc e                                                 Marmara Uni v. Fac ulty of Phar mac y, Istanbul, Tur key; Erciyes Uni v. F aculty of
                                                                                              Phar mac y, Kays eri, Tur key
We discovered a diar ylquinoline (TMC207 or R207910) with potent bactericidal
activity against drug-sensiti ve and drug-resistant M yc obac terium tuberc ulosis.           Background: Because the administr ation of infusi ons sometimes needs a long
Whol e genome s equenci ng of r esistant mutants suggested that the dr ug targets             period of time in combi nati on chemotherapy, it would appear that the best and
the energy s upply of myc obacteria by i nhibition of the ATP s ynthase. The                  easiest method is to give more than one drug in the s ame infusion sol ution.
oligomeric subunit c (AtpE) of ATP s ynthas e was validated as the target by                  Because of this, investigation of the chemical compatibilities of c hemotherapeutic
genetic, bioc hemical and binding assays. Unli ke other TB drugs, TMC207 is                   drug combi nations given in the s ame infusion solution befor e clinical studies is
equally ac tive against growing and dor mant TB bacilli, making it a good candi date          quite important. A new combi nation epirubicin and irinotecan were c hos en for
for shortening TB therapy.                                                                    investigation of chemic al incompatibility, i n order to search the applicability of
                                                                                              thes e two drugs in the same i nfusion s olution in a c ombination c hemotherapy.
In mice, four weeks of TMC207 monotherapy exceeds the bac tericidal ac tiviti es of           Methods: Visual c ompati bility was assessed by means of effer vesc ence, col our
isoniazid and rifampin by at least 1 log unit. Substitution of rifampin, isoniazid, or        change, precipitation and pH change after the drugs had been injected into s ame
pyr azinamide (the World Health Organization's first-line treatment regimen) with             infusion sol ution. Chemical interaction was further i nvestigated quantitati vel y using
TMC207 acc elerated bactericidal acti vity, leading to complete culture c onversion           a spectrophotometric method in infusi on sol ution in clinical concentrati ons. The
after 2 months of treatment in some combi nati ons, agains t 5 months for the                 molar ratio for the reac tion to proceed was also determi ned. All the interaction
standard regimen. Four months of treatment with rifampi n + pyrazi namide +                   study was repeated in phar mac eutic al for ms, imitating a real application.
TMC207 yiel ded the same relaps e rate as si x months of the standard regimen.                Results: No sign of inc ompati bility was obs erved upon visual examination. But a
Similar improvements were obs er ved when TMC207 was combi ned with drugs to                  chemical inc ompati bility was obs erved in the UV s pec tra of the drug mi xtures . The
treat MDR-TB, suggesting that us e of TMC207 may also significantl y reduc e the              molar ratio of epirubicin-HCl/irinotec an-HCl at which the interaction reached a
duration of treatment of MDR-TB.                                                              maxi mum was found to be 2:1. The chemic al interac tion occurred i mmediatel y
                                                                                              after admi xing and no visual or spectral change was noticed for 24 h after the
The bactericidal acti vity of TMC207 was confirmed in patients in a one week earl y           interaction had occ urred.
bactericidal ac tivity trial and the drug is now being inves tigated in a phase 2 trial in    Conclusions: Thes e drugs are chemic ally inc ompatible. The positive or negative
MDR TB patients                                                                               contribution of s uch chemical i nterac tions to the pharmacological effec t of the
                                                                                              combinati on might be of importanc e and while the applicability of these two drugs
                                                                                              in combi nati on is investigated in further phar mac ological studies, their chemical
                                                                                              interaction s hould als o be a consideration.




A Potential Tumor Cell-Pen etrating Peptide, CRGDCF(K[H-]KKK) 6, for the                     Preoper ative use of An algesia in Appendicites
Deliver y of Antisense and siRNA Oligonucleotides
                                                                                             ARAM FO
AOKI Y1,2, NISHIO S2 , MIYAMOTO T2, KUMAGAI M 1, HASHIZUME K2
                                                                                             Hadramout Uni versity, M ukalla, Yemen.
1
 Department of Internal Medicine, Matsumoto National Hospital, Mats umoto,
Japan; 2Department of Aging Medicine and Geriatrics, Institute on Aging and                  Background: Appendicitis is a common caus e of acute abdominal pain, earl y
Adaptation, Shinshu Uni versity Gr aduate Sc hool, Mats umoto, Japan                         analgesia was considered previously as it coul d mas k physical signs and henc e
                                                                                             delay the diagnosis and s urgical inter vention, now this has been challenged
Background: We have previ ousl y developed a tumor c ell-penetrati ng peptide                Methods: A prospecti ve, experimental study had been carried out in Ibn Sinna
(cRGD-hK) of 36 amino acid residues, which comprises a c yclic RGD motif for                 general Hospital from November 2006 to Marc h 2007, our aim was to determine
tumor homing and cell internalization, a DNA- or RNA-binding oligol ysine, and               the influence of Diclofenac s odi um DS( voltaren) on mas ki ng the diagnosis of
histidyl residues to facilitate the delivery i nto the c ytosol. T he length of antisens e   acute appendicitis.
or siRNA oligonucleotides is thought to match that of the oligolysine, presuming             The data collected by using well designed questionnaire with obser vation during
that both form c omplexes electros tatic ally in a 1:1 molar rati o (Figure). Then, we       the period of admission before the operation.
have been ass essing the possibility of suc h compl exes as antineoplastic agents.           Results: T he study incl udes 80 patients ( 40 as c ases and 40 as c ontr ols) and The
Methods: 1) With luciferas e expression plas mids as a reporter, we tested the               result reveal ed that most of the s ymptoms (fever, anorexia, nausea and vomiti ng)
functi onal potenc y of elements of cRGD-hK in cancer cells. 2) The effects of the           and si ngs (tenderness, obturator and Psoas signs, local guarding and rigidity), in
complexes of cRGD-hK with antisens e phos phorothioate DNA and siRNA                         additi on to the rate of perforati on and the vital sign were not hidden by
corresponding to the luciferase gene wer e compared i n vitro. 3) Using siRNA                DS(Voltaren) with P value >0.05.while other s ymptoms (pai n) and signs (rebound
corresponding to the c-raf gene, we ass essed the effects of cRGD-hK/siRNA                   tenderness, R ovsing and poi nting) had been hindered by the us e of DS (voltaren).
complexes on i ntrac ellular c-Raf protein l evels of pancreatic cancer c ells and on        The most common pres enting s ymptom in placebo and DS group was pain (100%)
the tumor growth i n nude mic e.                                                             which showed a mar ked decrease i n severity in thos e who rec eived voltaren as
Results: 1) The thr ee elements of cRDG-hK were i ndicated to func tion as                   analgesia ( 72.2%)
expec ted, by using bafilomyci n A1 and c ycloRGDfV. 2) The gene-silenci ng effect           Conclusion: Some of the s ymptoms and signs of acute appendicitis were mas ked
of the complexes with siRNAs was greater than that with antisens e                           by the us e of analgesia; while others were not .and overall Diclfenac s odium did
phos phorothioate DNA. 3) The pepti de/siRNA c omplexes lowered intr acellular c-            not i nfluence the decision of diagnosis or the management of ac ute appendicitis.
Raf protein levels of cultured c ells at less than 500 nM for 48h inc ubation. When
tumor-bearing nude mice were i ntraperitoneall y administered with the complexes
(5 μg RNAs three times a week for 4 weeks), the tumor growth was found to be
significantly (P<0.05) inhi bited in the third and fourth week.
Conclusions: It is s uggested that the cRGD-hK coul d functi on as a tumor c ell-
penetrati ng peptide for the deli ver y of siRNA and antisens e oligonucleotides , but
further s tudi es ar e needed.
                                         H
                                  CRGDCF(KKKK) 6 (cRGD-hK)

                                   + +
                                   _ _
                                           +
                                           _    +
                                                _    +
                                                     _     + _
                                                           _ +

                                   _   _   _     _     _   _   _
                                               siRNA

Fig. Schematic drawi ng of a putati ve c omplex of the tumor cell-penetrating peptide
(cRGD-hK) and s mall i nterfering RNA duplex (siRNA).




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                Page A-14
                                                             EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                   Celebrating the 100th Anni versar y of the
                                                                     Nobel Prize Award to Paul Ehrlich
                                                                         Nürnberg, October 3-5, 2008
Single Chain Antibodies (ScFvs) and Immunoconjugates: Computational and                  Exer cise as a Modalit y to Identif y Ther apeutic Molecules for Treatm ent &
Functional Appro aches                                                                   Prevention of Cancer-Associated Cachexia: Po ssib le Enhancement of Anti-
                                                                                         inflammator y C yto kines Through an Intermittant Activation of the Stress-
                 1              2                2               2            3
ARCANGEL I C , PUGNALI M , SPER ANDEI M , CANTALE C , BUCHER M ,                         Response Path ways
              4             1,4           2
GIANESE G , ROSATO V , GALEFFI P
                                                                                         ARDIES, CM
1
  ENEA, Dept. FIM-CAMO Rome, Ital y; 2EN EA, D ept. BAS-BIOTEC R ome, Ital y;
3                                   4
  Univ. C ologne C ologne, Ger many; Ylichr on S.r.l. c/o ENEA, Rome, Italy.             Northeas tern Illinois Uni versity, Chicago, USA.

Background: ScF vs in whic h the variable heavy and light chains are connected               Background: Repeated exercise is well-known to reduc e risk for canc er,
by a peptide linker maintain the binding specificity and affi nity of the parental           cardiovasc ular diseas e, type II diabetes, and a variety of neurol ogical disorders; a
antibody IgG. ScF vs coupled to highl y toxic mol ecules (immunoc onjugates) are             magic bullet if ther e ever was one! Cachexia affec ts approximately 50% of all
currentl y being devel oped for c ancer therapy. Ai ms: 1) To assess the effects of          cancer patients and is characterized by weakness , fatigue, anorexia, adipos e and
specific mutations on the stability, struc ture and dynamics of the scF v antigen            skel etal muscle atrophy, insulin resistanc e, and impaired i mmune func tion; a
binding site. 2) To develop an in silico proc edure for eval uati ng physicoc hemical        conditi on desparatel y in need of a magic bullet! Cachexia appears to be
properti es of two tumor-targeting anti-HER2 immunoc onj ugates. 3) To propos e              associated with elevated levels of PIF, TNF-α, IL-1, IL-6, and Interferon-γ.
plant-based expression s ystems for high-level scFv producti on.                             Exercise induces the production of sTNFr, IL-1ra, and IL-10 (anti-inflammator y
Methods: This study incl uded four scF vs (scFv(F8), scF v(ADDLs), scF v(FR5),               cytokines). Aim: To test the hypothesis that ac tivation of stress-respons e pathways
scFv(800E6)) and two tumor-targeting anti-HER2 immunoc onjugates (scFv(FR5)-                 may be i nvol ved in the protecti ve effect of exercise.
ETA, scFv(800E6)-ETA). For the computati onal procedures all the antibody                    Methods: Rats were familiarized with a rodent treadmill on four separ ate days
structures were deri ved by homology modeling and assess ed by molec ular                    over a period of two weeks and then forc ed to run for 60 minutes at a s peed of 27
dynamics (MD) simul ations . As regards the experimental s ection tobacco plants             m/min (a moderatel y hard wor kload for untrained rats). Three ani mals were killed
were trans formed for stabl e and transi ent expr essions. Specific expression vec tors      at each time point: 0, 15, 30, 60, 90, 120, 180, 240, and 300 minutes after the start
containing the gene enc oding for the scF vs of interest were us ed. Transgenic              of the exercise. Lungs from 3 animals at each ti me point were pooled and nuclei
plants wer e culti vated als o in hydroponic and aer oponic sys tems. DNA, RNA and           prepar ed by Dounce homogeniz ation and differential centrifugati on. Nucl ear
protein anal yses were perfor med in leaves , roots and r oot exudati ons.                   proteins were anal yzed by wester n blot using anti cJun/AP1 monoclonal antibody
Results: Structural and MD anal ysis indicated a strong correspondenc e between              (Ab-3, Oncogene Res earch Products).
structurall y–determined fl exibility of the binding site with the different functional      Results: Jun bi nding was appearent at 4 hours and one hour later was
behaviors proved by the wild-type and its mutants. Computational anal ysis of anti-          undetec table. This i ndicates that a 60 minute bout of running exercise is s ufficient
HER2 immunoc onjugates s howed that the pr esenc e of a toxin does not                       to enhanc e jun content but that this effect is of a rel ati vel y short duration.
significantly affect the major physic ochemical parameters and their structure. The          Conclusions: Based on these prelimi nar y res ults the possibility exists that a
highest level of scF vs expression was obser ved i n roots.                                  transient exercise/stress- medi ated ac tivation of the MAPK and/or JNK-MAPK
Conclusions: 1) The computati onal approaches repres ented a good tool for                   pathways (possibl y thr ough enhanc ed C a++ and ROS) is responsibl e, in part, for
structure-based design of antibody-bindi ng site, for anal yzing physicoc hemical            the anti-inflammatory effec ts of exercise as well as the enhanc ed ac tivity of
properti es of immunoc onjugates and for predicti ng the effects of the linked toxi n on     antioxi dant enz ymes and phase II enz ymes pr eviousl y obser ved by this lab.
structure, dynamics and func tionality of the antibodi es. 2) The proposed plant-            Because s keletal muscle is far more metabolicall y acti ve than lung during
based expressi on s ys tem s eems to represent a promising tool for a large-scale            exercise, one might expect that the degree of an exercise-induc ed ac tivation of
scFv production.                                                                             AP-1 in muscl e would be greater than that of lung. Development of drugs which
                                                                                             mimic this acti vation profile should produc e similar benefits.




Magic Bullet s in Removing Enterococcus faecalis Biofilms.                                   Vaccination with Recombin ant MHV68 Producing IFN Effectively Protects
                                                                                             Mice Again st Infection W ith W ild Typ e MHV-68 and Dram atically Reduces the
ARIAS-MOLIZ MT1, FERRER-LUQUE CM 1, ESPIGARES-RODRÍGUEZ E2 ,                                 Establishment of Long-term Spleen Laten cy.
PÉREZ-HEREDIA M 1 , BACA-GARCÍA P1
                                                                                             ARICO‘ E1, MONQUE DM 1, MOSCHELLA F1, D‘AGOSTINO G1, VENDITTI M 1,
1                                                      2                                               2        3        3              1              1
 School of Dentistr y, Uni versity of Granada, Spain; School of Pharmac y,                   KALINKE U , ALLEN D , NASH A , BELARDELLI F , FERRANTINI M .
University of Gr anada, Spain.
                                                                                             1
                                                                                              Istituto Superiore di Sanità, Rome, Ital y; 2 Paul Ehrlich Institute, Langen,
Background: Enteroc occus faecalis is the most c ommon and, occasi onally, the               Germany; 3U niversity of Edi nburgh, Edi nburgh, U K.
onl y single isolated bac terium from root canals of teeth with persistent periapical
periodontitis. Its inherent anti microbial resistanc e and ability to adapt to harsh         Background: Human gammaher pes viruses suc h as Eps tein-Barr Virus cause
environmental changes make E. faec alis responsible for many endodontic failur es;           lifelong infections and associated diseas es, by virtue of their ability to establish
moreover, suc h adverse c onditions may favour the growth of this bacterium as a             latent infection.
biofilm i n root canal walls.                                                                Mice infected with murine her pes virus 68 (MHV-68) represent a vers atile
The elimination and/or control of E. faecalis biofilms is a goal in root c anal therapy.     experimental s etting to study the bi ology of gammaher pes viruses and to test
Several irrigating solutions are us ed during the endodontic treatment; some of              vacci nati on str ategies against them.
them have been wi del y tested agains t planktonic bacteria. Because of the high             We recentl y obs erved that a clone of recombinant MHV-68 carrying the mous e
resistance of the biofilms to the endodontic irrigants, the ai m of our study was to         IFN1 gene (MHV-68mIFN1) shows a significant in vivo attenuation, which is
evaluate the effec tiveness of four irrigating solutions used in r oot canal teeth           mediated by the c ytokine released during the course of the infection, and affects
against E. faecalis biofilms .                                                               both the acute replication and s pleen latenc y.
Methods: Four irrigants - sodium hypoc hlorite, c hlorhexidine, ethylene-                    Methods: C57 BL/6 mic e rec eived two i ntranasal (i.n.) admi nistrations of 105
diami netetraac etic acid (EDTA) and citric acid - were tested at 1, 5 and 10 min of         earstwhile pfu of psor alen-UV-inac tivated wt or recombi nant IFN1-producing
expos ure to biofilms of E. faecalis ATCC 29212. The bi ofilms were grown                    MHV68. A group of mice rec ei ved i.n. 105 pfu of live-attenuated MHV68m IFN1
aerobic ally in the MBEC TM high-thr oughput device for 24h at 37ºC. They were               as a vacci ne. Four weeks later, vaccinated and a group of unvaccinated mice
expos ed to ten s erial twofold dilutions of eac h irrigating solution. The antibac terial   were infec ted (i.n.) with 4 x 105 PFU of MHV-68.
activity of the root canal irrigants was evaluated by determi ning the viable cell           Virus titres in the lungs and s pleen latenc y were measur ed by plaque and
counts and log killing of E. faec alis biofilm cultures. A c onc entration of an irrigant    infectious c entre ass ay respecti vel y. Virus non-specific B-c ell acti vation was
was considered to be effec tive when it produc ed a reduction of ≥ 5 logarithmic             assessed by FAC S. Mol ecular anal ysis of the viral genomes harboured in mice
units.                                                                                       spleens cells was performed by r eal ti me PCR. An ELISA was us ed to quantify the
Results: Sodium hypochl orite was the most effecti ve s olution at any dilution and          anti-MHV68 humoral immune res ponse.
time of exposure tested. Chlorhexidine and citric acid solutions showed less                 Results: Mice vacci nated with live-attenuated or partiall y inacti vated MHV-
antibacterial acti vity and needed more time to kill E. faecalis biofil ms. EDTA             68mIFN1 were protec ted agains t the challenge with wt MHV-68 in ter ms of acute
solution lac ks antibac terial ac tivity agains t E. faecalis biofilms even after 10 min     replication and long-ter m latenc y. This protection was ass ociated with a significant
contact ti me.                                                                               virus-specific IgG antibody res ponse.
Conclusions: Sodi um hypochl orite shoul d be elected as the best irrigating                 Conclusions: IFN1, produc ed at the site of the infection by the vaccinating virus,
solution as it showed the highes t effecti veness agains t E. faecalis bi ofilms.            acted as an adjuvant in sti mul ating an anti-MHV68 immune res pons e effecti ve in
                                                                                             eliciting protection agains t all phas es of MHV68 infection. We believe that the
                                                                                             ability of MHV-68mIFN1 to produce IFN1, thus efficientl y sti mulating the
                                                                                             immune s ystem whenever it reacti vates from latenc y, makes this recombi nant
                                                                                             virus a safer live-attenuated vaccine as compared to the latenc y-deficient clones
                                                                                             previousl y described by others.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                               Page A-15
                                                        EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                             Celebrating the 100th Anni versar y of the
                                                                Nobel Prize Award to Paul Ehrlich
                                                                  Nürnberg, October 3-5, 2008
Remifentan yl: Ho w it R elives Human and Earthly Pain, and New                    Differently Directed Changes in Interferon-γ Production Depending on
Persp ectives.                                                                     Radioadaptive Response.

ARIZA F1; TORRES G2                                                                               ARKHIPOVA EN, ALCHINOVA IB, KARGANOV MYu

1                                                                                                 Institute of General Pathology and Pathophysiol ogy, R ussian Academy of Medical
    Transplantati on Anesthesiolog y,Fundación Valle del Lili, Cali, Colombi a;                   Sciences , Moscow, Russia
2
    Cardiovascul ar Anesthesiolog y, F undación Valle del Lili, Cali, C olombia.
                                                                                                  Background: Interferon-γ (IFN-γ) is a pleiotropic c ytokine with anti proliferati ve
Backgro un d: Remifentan yl use has become a standa rd analgesic therapy during and
                                                                                                  and immunomodulator y ac tiviti es that are crucial for the r eg ulation of immune
after low-high complexity surgeries. Its indications have extended beyo nd surgery rooms
                                                                                                  respons es.
and today, it is one of the drugs that has importantly changed the way of administrating
                                                                                                  Methods: We examined a group of militar y pilots. T he examinees were di vided
anesthesia for a considerable numbe r of p rocedures specially for those that a re
ambulatory (a -da y surge ry). Howe ve r it is unkno wn w hat it´s impact will be o ver the       into 3 subgroups: ground personnel (9 persons, co ntrol group), 17 pilots with
emission of halogenated anesthetics which may be the origin of organic -halogenated               <1000 h flight time, and 12 pilots with >1000 h flight time. T he qualit y of reparation
pollutants (OHP) that are released into the atmos phere, resulting in ozone layer damage          is in many respects genetic ally determi ned; therefore, we used peripheral blood
and more o ver t hese compounds are recognized as neu rotoxic and end ocrine modulators.          lymphoc ytes from pilots for in vitro detection of a radioadapti ve res ponse (RAR),
Around the wo rld there a re man y institutions where it is not possible to administer total      which was evaluated by the number of c hromos ome aberrations.
endovenous anesthesia (T IVA) due to economical implications. Our objective was to                Results: No diff erenc es i n IFN-α serum c ontent after i nduction by NDV virus were
analyze the impact of the use of remifentanyl during surgery ove r the quantity of                detected. The adapti ve r esponse was obser ved in 7 indi viduals of the control
halogenated anesthetics, using intermediate infusions of remifentanyl (0,15 -0.25                 group (78%), in 10 pilots who had <1000 flight hours (59%), and in 4 pilots having
mcg/kg/min).                                                                                      >1000 flight hours (33%). The e xamined indivi duals were di vided into 2 groups
Me th ods: Over a period of one year an initial prospective study was unde rta ken to
                                                                                                  depending on the pres enc e of RAR, and IFN-γ production after radiation was
identify mean plasma concentrations of remifentanyl fo r a ta rget biespectral inde x (BIS) of
                                                                                                  measured. It was shown that at doses 0.05 Gy or 0.5 Gy no differences between
40-50, in a hispanic population at a level 4 hospital, based on the pha rmacokinetic model
described by Minto et al. T he second stage of the stud y was to identif y the total amou nt of   groups were detec ted. Exposure with these dos es s equentiall y in 48 h inter val
halogenated gases consumed during this period compa ring it to the consumption from the           resulted to differentl y direc ted c hanges : lymphoc ytes of indivi duals with RAR
previous year. Va riables are repo rted as numbe r of patients, medians and ranges, means         produc ed more IFN-γ than before while cells of persons without RAR made it less .
and SD. Nominal data were analyzed using the exact Fisher test. Ordinal and continual             Conclusions: The quality of adapti ve mechanists evaluated by RAR may be
non-normally distributed variables were anal yzed using Mann -Whitney Ra nk summ test.            useful for esti mation of indi vidual sensiti vity to radiation during radiotherapy in
Continuous variables were comp ared using Student t -test.                                        oncol ogy and in prediction of professional risk.
Results: 12.532 surgical procedures were done. T here were no significant differences in
comparing demogra phic variables betwee n studied groups. Remifentan yl mean plasma
concentration in this population was 4,76 (±0,45 ) ng/dl f or an a verage consumption of
0,17 (± 0,1 3) mcg/ kg/min. being significantly high for those patients whom had undergone
abdominal surgeries (p<0,0 25). Desflurane, sevoflurane and isorane consumptions we re
significantly reduced w hen remifentanyl was used [32 (±5 ), 23 (±2), 15 ((±1 ) vs. 13 (±3); 9
(±1); 6 (±1) ml/h r. (p< 0,001)]. T he total estimated amount of halogenated gases per month
was reduced to approximately 55% (40.192 vs. 17.099 ml).
Concl usio ns: 1.Standa rdized use of remifentan yl in surgical units which use balanced
inhaled anesthesia techniques, significantly reduces the usa ge of volatile halogenated
anesthetics, and protects environment. 2.Implications derived from these observations
could be useful for those institutions in various developing countries where T IVA is not
feasible for economical or infrastructural reasons.
3. New associations between remifentanyl and other d rugs like ketamine or mida zolam,
based on pharmacological interaction mechanisms could lower even more h alogenated
gas consumption during surgery. It is a necessity to continue searching new anesthetic
technics that are friendly with our planet and at the same time giving economic benefit
especially to those in poorer populations.




Conscious drug selection and dosing b y genot yping and ph enotyp ing of                          Cardiac tamponade from sling shot metal d arts in Chuuk: a r etrospective
alleles with mutations, deletion and/or duplication of the CYP2D6 g ene                           review of cases.

ARNETH B 1, SHAMS M 2, HIEMKE C 2, and HÄRTTER S2                                                 ARSEN AL JC, REMIT K, YICHIRO O.
                                      1                  2
Departments of Clinical Chemistr y and Ps yc hiatr y , Uni versity of Mai nz, Germany             C/- Chuuk State Hos pital, Federated States of Micronesia. jcars 3@ yahoo.com

Background: Pol ymorphisms of C ytoc hrome p450 2D6 (CYP2D6) have a                               We deter mined the immedi ate cause of death of patients with penetrating cardiac
significant effect on the pharmacokinetics of most antidepressants. Indi viduals are              injuries from slingshot metal darts. This retros pec tive review of c ases focus ed on
classified as poor metabolizers (PMs) due to inheritance of two mutant CYP2D6                     thos e 7 patients with penetr ating cardiac injuries from the period July 1999 to Jul y
alleles and develop higher pl asma drug conc entrations causi ng an increased ris k               2005. T here were 6 patients who underwent emergenc y thor acotomy regardless of
of side effects and toxicity when subj ected to standard recommended dos es of                    the type of operati ve approach. Fi ve of the 6 patients who were operated
CYP2D6 substrate drugs. In c ontras t indi viduals are classified as ultrarapid                   underwent left Lateral Thoracotomy and 1 patient under went Median Sternotomy.
metabolizers (UMs) due to inheritanc e of alleles with duplicated or multiduplicated              There were 11 patients who sustained cardiac injuries out of the 240 cas es
active genes and will thus under circumstances not reac h therapeutic pl asma                     reviewed. The patient's with cardi ac injuries had a higher mortality (27.3%) than
levels of CYP2D6 s ubstrates, leading drug resistance and false accus ation of non                thos e who have penetrating thoracic injuries (3.5%) witho ut ass ociated car diac
compliance. Our study aimed to devel op a rapi d and reliable proc edure us ed for                injury. Of the s even patients who had penetrating cardi ac injuries , 5 patients
early detection of PMs- associated mutations and/or deletions (CYP2D6 * 4, *3, * 6,               underwent left Lateral thorac otomy and 1 patient underwent median sternotomy.
*
  9 and * 5) and UMs- ass ociated CYP2D6 gene duplication or multi plication.                     All 6 patients had ches t tube thoracotomy insertion prior to surgery. There w ere 2
Methods: EDTA blood was drawn from twenty five pre-selected depressed                             deaths i n this review of penetrating cardiac injuries. The other patient with 7
patients [(14 men and 11 women), mean age ±SD (49.8 ±12.7)] treated with the                      multipl e slingshot injuries died of c ardiac tamponade with hypovolemic shoc k.
antidepress ant venlafaxine and having unus ual resulting plas ma conc entrations of
venlafaxine were chosen for genotypi ng anal ysis. Real-time PCR reaction with
subsequent fluorometric melting point analysis of the PCR produc t was used.
Gene deletion (*5) and gene duplication or multiplication were investigated based
on the meas urement of the fl uoresc enc e intensity quoti ent (N) of the CYP2D6
gene relati ve to the albumi n gene as an internal standard gene using a
quantitative PCR technique.
Results: One homoz ygous (*6/* 6), and thr ee heteroz ygous (two with* 4/* 5 and one
with* 4/* 6) were detected. Fi ve i ndi viduals were heteroz ygous (*4/non detec table
allele) and onl y one patient had heteroz ygote gene duplication [*4/gene duplication
(2x* 1)]. Six indivi duals had gene duplication. Melting curves were verified using
DNA samples of known genotypes and by sequencing the PCR products.
Conclusions: Our new genotyping proc edur e was evaluated agai nst the ratio
between the O-demethyl ated (ODV/V) metabolite of the CYP2D6 substrate
Venlafaxine (V) and determined by HPLC servi ng as phenotype of the genotyped
patients. T his genotyping procedure was r egarded as fas t and reliabl e for clinical
routine.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                   Page A-16
                                                             EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                    Celebrating the 100th Anni versar y of the
                                                                       Nobel Prize Award to Paul Ehrlich
                                                                         Nürnberg, October 3-5, 2008
The Long Term Follo w up Result s of H ydatid C ysts after Albendazole and                Efficacy of amo xicillin/clavulan ic acid in preventing infectious and
Mebandazole Tr atment                                                                     inflammator y complications follo wing imp acted m andibular third molar
                                                                                          extraction.
            1           2            2               2               2
ARSL AN A , ÖDEV K , PAKSOY Y , ARIKOGLU H , and ŞAHİN M
                                                                                          ARTEAGOITIA I1, DIEZ A2, BARBIER L12, SANTAMARIA G1 SANTAMARIA J12
1                                           2
  Gaziantep Uni versity, Gaziantep, T urkey, Selçuk U niversity, Meram Campus,
                                                                                          1
Konya, Tur key                                                                              University of the Basque Countr y. Lei oa, Spain 2Cruces Hospital. Os akidetza.
                                                                                          Spain
Background: Our experi ences gai ned over th e years of study on th e treatment of hy datid cyst d iseas e
showe d that women ar e more susce ptibl e to hydatid cyst dise ase then me n. Anthelmi nthic dru gs such as
albe ndaz ole or m ebe ndaz ole ar e more effective i n lu ng cysts than the liv er cysts when pr escrib ed ora lly.             Background: The ai m of this clinical trial was to eval uate the efficac y of
Initially w e und ertook exp erime nts to explai n this phe nome no n.                                                           amoxicillin/clavulanic acid 500/125 in the reducti on of infecti ous and infl ammator y
Methods: Cystic liver an d lun gs wer e col lected fresh ly from the mun icip al sl aug hter ho use an d from the                complications after extrac tion of an i mpacted mandibul ar third molar. Our
patients. Each cystic flui ds col lected a nd vi abl e scolec es were poo led a nd us ed for an alysis. Scol icid al ag ents
such as hyperto nic s ali ne s oluti on, 0.5% silv er n itrate, 25% etha nol, and 10% al ben dazo le s uspe nsio n wer e         hypothesis is there are more infec tious and inflammator y c omplications in patients
used at a tim e to determ ine th e effect of scolic ida l ag ents kin etical ly on th e via bil ity of scolec es from min utes   treated with placebo than in those treated with antibi otic, with a maxi mum ratio
to 72 hours assess ed by eos in Y dye sol ution exc lusi on a nd inc lusi on.                                                    difference of 0.067.
Clinical use of in vitro findings: Initially, sh eeps h avin g hy datid cyst d iseas e w ere sel ected by u ltrason ic
examin ation of the liv er a nd lun g of th e a nima ls br oug ht to sl aug hter h ouse. Th e a nima ls w ere grou ped for       Methods: A doubl e-blind pl acebo-controlled randomized clinical trial. The sample
albe ndaz ole a nd meb and azo le treatment. And eac h anim al cysts were treate d percuta neo usly with e ither 10 %            was derived from the population of subjects attending Cruces Hospital for
albe ndaz ole or me ban dazo le sus pens ion so lutio n. Based on sh eep exper iment the pati ents treated with sa lin e         evaluation and extrac tion of a impacted mandibular third molar. A maxillofacial
solutio n by mea ns of PAIR-PD (perc utane ous punctur e, asp iratio n, in jectio n a nd irrig ation, and r easp iratio n)
and al ben dazo le by PAI (percutan eous as pirati on an d in jectio n) proce dure.                                              surgeon performed the operations under local anesthesia. T he surgical technique
Results: Among th em hyp ertonic s ali ne s oluti on, etha nol, a nd si lver n itrate treatment ex erted imm edi ate             was the s ame in all cases, and the follow-up period was 8 weeks . Patients were
effect within ha lf an h our whi le alb end azol e sh owe d its effect after 4 8 h ours. Bec ause of thes e fin din gs w e       treated with postoperati ve plac ebo or amoxicillin/clavulanic acid 500/125 mg 3
used pre and p ost percuta neo us oral alb end azol e or meb and azol e treatment for 72 h ours pri or to PAIR-PD or
PAI involv ement. Thus, tur gid cyst bec omes fl accid an d e nab les punctur e w ithout s pil lin g the cystic co ntent
                                                                                                                                 times a day during 4 days. The outc ome variable was infectious and infl ammator y
which pr events comp licati ons, diss emin ation a nd recurr ence. Percuta neo us dra ina ge an d irrig ation a ppl icatio n     complications . Sex, age, s moking, molar depth, angulation, need for sectioning,
showe d that protosc olec es meta bol ize an d co nvert thes e drugs into activ e form to affect the ir vi abi lity              ostectomy, and operation ti me were rec orded. Anal ysis was by intention to treat.
irreversi bly. Gener ally, p atients ar e referre d to hos pital with a lrea dy existi ng cysts. PAI of the cysts with
antihe lmintic drugs or PAIR-PD of scol icid al a gents or sa lin e sol ution c ure the cyst with a min imum morb idity.         Results: A total of 490 lower third molars were surgically removed (259 anti biotic
The kinetic in vitro stu dies s how ed that scl erosi ng a gents, such as ethyl alco hol, si lver n itrate, sali ne so lutio n   and 231 plac ebo), The patients' mean age was 24,15 (23,70-24,59), the frequenc y
kill the protosc olec es imme diate ly an d al ben dazo le and m eba ndoz ole s uspe nsio n so lutio n starts affecting 4 8      of postoperative infectious and inflammator y complications was 1.9% in the
hours later a nd kil ls the protosco lec ess compl etely after 72 ho urs. Howev er, sclerosi ng ag ents can caus e
chemica l scleros ing ch ola ngitis w hich l imits their use.
                                                                                                                                 antibi otic and 12.9% i n the placebo group (OR 7.6, 95%CI 2.9-19.9; P < .001). The
Conclusion: The proce dure descr ibe d ab ove offerred safe alter native to s urgic al tre atment of hy datid cysts.             number needed to treat was 10 (7-16). Unadjusted r elati ve risk was 0.15 (0.06-
Over 10 years foll ow up our proc edur e d id not exper ienc e a ny m ortality whic h is h igh er w ith sur gica l               0.38) (P < .001). Absol ute reduction ris k was 0.11(0.066-0.155)]. T herefore, the
proced ure. Comp licati ons such as an aphy lactic reacti on, side effects and recurr ence ar e minim al. Hospita l
stay is red uced cons ider ably an d the patie nt can r eturn th e w ork the fo llo win g d ay. Hosp ital c ost is re duce d     hypothesis c annot be rej ected. In third mol ars submuc ous (p=0.091 and NNT 17
consid erab ly. How ever, the best ther apy is to pr event cyst formatio n from the protosc olec es at ear ly ph ase of          with IC95% 8-infinite). Multivariate anal ysis shows treatment with antibiotic (OR =
infection or to preve nt cyst growth by preventin g the differentiati on of germin ative membr ane. Therefor e, it               8.66 (3.17-23.67); P < .001) and age (OR = 1.08 (1.00-1.16); P = .029) are the
looks pl ausi ble to dev elo p vaccin ation targ eted to germi native mem bran e differenti ation wh ich circumv ents
immune system in the i nfected pati ents.                                                                                        onl y variables to be i ncluded in the logistic regression model. Possi bility of
                                                                                                                                 infection: P( x)=1/1+e -(-3,74+0,074 EDAD -2 ,075 ANTI BI OTIC(1)) Severe complications occ urred
Refer ences:                                                                                                                     in one patient i n plac ebo group
1. Arslan A, Ödev K, Baykan M, Arıkoğlu H, Çataloluk O, Paksoy Y, Bulun M: Ekinokokkus gran ulos us‘u n
bulaşm ası, org an tutu lumu, te dav isi v e kar apınar lı iç in alınması ger eken tedb irler. Kara pınar S empozy umu,          Conclusions: Amoxicillin/clavulanic aci d is efficacious in reducing the incidenc e of
26–2 7 Ekim 200 0, Karapınar, Kony a, Sempozyum kita bı s: 281-29 2.                                                             postoper ati ve i nfecti ous and infl ammator y complic ations following third molar
2. Ödev, K., Paksoy, Y., Arslan, A., Aygün, E., Sahin, M., Karak öse, S., Baykan, M., Arikog lu, H., Aksoy, F.,                  extraction but s hould not be pr escribed in all cases . Preventive antibi otic treatment
2000. Son ogra phic ally gui ded p ercutan eous treatme nt of hepatic hy datid cysts: Lon g-term results. J. Clin.
Ultrasou nd. 28, 469- 478.                                                                                                       would not be indicated for third mol ars submucous. Age should be taken into
3. Paksoy, Y., Ödev, K., Sahin, M., Dik, M., Ergul, R., Arslan, A., 2003. Percuta neo us son ogra phic ally gui ded              account, i nfec tion risk incr eases 8% ever y year in all patients.
treatment of hydatid cysts in sh ee p. Direct inj ectio n of meba ndoz ole a nd a lbe ndaz ole. J. Ultraso und. Med. 2 2,
797-8 03.
4. Paksoy, Y., Ödev, K., Sahin, M., Arsla n, A., Koc, O., 2005. Percutan eous treatment of liver hydati d cysts:
comparis on of direct in jectio n of albe ndaz ole a nd hyp otonic sa lin e soluti on. AJR. AM. J. Roentgenol. 18 5,
727-7 34.
5. Arslan, A., Arikogl u, H., Paksoy, Y., Odev, K., Koc, O., 2006. Comment on percuta neo us treatment of liv er
hydatid cysts. Reply. Am. J. Roentgen ol. 186, 11 99-1 200.




Manganese (Mn) Transport at the Blood-Brain Bar rier: Implications for                                                           Targeting Dexamethasone-Loaded anti-E-selectin Lipo somes Pr events
Parkinson's-Like Disease                                                                                                         Glomerulonephritis Progr ession: The Potential of Vascular B ed-Specific
                                                                                                                                 Drug Deliver y
ASCHNER M 1,2 , BENEDETTO A1, AU C 1
                                                                                                                                 ÁSGEIRSDÓTTIR SA, KAMPS J, HEERINGA P, RUITERS MHJ, MOLEMA G
1                               2
 Dept. Pediatrics and Dept. Pharmacol ogy, Vanderbilt Uni versity Medical Center,
Nashville, TN, USA                                                                                                               University Medical Center Groningen, Uni versity of Groni ngen, T he N etherlands ,
                                                                                                                                 Dept of Patholog y & M edical Biology, Laboratory for Endothelial Biomedicine &
Background: Though an essential trace element, expos ure to high Mn levels has                                                   Vascular Drug Targeting Res earch
been implicated in a Par kinson‘s-like diseas e, manganism. We hypothesized that
symptoms ass ociated with thes e two dis orders reflect perturbations in s hared                                                 Background: Glomerul onephritis is a renal dis eas e characterized by glomerular
molec ular pathways. Accordingl y, studies were c arried out in C. elegans to test the                                           inflammation which is frequentl y treated with glucocorticoids . However, their us e
hypothes es that (1) di valent metal transporter (DMT1) is a putati ve M n transporter,                                          has limitations because of s ys temic side effects.
and (2) Mn preferentiall y targets dopaminergic (DAergic) neurons.                                                               Aim: To test the hypothesis that targeted delivery of dexamethasone by
Methods: Bristol wild-type (WT) C. elegans N2 strai n was used unless other wise                                                 immunoliposomes to glomerular endothelium decreas es renal injur y, while
indicated and was grown at 20°C in Petri dishes on nematode growth medium                                                        preventing s ystemic side effects of dexamethas one.
inoculated with OP50 E. coli. For lethality ass essment, live and dead wor ms were                                               Methods:        Glomerulonephritis was induc ed i n C57bl/6 mic e and monitored for 2
counted on eac h plate and rec orded. Mn content was measured by atomic                                                          weeks. D examethas one-containing i mmunoliposomes and free dexamethas one
absorption s pectrometry. Neurodegeneration was assess ed by confocal                                                            were injected i ntravenously. Gene expr ession was quantified in renal endothelial
microscopy.                                                                                                                      subsets after lasermicrodissec tion. Disease par ameters anal yzed included the
Results: The C. elegans genome enc odes three DMT1 orthologues (SMF-1,                                                           extent of glomerular crescent formation, albuminuria, and blood ureum nitrogen
SMF-2, SMF-3). C. elegans deletion- mutants s m        f-1(eh5) and s m    f-3(ok 1035)                                          and plas ma glucose l evels.
exhibited resistanc e to Mn exposure compared to WT, while s mf-2(gk1330) was                                                    Results: E-sel ectin was expressed s electi vel y by glomerular endothelial c ells after
more sensitive. C orroborating these obs er vati ons, after expos ure, M n content in                                            inducti on of glomerulonephritis. Cons equentl y, acc umulati on of anti-E-selec tin
sm f-2(gk 1330) was greater than WT, s mf-1(eh5) and s mf-3(ok 1035), the latter                                                 (AbEsel) liposomes was 3.6 ti mes higher than non-targeted IgG lipos omes in
taking up the leas t Mn of all mutants . SMF-1::GF P and SMF-3::GFP were found                                                   diseas ed ki dney. In glomeruli dexamethasone-AbEsel liposomes co-loc alized with
co-express ed in the gut and the maj or epidermis hyp7, li kel y acc ounting for most                                            endothelial cells. Targeted delivery of dexamethasone-Ab Esel liposomes reduced
Mn uptake, while SMF-2::GFP was mai nly express ed in the " mc1-3" and " vpi1-6"                                                 glomerular endothelial expression of P-selectin, E-selec tin and VCAM-1 by 60 to
cells. Confocal microsc opy reveal ed that Mn specific ally targeted DAergic                                                     70%. Other renal microvasc ulature was not affected b y targeted dexamethas one
neurons, while sparing others (GABA, glutamate, and ac etylcholine). The DA                                                      deliver y and unlike administration of fr ee dexamethasone, site selec tive deli ver y of
transporter knoc k-out dat-1(ok 157) and the D A receptor dop-2(vs105);dop-                                                      dexamethasone-Ab Esel       liposomes      di d  not    increase    bl ood    glucos e.
1(vs100);dop-3(vs106) triple mutant were hypersensitive to Mn exposur e.                                                         Dexamethasone-AbEsel liposomes reduced al buminuria at day 7 and ameliorated
Combi ned with measur ements of DA content in these str ains, our results                                                        renal injur y at day 14 as evidenced by a reduction of blood urea nitrogen levels,
established that elevated DA levels sensitize the worm to Mn toxicity.                                                           decreas ed glomer ular crescent formation, and down regulati on of diseas e
Conclusions: (1) DMT1 in a putati ve blood-brain barrier Mn transporter and a                                                    associated genes .
target for trans port i nhibiti on i nto the mammalian brai n under co nditions of                                               Conclusion: 1) E-selectin is an exc ellent target for selecti ve deliver y of potent
exc essi ve Mn exposure. ( 2) DAergic neurons are exquisitel y sensiti ve to Mn and                                              anti-inflammatory drugs to glomerular endothelium. 2) Rec ent new application of
extracellular DA is i nvol ved in Mn-induced neurotoxicity. (3) C. elegans is ideall y                                           this powerful strategy i ncludes enc apsul ation of gene s pecific s mall interferenc e
suited for studi es on genetic pathways involved in Mn toxicity and PD.                                                          RNA to knoc k-down genes important for diseas e devel opment.

Supported by R01 ES10563 & D oD W81XWH-05- 1-0239.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                                          Page A-17
                                                                   EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                           Celebrating the 100th Anni versar y of the
                                                                              Nobel Prize Award to Paul Ehrlich
                                                                                Nürnberg, October 3-5, 2008
Randomized Controlled Clinical Trial of Day-Care Based and Hospitalized                          Transcription of Major Histocompatibility Complex Class I (K b) and Transporter
Management of Severe Childhood Pneumonia by Injection Ceftriaxone in Dhaka,                      Associated with Antigen Processing 1 and 2 genes is up-regulated with age.
Bangladesh
                                                                                                 ASSOUNGA AG1,2, WARNER CM 1
ASHRAF H 1, ALAM NH 1, GYR N 2
                                                                                                 1
                                                                                                   . Department of Biology, Northeastern Uni versity, Boston, MA 02115.
1                                                                                                2
  International Centre for Diarrhoeal Disease R esearc h (ICDDR,B), Dhaka,                         . Department of Medicine, Uni versity of KwaZulu-Natal, Durban, South Africa.
                2
Bangladesh, Uni versity of Basel, S witzerland
                                                                                                 Background: The transporter associated with antigen processing 1 and 2 (TAP1 and
Background: Although hos pital-bas ed treatment of severe pneumonia in children                  TAP2) genes belong to the ATP-binding cassette family of transporter genes. They
is desirable, but practical barriers often prevent c hildren in ar eas with the highest          provide peptides necessary for the assembly of MHC class I molecules by
rates fr om recei ving hos pital c are. We have shown rec ently in an uncontrolled tr ial        transporting these peptides into the endoplasmic reticulum. As MHC class I protein
a success i n day-care treatment of sever e pneumonia in children; however, a                    expression increases with age, we have explored the effect of age on the transcription
                                                                                                                    b
randomized, c ontr olled trial is rec ommended.                                                  of MHC class I (K ), TAP1 and TAP2 genes in C57BL/6 mice.
                                                                                                 Methods: Blood and spleen l ymphoc ytes were isolated and from mice aging from 3
Methods: The study was c onducted at the R adda Clinic, Mirpur, Dhaka and                        months to over 24 months. RNA was extracted and mRNA for Kb, TAP1, TAP2 were
Mirpur Shishu (Paedi atric) Hospital, Mirpur, Dhaka as the two primary sites for the             quantified using slot-blot hybridization followed by a densitometr y.
day-care management and inpatient management, respecti vel y. Children of either                 Results: There is a parallel age related increase (1.5-fold) in blood l ymphoc yte
sex aged 2-59 months with severe pneumoni a accordi ng to World Health                           mRNA of thes e genes from 3 months to 21 months. In mice over 24 months old there
Organization (WHO) criteria without associated co- morbi dities wer e randomized to              is a decrease in Kb and TAP1 mRNA, but an increase in TAP2 mRNA. In s pleen
receive either day-care treatment at the Radda Clinic, or hospitalized treatment at              lymphoc ytes an age-related increase in all three mRNA species occurs throughout
Mirpur Shishu Hospital. Children at the clinic recei ved day-car e management with               life. While MHC class I and Tap genes follow about the same age related changes,
appropriate anti biotics like injecti on ceftriaxone, feeding and supporti ve c are from         MHC class I mRNA is about 50 times more abundant than either TAP1 or TAP2
08:00-17:00 ever y day, while mothers were educ ated on continuation of c are at                 mRNA.
home during the night. Chil dren at the hos pital recei ved hospital care with similar           Conclusion: Transcription of MHC class I (Kb ) and peptide transporter (TAP1 and
antibi otics like inj ection ceftriaxone, feeding and s upporti ve c are for 24 hours ever y     TAP2) genes is up-regulated with age. It is possi ble that c oordinated expressi ons of
day rather than 9 hours at the clinic until i mprovement.                                        MHC class I and TAP1 or TAP2 may predis pos e an animal to better antigen
                                                                                                 presentati on and a longer life.
Results: From September 2006 to Dec ember 2007, 251 children (125 at clinic as
day-care, 126 at hospital as hospital care) with severe pneumonia without
associated co- morbi dities were enrolled, 83 (33%) were hypoxaemic with a mean
(SD) oxygen saturation of 94 (5)%, which i ncreas ed to 98 ( 1)% on oxygen therapy.
Day-care management was s uccessful in 109 children [87% (95% CI 80% to
92%)], as opposed to the succ ess rate of 98% (95% CI 94% to 100%) by hos pital
management. Fifteen [12% (95% CI 7% to 19%)] chil dren in the day-care group
had to be referred to hospital for supporti ve management, but none in the hos pital
group.

Conclusion: Most (87%) childr en with s evere pneumonia without associ ated co-
morbiditi es can be successfully managed on a day-care basis at a day-care clinic.
However, few (12%) c hildren required referral to hospital.




The changes in renal function after a single dose of intravenous furo semid e                 How to avoid drug—drug inter actions
in patients with compen sated liver cirrhosis
                                                                                              ÅSTRAND B 1, MONTELIUS E2 , ANTONOV K3 , HOVST ADIUS B1 AND
ASSY N 1 ,2,5, KAYAL M,3 MEJIRISKY Y,3 GOR ENBERG M,4 HUSSEIN O,2 AND                         PETERSSON G2
SCHLESINGER S2
                                                                                              1
                                                                                               School of Pure and Applied N atural Scienc es, U niversity of Kal mar, Kal mar,
1
  Liver Unit, Sieff H ospital, Safed, Isr ael, 2Department of Inter nal Medicine A, Si eff    Sweden
Hospital, Safed, Israel                                                                       2 School of H uman Scienc es, Uni versity of Kal mar, Kal mar Sweden
3
  Department of Internal Medicine B, Sieff Hos pital, Safed, Israel. 4Department of           3 The Associ ation of Pharmaceutical Industr y, Stoc kholm, Sweden
Nuclear Medicine, Sieff Hospital, Safed, Isr ael. 5F aculty of Medicine, Technion
Institute of Tec hnolog y, Haifa, Israel                                                      Background: It may be favorable to us e a combinati on of drugs, if the
                                                                                              combinati on is well documented, to enh anc e the effect or to r educ e adverse
Background:Pati ents with compensated Child-A cirrhosis have sub clinical                     effec ts. However, in the c ase of pati ents visiting several different physicians, who
hypovolemia and diur etic treatment could res ult in r enal i mpairment.                      are prescribing less appropriate c ombination of drugs due to bei ng unaware of
Aim: To evaluate the changes in renal functional mass as reflec ted by DMSA                   each other, the outcome may be negati vel y influenc ed. Th e pol yphar mac y may
uptake after single inj ection of intravenous furos emide in pati ents with                   even result i n serious adverse drug events .
compens ated li ver cirrhosis.                                                                Methods: In Sweden, the government has legislated and funded a nationwide
Methods: Eighteen cirrhotic patients were di vided in two groups; eight patients              mandator y databas e for all dispens ed prescriptions. The i nfor mati on content is
(group 1, age 56 ± 9.6 yrs, Gender 5M/3F, 3 alcoholic and 5 non alcoholic) were               availabl e during 15 months for prescribers and dispensing pharmacists , as well as
given low intravenous 40 mg furos emi de and ten other pati ents (group 2, age 54 ±           for the r egistered indivi dual. Prior to the launc h of the nationwide databas e in
9.9, Gender 6M/4F, 4 alcoholic and 6 non alcoholic) were given high 120 mg                    Sweden, studies were perfor med to estimate the preval enc e of potenti al drug —
furosemide res pecti vel y. Renoscintigraphy with 100MBq Of Tc 99 DMSA was                    drug inter actions (DDI) in a general population and to evaluate the historical
given intr avenousl y before and 90 minutes after furos emide admi nistration and             change i n risk over three decades.
SPECT imaging was deter mined 3 hours l ater. All patients were kept under low                Results: On average each i ndi vidual filled 14.6 prescripti ons during a 15 month
sodium diet (80mEq/d) and all diuretics were withdrawn for 3 days . 8-hours UNa               study period (2003-2004). The risk of recei ving a potential DDI was esti mated as
exertion, Calculated and meas ured Creati nine clear ance (CCT) were performed                the cumulati ve inci dence 0.26 overall. T he relati ve risk for women was estimated
for all patients.                                                                             as 1.3. F or more s evere potential DDIs the cumulati ve incidence was estimated as
Results: Intravenous fur osemide increased the mean renal DMSA uptake in 55%                  0.02. T he risk of recei ving a potenti al DDI was positi vel y c orrelated to age and
of patients with c ompensated cirrhosis and thes e changes persist up to three                pol ypharmac y. The change in risk over three dec ades increas ed for type C
hours after injecti on. T his increase was at the s ame extent in either low or high          (relative risk RR 1.18), but decreased (RR 0.71) for the more s evere type D
doses of furosemide. (From 12.8% ± 3.8 to 15.2% ± 2.2, p < 0.001 in Gr I as                   interactions. Pol yphar mac y i ncreas ed with more than 60% during the three dec ade
compared to 10.6% ± 4.6 to 13.5% ± 3.6 in Gr 2, p < 0.001). In 8 pa tients (45%, 3            study period. Fifteen months after launch of the new Nati onal Phar mac y Register
pts from Gr 1 and 5 pts from Gr 2) DMSA uptake remai n unchanged. The mean 8                  in Sweden, the prevalenc e of i ndi viduals with dis pensed drugs was 71%
hrs UNa excretion after intravenous furosemide was above 80 meq/l and was                     (6,424,487/9,047,752). F or elderly (80-89 years) the mean number of dispensed
higher in Gr 2 as compared to Gr 1 respecti vel y (136 ± 37 meq/l) VS 100 ± 36.6              prescriptions was 27.8 during the first 15 months.
meq/l, P = 0.05). Finally, bas al global renal DMSA uptake was decreas ed in 80%              Conclusions: The new National Phar mac y Register will provide health c are
of pati ents; 22.5 ± 7.5% (NL > 40%), as compared to nor mal calculated creatinine            professionals with a powerful tool to s ystematicall y review all prescriptions. Alert
clearanc e (CCT 101 ± 26), and measured CCT of 87 ± 30 cc /min (P < 0.001).                   systems integrated in electronic healthcare records may be used to detect
Conclusion: A single furosemide injecti on incr eas es renal functional mass as               potential DDIs. To gain approval among physicians, the alerting shoul d foc us on
reflected by DMSA in 55% of patients with c ompensated cirrhosis and identify 45%             the more severe and clinicall y relevant DDIs. More indi vidual-oriented information
of patients with reduc ed uptake and who could develop renal i mpairment under                (laboratory, genetic, allergies) may in the future be proc essed befor e prescribing of
diuretics. Whether or not albumi n infusion exerts bene ficial effect in those patients       drugs, to better c ustomize the therapy for the si ngle indi vidual.
with r educ ed DMSA uptake remai ns to be determined.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                 Page A-18
                                                          EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                  Celebrating the 100th Anni versar y of the
                                                                     Nobel Prize Award to Paul Ehrlich
                                                                       Nürnberg, October 3-5, 2008
Distinct Cell C ycle Proteins Control Sch wann C ell Prolifer ation in Health and       Magic Bullet s for the Treatment of Oxidative Stress-Induced
Disease                                                                                 Neurodegener ative Disord ers

ATAN ASOSKI S                                                                                ATLAS D

University of Bas el, Basel, Switz erland                                                    Dept. Biological Chemistr y The Hebrew Uni versity, J erus alem 91904, Israel

Background: Proliferating Schwann cells, the glial cells of the peripheral ner vous          A limited capacity to self-repair and regenerati on of cellular damages is the
system, are a pr omi nent feature during earl y de vel opment and after damage to            hallmark of many c entral and peripheral regions of our body. In the l ast few years,
peripheral nerves. Altered Schwann cell prolifer ation is also associated with               evi dence has acc umulated that shows major damage to much type of c ells that
diseas es and pathological states i ncludi ng inherited peripheral neuropathi es,            comes from oxidation. The s ource of free radic als and oxidizing agents are part of
peripheral ner ve tumors, and peripheral neuropathies sec ondar y to diab etes,              the normal cellular mac hiner y, however, failing to remove exc ess of free radicals
cancer chemotherapeutic agents , or toxins . To gain more insight into the                   or the oxidizing agents creates a situation called oxi dati ve stress ( OS), responsi ble
molec ular proc esses governing Sc hwann cell proliferation in health and dis eas e,         for a devastating complications which end in cell death. In the las t decade, clinical
we examined the Sc hwann cell c ycle and its regul ation in vivo.                            trials with various anti oxidants failed mai nly du e to the fac t these c ompounds do
Methods: We have examined the expressi on, regulation, and localization of                   not penetrate the cell.
cyclins, c yclin-dependent ki nas es (cdk), and c ell cycle inhibitors in Schwann cells      We developed two new series of compounds consisting of small molecular weight
of developing and adult peripheral nerves using i mmunohistoc hemistr y. In                  thiol compounds that can cross the pl asma membrane into the cell as well as the
additi on, we used appropriate mutant mice to examine the functi onal requirement            blood brai n barrier (BBB).
for the respecti ve cell c ycle proteins in Schwann cell proliferation.                      i) AD4 (N-acetyl c ystei ne amide) is one of the first series of c ompounds that was
Results: Proliferating Schwann cells during development express cyclin D1 in the             shown to very efficient in vitro and in a large number of animal models, reversing
cytoplas m. After injur y, c yclin D 1 becomes l ocalized to the nuclei of pr oliferating    the effects c aus ed by oxidati ve s tress (Ref. 1-10). Among its promi nent pr operti es,
Schwann cells. C yclin D1-deficient animals reveal ed that devel opmentall y                 it is an inhibitor of MAP Kinas es such as ERK1/2, JNK, and p38 in vitro it
regulated pr oliferation is not affected by the abs enc e of c yclin D1, whereas injur y-    scavenges ROS (Reac tive Oxygen Species), regenerate GSH, chelates copper
induc ed proliferation is impaired. We further found that the cell c ycle inhi bitor p21     ions, and shows restor ation of oxidative stress mar kers such as cell viability (MTT
appears first in the c ytoplas m of Sc hwann c ells at postnatal day 7 when most cells       assay), pr otein carbonyls, nitros ylati on (3- nitrotyrosine), and lipi ds.
have already ceased di viding. After ner ve injur y, however, p21 is localized mainl y       ii) This novel s eries of low molecular weight peptides is called ‖Redox-Cluster‖
in nuclei of dedifferentiated Schwann cells. Consistentl y, p21-deficient Sc hwann           (RC). They offer a prolonged action base on unique properties of cell entry and
cells do not undergo proper growth arrest in later phas es of ner ve development. In         trapping insi de the cell.
contrast, after ner ve injur y, nuclear p21 is required for correct cell cycl e control at   These compounds were also s hown to be potent inhi bitors of the MAP Kinas es
the peak of Sc hwann cell proliferation. We next i nvestigated the requirements for          ERK1/2, JNK, and p38 in vitro. T hey are scavengers of ROS (reacti ve oxygen
cdk2, 4, and 6 during Sc hwann cell proliferation. We s how that onl y cdk2 and 4            species, and show restorati on of all oxidati ve stress mar kers such as Cell viability
are express ed in peripheral nerves. Our data from cdk-deficient mic e indicate that         (MTT assay), protei n carbonyls, nitros ylation (3-nitrotyr osine) and lipid
postnatal Schwann cell proliferati on is abolished in the abs enc e of cdk4 but not in       peroxidation (levels of HNE). In studies in vivo a significant impr ovement in
the abs enc e of cdk2 or 6.                                                                  oxi dati ve mar kers was obs erved.
Conclusions: We find that distinct components of the c ell c ycle mac hiner y that           The two families of glutathione pr ecursors are potenti ally promising ―magic‖ bullets
regulate Sc hwann c ell proliferation during development differ fundamentall y from          for the treatment of neurodegenerati ve disorders, as well as other degenerative
thos e acti vated following ner ve injur y or i n peripheral neuropathies.                   and multi-system disor ders, s uch as di abetes and as thma.




Small Lipoprotein A-I subclasses (42,000-70,000) are Promising Biomar ker s                  Investigation of SGK-1 and Dexr as1 expression in Human Embr yonic Kidney
in Cardiovascu la Disease                                                                    (HEK 293) cells

ATMEH RF, KASASBEH AZ, ABUODEH MR                                                            GHASSEM ATTRAZ ADEH YAZDI1, MICHAEL SHIPSTON 2, F ERENC ANTONI

Jordan University of Science and Technology, Irbid, Jordan                                   Department of Neuroscienc e, U niversity of Edi nburgh, EH8 9JZ, Sc otland, United
                                                                                             Kingdom, 1 Hor mozgan Uni versity of Medical Sciences , Iran, 2Membr ane Biol ogy
Background: The predictive value of the high density lipoprotein (HDL)                       Group, Uni versity of Edinburgh Medical School, H ugh Robs on Buildi ng,
cholesterol as a biomarker for cardiovascular disease is now questionable. HDL               Edinburgh, EH 8 9XD. Sc otland, U nited Kingdom
contains several subclasses of varying size, composition, and function. Aims: 1)
To detect and quantitate the apolipoprotein A-I-containing lipoproteins (LpA-I)              Adrenal corticosteroids are i nvol ved in multiple as pec ts of CNS function — for
directly from fresh plasma instead of HDL subclasses. 2) To study the plasma                 example, the feedbac k inhi bition of the hypothalamic-pituitary adrenoc ortical axis.
distribution of these LpAI subclasses in normolipidemics and hyperlipidemics. 3)             Typic ally, the ac tion of gluc ocorticoids is mediated by r apid i nduction of mRNA and
To follow up the changes in their distribution after a fatty meal and during                 protein s ynthesis. Pot enti al mediators of glucocorticoid ac tion include dexras 1,
pregnancy.                                                                                   (activator of G protein signaling 1) first reported as a rapidl y induced mRNA in
Methods: Fresh plasma from 90 normolipidemics and 20 hyperlipidemics was                     pituitar y tumour cells. The rapi d induc tion of dexras 1 in response to
subjected to gradient polyacrylamide gel electrophoresis followed by                         glucocorticoids in pituitar y cells raises the possi bility that it may be invol ved in
electrotransfer onto agarose gel-containing anti-apoA-I. Similarly, plasma from 8            negati ve feedbac k regulati on of cortic otropi n secretion. In the pres ent study we
normolipidemic sunjects was withdrawn after fasting and 4 and 6 h after a fatty              examined the induction of dexras1 in human embryonic kidney (HEK293) cells
meal. Two pregnant women were followed 4.5, 8.0, and 11.5 months during                      which have been used as a model for glucocorticoi d-mediated regulation of
pregnancy and after delivery.                                                                corticotropi n secretion.
Results: We detected 12 different LpAI subclasses of molecular mass ranging                  To verify that HEK 293 cells contai n func tional glucocorticoid receptors (GR)
from 42,000 to > 354,000 with Stokes radius of 2.96 to > 5.8 nm. The percentage              activated by dexamethasone, MMTV-LTR plas mid, which responds to acti vated-
of the smallest subclasses SlpAI2, SlpAI3, and SlpAI4 (50,000, 45,000, and 42,000,           GR by enhancing luciferase expressi on via the MMTV promoter, was tr ansfected
respectively) was low in normolipidemics (7.8, 3.4, and 2.7%) and significantly (P           into the HEK 293 cells. 48 h after transfec tion, the cells were treated with
< 0.01, 0.05, 0.05, respectively) higher in hypelipidemics (18.9, 8.3, 5.2%,                 dexamethasone. Anal ysis of the time-course of dexamethasone ac tion on MMTV
respectively). In normolipidemics, the level of SLpAI2 increased significantly (P <          regulated luciferase ac tivity revealed that luciferas e induc tion was maximal at 120
0.05) than the fasting level 4 h after fat ingestion, and decreased after 6 h (14.1,         min. Exposur e to various c oncentrati ons of dexamethasone for 120 min produced
20.3, 19.5 mg/dL, respectively). The percentage of SLpAI1 increased during                   an increas e of MMTV regulated luciferas e acti vity in HEK 293 cells in a
pregnancy and decreased after birth (7.8, 9.7, and 6.6%), and SLpAI 2 was                    concentrati on -dependent manner. Maxi mum effec t was obtained with 100 nM
detected (4.6%) after 8 months and disappeared after birth. A strong positive                dexamethasone (up to 5-fold).
correlation was observed between the SLpAI subclasses and plasma                             The expression of dexras1 was assess ed by Northern blot. The results of Northern
triacylglycerols (TAGs) in all normolipidemics (r = 0.41, P < 0.01) and in males of <        blot s howed a 5 kb dexras 1 and a 2.6 kb s erum and gluc ocorticoid-induc ed protein
25 y (r = 0.70, P < 0.01).                                                                   kinase (SGK-1) mRNA (a positi ve c ontrol for glucoc orticoid i nduc tion) s pecies in
Conclusions: 1) Significant differences in the distribution of SLpAI subclasses in           HEK 293 c ells. Dexamethasone had no significant effec t to amount of dexras 1
plasma were detected in normo- and hypelipidemics. 2) Their plasma level                     mRNA induction for var ying ti mes (0-120 min) but i ncreas ed SGK-1 mRNA with
increases with the increase of plasma TAGs. 3) They may be related to the                    maxi mum effect at 30 min. T he analysis of SGK-1 protei n in HEK 293 cells also
lipolysis of the TAG-rich lipoproteins. 4) The variation of SLpAI2 level was more            showed significant increas e in response to dexamet has one.
evident than the others and it may be a good candidate as a biomarker for                    The results s uggest that; 1) HEK 293 cells respond to dexamethas one via an
cardiovascular disease.                                                                      endogenousl y express ed GR; 2) D exras1 is not a glucocortic oid-induced protein in
                                                                                             HEK 293 c ells; 3) SGK-1 is i nduc ed by dexamethasone i n HEK 293 c ells.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                Page A-19
                                                           EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                 Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                      Nürnberg, October 3-5, 2008
     Value of pharmacokin etic/pharmacod ynamic in do se m anag ement of               The Role of Endothelial Cell Heterogeneit y in the Search for Anti-angiogenic
     ceftazidim e and imipen em in ICUs                                                Agents

     AUBERT G1, CARRICAJO A1, AUBOYER C 2, ZENI F3                                               AUERBACH R, AUERBACH W
     1                                                                         2
      Antibiol ogy Laborator y, Uni v. North H osp. - Saint-Etienne - France; Intensive          Laboratory of D evelopmental Bi ology, Department of Zool ogy and Institute on
     Care Unit, Uni v. North Hosp. - Sai nt-Etienne - Franc e; 3Intensi ve C are Unit, Uni v.    Aging, University of Wisconsi n, Madison, WI 53706 USA
     Bellevue Hos p. - Saint-Eti enne. - France.
                                                                                                 Background: In the late 1800s, Paul Ehrlich documented the clear differenc es
     Background: The purpos e of our study was to assess the value of serum ass ay               among endothelial cells from different organs by describing the uniqueness of the
     of ceftazidi me (CAZ) and imipenem (IMI) in patients in the intensi ve care unit (ICU)      brain-associ ated vascul ature, the blood-brai n barrier. Nevertheless, endothelial
     of the Sai nt-Etienne Uni versity Teac hing Hospital and in other ICUs in the region        cell heterogeneity, aptl y demonstrated by the blood-brain barrier, was not
     with r egard to optimis ation of treatment management.                                      generally appreci ated until the last decade and even now has not become a major
     Methods: Between 01/11/05 and 29/02/08, in patients hospitalised in ICUs, not on            consideration in developi ng angiogenic and anti-angiogenic therapi es.
     dialysis and undergoing treatment with CAZ given in a conti nuous infusion or with          Recognizing the importanc e of this heterogeneity is critical for the development of
     IMI (non-conti nuous), s erum ass ay of the respecti ve antibiotics were perfor med 36      "m  agic bullets" against cancer or immune-mediated diseas es, inasmuch as there
     hours after the s tart treatment using a single s erum s ample for CAZ and                  is no single uniform target in the vasc ulatur e.
     determination of trough and peak conc entrations for IMI. Assays were performed             Methods:        There are multipl e methods for studying angiogenic and anti-
     using the microbiolog y tec hnique with results 18 hours after sample c ollection.          angiogenic agents , ranging from in vitro models to various in vivo assays. Critical
     CAZ 2 g was given s ys tematic ally i n a bol us at the start of treatment.                 to the c hoice of targets is the recognition of endothelial cell heterogeneity. We and
     Results: Assays were performed in 92 and 105 patients respecti vely for CAZ and             others have developed numerous endothelial c ell lines from various microvasc ular
     IMI. Mean patient age was 66 years (19 to 89 years) and mean weight was 73 kg               organ beds, from large arteries, from s pecific vei ns, and from tumors.
     (33 to 122 kg). The dosage was between 1 g and 6 g/24 h for CAZ and between 1               Results: Responses to different agents in vitro varies with the source of
     g and 6 g/24 h for IMI. T he mean ser um CAZ c onc entration was 46.9 mg/L (7.4 to          endothelial cells used in the ass ays. In vivo res ults depend on the site of inj ection,
     162.3 mg/L). Ser um CAZ c onc entrations were as follows: 35 to 65 mg/L in 37% of           the physic al topography, and the organ environment. The vasc ulature of
     patients, < 35 mg/L in 43.2% and > 65 mg/L in 19.8%. Infection was established in           devel oping embryos differs fr om that of adults, and there are critical distinctions
     51 patients, with 42 strains of P. aerugi nosa detected. The serum concentration /          between vessels of the l ymphatic s ystem and thos e of the bl ood vasc ulatur e.
     MIC ratio was >= 5 for 84.3% of pati ents and > 10 for 65.6% of pati ents . Trough          Examples of both in vitro and in vivo studies of effects of various pharmacological
     concentrati ons of IMI wer e < 0.5 mg/L for 14.7% of patients, between 0.5 and 2            agents will be pres ented.
     mg/L for 43.2% , and > 2 mg/L for 42.1%. The mean peak concentration of IMI was             Conclusions: Endothelial cell heter ogeneity pres ents an important challenge to
     19.9 mg/L ( 3 to 78 mg/L). Infecti on was rec orded in 47 patients, i ncluding 34           the development of phar mac ological reagents ("Magic Bullets") when attempting
     enterobacteria and 11 P. aerugi nosa. Anti biotic dosage was adj usted respecti vel y       to target the vasc ulature of specific organs, differ ent tumors, or selected sites of
     for CAZ and IMI in 19.8% and 28.4% of patients based on the initi al ass ay results .       inflammation. Paul Ehrlich pioneer ed i n an area of pathology whos e i mportanc e is
     Conclusion: Our study shows that assays are needed in ICUs to confirm the                   onl y now beginning to be appreciated.
     efficac y of ti me-dependent antibi otics (ß-lactams), to avoid treatment toxicity, to      Dedication: This contribution is pres ented i n honor of Dr. Judah Fol kman, long -
     achieve efficac y as rapidl y as possible and to avoi d s election of resistant mutants,    time c olleague and friend you died unexpec tedly earlier this year.
     particularl y in s trains having limited s usceptibility to antibiotics.




     Targeted deliver y of cisp latin using polym eric nanoparticles                             The Effect of Melatonin and Zinc on the Immune Response in Experimental
                                                                                                 Toxoplasm a Retinochoroiditis
     AVGOUSTAKIS K 1
                                                                                                 AVUNDUK AM 1 , AVUNDUK MC 2, BALTACİ AK 2, MOGULKOC R 2
     1
         Univ. of Patras , Patras, Greec e
                                                                                                 1
                                                                                                     Karadeniz T echnic al Uni versity. Trabzon, Tur key
                                                                                                 2
     Background: Cisplatin us e is ass ociated with s erious si de effects. T he more                Selçuk Uni versity. Konya, Tur key
     selecti ve deliver y (targeting) of cisplatin to tumor cells would reduc e drug toxicity,
     improving its ther apeutic index. Ai m: To develop long -circulating cisplatin-loaded       Background: Toxopl asmosis is a zoonotic diseas e and the most common caus e
     nanoparticles and test their antic anc er acti vity in mic e cancer models.                 of infection of the human retina . In humans and ani mals, the primar y control
     Methods: A) Nanoparticles preparation: Cisplati n-loaded pol y(lacti de-co-                 mec hanis m of Toxoplas ma gondii (T. gondii) infec tion invol ves T l ymphoc ytes.
     glycolide)-pol yethyl enegl ycol (PLGA-PEG) nanoparticles were prepared by a                One of the potential beneficial approac hes to i mprove the immune defens e agai nst
     double emulsion method. B) Determination of cispl atin profiles in blood circulation:       T. gondii infestation is zinc s upplementati on, since due to its c atal ytic and
     BALB/c mice were administer ed intravenousl y with PLGA-PEG/cisplatin                       regulator y functions; zinc can enhanc e resistance to infec tions . Other potential
     nanoparticles and at pr edeter mined ti me intervals bl ood samples were obtained           approaches that deser ve further investigation in this res pec t are the effect of
     and ass ayed for plati num. C) Evaluation of the tolerance of BALB/c mice to the            melatoni n (MEL) deficienc y and artificial MEL s upplementati on on the i mmune
     PLGA-PEG/cispl atin nanoparticles: 5 mic e groups (n=3) recei ved 3 intravenous             respons e to T. gondii infection. MEL is a hormone secreted by pineal gland that
     injections at 7-day intervals. T hree groups of mice rec ei ved PLGA-PEG/cisplatin          has both direc t and i ndirect effects on the immune s ystem. In the current study, we
     nanoparticles with a cisplati n content of 2, 5 or 10 mg cisplatin/Kg. The fourth           investigated the impac t of MEL and/or zinc deficiencies and artificial MEL and/or
     group of mic e recei ved blank nanoparticles and the fifth group of mic e (n=3)             zinc supplementations on i mmune and inflammator y res ponses in the rat model of
     received 100 μl saline. D) Evaluation of anticanc er acti vity of PLGA-PEG/cisplatin        toxoplasma reti noc horoiditis.
     nanoparticles: HT 29 tumor cells were injected s ub-cutaneousl y into the l eft flank       Methods: Eighty-four Sprague Dawley male rats were di vided into 12 equal
     of SCID mic e. Fifteen days later, the mic e (n= 6-8) were injected intravenously 5         groups. All groups, exc ept controls were infected with T. gondii parasite by
     times at weekly intervals with free cisplatin or cisplatin-loaded nanoparticles at the      intraperitoneal injecti on. Combinati ons of zinc deficient diet, pineal ectomy (Px),
     same dose (5 mg/kg on a cisplatin basis).                                                   artificial zinc, and MEL were supplied during a 1 month period. At the end of the
     Results: The entrapment of cispl atin in the nanoparticles resulted in a significant        experiment, retinal and choroidal total lymphoc ytes, CD3+, CD4+, and CD8+ cell
     prolongati on of cis platin pres ence in blood. Balb/c mice tolerated 3 weekl y             numbers were counted in histological sec tions .
     intravenous inj ections of a relati vely high dose of blank PLGA-mPEG                       Results: The highes t amount of c ellular infiltration (l ymphoc ytes , CD3+, CD4+,
     nanoparticles (500 mg/Kg) and 3 weekl y intravenous inj ections of a high dose of           CD8+ cells) in the choroi d and retina were detected in infected+MEL+zinc treated
     nanoparticle-entr apped cis platin (10 mg/Kg). The cis platin-loaded PLGA-mPEG              rats and the least amount of cellul ar infiltration was obs erved in Px+zinc deficient
     nanoparticles was effecti ve at del aying tumor growth in HT 29 tumor -bearing SCID         diet treated rats. Although single zinc or MEL s upplementati on had no significant
     mice. The group of mice treated with cisplatin-loaded nanoparticles exhibited               impac t on the cellular infiltration in the retina and choroi d in Px rats , combined
     higher sur vival rate compared to the free cisplatin group                                  therapy significantly impr oved these r esponses .
     Conclusions: 1) PLGA- mPEG/cisplatin nanoparticles could prolong cisplatin                  Conclusion: Artificial suppl ementation of MEL and zinc shoul d be c onsidered as
     residenc e in blood and they were well tolerated by nor mal Bal b/c mice even when          an adjuncti ve therapy to cl assic treatment of Toxoplas ma retinochoroiditis
     relativel y high doses were administered to mice. 2) The PLGA- mPEG/cisplatin               especi ally in immunos uppressed and elderly patients if our data will be confirmed
     nanoparticles reduced tumor growth in SCID mice with HT29 xenografts, and                   in a clinical s etti ng.
     thes e mic e exhibited higher sur vi val rate than free cisplatin.
                                                                                                 Authors‘ disclosure statement: Supported in part by a R esearch Fund of Selcuk
                                                                                                 University Grant no: TF 98/042. Published in Ophthal mologica. 2007;221(6): 421-5
Au                                                                                               S.Karger AG, Basel. Pres ented in part at the World Ophthal molog y Congress, Feb
                                                                                                 19 - 24, 2006,São Paulo, Brazil.




     All abstracts are listed in alphabetical order of the presenting author.
     Abstracts                                                                                                                                                              Page A-20
                                                     EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                            Celebrating the 100th Anni versar y of the
                                                               Nobel Prize Award to Paul Ehrlich
                                                                 Nürnberg, October 3-5, 2008
Preventing Immune Evasion as a Strateg y for Enhancing th e Effectiveness         Levetiracetam in the Treatment of Neuropathic Pain: Eviden ce from C ellular
of Herpes Simplex Virus Vaccin e.                                                 and Behavioral Pain Models

AW ASTHI S, LUBINSKI J M, AND FRIEDMAN H M                                                       AYAR A1 , OZCAN M 2
                                                                                                                                                          1             2
Infec tious Disease Division, Department of Medicine, School of Medicine,                        Firat University, Fac ulty of Medicine, Departments of Physiolog y, Biophysics,
University of Penns yl vania, Philadelphia, PA, U SA.                                            Elazig, Turkey.

Background: Herpes si mplex virus-1 (HSV-1) and HSV-2 gl ycopr otei n C (gC) are                 Background: Neuropathic pai n is a c ommon, complex, cos tly pain condition,
immune evasion proteins that inhibit compl ement acti vati on by bi nding                        "initiated or caused by a primar y lesion or dysfunction in the ner vous s ystem‖,
complement c omponent C3b. We pr eviousl y r eported that mice passi vel y                       presenting a major healthc are and s ocial probl em. Despite an i ncreasing number
immunized with a gC1 monoclonal antibody (MAb) were protected from HSV-1                         of trials there is currentl y no s atisfactorily effecti ve therapy for neuropathic pai n.
challenge onl y if the MAb bl oc ked the interaction between gC1 and C3b. We also                Similarities between the pathophysiol ogical phenomena obser ved in some
demons trated that immunizing mice with gC1 protein induced anti bodies that                     epileps y models and in neuropathic pain models justify the us e of anticonvulsants
bloc ked C3b bindi ng and protected mic e against HSV-1 challenge by bloc king                   in the s ymptomatic management of neuropathic pai n. We attempted to eluci date
immune evasion.                                                                                  the effec tiveness of levetiracetam in neuropathic pain state in mice model and
Method: We use the mous e flank (epider mal disease) model to eval uate whether                  subsequentl y c ellular mec hanis ms invol ved in a cellul ar nocic epti ve model.
immunizing with gC1 pr otei n increases the effic ac y of a gl ycoprotei n D (gD1)               Methods: The in vivo nocicepti ve behavi oural ―hot-pl ate tes t‖ was performed in
subunit vaccine when challenged with HSV- 1. We first defined a gD1 immunizing                   normal and di abetes (streptozoci n 200 mg/kg i.p.)-induced adult male Balb/C
dose that produced partial protecti on against HSV-1 c hallenge. Our rationale for               mice.
partial protection was that the gD2 subunit vacci ne us ed in human trials provided              Subs equent to behavior al tes ting, electrophysiological measur ements and calcium
onl y limited protection, and we wanted to reproduce these res ults in mice. We then             imaging experi ments were perfor med on cultured neurones of the rat dorsal root
immunized mice with either gD alone or with both gD and gC and challenged with                   ganglia (DRG) using the whole cell patc h-clamp tec hnique and the fura-2
HSV-1.                                                                                           ratiometric fluorescence microsc opy. Data wer e anal yz ed using Kr us kal -Wallis
Results: We found that when gC1 was added to gD1 immunizations, mic e were                       One- way Analysis of Variance (ANOVA), D unnett‘s and Students‘ t tests , where
significantly protected from epidermal disease c ompar ed with gD alone.                         appropriate.
Importantl y, the combined immunizations were more effecti ve than gD alone in                   Results: While l evetirac etam had (60-900 mg/kg) no significant effect on the
preventing infec tion of dorsal root ganglia. Passi ve immunization of anti -gC1 IgG             nocicepti ve thr eshol d in normal mouse muc h lower dos es (≤200 mg/kg)
obtained from mic e immuniz ed with gC1 protein protected complement intact                      significantly res tored the pai n thres hol d latenc y in diabetic mice, i n a dose-
mice, but not C3 knoc kout mic e.                                                                dependent manner. Current clamp rec ordings from DRG cells indicated that
Conclusion: We conclude that immunizing with gC1 bloc ks immune evasion and                      levetiracetam caused membrane hyperpol arisation and r educ tion of multiple action
enhanc es the efficac y of a gD1 s ubunit vaccine.                                               potential firing. Estimation of reversal potentials of levetirac etam-induced
                                                                                                 hyperpolarizing currents indicated invol vement of K+ channels. Furthermor e,
                                                                                                 levetiracetam dose-dependently suppress ed the depolarisation ( high KCl) -
                                                                                                 induc ed intracellul ar calcium signals in DRG neurons .
                                                                                                 Conclusions: R esults obtained from in vi vo behavi oral tes ts and c ellular
                                                                                                 electrophysiological and ratiometric fluor escenc e measurements in rat sensor y
                                                                                                 neurons with agreement l end support to the validation of the promising therapeutic
                                                                                                 potential of the new antic onvulsant levetiracetam for the management of
                                                                                                 neuropathic pain.




Insulin, IGF-1 and Rosiglitazone: Ho w Do Th ey Effect The Glucose                               Antimicrobial R esistance b y Mycoplasma species in Farm Anim als.
Metabolism and Insulin Resistan ce in Human SH-SY5Y C ells with Alzheimer
Key Proteins?                                                                                    AYLING RD.
                        1               2
AYC AN (YERER) MB , ECKERT A                                                                     Veterinary Laboratories Agenc y ( Weybridge), Addlestone, Surrey, UK. KT15 3NB
1
  University of Erciyes, Fac ulty of Phar mac y, Department of Pharmac olog y, 38039,            Background: More than 125 Mycoplas ma species have been identifi ed, many
Kayseri, Tur key.                                                                                being pathogens in farmed ani mals. Myc opl asmas lack a cell wall, hence they are
2
  Ps ychiatric Uni v. Clinics, Neurobiol lab. for Brain Aging, Bas el, Switz erland.             not susc epti ble to penicillins and some other anti microbials. M ycopl asma
                                                                                                 infections are usually treated with tetrac yclines, macrolides, c hloramphenicols,
Backgro un d: IGF are potent neu rotrophic and neurop rotective facto rs that re ve rse the      aminoc yclitols and the fluoroqui nolones, however anti microbial resistance to all of
effects of Ab25–35-, 1–40- and am ylin induced neurotoxicity in hi ppocampal cells. IGF-I        thes e has been reported. The treatment of myc opl asma infections is rarel y
protects hippocampal neurons against Ab-induced toxicity. F urthe rmore, insulin resistance      successful and often requires repeated treatments . Although Mycoplas ma species
leads to a functional decrease in insulin receptor (IR) -mediated signal transduction in the     are not us uall y z oonotic, this repeated us e of antimicrobi als and development of
brain, again consistent with the hypothesis that hy perinsulinemia or insulin resistance may     resistance may have i mplications for the critically important antimicrobials used in
potentiate the risk of AD. In this study, the effects of insulin, IGF-1 and rosiglitazone on
                                                                                                 human health. MIC data from U K bovine and ovi ne mycoplas ma isol ates and
normal and transfected SH -SY5Y cells (P301L a nd hT au40) with ke y p roteins involved in
                                                                                                 resistance mechanis ms in M. bovis is presented.
Alzheimer‘s disease (AD) have b een i nvestigated.
                                                                                                 Methods: In vitro microbroth dilution mini mum inhi bition c onc entrations (M IC‘s)
Me th ods: T he cell lines were cultured in different glucose mediums (0 -100m M) to identify
                                                                                                 were determined for 14 anti microbials agains t M. bovis and M. ovipneumoniae
the effects of insulin (5µg/ml), IGF -1 (1-1 0-100ng/ ml) and rosiglitazone (20µ M) in altered
glucose concentrations. AT P synthesis and the mitochondrial m embrane potential                 isolates. Sensitive M. bovis isolates were s ubc ultured at sub-MIC levels in 14
(MMP,ΔΨm) were measu red.                                                                        different anti microbials to induce anti microbial resistanc e in vitro. Where
Results: Insulin was found to increase the AT P and the MMP significantly in SH -SY5Y            anti microbial resistance had been induced this variant DNA was us ed to PCR the
cells in all glucose levels. However, insulin didn‘t cause any changes in low glucose            domains II and V of the 23S rRNA gene and the gyrA and parC genes, which were
medium in P301L cells whereas it increased these values in high glucose environment              then sequenc ed.
(p<0.05 ). In hT au40 cells insulin increased the AT P synthesis in low glucose medium           Results: Is olates of M. bovis and M. ovipneumoniae gave high i n vitro MIC values
whereas the AT P synthesis reduced in these cells in high glucose medium (p<0.05).               against most classes of anti microbials, exc ept the fluoroquinol ones , although high
Rosiglitazone slightly lowered the AT P sythesis and the MMP in normal cells in low
                                                                                                 fluoroquinolone MIC‘s have since been recorded in fi eld isol ates . The rate of
glucose medium, and slightly increased in high glucose medium. In hT au40 (p<0 .05) and
                                                                                                 devel opment of antimicrobi al resistance varied between isolates and between
P301L (p<0.0 01) cells rosiglitazone alone decreased the AT P synthesis whereas the cells
were pre vented significantly from this reduction by the combination of insulin and              anti microbials, but resistance was induc ed by all isol ates to all antimicrobials.
rosiglitazone (p<0.05 ) which re veals that there is a probable insulin resistance in            Fluoroquinol one resistant isolates showed a single base change (G to A) at
transfected cells where the AT P synthesis may be promot ed by rosiglitazone in the              position 259 and (C to T) at position 248 in the gyrA gene, obser vati ons that have
presence of insulin. 100ng/ml IGF-1 , increased the AT P levels in low glucose medium and        been shown to c onfer fluoroquinol one resistance i n E. coli.
this increase was greater then the insulin had in normal SH-SY5Y Neuroblastoma Cells. 1          Conclusions: It is unli kel y that the genetic exchange of resistanc e genes occurs
and 100ng/ml of IGF-1 had slightly lowered the AT P levels in high glucose level medium          between Mycoplas ma species and z oonotic organisms, however the treatment of
whereas 10ng/ml of I GF -1 had n o significant effect on the AT P synthesis in SH-SY5Y           Mycoplas ma infecti ons in far med animals requires careful sel ection and monitoring
cells. In P301L cells, in the 0 and 25mM glucose medium 100ng/ml I GF -1 lowere d the            of antimicrobial us e to maxi mise the effecti veness of treatment and to evade the
AT P synthesis significantly. In the hT au40 cells there were not an y significant effect of     further development of anti microbial r esistanc e.
IGF -1 in AT P synthesis. When we compare the MMP , all concentrations of IGF-1 had
lowered the MMP in 0 and 100 m M glucose mediums but increased the MMP especially in
25mM glucose (p<0.05 ) in normal SH -SY5Y Neu roblastoma Cells. Howe ver, it didn‘t ha ve
any effects on P301L cells. IGF-1 only lowered the MMP in hT au40 cells in high glucose
(100m M) mediums.
Concl usio n: Since brain possesses high energetic requirements, any decline in brain
mitochondria electron chain could have a se vere impact on b rain function and pa rticularly
on the etiology of neurodegenerative diseases. T herefore, to identify the relationships
between insulin, IGF -1 a nd rosiglitazone combination could be beneficial to find out no vel
treatment alterna tives for the insulin resistance in late on -set of AD.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                    Page A-21
                                                           EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                  Celebrating the 100th Anni versar y of the
                                                                     Nobel Prize Award to Paul Ehrlich
                                                                       Nürnberg, October 3-5, 2008
Cell H ydr ation as a Univer sal and Extra-Sen sitive Biom arker for                    EM703 a New Der ivative of Er ythrom ycin, Inhib its Lung Fibrosis Induced b y
Determination of the Functional State of the Organism                                   TGF- Signaling in Murine and Human Lung Fibroblasts

AYR APET YAN S                                                                               AZUMA A, LI YJ, YU CH, ABE S AND USUKI J

UNESCO Chair-Life Scienc es Inter national Postgraduate Educ ational Center,                 Department of Internal Medicine - Pulmonary M edicine, Infec tion, and Oncol ogy,
Yerevan, Armenia                                                                             Nippon Medical School, T okyo, JAPAN

Background: Although the functional significance of intrac ellular water is widely           Background: 14-membered ring macrolides have been effecti ve in c hronic airway
accepted, its messenger role in signal transduction and generation of different              inflammation and also prevented lung injury and fibrosis in bleomyci n -challenged
diseas es is not adequatel y s tudied by the res earchers. M y presentation contai ns a      mice via anti-inflammator y effects . EM703 is a new compound of Er ythromycin
review of our data on the metabolic regulation of cell hydration and its                     (EM) without bac tericidal effects. We investigated the anti-inflammator y and
physiological significanc e in nor m and pathology.                                          antifibrotic effects of EM703 in 1) experimental murine fi brosis model i nduc ed by
Methods: Neuronal, muscle and reproducti ve cells, isolated tissue and organs of             bleomycin and 2) murine and human lung fibroblast c ell lines.
animals and woman breast c anc er tissues s erves as a subject for inves tigations.          Methods: 1) Seven weeks old male ICR mic e (eight mice/group) were used.
The light microscopic, whole cell and patc h clamp, isotope, standard biochemic al           Bleomycin was administered intravenousl y to mice at day 0. EM703 was orall y
and genetic engineering methods were used.                                                   administered dail y to mice. All groups were examined for cell populations in
Results: The data showed a clos e correlation between cell hydration and number              bronchoal veolar lavage fl uid, and for inducti on of mRNA of Smad3 and Smad4 in
of functionall y ac tive membrane proteins, having enz ymatic, c hemor ecepti ve and         lung tissues by RT-PCR at day 7. Fibroblas tic foci wer e assessed histol ogically
ionoforetic properties. The data on the Table show that the number of ouabain                and hydr oxyproline content was c hemically deter mined in l ung tissues at day 28.
receptors (Na/K pump units) depends on membra ne surface, which changes upon                 2) We also ass essed proliferation and s oluble c ollagen producti on, and examined
the effec t of factors causing the increas e of membr ane per meability. The                 inducti on of mRNA of s mad3 and s mad4 by RT-PCR in lung fibroblast cell line
correlation between the Na/K pump regul ating the cell hydration and intrac ellular          MLg2908. We examined smad3, smad4, smad7 and phosphor ylated smad2/3 (P-
signaling system was also s hown. It makes the cell hydration as a uni versal and            smad2/3) protein assay by wes tern blotti ng in lung fibroblas t cell lines.
extrasensitive cell mar ker deter mining the cell functional state and a sens or for         Results: Bleomycin-induc ed lung fibrosis, infiltration of macrophages and
different extraweak environmental signals. By specific mRNA-induced expression               neutrophils into the airspac e were inhibited by EM703. Expression of s mad3 and
in oocites membrane was shown that the cyclic nucl eoti de–dependent Na/Ca                   smad4 mRNA was clearly attenuated by bleomyci n, but rec overed by EM703.
exc hanger plays a crucial rol e in c ell volume r egulation, when the Na/K pump is          EM703 als o inhibited fibroblast proliferation and the collagen pr oduc tion in lung
inacti ve (cell pathol ogy).                                                                 fibroblasts i nduc ed by TGF. Expression of s mad3 and s mad4 mRNA in murine
Conclusions: 1) The number of functionall y acti ve protei n molecul es in cell              lung fibroblasts disappeared by TGF , but recovered by EM703. EM703
membrane depends on cell acti ve s urfac e. 2) There are a negati ve feedbac k               inhibited expression of phosphor ylated-s mad2/3 and smad4 protei n in murine lung
between Na/K pump and c ell hydrati on and a positive feedbac k between                      fibroblasts induced by TGF.
membrane per meability and c ell hydr ation. 3) The c ell overhydration is a marker          In human l ung fibroblast, EM703 inhibited the augmentation of Smad3 mRNA
for cell patholog y. 5) The Na/K pump regulating the c ell hydration is a uni versal         induc ed by TGF . Inhi bited Smad3 mRNA by TGF was augmented by co-
and extras ensiti ve sensor for various environmental factors.                               incubation with EM703.
                                                                                             Conclusions: Thes e findi ngs suggest that EM703 improves bleomycin-induced
     [3 H] Ouabain                           Incubation medi um
                                                                                             pulmonary fibrosis i n mice by actions of anti inflammation and r egulation of TGF
    concentration    Hyp otonic   Isotonic   Hyperto nic        Ach 10-4 M    GABA 10-4 M    signaling, which is ass ociated with inhibiti on of phosphor ylation of Smad2,3
        1x10-9       32.02.2     21.11.4     12.20.9        30.541.55      27.633.17    through rec over y of Smad7 level.

        1x10-8       79345.6     50830.1    28319.4         270.9328.53   174.4813.54

TABLE: Binding of [3H] Ouabain ( x108 molec ules/mg dr y weight) to Helix Pomatia
Cell Membrane i n different mediums




Effects of Alcohol and Sucrose Intake on Rat Liver C yp 2e1

AZZALIS L A1, FONSECA FLA2, SCHINDLER F2, GIAVAROTTI L1 , MONTEIRO
HP3, VIDELA LA4, JUNQUEIRA VBC 5
1                                                          2
  Universidade Anhembi Morumbi, São Paulo, Brasil, Fac uldade de Medicina do
ABC, Departamento de Hematologia e Onc ologia, Santo André, Brasil,
3
  Departamento de Bi oquímic a, UNIFESP, São Paulo, Brasil, 4 Programa de
Farmac ologia Molec ular y Clínica, Ins tituto de Ciencias Bi omédicas , Fac ultad de
Medicina, U niversidad de Chile, Santiago, Chile. 5Disciplina de Geriatria,
Departamento de Medicina, UNIFESP, São Paul o, Br asil.

Background: Ethanol metabolism by C YP2E1 l eads to a significant r eacti ve
oxygen s pecies (ROS) release, accompanied by the defense s ystems decr ease
against oxidati ve stress . Since expressi on of CYP2E1 is ver y muc h influenc ed by
nutritional factors, specially carbohydrate consumption, and various results
concerni ng the expression of CYP2E1 were obtained with low-carbohydrate
alcohol liquid diet or the intragastric tube feeding model that also utilizes a low
carbohydrate diet, this study describes the effects of ethanol and sucrose
treatment on CYP2E1 levels using an ad lib model.
Methods: Male Sprague-Dawley rats were fed ad. lib. for 1, 2, 3 or 4 weeks a
commercial diet (Purina Ind., Brazil) plus a 25% ethanol-20% sucros e solution.
Control groups were isocal orically pair-feed to the leadi ng ethanol-consuming
animals, or recei ved isoc aloric amounts of sucros e for pairing onl y ethanol
calories. Eighteen hours before s acrifice ethanol was withdrawal and animals had
onl y free acc ess to tap water or they were offered food and water ad. lib.
Results: Our results have s hown that ethanol administration was associated with
CYP2E1 induc tion, otherwise CYP2E1 stabilization was more ass ociated to
sucrose c ons umption.
Conclusions: Our findi ngs indicate that dietar y deficienci es, es peciall y low
carbohydrate intake coul d be crucial i n the CYP2E1 stabilization.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                           Page A-22
                                                                  EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                        Celebrating the 100th Anni versar y of the
                                                                           Nobel Prize Award to Paul Ehrlich
                                                                             Nürnberg, October 3-5, 2008
Significant Inter action s bet ween som e Antibiotics and Antimalarial Drugs                  Toxins and Adhesion Factors Associated with Staphylo coccu s Aureu s
                                                                                              Isolated from Ur inar y Tracts Infe ctions
BABALOL A C.1, OLANIYI A.1, IWHEYE G., FASHEDEMI T.1, AJOKU C.1 ,
             2
ADENIYI B                                                                                     BABA MOUSSA L 1, ANANI L2, HAÏKOU N 1, HOUNSOU F2, COUTURIER M 3,
                                                                                              SANNI A1, MONTEIL H 3 , PREVOST G3
1                                                2
  Department of Pharmaceutical C hemistry, Department of Pharmaceutical
                                                                                              1
Microbiology, F aculty of Pharmac y, Uni versity of Ibadan, Nigeria                             FAST-Uni versity of Abomey-Calavi, Bénin; 2 LNSP, Cotonou, Bénin ; 3UPRES
                                                                                              EA-3432, Institut de Bac tériologie, Strasbourg Franc e
Background: Coadministration of anti bacterial and antimalarials is common in the
tropics as a result of frequent ass ociation of malaria with other infecti ons. The           Background: Staphyloc occus aureus infections are widel y pr evalent in West
combinati on of ampicillin and cloxacillin (AM-CL) prescribed i n many infections             Africa and are often associ ated with urinary trac t infections (UTIs). Virulence
produc es a broad-spectrum antibi otic acti vity agains t both Gram-positi ve and             factors from S. aur eus have rar ely been described for s uch infections. The
Gram-negative bacteria. T he c urrent studies investigated effects of                         purpos e of the current study was to determi ne the prevalence of toxins and
chloroquine(CQ), proguanil(PG), quinine(QN) and artesunate(ART) on the                        adhesion factors obtained from S. aur eus isolated from presumed primar y UTIs at
bioavailability and acti vity of cloxacillin(Clox) and ampicillin(Amp) in vivo and in         the Cotonou Uni versity Hospital (CUH) in Benin as compared with the Stras bourg
vitro.                                                                                        University H ospital ( SUH) in France.
Methods: 7 healthy volunteers recei ved single oral doses of Clox al one followed             Methods: Both ambul ator y and hospitalised patients were included in the s tudy.
by Clox + PG, another 8 vol unteers rec ei ved AM-CL alone and AM-CL+ CQ, while               Sixty-fi ve independent strains of S. aureus from CUH and 35 s trains from SUH
another 14 volunteers recei ved AM-CL al one and AM-CL + ART in a cr oss-over                 were obtained over a four-month period. Virulenc e factors were c harac terised by
manner. Total urine voided was collec ted at various time inter vals. Clox in urine           immunodetecti on or multiplex pol ymeras e c hain reacti on, and meticillin
was determined by HPLC. Effect of CQ and PG on dissoluti on of Amp and Clox                   susceptibility was r ecorded. Approximately 50% of all isolates produced at least
and on their antibac terial ac tivity against E. c oli and S. aureus was investigated.        one enterotoxin.
Results: CQ showed a significant decreas e in total amount (Du∞) and % dose of                Results: No isolate from SUH produc ed PantoneValentine leuc ocidin (PVL),
Clox excreted in urine by 64%, while PG led to 48% decreas e. Ongoi ng study                  whereas 21.5% of the S. aureus isolates from CUH produc ed PVL (P < 0.01). Si x
reveals 90% reduction of Clox by QN. In vitro dissolution revealed >40% reduction             of 14 (43%) PVL positi ve isolates were meticillin-resistant. At SUH, the incidenc e
in % Amp and Clox diss olved in the pres enc e of CQ. Similar r esults were obtained          of MRSA (57%) was significantl y higher (P < 0.01) than at CUH (14%). Genes
with PG. The MIC of Amp was incr eased two- to four-fold from 5.42 µg/ml to 10.83             encoding clumping factor B, and elasti n and laminin binding proteins were
& 21.66 µg/ml by CQ, an indication of reduction in bactericidal activity of Amp.              detected in almost all isolates (80%), irrespecti ve of the geographical origin.
Similar results were obtai ned between Clox and CQ. 9 out of 14 subjects ( 64%)               Conclusions: The results for elastin binding protein differ ed significantl y from
showed 39% decreas e in urinar y excretion of Clox while onl y 5 (36%) showed an              published data regarding isolates from other clinical origins. Staphyl ococc al toxins
increase of 27% when AM-CL were coadminister ed with ART.                                     and adhesi on factors may be important in the physi opathology of UTI.
Conclusion: These results indic ate significant drug -drug interacti ons between
AM-CL and antimalarials in a way that correlates in vitro with in vi vo findi ngs. The
urinary pharmacoki netic s tudies indicate mar ked reducti on i n bi oavailability of Clox
when coadministered with CQ, PG and QN and ART. The in vivo i nterac tions may
be due to interferenc e with the dissoluti on of Amp and Clox in the body by the
anti malarials. Though clinical implications of the findings are inconcl usive, there
shoul d be c aution in prescribing these class es of drugs together to avoid s ub-
therapeutic levels, which c an lead to treatment failure and drug resistance.




Panton-Valentine leu cocidin as a m ajor virulence factor asso ciat ed to                   Transderm al Drug Deliver y into and Beneath the S kin - Application to Anti
furuncles                                                                                   Inflammator y Drug s

BABA MOUSSA L1, PREVOST G2, COUTURIER M 2, MONTEIL H 2, MOREAU B3,                          BABU RJ1, CHADHA GS1, PARSONS D L1, PATLOLLA RR 2, SINGH M 2
PRADINAUD R 4, COUPPIE P4
                                                                                            1                                                                   2
                                                                                             Harrison School of Phar mac y, Auburn Uni versity, Auburn, USA; College of
1
 FAST-Uni versity d‘Abomey-Calavi, Bénin ; 2UPRES EA- 3432, Institut de                     Phar mac y, Fl orida A&M Uni versity, T allahassee, USA.
Bactériol ogie, Stras bourg ; 3Bac tériologie ; 4D ermatologie, C.H.G. Cayenne,
France                                                                                      Background: α-Melanoc yte-sti mulati ng hormone (α-MSH) is an endogenous
                                                                                            neuro-i mmunomodulatory peptide that has potent anti-inflammator y effec ts. We
Secretion of the Panton-Val entine leucoci din by S. aureus is associ ated to               hypothesiz e that this compound can be developed as a topical formulati on for the
furuncles in more than 90% of isolates and in cas es of furuncl es. Besides , it also is    therapy of ps oriasis and c ontact der matitis. The stability of α-MSH, and in vitro and
associated with c ommunity pneumonia. However, most investigations considered               in vivo percutaneous abs orption from various dermatological vehicles was
onl y limited s eries of virulenc e factors being pr oduced by thes e isol ates.            investigated in rats. The anti-inflammator y effect of topicall y deli vered -MSH was
To determine whether Panton-Valentine Leucoci din is an essential fac tor                   tested on an allergic contac t dermatitis (ACD) mouse model.
associated, we determined production of toxins or the presence of genes enc oding           Methods: The stability of -MSH in ethanol-water (1:9) at various temperatures
enterotoxi ns, leucotoxins, epidermolysi ns, Epider mal Differentiation Inhi bitor          (40 to 70oC) and pH c onditions (pH 1.0 to 10.0) was examined. T he in vitro
(EDIN) factors, and 8 adhesion factors for 16 isolates fr om VIH - patients with            permeati on and s ki n retenti on kinetics of -MSH as a saturated s olution i n various
furuncles , 9 isolates fr om VIH + pati ents, and 30 isolates from s econdary s kin         dermatol ogical vehicles were studied usi ng hairless rat s kin and Franz diffusion
infections, all independent isolates from the Cayenne‘s hospital (French Guiana).           cells. The in vivo s kin penetration of α-MSH was studied i n rats by a der mal
Antibiotic resistant was also performed.                                                    microdial ysis tec hnique. The anti-inflammator y effect of sel ected topical
Only one of the is olates from furuncles do not produce Panton-Valentine                    formul ations (0.25 and 0.5% of -MSH in 10% n-methyl-2-pyrrolidone and 50%
leucocidi n, while onl y three isolates from the secondar y s kin infec tions produc e      ethanol) was evaluated in the ACD mouse model.
this toxin. The difference between these inci denc es is statistic ally significant         Results: Stability s tudi es indicated that α-MSH possess an ~110 day shelf-life
(Student T test, p<0.001). Concurrently, 14/30 isolates from s econdary s kin               (time of 10% degradati on) as determined by H PLC. F urther, -MSH demons trated
infections and 12/25 isolates from furuncles produced at least one enterotoxi n.            good stability in the pH region between 3.0 and 8.0. Permeati on studies indic ated
One is olate amongst all produces Toxic Shoc k Syndrome T oxin-1, 5/30 isolates             that ethanol, transc utol and propyl ene g l ycol (PG) and ethanol vehicles had
from sec ondar y s kin infections produce epidermolysi n A, and 1/25 from furuncles         maxi mum permeation of α-MSH through the skin (between 5.0 and 7.5 g / 24 h).
produc es epidermol ysin B. N o isolate carries any gene encoding Epider mal                Ethanol demonstrated the maxi mum s kin retention (2.0 g /mg) as compared to all
differentiati on Inhibitor factors A, B, or C. Concerning adhesion factors, neither the
                                                                                            other vehicles for whic h the s kin retention was < 1.0 g /mg. Dermal microdial ysis
presenc e of genes enc oding clumping factor B, collagen binding protein, bone-             results show the ethanol formul ation pr oduced a maxi mum conc entration (C max ) in
sialoprotein, l amini n bi nding protein, elas tin-binding protein, fibronectin and
                                                                                            dermis of 6.4672.11 ng/ml as c ompar ed with the PG formulati on with a C max of
fibrinogen binding proteins significantl y differs i n each group of is olates. In
                                                                                            2.5651.284 ng/ml, demons trating 2.5 fol d higher der mal levels by the ethanol
additi on, ther e was no significant statistical differenc e for the occurrence of
virulence for isolates fr om VIH- or VIH+ pati ents.                                        formul ation. -MSH for mulations demonstrated significant anti-inflammator y
Therefore, Panton-Val enti ne leuc ocidin can be presumed as the mos t essential            activity in an ACD mous e model. T he data indicate that like dexamethasone, α-
causative factor for furuncles, and immune disorders as acquired                            MSH was effecti ve i n reducing the ACD response.
immunodeficienc y does not s eem to c ontribute to any susc epti bility in devel oping      Conclusions: Stabl e -MSH can be formulated for effecti ve topical deli ver y into
furuncles .                                                                                 skin layers to demons trate significant anti-inflammator y ac tivity in an ACD mous e
                                                                                            model.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                             Page A-23
                                                                   EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                          Celebrating the 100th Anni versar y of the
                                                                             Nobel Prize Award to Paul Ehrlich
                                                                               Nürnberg, October 3-5, 2008
Is GRP78/BiP, a Mast er-Regulator of Defensive Unfolded Protein R esponse,                      The Strong Gro wth Advantag e in Stationar y Phase Phenomenon in Mixed
a New ―Mag ic T arget‖? GRP78/BiP-targeting C ytotoxin and ER Stress-                           Cultures of Antimicrobial Resistant Esch erich ia and Salmonella
Inducing Drugs Synergiz e to Kill C ancer Cells
                                                                                                BACUN-DRUZINA V1, BUTORAC A1, HALEC I1, GJURACIC K2
BACKER MV1, PATON AW2, PATON JC 2, B ACKER JM 1
                                                                                                                                                                        2
                                                                                                Faculty of Food T echnolog y and Biotechnolog y, Z agreb, Croatia; GSK R esearch
1
  Sibtec h, Inc., Brookfield, CT 06804 USA; 2Sc hool of Mol ecular and Biomedical               Centre Z agreb Ltd., Zagreb, Croatia.
Science, Uni versity of Adel aide, Adelaide, SA, 5005, Australia
                                                                                                Background: During prol onged stationary phas e mutants with increas ed fitness
Background: Diverse i nsults increase the amount of unfolded/misfol ded proteins                express growth advantage in stationar y phase‖ (GASP) enabling them to grow
in the endopl asmic r eticulum (ER). T his leads to acti vation of a defensi ve unfolded        and displac e the parent as the majority population. Aims: 1) To study the GASP
protein respons e, which is c ontrolled by ER-resident pr otein, GRP78/BiP. Whether             phenotype in mixed c ultures of Escherichia coli and Sal m        onella enterica s erovar
the c ell dies or sur vi ves the ER-stress is determined by the amount of available             typhi murium wild type and strains harboring either the resistance to nalidixic acid
                                                                                                      R                              R
GRP78/BiP. Recent discovery that GRP78/BiP is the onl y known substr ate for the                (Nal ) or to streptomycin (Str ). 2). To evaluate the i nfluence of multidrug
proteolytic A subunit (SubA) of a novel bacterial AB5 toxin provides the first                  resistance on the appearance of GASP phenotype. 3) To detec t if there are
opportunity for targeted destr uction of GRP78/BiP. Aims: 1) T o es tablish if                  genomic differenc es between both aged wild-type and used resistant mutants
targeted delivery of SubA into tumor c ells is s electi vel y c ytotoxic. 2) To establish if    (Str R and N al R ) of E. coli, the pulsed-field gel electrophoresis (PFGE) of total
destructi on of GRP78/BiP l eads to enhanced toxicity of ER-stress inducing dr ugs.             genomic DNA was c onducted.
Methods: SubA was geneticall y fus ed to human epi der mal growth factor (EGF)                  Methods: In a typical GASP competition experi ment, c ells from a 10-day- old
and the res ulting EGF-SubA was express ed in E.coli. T umor cells (MCF7, PC 3,                 culture are inoculated as a numerical minority (1:100 vol/vol) into a young (1-day-
F98, F98-EGFR, MDA231luc) wer e treated with EGF-SubA alone or in                               old) culture. The genomic DNA from randoml y chos en colonies was digested with
combinati on with various drugs. Bioc hemical and cell biol ogy methods were used               SfiI and res ol ved by puls ed-field gel electr ophoresis (PFGE).
to char acterize cellul ar EGFR levels, GRP78/BiP cleavage, ac tivation of unfolded             Results: In the mi xture c onsisti ng of the aged S. enterica Str R and young E. coli
protein respons e, apoptosis, and c ell viability.                                              Nal R , strong phenotype GASP mutants of S. enterica dominated after the third day
Results: Exposure of cells expressing high levels of EGFR to EGF-SubA res ults                                                 but
                                                                                                of mutual growth. Li kewis e, R with i nversed bacterial r esistanc e, in the mi xture of
in rapid (~2 hours) EGFR-mediated destruction of GRP78/BiP. Des pite the                        10-day- old S. enterica Nal c ultur e with young culture of E. c oli Str R , the S.
ongoing cleavage, in most cells it results in significant upregulation of GRP78/BiP             enterica NalR GASP mutants of str ong phenotype dominated the mixture after the
level by 24h of treatment. EGF-SubA is highl y c ytotoxic to growi ng and c onfl uent           fifth day and wer e maintained at about 1x108 CFU/ml. Electrophoretic karyotype of
cells with high level of EGFR (> 105 EGFR per cell), with IC 50 in the range of 3 to            the 10-days-old GASP mutants of E. coli strains c arrying the resistance revealed
40 pM, while EGFR-negati ve cells are at least 500-fold less sensiti ve. EGF-SubA               additi onal bands when compared to the wild-type.
strongly s ynergizes with thapsigargin, an ER-stress inducing drug, with ~10-fold               Conclusions: 1) T he s trong GASP phenotype was obtained i n mi xed c ultures with
enhanc ed effic ac y of the drug combination, rel ati ve to eac h c ompound alone. Less         the aged mutant s trains, but not when the isogenic antibiotic-sensiti ve strains were
prominent s ynergism is obs er ved with drugs that ar e less str essful for ER.                 used. 2) The cells in mixed cultures of doubl e mutant E. coli strain Nal R Val R bear
Conclusions: GRP78/BiP might be a new ―Magic Target‖. Its targeted destruction                  multipl e us eful GASP mutati ons that i ncreas e its R     fitness in c omparison with
                                                                                                                             R
with subnanomolar concentrati ons of EGF-SubA is extremel y c ytotoxic, while non-              mutants E. coli strain Str and S. enterica s train Str and s howed strong GASP
toxic conc entr ations of EGF-SubA dis arm cellul ar defens e and allow to us e                 phenotype. 3) The PFGE analysis demons trated the significant chromos omal
virtually non-toxic amounts of ER-stress induci ng drugs.                                       rearrangements in 10-day- old bacterial anti biotic-resistant mutated cells that
                                                                                                correspond with the appearance of s trong GASP phenomenon.




AntiNeoplaston: Synthesis, Biological, and Clinical Eva luation in Eg ypt                        Antibacterial, Antisecretor y and Antihemorrh agic Activity of Azadirachta
                                                                                                 indica U sed to Treat Choler a and Diarrhea in India
BADRIA F A1*, ABOU-ZEID L2, MOWAFY A3, HAWAS S4, KHAFAGY Y5 . MABED
M6                                                                                               BAG PK 1 , THAKURTA P1, BHOWMIK P1 , MUKHERJEE S1, HAJRA TK1, PATRA
                                                                                                 A2
                  1                        2                     3
Phar macognos y , Medicinal C hemistry , and Biochemistr y D epartments, Faculty
                                                                                                 1
of Pharmac y                                                                                      Department of Biochemistry, 2 Department of Chemistr y, Uni versity of Calcutta,
Immunolog y4, Surgery5, and Oncolog y6 Departments, F aculty of Medicine                         Kol kata, Indi a
Mansour a Uni versity, M ansoura, Egypt 35516
                                                                                                 Background: Indigenous uses of Azadirac hta indic a A. juss (Maliac eae) (loc ally
Backgro un d: Antineoplastons, first described by Burzyns ki, are natu rally occurring           known as neem) leaves in different parts of Indi a for curing gastrointestinal
peptides and amino acid derivatives, which control neoplastic growth. Antineoplaston A -         disorder s uch as diarrhea and cholera is wide spread. The objecti ve of the pres ent
10 (3-phen ylacetyl amino-2,6-pipe ridinedione) is the first chem. identified antineoplaston.    study was to eval uate the antibacterial and antisecretor y acti vity of neem extract
We previously reported the utility of antineoplaston-A 10 (3-phen ylacetylamino-2,6 -            against Vi brio cholerae, a caus ati ve agent of watery diarrhea s uch as cholera.
piperidinedione) as an endogenous cancer protector and immune modulator in breast                Methods: Methanol extr act of neem leaves wer e tested using the strains of multi-
cancer patients.                                                                                 drug resistant V. chol erae belonged to O1, O139 and non-O1, non-O139
Me th ods: Antineoplaston A-10 level was measure d in the urine of 31 breast cancer
                                                                                                 serotypes. Anti bacterial acti vity of the extract [10, 25, 50, 100, and 200 mg/ml (200
patients and 17 normal women using high performance thin layer chromatograph y
(HPT LC).                                                                                        μl/well)] was determined by agar-diffusi on assay. Minimum inhi bitor y c onc entration
Four new piperidinedione A 10 analogs were synthesized and tested for their antimitotic          (MIC) and minimum bactericidal conc entration (MBC) were assess ed using the
activity on a human breast cancer cell line against the prototype A 10 and the antibreast        broth microdilution method. The effect of the extract on fluid s ecretion and
cancer drug tamo xifen. Moreo ver, the D NA binding capacity of such compounds. was              hemorrhage i n intestine induced by V. cholerae was studi ed using mouse model
evaluated against A 10.                                                                          (Male BalB/C mic e, 23.8 ± 1.1 g body weight). Mice of each of groups 1 and 2
Apoptosis was measured in patients with breast cancer at time of diagnosis and to                (n=2 per group in duplicated) were admi nistered 500 μl of bacterial inocul ums
correlate u rinary antineoplaston A-1 0 levels with neutro phil apoptosis and to describe the    (2×109 CFU/ml) orally. After 1 h mic e were fe d either with 200 μl of methanol
direct effect of A-10 in vitro on neutrophil apoptosis in breast cancer patients. T he           (group 1) or crude extract (group 2; at oral dos es between 100 and 1800 mg/Kg in
participants were patients with a histologically confirmed diagnosis of breast c ancer.          200 μl of methanol) orally. Mice were sacrificed after 24 h incubation and fluid
Only those cases without p revious t reatment for breast cancer we re included. Neutrop hil
                                                                                                 accumulati on (FA) ratio was measur ed.
apoptosis was assessed in breast cancer patients both morphology and by DNA
                                                                                                 Results: Crude extract s howed inhibitor y acti vity against multi s erogroup strains of
fragmentation and studied relative to healthy cont rols. Antineoplaston A -10 was
                                                                                                 V. cholerae by agar-diffusi on assay with significanc e (p<0.05). MICs reac hed by
measured using high performance liqiuid chromatog raphy in urine samples collected from
the patients. Urine samples f rom no rmal w omen served as controls. Direct eff ect of           50% (MIC 50) and 90% (MIC 90), and MBC for the extrac t were 2.5 mg/ml, >5mg/ml,
antineoplaston A-10 on ne utrophil apoptosis was tested in vitro afte r adding A-10 at a         and 10 mg/ml respec tivel y. Administration of the extract (1800 mg/kg) did not
concentration. of 10 ng/mL to the cellular suspensions of breast cancer patients. Non -          produc e any sign of toxicity in mic e. Group 1 mice showed fluid accumulation (FA
treated samples served as controls.                                                              ratio; between 0.11±0.01 and 0.16±0.02) and hemorrhage in the intesti nes. Neem
Results: Significantly lower antineoplaston A -10 levels were detected among patients            extract showed acti vity with inhi bition (of fluid secr etion) values of 27.7 ± 7.8, 41.1
with breast cancer. (E) -3-(4-Nitrocinnamoylamino)-2,6-pipe ridinedione and (E)-3 -(4 -          ± 3.4, 43.3 ± 1.3, 57.0 ± 5.9, and 77.9 ± 7.2 % at dos es of 100, 200, 300, 450, and
hydro xycinnamoylamino) -2,6-pipe ridinedione        were      seve ral -fold more     p otent   1800 mg/kg res pecti vely. Oral administrati on of the extr act inhibited hemorrhage
antiproliferative agents than A 10 and tamo xifen. T hey also had significantly higher           induc ed by V. cholerae in mouse intes tine at a dos e ≥300 mg/kg (visually
capacity to bind DNA than A 10. Conve rsely, (E) -3-(cinnamo ylamino)-2 ,6-piperidinedione
                                                                                                 obser ved).
and (E) -3-(4-methoxycinnamo ylamino)-2,6 -piperidinedione had wea ker biological profiles       Conclusions: 1) Methanolic extract of A. indic a leaves was an effecti ve
than the lead compound A 10. De tailed synthetic, spectroscopic, and biological data are
                                                                                                 antibacterial agent against V. cholerae, and significantly r educ ed the fluid
reported.
Significantly higher neutrophil apoptosis levels were detected among patients with breast        secretion and hemorrhage induced by V. c holer ae in mous e intestine. 2) The
cancer with a P value <0.001. Urinary antineoplaston A -10 level is significantly neg.           active extract may be empl oyed for the treatment of c hol era and as potential
correlated with high apoptosis levels (P<0.0001 ). In vitro, antineoplaston A -10 was f ound     source to develop novel antimicrobial compound and antisecretory drug useful to
to inhibit significantly the neutrophil apoptosis with a P value <0.0001.                        treat c holera and diarrheal patients.
Concl usio ns: Antineoplaston A-1 0 ma y pro vide rational basis for designing trials to         Authors‘ disclosur e statem ent: This wor k has been published in J.
employ its immune modulatory potentials as adjuvant thera py in breast cancer patients.          Ethnophar mac ology: Thakurta, P., P. Bhowmi k, S. Mukherjee, T. K. Hajra, A.
T hese findings confirm the presence of immune defects among patients with breast                Patra and P. K. Bag. 2007. Antibac terial, antisecr etor y and antihemorrhagic
cancer and such results should stimulate the development of ne w strategies to induce and        activity of Az adirachta indic a used to treat cholera and diarrhea in Indi a. J.
augment immunity fo r the treat ment of b reast cancer.
                                                                                                 Ethnophar mac ology (Els evier) 111 (3): 607-612.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                    Page A-24
                                                         EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                      Nürnberg, October 3-5, 2008
The Frequency-Dependent Effect of Infrasound on Bull Sperm Velo cit y                 Synergistic Action of Resveratrol and its Polyh ydroxylated Derivative with
                                                                                      Cytar abine in HL-60 Hum an Prom yelo cytic Leukemia Cells
BAGHDASARYAN N, AYRAPETYAN S
                                                                                      BAGO-HORVATH ZS1, SAIKO P1 , MURIAS M 2, ERKER T2, HANDLER N 2,
UNESCO Chair-Life Scienc es Inter national Postgraduate Educ ational Center,          MADLENER S3, J AEGER W2, GRUSCH M 4, FRITZER-SZEKERES M 1,
                                                                                                    3            1
Yerevan, Armenia                                                                      KRUPITZA G , SZEKERES T
                                                                                             1
Background: The rheotaxic properties of sperm indic ate the existence of                      Clinical Institute of Medical and Chemical Laboratory Diagnostics, M edical
specialized mechano-sens ors in sperm membr ane detecti ng the flow directi on of            University of Vienna, Aus tria, 2Department of Pharmaceutical C hemistry, Fac ulty
                                                                                                                                                 3
the ej aculate. T herefore it is suggested that these sensors could ser ve as one of         of Life Sci enc es, Uni versity of Vienna, Aus tria, Institute of Clinical Patholog y,
targets for infrasound (IS) effect and woul d have s ensiti vity t o IS frequenc y. The      Medical Uni versity of Vi enna, Austria and 4Department of Medicine I, Institute of
overall aim of the present work was to study the frequenc y-dependent effec t of             Cancer Res earch, Medic al Uni versity of Vienna, Austria
30dB IS on sper m velocity, 45Ca uptake and i ntracellul ar level of c-AMP and c-
GMP.                                                                                         Background: Res veratrol, a naturall y occurring stilbene derivati ve, is a potent free
Methods: Experiments wer e performed on preliminary fr ozen bull sper m                      radical scavenger exerting a multitude of bioc hemical and antineopl astic effec ts.
incubated in 2.9% Na-citrate water soluti on at 30oC. IS treatment of sper m                 Resveratrol was identifi ed as an i nhibitor of ribonucleotide reductas e (RR), a key
samples was done by means of IS waves generating by a s pecial setup. 45Ca-                  enz yme of DNA s ynthesis and an e xcellent target of cancer chemotherapy.
uptake and i ntracellul ar cyclic nucleoti des c ontents (cAMP and cGMP) were                Inhibitors of RR have been pr eviousl y s hown to exert s ynergistic combination
measured by Wallac 1450 liquid scintillation counter. The sperm velocity was                 effec ts with c ytarabine, a well-established antileukemic agent. We investigated the
recorded by means of digital video camera (SONY, Japan) set on biological                    biochemical effects of res veratr ol and its polyhydroxyl ated deri vati ve, 3,3‘,4,4‘,5,5‘-
microscope MB-14B c onnected to PC and sperm direct vel ocity was es timated by              hexahydroxys tilbene (M8) on the in situ RR activity, cell cycle distribution,
Pinnacl e studio program.                                                                    inducti on of apoptosis and inhibition of NF-kappaB. Furthermore, it was tested
Results: It was shown that 2Hz, 30dB 5min IS treatment c aused the pronounced                whether a combination of res veratrol or M8 with cytarabine c ould yi eld s ynergistic
elevati on effect (25%, ** *: p< 0.001) on the s per m vel ocity (Figure 1), which was       cytotoxic and apoptotic effects i n human HL-60 promyel oc ytic leukemia cells.
accompanied by decreasi ng of intracellul ar level of c-GMP by 260.7%                       Methods: Cytotoxic effects of res veratrol, M8 and c ytarabine al one and in
(*: p<0.05), and increasing of intrac ellular level of c-AMP 43.57% (*: p<0.05) and         combinati on were analyz ed using growth inhibition and clonogenic ass ays.
45
   Ca-uptake 2856% (*: p<0.05) by s perm.                                                   Synergistic combination effec ts were identified by the Calc us yn software. In situ
                                                                                             inhibition of RR was determi ned by the i ncorporation of 14C-labelled c ytidine into
                                                                                             the DNA of res veratrol-treated HL-60 c ells. Concentrati on of NTPs and dNTPs
                                                                                             was meas ured by a HPLC method. Induction of apoptosis was studi ed using a
                                                                                             Hoechst/propidium iodide staini ng method. Inhibition of TNF-alpha induced
                                                                                             activation of NF- kappaB was shown by EIA and Western blotti ng and c ell c ycle
                                                                                             distribution was anal yzed by F ACS.
                                                                                             Results: Res veratrol effecti vel y inhibited inc orporati on of 14C-labelled c ytidine into
                                                                                             DNA. Furthermore, incubati on of HL-60 cells with res veratrol significantl y
                                                                                             decreas ed intrac ellular dCTP, dTTP, dATP and dGTP c oncentrati ons. M8
                                                                                             depl eted intrac ellular NTP pools and dTTP as well as dATP pools. Moreover, M8
                                                                                             inhibited the ac tivation of NF-kappaB and arrested HL-60 cells in the S-phase of
                                                                                             the cell c ycle. Bas ed on these res ults, we investigated the combination effects of
                                                                                             resveratrol and M8 with cytarabi ne. In growth inhibition, apoptosis and clonogenic
                                                                                             assays, res veratrol and M8 acted s ynergistically with c ytarabine in HL- 60 c ells.
                                                                                             Conclusions: Bas ed on the synergistic cytotoxic and pro-apoptotic effects, the
Fig 1. T he effect of 1- 10 Hz 30dB IS on bull sper m v elocity. * : p<0.05; **: p<0.01;     combinati on of c ytarabine with res veratr ol or M8 could bec ome a viable option i n
***: p<0.001                                                                                 the c hemotherapy of leukemia and therefore des erves further testing.
Conclusions: The 2Hz, 30dB IS 5min pretreatment caus ed: 1) activation of
sperm vel ocity; 2) increase of 45Ca uptake, 3) increase of intracellular contents of
cAMP and decrease of cGMP.


The action of a new drug th at targets a cancer‘s blood supply: the stor y of                Are Grap efruit, Or ange, Lim e, Pummelo and Apple the Forbidden Fruits of
DMXAA (ASA404)                                                                               Drug Interactions?

BAGULEY BC                                                                                   BAILEY DG, DRESSER GK, KIM RB

Auc kland Cancer Society Research Centre, the Uni versity of Auc kland, Private              University of Western Ontario, London, Canada
Bag 92019, Auc kland 1142, New Zealand.
                                                                                             Background: Intestinal drug metabolism and carrier-medi ated drug trans port are
DMXAA (ASA404; 5,6-dimethyl xanthenone-4-ac etic acid), developed in this                    accredited as fundamental factors affecting s ystemic drug availability. This
laborator y [1,2], is currentl y undergoing Phas e III clinical trial, in combination with   presentati on will initially revi ew i nacti vation of CYP3A4 by grapefruit, Seville
the c ytotoxic drugs carbopl atin and paclitaxel, in patients with non s mall cell lung      orange, lime and pummelo juices and i ncreas ed drug bi oavailability. T he foc us
cancer. DMXAA was originally developed as the most acti ve member of a series of             will be recent findings of inhibition of OATP1A2 medi ated drug uptake by
anal ogues of the dr ug flavone acetic acid [3]. DMXAA has both a direct, earl y             grapefruit, orange and appl e juices and decreased absorpti on.
effec t on tumour bl ood vess els, leading to inhi bition of tumour blood fl ow, and a       Methods: A series of in vitro and clinical pharmacokinetic studies were c onducted.
later effect that is mediated by loc al releas e of c ytoki nes and other vas oac tive       Results: OATP1A2 was express ed in healthy human s mall intestine and co-
molec ules. It is this double action that maintains a s ufficiently l ong duration of        localized with efflux tr ansporter, MDR1 (P-glyc oprotein), to the apic al membrane of
tumour blood fl ow arrest to induc e irreversible damage and vasc ular collaps e [1].        enteroc ytes . OATP1A2 was the onl y shown uptake tra nsporter of the
The effec t DMXAA on tumour vasc ular endothelial cells appears to be mediated by            antihistami ne, fexofenadine, whic h is also a s ubstrate for MDR1. Grapefruit and
p38 MAP ki nas e but the signalling pathway in tumour macrophages that leads to              orange juices at 0.5% normal strength hal ved the in vitr o acti vity of OATP1A2.
increased production of cytokines has not yet been identified. DMXAA facilitates a           These juices at 10-fol d higher concentrati on produc ed much less in vitro inhibition
positi ve feedbac k loop where c ytoki nes fr om tumour macrophages r educe blood            of MDR1, demons trating potent and preferenti al in vitro inhibition of OATP1A2.
flow and the res ulting tissue hypoxia acti vates macrophages. The clinical                  Clinically, grapefruit and orange juice at high volume (1200 ml) mar kedl y
antitumour acti vity of DMXAA is improved by c o-administrati on of c ytotoxic drugs         decreas ed oral fexofenadine bioavailability to 30% of that c ompar ed to water.
and rec ent experimental results suggest that c ytotoxic drugs can acti vate tumour          Grapefr uit juice c ons umed at a nor mal vol ume (300 ml) hal ved s ystemic
macrophages through toll-like receptors such as TLR4. Thus, the potential                    fexofenadine availability consistent with a mechanis m involvi ng selecti ve direct
antitumour ac tivity of tumour macrophages may be increase firstl y by DMXAA                 inhibition of intestinal OAT P1A2. The major fl avonoids in grapefruit and orange
itself, secondl y by hypoxia and thirdly by cytotoxic drugs. Studies with DMXAA              juice, res pecti vel y naringin and hesperidin, caus ed concentration-dependent in
may not onl y provide a basis for improved clinical ther apy but also facilitate             vitro inhi bition of OATP1A2. The IC 50 for naringin of 3.6 M was several hundred-
understanding of host events involved in the tumour response.                                fold lower than that pr oduci ng equal i nhibition of MDR1. An aqueous s olution of
                                                                                             naringin (300 ml) at the same conc entration as that measure d in the tested
1. Baguley BC. Anti vascul ar therapy of c anc er: DMXAA. The Lanc et Oncology               grapefruit j uice (1200 M) decr eased fexofenadine bioavailability to 75% of that
   2003, 4, 141-148.                                                                         with water, which accounted for half the reduc tion obs er ved with grapefruit juic e.
2. Tozer GM, Kanthou C, Baguley BC. Disrupti ng tumour blood vessels. N ature                Conclusions: Thes e results support a new mec hanism of food-drug interactions.
   Reviews Canc er 2005, 5, 423-435.                                                         The fact that naringin was clinicall y acti ve substance likel y represented the first
                                                                                             report of a single dietar y ingredient producing a drug inter action in humans by
3. Rewcas tle GW, Atwell GJ, Li ZA, Baguley BC, D enny WA. Potenti al antitumor              modulati ng drug transport acti vity. This type of interaction appears relevant to a
   agents. 61. Str ucture-ac tivity relationshi ps for i n vi vo c olon 38 acti vity among   range of medications , particularly for thos e drugs that are ess enti al for treatment of
   disubstit uted 9-oxo-9H- xanthene-4-acetic acids . J Med Chem 1991, 34, 217-              serious medical conditions.
   222.
Supported by the Auckland C ancer Society.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                 Page A-25
                                                              EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                     Celebrating the 100th Anni versar y of the
                                                                          Nobel Prize Award to Paul Ehrlich
                                                                            Nürnberg, October 3-5, 2008
Cytokin e-associated neutrophil extr acellular traps and antinuclear                       A Novel Anti-Angiogenic Glycother apeutic for Breast Cancer
antibodies in Plasmodium f alcip arum infected ch ildren under the age of s ix
                                                                                           BANERJEE DK
BAKER V1, IMADE G2, MOLTA N 3, PAM S2 , OBADOFIN M 2, SAGAY S2 , EGAH
   2        2             4            5         6          6           7             7
D , IYA D , AFOLABI B , BAKER M , FORD K , F ORD R , ROUX K , KELLER T                     Department of Bi ochemistr y, School of Medicine, U niversity of Puerto Rico,
                                                                                           Medical Sciences Campus , San J uan, PR. USA
1
  Chipola C ollege,FL, 2 JUTH Medical Sc hool, Nigeria; 3Jos U niversity, Nigeria;
4                                                   5                       6
  Nigerian Institute of Medical Res earch, Nigeria; Jac kson Hos pital, F L, World         Compl ex etiolog y of br east canc er needs personalized treatment. Angiogenesis is
Health Mission, PA, 7Florida State Uni versity, F L                                        critical for the breas t tumor growth. Our obj ecti ve has been to evaluate the
                                                                                           requirement of lipid-linked oligosaccharide (LLO), a pr e-requisite for asparagine-
Background: In Plas modium falciparum-i nfec ted children, rel ationships between          linked glyc oproteins i n angiogenesis and as k if anti-angiogenic glycotherapeutics
blood cell histopathology, blood plas ma c omponents, development of                       could eliminate the breast tumor growth. Our angiogenesis model is a non-
immunoc ompetence and disease s everity remain poorly understood. Blood from               transfor med capillar y endothelial cell line with pres erved physiol ogy and anatomy
Nigerian children with uncomplicated mal aria was anal yzed to gain insight into           responding adequately to cellular microenvironment. cAMP-signaling enhanc es
thes e relations hips. This investigation presents evidenc e for circulating neutrophil    LLO bios ynthesis and results in acc elerated c ell c ycle progression, increased
extracellular traps (NETs) and antinucl ear IgG antibodies (ANA). T he pres enc e of       capillary l umen       formation and HSP-70              and HSP-90 expression.
NETs and ANA to double-stranded DNA along with the c ytoki ne profiles found               Mannos yl phospho dolichol s ynthas e (DPMS), a key regulator in the LLO
suggests autoimmune mechanis ms that coul d produc e pathogenesis in children,             bios ynthesis is also increased. Cloning and sequencing identifies a PKA motif in
but i mmunoprotecti on in adults.                                                          DPMS gene and supports PKA-dependent phos phor ylation. On the other hand,
Methods: Peripheral blood smear slides and blood s amples obtained from 21                 tunicamycin (TM, an anti biotic and an LLO inhibitor) inhibits the cell surfac e N -
Nigerian children under si x years of age, presenting with uncomplicated malaria           glycan expr ession, down regulates the e xpression of Bcl-2, D-type c yclins, c dks
before and seven days after initiation of sulfadoxi ne-pyrimethamine (SP) treatment        and the transcription factor E2F c ausing a G1 arrest and halts angiogenesis.
were anal yzed. The slides were stain ed with Giemsa and with DAPI. Levels of the          Increas ed cas pas e-3, -9 and -12 expressi on, DNA laddering and Annexi n V
pro-inflammator y c ytoki nes IFN-γ, IL-2, TNF-α, CRP, and IL-6, selec t anti-             binding all support apoptosis. High expression of GRP-78/Bi p indicat ―ER stress‖
inflammator y c ytokines TGF-β and IL-10, and ANA were determi ned by                      and the induction of unfolded protein respons e. DPMS acti vity as well as its
immunoassay.                                                                               protein expression is lost, and VEGF 165 loos es the c ellular pr otec tion i n TM treated
Results: The c hildren exhibited circulati ng NETs with adherent par asites and            cells. TM inhibits angiogenesis in Matrigel ™ implants and also reduc es the
erythroc ytes, el evated ANA levels, a Th2 dominated c ytokine profile, and l eft-         humaniz ed breast tumor growth in athymic nude mice by 50-65% i n 1-3 weeks
shifted leukoc yte differenti al counts . Nonspecific ANA levels were significant in       when administered or ally or intravenously. Therefore, TM has a potential to
86% of the children pr e-treatment and in 100% of the children seven days after SP         succeed in the clinic as an exc ellent breas t cancer therapeutic.
treatment, but in onl y 33% of age-matc hed control s amples c ollected during the         Supported i n parts by grants from NIH/U54-CA096297 and Sus an G. Komen
season of low par asite trans mission. Levels of ANA s pecific for dsDNA were              Breast C ancer F oundation BCTR58206.
significant in 81% of the children both pre-treatment and post-treatment.
Conclusions: The results of this inves tigation suggest that NET formation and
ANA to dsDNA may induce patholog y in falciparum-infec ted c hildren, but acti vate
a protecti ve mechanis m agains t falciparum mal aria in adults. The significance of
in vi vo circulating chromatin in NETs and dsDNA ANA as a causative factor in the
hypores ponsi veness of CpG oligonucleoti de-based malaria vaccines is discuss ed.




Sear ch for diagnostic and prognostic m arkers for Glioblastoma                             Synthesis and Bio logical Evalu ation of Anticancer β-Lactams
multiforme(GBM).
                                                                                            BANIK BK 1, BANIK I2 AND BECKER FF 2
BANERJEE HN
                                                                                            1
                                                                                             Department of Chemistr y, T he U niversity of Texas-Pan American, Edinburg,
                                                                                                 2
Department of Bi ological & Phar maceutical Scienc es. Di vision of M athematics,           USA; Uni versity of Texas M. D. Anderson Cancer Center; H ouston, USA.
Sciences , Tec hnology and Phar mac eutical Sciences. R oom# 425, J enkins
Science Buildi ng. Elizabeth City State Uni versity, U niversity of North C arolina.        Background: Sys tematic anal yses of pol yar omatic deri vati ves through an
                                                                                            examinati on of their anticancer effects i n vitro and in vivo has been demonstrated.
Background: Glioblastoma Multiforme are brain tumors whic h are a                           During the cours e of our studies, it has been anticipated that conformationall y
heterogeneous group of central ner vous s ystem neoplas ms that arise within or             restricted analogues might increas e the potenc y. We have envisioned that
adjac ent to the brain. Some are c urable by surgical resec tion, but many c annot be       stereodefi ned β-lactams will ser ve as i mproved confor mati onall y c ons trained
eradicated by current treatments, and, when they are, dis abling neur ological injur y      anal ogues of our open-chai n diamides that have already been shown to poss ess
often ensues. At present these brain tumors are detected by imaging onl y after the         anticancer ac tivity. Sinc e many β-lactams (penicillin-types of compounds) have
onset of neur ological symptoms and no earl y detec tion strategies are in used.            been widel y accepted as safe antibac terial agents for more than 50 years, our
Imaging, the obvious screening strateg y for glioblastomas is extraordinaril y costl y,     hypothesis is that novel drugs can be s ynthesized and s elected within this class
especi ally given the relati ve rarity of these tumors in c omparison with br east or       that will have both an enhanced anticancer ac tivity and low toxicity to nor mal
prostate canc er. Genetic tes ting is a better option and is desirable as a screening       tissues.
tool bec ause it is generall y based on a simple blood test. The current                    Aims: 1) To s ynthesize -lactams derived from polyaromatic i mines . 2) To study
histopathological approach to the diagnosis and classificati on of glioblastomas is         their effec ts in vitro and in viv o. 3) To s tudy the mechanism of ac tion of the lead -
not s atisfac tor y in many respects.T herefore,a gene and protei n based diagnostic        lactams
and classificati on s ystem is best for prognosis and therapeutic approaches.               Methods: Synthesis of -lactams was achieved following c ycloaddi tion r eaction of
Methods: The following tec hniques were used to analys e glioblas toma speci mens           imines with aci d chloride in the presence of triethyl ami ne. In vitro cytotoxicity and
and cell lines and search for bi omarkers a)Microarray anal ysis b)SELDI s tudies c)        in vivo study were performed using NCI protoc ol. Ames assay, topoisomeras e
Laser Optical tweez ers d)DIGGE and e)Real Time PCR f)ELISA.                                inhibition and c ell c ycles were also investigated.
Results: Several novel protei ns and genes were identified that differentiall y             Results: A number of racemic and opticall y acti ve novel β-lactams were
express in Glioblastoma specimens in comparison to normal c ontrol br ain                   synthesized. Synthesis of thes e agents usi ng microwave irradi ation was
samples.                                                                                    perfor med. An unprec edented stereoc hemical results was identified. Some of
Conclusions: There are several genes that ar e either down regulated,loss of                thes e compounds demonstrated promising antitumor activity in vitro and in ani mal
functi on or upregulated that we obs erved in our res earch.We are wor king farther         tumor model s ystems . In some instances the antic ancer ac tivity exceeded that of
on the importanc e of thes e genes and their protei ns in their role as a significant       cisplatin in vitro. Thes e agents demonstrated a bloc kade of the G2/M chec kpoint in
biomarker by anal yzing more patient populati on.                                           cancer cell lines, but they had no effects on topois omerases . The ac tive
                                                                                            compounds were non-mutagenic.
                                                                                            Conclusions: 1) Synthesis of several novel anticancer β-lactams achi eved. 2)
                                                                                            One of them demonstrated antitumor acti vity in animal model. 3) Although the
                                                                                            mec hanis m of action of the lead compounds was not es tablished, our res earch on
                                                                                            cell cycle anal ysis offered intriguing possibilities.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                               Page A-26
                                                         EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                               Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                      Nürnberg, October 3-5, 2008
Synthesis of a Ser ies of 8-(Substituted-Phen yl)xanthines and a Stud y on the       The Modified Vaccin ation Technique develop ed b y B arabas provides the
Effects of Substitution Pattern of Phen yl Substituents on Affinit y for             "magic bullet" for the prevention and cure of chronic ailm ents su ch as
Adenosine A1 and A2A Receptors- The Magic of Theoph ylline                           autoimmune disord ers and cancer sp ecifically, without side effects

BANSAL R 1, KUMAR G1, GANDHI D 1, YOUNG CL2, HAR VEY AL2                                                                  BARAB AS AZ1, COLE CD 2, LAFRENIERE R 1
1                                                        2
 Panjab Uni versity, Chandigarh, India;                      Uni versity of Strathcl yde, Gl asgow, United                1 Department of Surger y, Uni versity of C algary, H ealth Sciences C entre, Calgary,
Kingdom                                                                                                                   Alberta, Canada
                                                                                                                          2 Department of Neuros urgery, Uni versity of Utah, Salt Lake City, Utah, USA
Background: Adenosi ne r ec eptor s ar e promising therapeutic targets in a wide
range of conditions, i ncludi ng cerebr al and c ardiac ischaemic diseases, sleep                                         Background: Barabas has devel oped and implemented a new vaccination
disorders, i mmune and inflammatory disorders and c anc er. 8- Phenyltheophylli ne                                        method called modified vaccination technique (MVT) - in experi mental ani mals -
is the par ent member of a vari ety of potent adenosi ne rec eptor                                                        that c an be employed both prophylactic ally and therapeuticall y with equal
antag onis ts. H erein we report the s ynthesis of a new s eries of 8-(subs tituted-                                      effec tiveness to c ombat chronic disorders such as autoi mmune dis eas es, cancer
phenyl)xanthines and the effects of substit ution pattern of phenyl substituents on                                       and exogenous antigen induc ed mis haps. This brea kthrough discover y of antigen
adenosine rec eptors binding affinity.                                                                                    presentati on by the MVT allows the inducti on of a predeter mined beneficial
Methods: The three series of compounds A, B and C wer e prepared by treating                                              immune respons e outcome.
5,6- di ami no- 1,3- di methyl ur acil with al kyl ami noal kyl s ubsti tuted deri vati ves                               Presently available vaccination programs are preventati ve (through ac tive
of vanilli n, i sovanillin and 3-hydroxybenz al dehyde and s ubs eq uent                                                  immunizati on) or therapeutic (through passi ve i mmuniz ation) and neither
c ycli zati on i n thi onyl c hlori de.                                                                                   technique can evoke specific downregulator y (in autoimmune disorders) or
                                                 O
                                                                        OCH2CH2 NR                          OCH2 CH2 NR
                                                                                                                          upregulatory (in cancer and chr onic infec tions) immune res ponses to correct
             O              OMe                                                        O
                   H                  H3 C
                                                             H                               H                            chronic dis orders.
H3 C                                                         N               H3C
        N          N                         N                                     N         N                            Methods: When vaccinating, using the MVT, appropriatel y ass embled i mmune
                   I            OCH2 CH2NR
                                                                            OMe
                                                                                                                          complexes - made up of the specific target antigen and s pecific homologous
                                        O        N           N                O        N     N
    O              N
             N
                                                                                                                          pol yclonal or monocl onal anti bodi es against the target antigen - have to be
             CH3
                       A                           CH3       B                         CH3       C                        prepar ed (the " magic bullets") for the preventi on and treatment of presentl y drug
                                                                                                                          onl y treatable conditions s uch as autoimmune diseases and canc er. T he goal is to
Results: T he c ompounds wer e eval uated for their affi nity for A1 and A2 A                                             inject exactl y the right immune c omplex (the "magic bullet") i nto recipients to
rec eptor s usi ng [3 H]DPC PX and [3H ]ZM- 241385 as r adiolig ands . T abl e                                            evoke the producti on of the same class of immunoglobulin (i.e., antibody) with the
summariz es the obs er ved affi niti es of vari ous newl y s ynthesi z ed 8-                                              same specificity agains t the t arget antigen that resides in the inoc ulum.
phenyl xanthi ne deri vati ves in radi olig and bi nding ass ays at human A 1 and                                         Results: Using the MVT antibody information transfer is ac hieved res ulting in
A2 A r ec eptor s. T he pr es enc e of a methoxy s ubs tituent at 3 or 4- positi on of                                    predetermi ned immune respons es. Through the utilization of the MVT the
phenyl ri ng i n A and B al ong with an ortho pol ar side c hai n i ncr eases                                             potential of downr egulating or upregulati ng immune events - in humans with
sel ecti vi ty for A2 over A1 r ec eptor s. H owever abs enc e of a methoxy gr oup                                        certain autoimmune disorders and cancer - to achieve self cure and regained
as i n C r es ults i n al most eq ual s el ec ti vi ty for both s ubtypes.                                                toleranc e to s elf is in sight.
                                                                                                                          Conclusion: We believe absol utel y in the immune s ystem's natural ability to
Table: Adenosine A1 and A2A binding affinities of c ompounds                                                              correct mistakes. It requires onl y in certai n instances tailor made "instructions" for
                                                                                                                          eliciting and maintaining redirected benefici al immune-respons e outcomes .
        Comp. N o.          A                B                   C         DPCPX                 ZM 241385
                A1         >100         >100                     2.1      0.095                      0.54
            K i (µ M)
               A2A                                                                 1
                            0.7              2.7                 0.8       0.13                      0.064
            K i (µ M)

Conclusions: It c an be concl uded that s ui tabl e sel ecti on and positi oni ng of
ar yl s ubs tituents may l ead to devel opment of potent and s el ec ti ve xanthi ne
bas ed adenosi ne r ec eptor antag onis ts .

Bacteriocin subst ance producting b y L actococcus lactis against Listeria                                                Allo steric modulation of hormone r eceptors in can cer tr eatment
monocytogenes isolated in read y-to-use fish fillets
                                                                                                                          BARKER S, PUDDEFOOT JR, VINSON GP.
BARILE M.1, PALOMBA S.2, CERES C.1, MURRU N.1, PEPE O2
                                                                                                                          School of Biological and Chemical Scienc es,
1
 Department of Zootec hical Sci enc es and F ood Ins pec tion, Fac ulty of Veterinar y                                    Queen Mar y, U ni versity of London, London, United Kingdom.
Medicine, N apoli, Italy; 2Department of Food Sci enc es, Fac ulty of Agricultural,
Portici, Italy                                                                                                            Background: Trilostane was originally developed and used as an inhibitor of 3-
                                                                                                                          beta hydroxysteroid dehydrogenas e in treating Cushing‘s s yndrome. It was later
Background: Bacteriocins produced by lactic acid bacteria are i mportant in                                               found to confer clinical benefit in breast canc er patients. Our studies began with
preventing the growth of pathogenic bacteria especiall y in ready to use fish that                                        the obser vati on that trilostane (at micromolar conc entrations) could effect the
could be potentiall y contaminated by pathogens.                                                                          functi on of the estrogen rec eptor (ER) but without displ acing the nati ve ligand,
Methods: 26 Carnobacterium piscicola, 5 Carnobacterium div ergens and 24                                                  estradiol, from its ligand bindi ng site. We have proposed that trilostane can act as
Lactobacillus spp. str ains wer e screened against 22 strains of Listeria                                                 an allosteric modulator of the ER and our res earch has foc used on tr ying to find an
monocytogenes, 5 strains of Yersini a enteroc olitica, 1 strain of Staphylococc us                                        explanation for its efficac y i n the treatment of postmenopausal br east c anc er.
aureus , 1 strain of Pseudomonas s pp, 1 str ain of Sal monella spp. All producer and                                     Methods: In these studies we have used a wi de range of bioc hemical and
indicator strains wer e isolated from farmed gilthead fish fillets (Sparus auratus)                                       functi onal methods, including ligand-binding ass ays, gene reporter assays , gene
held in the culture collec tion of Department of Zootec hical Sciences. The                                               expression microarrays and cell proliferati on assays , in order to try to define an
microorganisms were previousl y i dentified by biochemical method and s ubmitted                                          alternative mec hanism of action for trilostane in the context of breast c anc er.
to antagonistic ac tivity experiments in solid and liquid media by using the spot on                                      Results: Trilostane does not dis place radiolabelled es tradi ol from estrogen
lawn and the agar well diffusion assay, respecti vel y. LAB strai ns producers of                                         receptor preparati ons. It inhibits MCF-7 breast canc er cell proliferation at
bacteriocin-like s ubstanc e in solid media, were then tested for bacteriocin                                             concentrati ons of 1μM and above. It has direct inhi bitor y effec ts on ER function
produci ng in broth and sensiti vity to heat (60-80°C/1h; 100°C/30 min.; e 121°C/10                                       and events medi ated through oestrogen respons e elements (EREs) and ac tivating
min), and to proteolitic enz ymes. Bacterial growth cur ves and bacteriocin                                               protein- 1 motifs. In a study of the effects of trilostane and tamoxifen, on MCF-7
produc tion were studi ed for LAB strai n produc er.                                                                      cells using microarrays, striking differ ences were found in gene expression
Results: On 25,4% of LAB strains showing anti microbial acti vity i n solid medi a,                                       profiles. Interestingly, trilostane was found to sel ecti vel y up-regulate the
onl y 1 s train, bel onging to Lactococc us lactis Sa 31, was able to produc e                                            expression of the beta subtype of estrogen rec eptor (ERβ), an effect replicated in
bacteriocin in liquid medium. Lact. lac tis Sa31 showed a narrow spectrum of it                                           vivo. This is significant as tamoxifen-resistant breas t canc er is often associ ated
activity towards Listeria monocy togenes (clear inhibition z ones 5-8 mm). No                                             with r educed ERβ protein expressi on and trilostane can inhibit proliferation of
inhibition zones were obs erved over Staphyloc occus aur eus, Sal monella s pp.,                                          tamoxifen-resistant MCF-7 cells in vitro.
Pseudomonas e Yersinia enter ocolitica. The increased bacteriocin production of                                           Conclusions: Our data support previ ous clinical fi ndings that trilostane can
Lact. lactis Sa 31 was obser ved after 11 h of inc ubation at 30°C in Elliker agar with                                   influence the effecti veness of tamoxifen and may subvert tamoxifen resistanc e,
maxi mum titre of bacteriocin of 2,560 Ar bitrary Units ml -1. The bacteriocin was                                        and demons trates in a wider context that allosteric modulation of rec eptor function
sensiti ve to tr ypsi n, pronase E and papain and was inac tivated by temperatures ≥                                      may be a us eful ther apeutic appr oach in circumstances in which the r eceptor
100°C.                                                                                                                    ligand binding domain is occupied by its cogn ate ligand or an antagonist. This
Conclusions: The bacteriocin produced by Lactoc occus lactis showed a relevant                                            concept has potenti al application in clinical situations in which either nucl ear
anti microbial acti vity against different strains of Listeria monocytogenes and could                                    receptors or surface membrane located rec eptors are implicated in cancer
be employed as biopreser vati ve in ready-to-us e fish fillets.                                                           progression.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                           Page A-27
                                                       EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                              Celebrating the 100th Anni versar y of the
                                                                 Nobel Prize Award to Paul Ehrlich
                                                                   Nürnberg, October 3-5, 2008
Elesclomol: A Novel Oxidative Stress Inducer for the Tr eatm ent of Metastatic      Blood-brain barrier P-glycoprotein function in aging and neuro logical
Melanoma                                                                            disease: in vivo measur ements in human s with [ 11 C]-verapamil PET.

BARSOUM J1 , F OLEY KP1, WILLIAMS A1, J ACOBSON E1 , O‘DAY S2, 4783- 03                     BARTELS AL, WILLEMSEN ATM, BART J, KORTEKAAS R, LEENDERS KL
STUDY GROUP                                                                                 Depts of Neurolog y and N uclear Medicine & Molec ular Imaging, Uni versity Medical
                                                                                            Centre Gr oningen, The Netherlands
1
 Synta Pharmaceuticals Corp., Lexington, MA, USA; 2The Angeles Clinic and
Research Institute, Santa Monica, CA, USA.                                                  Introduction: The blood-brain barrier (BBB) possess es a highly conc entrated
                                                                                            transporter, the P-glycoprotein (P-gp) trans porter. It protects the brain from several
Background: Elesclomol (E) is a small molecule drug candidate that stimulates               endogenous and exogenous compounds by acti ng as an acti ve cell membrane
production of reactive oxygen species (ROS) and selectively induces cancer cell             efflux pump. C hanges i n P-gp func tion have been i mplied in several neurological
apoptosis. The clinical efficacy of E in combination with paclitaxel (P) was                conditi ons. Upregulation of P-gp has been found to prevent uptake of drugs in
investigated in a double-blind, randomized, controlled Phase 2 trial in melanoma.           brain tumours and in epileps y, l eading to therapy-resistance. Decreased P-gp
Methods: The in vitr o acti vity of E was asses sed in human Hs294T mel anoma,              functi on has been associated with Amyl oid-β transport and progression of
Ramos l ymphoma and HSB2 leukemia cells. Preclinic al efficac y was tested in an            Alzheimer‘s diseas e (AD). Also, it has been related to toxi n exposure and the
M14 melanoma human xenograft model in mice. Stage IV metastatic melanoma                    devel opment of Parki nson‘s diseas e (PD). Finally, age-ass ociated decline in P-gp
patients were randoml y assigned to 213 mg/m2 E plus 80 mg/m2 P (E+P) or                    functi on may facilitate the accumul ation of toxic s ubstanc es in the brain. We
80 mg/m2 P as a weekly one- hour intravenous infusion, three of ever y four weeks           studied in viv o BBB P-gp func tion in ol der healthy s ubjects and in patients with PD.
until dis eas e pr ogression per RECIST or death. The pri mar y efficac y endpoint was      Methods: BBB P-gp functi on was studied using positron emission tomography
progression-free sur vival (PFS).                                                           (PET) and [11C]-verapamil. 17 healthy volunteers with age 18-86, 22 patients with
Results: E rapidl y increas ed ROS production in canc er cells. Sustai ned oxidative        Parki nson‘s disease in differ ent disease stages and si x s ubjec ts with parkins onis m
stress led to ac tivation of the intrinsic mitochondrial apoptosis pathway, with            (PSP and MSA) wer e scanned. Distribution volume (DV) of the trac er in the br ain
cardiolipin oxidati on, decreased mitochondrial membrane potential, c ytoc hrome c          was calc ulated usi ng Logan anal ysis. Statistical Parametric Mapping (SPM2) was
release and cas pas e acti vati on. Antioxidants bl oc ked all activities of E. ROS         used to s tudy specific regional differenc es between the subject groups.
increase and apoptosis preferenti ally occ urred in cancer c ells relati ve to nor mal      Results: Older subj ects s howed significantly increas ed brain tracer uptake as
cells. E inhibited tumor growth as a single-agent and s ynergized with P in causing         compared to the younger healthy s ubjec ts, i ndicating decreas ed BBB P-gp
tumor regression in human xenograft tumor models. At 21 US sites, 53 s ubj ects             functi on. In PD, the more advanc ed patie nts s howed incr eased tracer uptake in
were randomized to E+P and 28 subjects to P. The addition of E to P yielded a               frontal regions, while de novo patients showed decr eased uptake. In patients with
doubling of median PFS (112 v 56 days) and a 41.7% ris k reduction for diseas e             par kinsonism, increas ed uptake in bas al ganglia regions was obs er ved.
progression/death (haz ard ratio = 0.583, P = 0.035). Respec tive response rates            Conclusion: Decreas ed blood-brai n barrier P-gp function with aging could be a
were 15% v 3%. Median overall survi val was 11.9 v 7.8 months. E+P was well                 mec hanis m by which age acts as the main ris k fac tor for the development of
tolerated.                                                                                  neurodegenerative disease. It also means that s everal drugs will reac h higher
Conclusions: 1. E increas ed ROS levels in cancer cells, leading to apoptosis.              concentrati ons in the brai ns of el derly pati ents. T he knowledge that P-gp function
Cancer cells produce elevated ROS relati ve to normal c ells, and are therefore             may be modulated by drugs is als o important, as P-gp modulators may provide
susceptible to further ROS i ncreas es which pus h the cell beyond its oxidative            opportunities for inter vention, not onl y in tr eatment resistant cancer or epileps y but
stress tol erability limit. In this way, E takes advantage of a fundamental hallmar k of    also in the progression of neurodegenerati ve diseases.
cancer to sel ecti vel y kill cancer cells with littl e effect on normal c ells. 2. In a
randomized, blinded Phase 2 clinic al trial, E+P resulted in a clinicall y meani ngful,
statisticall y significant increase i n PFS, with an acc eptable toxicity profile and
encouraging survi val data. A global Phase 3 trial (SYMMETRYS M) of E+P v P in
chemotherapy-naïve stage IV metastatic melanoma pati ents is ongoi ng.




Laser-based Stable Isotope Tr acing in Human Breath                                         Er ythrom ycin: Magic Bullet for Sor e Throat and its Consequ ences in
                                                                                            Children
BARTLOME R and SIGRIST M W
                                                                                            BASNET NB 1,2, BASNET SB1
ETH Zurich, Zurich, Switzerland
                                                                                            1                                                                            2
                                                                                             Children‘s Medic al Diagnosis Center (CMDC), Kathmandu, N epal; Durga
Background: Stable isotopes have bec ome widel y available at affordable prices.            Bhawani Polycli nic, Kathmandu, Nepal.
They are increasi ngly used as tracers in clinical research and diagnosis. In breath
anal ysis, anal ytical methodology has relied upon expensi ve and cumbersome                Background: Most peopl e suffer from sore throat particularly in the first two
instrumentation such as isotope rati o mass spec trometr y (IRMS). In the last              decades of life. Previous studi es have proved that sore throat is a major cause of
decade, isotope rati o infr ared spec trometr y has bec ome a s erious c ompetitor to       sickness and a potentiall y dangerous sourc e of infection i n childr en. We evaluated
IRMS. Las ers have recentl y been employed to meas ure the 13C/12C isotope ratio            the clinical situation of sore throat in Nepal ese children, and reviewed literature to
of exhaled c arbon dioxide. However, breath sampl es mus t be prepared to reduc e           assess erythromycin as its magic remedy.
the water vapor content. In this wor k, we measured— without sample                         Methods: We prospec tivel y evaluated the clinical data of children below 19 years
prepar ation— the D/H isotope ratio i ncreas e in exhaled water vapor following the         who visited Children‘s Medical Diagnosis Center and Durga Bhawani Pol yclinic
intake of a deuterated tracer (D 2O).                                                       from J anuar y 2006 to Dec ember 2007. All patients were cons ulted and the fi nal
Current sensiti ve trace-gas detec tion schemes for i nfrared laser s pectr oscopy          diagnosis was made by a consultant pedi atrician and pediatric cardiol ogist. We
cannot handle condensable vapors. For this pur pose, we developed a high-                   also analyz ed all the publications indexed in Pub Med upto June 27, 2008 on key
temperature multi pass c ell.                                                               words children and s ore throat and erythromycin conduc ted in various studies.
Methods: A healthy vol unteer drank 5.14 mL of 99.9 % pure D 2 O diluted in 200             Results: Of the total 1175 examined patients 159 (13.53%) had tonsillitis.
mL tap water. T he D 2O intake corresponds to a dosage of 16 mg deuterium/kg                Amongst 159 patients 63.53% were between 4 to 10 years. The male femal e sex
body weight, a safe level that is within the dosage range us ed in clinical studi es.       ratio was 1.48. Our clinical experienc e s uppl emented by reported 150 literatures
Breath sampl es were then collec ted at 24-hour inter vals i n a c ommercial bag            have showed that er ythromycin has been used in all age groups and s ex, is
heated above 323 K until original bac kground levels wer e obtained. T he high-             effec tivel y acc eptabl e, well tolerated orall y, and als o availabl e in parenteral for m. It
temperature multipass cell was filled with a c ollected breath s ample without any          has been available worldwide after its discover y by McGuire and c owor kers in
preliminar y s ample preparati on. Roto-vi brational s pectra were r ecorded by fi nel y    1952 from a s train of Streptomyces er ythreus, originally obtained from s oil in the
tuning a mi d-infrared laser between 2788.60 and 2789.80 cm-1.                              Philippines. It is able to eradic ate a broad range of bacteria including streptoc occi,
Results: The D/H is otope ratio is usuall y express ed in parts per thous and relative      staphylococci, listeria, legionella, diphtheria, pertussis, tetanus, s yphilis and
to a standard, usi ng the common notation 2H = (R sample-R standard )/R standard  1000,
where R is the ratio of the heavi er to the lighter stable isotope of hydrogen. We          Camphylobacter jejuni without any fatal toxic effect. It exerts effec tive
perfor med repetiti ve measur ements before D 2O intake and determined  H with a
                                                                               2
                                                                                            concentrati on on tonsilar tiss ues. Er ythromyci n has both curati ve (e.g. sore throat)
standard deviation of 23 ‰. Following D 2O intake, we measured a significant 2 H           and prophylactic (e.g. rheumatic fever, rheumatic heart disease, acute
increase in exhaled breath of 768 ‰. We will pres ent ti me-dependent                       glomerulonephritis) benefits i n man. It has been proved effec tive in treati ng sore
measurements after D 2O intake and derive the total body water.                             throat and other infections thereby decreasing its untoward life thr eatening
Conclusions: Following the i ngestion of onl y 5 mL D 2O, an infrared l aser                cardiac, r enal, respirator y and neuronal c omplications, as well as the cos t of
spectrometer determines — without sample preparation — the D/H isotope ratio                treating cardi ac consequenc e, suc h as val vular surgeries.
increase in exhaled water vapor for the first time. T his opens the door to many            Conclusions: Er ythromyci n has reliable chemotherapeutic effect in treati ng sore
clinical applicati ons. The laser spec trometer has a great potential to bec ome a          throat and preventing its cardiac, respirator y, renal and neuronal cons equences in
portable and affordable devic e.                                                            children of developing and i ndus trialized nations thus proving its role as a ‗magic
                                                                                            bullet‘.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                    Page A-28
                                                               EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                       Celebrating the 100th Anni versar y of the
                                                                          Nobel Prize Award to Paul Ehrlich
                                                                             Nürnberg, October 3-5, 2008
Application of Saffron and It s Ingred ients as a Kno wn Ph armacological Herb               Ear Surg er y – Place for Topical use of Mitomycin C
from Ancient Times and the Mech anism s of Their Action
                                                                                             BATTELINO S, ZARGI M
BATHAIE SZ1 , MOUSAVI SZ2, HOSHYAR R 1 AND ASHRAFI M 1
                                                                                             University Medical Centre Ljubljana, Lj ubljana, EU-Slovenia.
1                                                                             2
  Faculty of Medic al Sciences, Tarbiat Modares Uni versity, Tehr an, Iran; Fac ulty of
Medicine, T ehran Uni versity of Medical Sci enc es, Tehran, Iran.                           Background: Stenosis and atresias of the external auditory canal (EAC) are rare
                                                                                             conditions difficult to manage with success. The main reason to operate is severity of
Background: Saffron (Crocus sativus L.), whic h is named as "red gold" i n Iran,             the hearing impairment and threatening cholesteatoma of the EAC. Some authors do
has been used not onl y as a food additi ve and a dye, but also as a medicinal herb          not find classical surgical methods sufficient and they favor the use of KTP/532 laser, or
in most parts of the world from anci ent ti mes. In the last two decades, it has been        larger surgical procedures. The proven ability of Mitomycin C (MMC) to inhibit
reconsidered bec aus e of its various biological pr operti es. Chemic al anal ysis has       fibroblasts in vitro has stimulated its use it in treatment to prevent stenosis and
shown the presence of more than 150 c omponents in s affron stigmas . In this                adhesion, also in otorhinolaryngology. The objective of the study was to evaluate the
presentati on we revi ewed the historical and medicinal uses of s affron in different        opening of the external auditor y canal in fibrotic atresias (congenitally and secondary)
parts of the world, es peciall y in Iran where saffr on was culti vated for the first time   and the hearing improvement, after the surgery and concomitant use of topical MMC.
(in addition to the wild type), and where nowadays is the biggest saffron producer           Methods: Ten patients, all together fourteen ears, with fibrotic external auditor y
(more than 80% of world s affron culti vation). T hen, the new findings from our             canal (EAC) atresias due to chronic external otitis, post-traumatic, post irradiated,
laborator y as well as other research groups, about its medicinal properties and             or congenital c ause were i ncluded. During the surgical proc edure – meatopl asty
various cellul ar and mol ecular mec hanisms of action were discussed.                       onl y endaur al approach was used, and 1 mL of MMC (0.4 mg/mL) was applied for
Methods: Web of Science and Medline were s earched for s affron and its                      4 minutes to the EAC. In 5 ears the applicati on of MMC was repeated one to si x
constituents. For anci ent us es of s affron, manual searc hing of Persian books and         month later, than 1mg/mL conc entration of MMC was used. During the application
searching inter net res ources, among with manual searching of their referenc es             of MMC the tympanic membrane was protected with a thin layer of dry Gelfoam.
were done. In our experiments , after purification of i mportant constituents from           Audiometric evaluation included pre and postoperati ve air -conduction thresholds
Iranian saffron, their application on br east and gastric cancer were studied in             (AC) and bone-conduction thres hol ds (BC).
model animals; their molec ular mec hanis m of action was also investigated using in         Results: Between 9 and 56 months after the surgery and concomitant application
vitro experiments.                                                                           of MMC the microscope visual contr ol of opening of EAC was ass essed. The
Results: Saffron was known from mor e than 3000 years ago by Iranians,                       hearing improvement was obser ved by using preoperati ve and postoperati ve pure
Assyrians Babyl onians and Minoans; not onl y for its application as a food additi ve,       tone threshold audi ograms (PTA). In 10 ears (72 %) the ear canals reminded open
dye and perf ume, but also for its us age as a medicinal herb (alone or in                   with a postoperati ve air-bone gap of 10 dB or less. No sens orineural hearing loss
combinati on with other drugs) to treat a wide range of diseas es. Newl y, its               was detected after surgery.
application in a variety of disorders involvi ng neuronal, car diovasc ular and other        Conclusions: 1) MMC used on a limited number of ears during surgery was effective
systems as well as cancer has been investigated. The c ellular and molec ular                in preventing scaring in fibrosis ending with atresias of EAC. 2) No complications or
mec hanis ms of its acti on are also under s tudy. Saffr on's more powerful                  sensorineural hearing loss were encountered after surgery and MMC application. 3)
components are carotenoids and monoterpene aldehydes. Struc ture-function                    The results of our study are comparable with the published reports and according to the
relations hip studies show that some properties are related to deglycos yl ated              follow up period our outcome results with a success rate of 72% could be considered
derivati ves, while others belong to more glyc os ylated ones.                               as final.
Conclusion(s): Saffron as an important medicinal herb is a good candidate to be
considered for new drug design.




Cefquinome and Amoxicillin in Go ats: PK/PD Integration                                                    Cell-free o ctamer ic h emoglobin as a blood substitute

BATZIAS GC                                                                                                 BAUDIN-CREUZA V1, VASSEUR C 1, DOMINGUES E1, HO C 2, MARDEN MC 1
                                                                                                           1
Laboratory of Veterinar y Phar mac ology, Faculty of Veterinar y Medicine, Aristotl e                        INSERM U779, U niversity of Paris XI, 78 rue du Général Leclerc, 94275 Le
University of T hess aloni ki, 54 124 Thessaloni ki, Greece                                                Kremlin-Bicêtre, France.
                                                                                                           2
                                                                                                             Department of Biological Sciences , Carnegie Mellon U niversity, Pitts burgh, PA,
Backgro un d: Cefquinome (CF Q) and amo xicillin (AMO) are beta-lactam antibiotics,                        USA.
belonging to the groups of cephalosporin and penicillin, respectively. CFQ and A MO are
intended for the t reatment of respiratory infection diseases, caused by Mannhei mia                       Background: Consi derable progress has been made in the devel opment of red
haemolytica and Pasteurella multocida and for the treatment of mastitis in livestock. T he                 blood cell substitutes, in particular with hemoglobi n (Hb) based oxygen carriers
aim of this study was to obtain and integrate pharmacokinetic (PK) and                                     designed to c orrect oxygen deficienc y. T he two major problems are enc ountered
pharmacodyn amics (PD) data of CF Q and A MO in the goats.
                                                                                                           with acellular Hb in the plas ma: the optimum oxygen affinity for adequate oxygen
Me th ods: T he pha rmacokinetic profile of CF Q an d A MO in goats was investigated
                                                                                                           deliver y to tissues and the dissociati on of Hb tetramers (α2β2) into dimers (αβ).
following intravenous (i. v.) and intram uscular (i.m.) a dministration at the doses of 1 mg/ kg
                                                                                                           Clinical trials with different Hbs obtained either by chemical modification or by
and 15 mg/ kg, respectively. At t he same time, the minimum inhibition (MIC) and minimum
bactericidal (MBC) concentrations of CF Q and A MO against two reference strains of                        protein engineering to prevent tetramer dissociation, have shown a vasoacti vity in
Mannheimia ha e molytica and Pasteurela multocida were dete rmined in cation-adjusted                      plasma. Increasing the molec ular size of the carrier has been pr opos ed to reduc e
Mueller- Hinton Broth (MHB) and goat blood plasma. T he bactericidal activity of CFQ and                   the undesirable vasoac tive properties. In order to eliminate problems related to the
AMO was tested a gainst Mannhei mia hae molytica and Pasteurella multocida using the                       small size of acellular oxygen carriers, we have made the next logical step of
time killing method.                                                                                       going from a tetramer to an octameric form of Hb by introducing the c ys teine
Results: After i.v. administration of CFQ, the pharmacokinetics of CFQ indicated a small                   residues oriented towards the exterior of the β-s ubunits.
volume of distribution (Vss=0.204±0.02 L ), a rapid clearance (2.433±0.59 mL/min) and                      Methods: The mutation βG83C was introduc ed by site-directed mutagenesis into
half-life of 1.36 ± 0.2 h. Aft er i.v. administration of A MO, the short terminal half -life (t1/2z ) of   the c o-expressi on vecteur pHE7 contai ning human , β-globin cDNAs and an E.
2.0 ± 0.47 h was the net result of ratio of the relatively small volume of distribution to the
                                                                                                           coli methioni ne aminopepti dase cDNA. T he rec ombinant Hb βG83C (rHb βG83C)
total clearance. After i.m. dosing absorption of C FQ was complete (F≥100 ) and ra pid and
                                                                                                           have engineered in JM 109 s trains of E.coli and purified by ion exchange
terminal half-life was 1.54±1.4 h. Ho we ver, afte r i.m. administration of A MO, the half -life in
goats (7.89±2.26 h ) was much highe r than aft er i. v. administration of A MO. T he difference            chromatography.
in half-lives between i.v. and i.m. administration of AMO sugge st that the i.m                            Results: The analysis of purified rHb β G83C by exclusion siz e chr omatography
administration of AMO in goats produce a flip-flop phenomeno n.                                            shows the presence of a single mol ecular s pecies corresponding to a molec ular
In broth MI C and MBC of A MO against M. hae molytica were 0.18 8 μg mL-1 and against P.                   weight of 129 kDa ie a dimer of tetr amer. The CO rebinding kinetics after fl ash-
multocida we re 0.250 μg m L-1 . MIC and MB C of A MO against M. hae molytica in goat                      photol ysis of this octameric rHb are similar to that of tetrameric Hb with two
plasma were MIC = MBC=0.1 88 μg mL-1 and against P. multocida in blood plasma of goats                     phas es c orresponding to two allosteric s tates (R and T states). The different
were MIC= MB C=0.375 μg mL-1 . MI C and MBC of CF Q, when dete rmined in MHB, we re                        studies s how that this octameric rHb was stable and did not dissociate easily into
0.047 μg mL-1 , against M. haemolytica and P. multocida. MI C and MBC of CF Q against M.                   small mol ecular species at low concentration. This rHb was resistant toward
haemolytica in goat plasma were 0.094 µg mL -1 . T he respective value against P.
                                                                                                           potential dis ulfide reducing agents pres ent in fres h human plas ma. Moreover
multocida was MI C= 0.047 an d MBC = 0.094 μg mL-1 in goat plasma. MI C and MBC data
                                                                                                           thes e octamers does not i nterac t with haptoglobin, a plas ma glyc oprotein that
against two refe rence strains M. hae molytica and P. multocida indicated that the A MO
generally e xhibited higher MIC an d MBC when compa red with CF Q. CF Q an d A MO we re                    binds dimers to participate in the elimination of Hb from blood circulation.
shown to be time dependent bactericidal antibiotics against target pathogens and the                       Conclusions: This mol ecule is thus potentially useful as blood substitutes. The
killing occurring at a concentration close to the MIC. T he rate of killing was not                        objecti ves are now to evaluate the effects of this rHb on the biolog y of the
significantly influenced by the increase of antibiotic concentration.                                      endothelial cell.
Concl usio ns: T aking into consideration the PK and PD parameter of A MO in goats, the
concentration of the drug in the plasma remain above the MIC (=0.375 μg/mL) of M.
haemolytica and P. multocida in the goats for 12 hours afte r i.m. administration, and it can
be concluded that a once-daily amo xicillin i.m. administration at the dose of 15 mg/ kg b. w,
yields therapeutically effective d rug le vels. Furthe rmore, according to PK a nalysis and PD
data of CFQ, it can be concluded that, for susceptible bacteria , a twice-daily cefquinome
i.m. administration, at the dose of 1 mg/kg b.w., will yields therapeutically effective drug
levels.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                          Page A-29
                                                                 EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                         Celebrating the 100th Anni versar y of the
                                                                            Nobel Prize Award to Paul Ehrlich
                                                                              Nürnberg, October 3-5, 2008
Inhaled H 2S for susp ended animation in anesthetized and ventilated mice                      Light (LM) and electron microscop e (EM) examination of the effects of
                                                                                               methotrexate (MTX) on the endosalp inx.
BAUMGART K 1, WAGNER F1, SIMKOVA S1, WEBER S1, C ALZIA E1,
                      1                   1                    2               2
RADERMACHER P , SENFTLEBEN U , HUBER-LANG M , KNÖFER L M ,                                     BAYRAM M 1, OZOGUL C 2, DURSUN A3, ERCAN ZS4 , ISIK I3 , DILEKOZ E4
GEORGIEFF M 1
                                                                                                                                       1
                                                                                               Gazi Uni versity, F aculty of Medicine, Department of Gynecolog y and Obs tetrics,
1
  Klinik für Anästhesiol ogie und 2Unfallchirurgie, Uni versitäts klinikum, Ulm,               2
                                                                                                 Department of H ystol ogy and Embr yol ogy, 3Department of Pathol ogy,
                                                                                               4
Germany                                                                                          Department of Pharmacol ogy

Background: Several organisms c an reversibly slow down their vital functions in            Background: To examine the effec ts of increasi ng dos es of mtx on the fallopian
order to sus tain otherwise lethal environmental stress, a phenomenon called                tubes.
hibernation. In mice, inhaling hydr ogen sulfide (H 2S) induced suc h a hibernation–        Methods: The study was carried out on 24 female rats (Albino Wistar type, 250-
like s tate c alled „s uspended animation― with decreased heart rate, res pirator y         300 g). Different dos es of Mtx were given to the randoml y di vided 4 groups by IP
                                                                              1
minute volume and metabolic expenditur e, and consec uti vel y hypothermia . H2 S           injection: 1mg/kg to group 1, 5mg/kg to group 2, 10 mg/kg to group 3 and
also is an endogenous gas eous mess enger and signaling molec ule, and both pro-            physiological serum to group 4 as the control group. After ten days, the fallopian
and antiinfl ammator y effects were reported. Si nce in rodents anesthesi a per se          tubes of the rats were removed for examination separately by LM and EM for
causes hypothermi a, we tested the hypothesis whether inhaled H 2S may also                 comparison.
induc e suspended ani mati on in anesthetized, mec hanicall y ventilated and                Results: LM showed that in group I the s urfac e epithelial cells were normal and
instrumented mice.                                                                          the l amina propria was infiltrated by numerous inflammator y cells with a
Methods: 15 hours after laparatomy animals received 100 ppm H 2S or vehicle for             prevalence of pol ymorphonuclear leuc oc ytes. Findings in groups 2 and 3 were
5 hours with core body temperatur e maintained at 38° or 27°C. Left ventricular             similar: the lamina propria was infiltrated with granuloc ytes i n one specimen from
pressure- volume loops were assess ed using a pressure-c onductanc e catheter.              each of the two groups, and granuloc ytes were also obser ved among epithelial
Cytoki ne (TNF-α, IL-6) and c hemokine (MCP-1, MIP-2) formation,                            cells. In the control group all surface str uctures wer e found to be in a nor mal
myeloperoxi das e and NF-B activation wer e measured in lung homogenates,                  conditi on. EM showed cilial loss in the epithelial cells and centr al crystol ysis in
mitoc hondrial respiration was meas ured in liver biopsies (high-resolution                 mitoc hondria in all group 1 specimens. Findings in groups 2 and 3 were si milar.
respirometry).                                                                              The c ytopl asm of the epithelial c ells seemed to be dense, there was promi nent
Results: In contrast to awake mice, H 2S al one did not affec t hemodynamics.               crystol ysis in the mitochondria, and vac uolisati on in the c ytoplas m seemed to be
Within 3 hours, however, both hypothermi a alone and combi ned with inhal ed H 2 S          augmented. Cilial loss was prominent, and the basal membrane was irregular.
decreas ed blood pressure, heart rate and thus car diac output, while stroke                Epithelial cell nuclei were in disorder. Li pid granules were obser ved extensi vel y in
vol ume, ejection fracti on and end-diastolic press ure were unchanged.         While       epithelial c ells. Eosinophils s eemed to be d ominant in connecti ve tissues below
inhaled H 2S and hypothermi a alone c omparably attenuated lung chemokine tissue            the epithelium. In all c ontr ol group specimens the epithelium seemed to be nor mal
levels, onl y combining hypothermia and H 2S significantly decreased tissue IL-6            with all organelles in pl ace; the condition of intercellular juncti ons, ciliated
levels. Strikingl y, H 2S significantl y increas ed NF-B acti vati on during               epithelium and all mitoc hondria also seemed to be normal, and the basal
normothermia. Combini ng hypothermia and inhaled H 2S attenuated the                        membrane was obser ved to be in order.
cytochrome-c-induced sti mulation of mitoc hondrial res piration.                           Conclusion: The ultras tructural der angements res ulting from administration of
Conclusions: 1) In contrast to awake mice anesthesia blunted the hemodynamic                Mtx in dos es in excess of 1mg/kg can caus e a reduc tion in the surfac e
effec ts of inhaled H 2 S alone. 2) H 2S has anti-inflammatory properties beyond            epithelium's ability to make rhythmic las hing movements and can i mpair the
hypother mia alone. 3) Reduc ed c ytoc hrome-c-induc ed stimulation of mitoc hondrial       patenc y of the fallopi an tubes. All thes e disturbances coul d be involved to s ome
respiration suggests attenuated mitochondrial damage.                                       degree in the c aus ation of infertility and rec urrent ectopic pr egnanc y. Therefor e,
                                                                                            the dos age of Mtx should be li mited to use of the lowest effecti ve dos e to avoid
References: 1. Bl ac kstone et al, Science 2005;308:518                                     thes e adverse effects.
Ackno wldeg ement: Supported by the Deutsche Sepsis-Ges ellschaft




Gastrointestin al Prokin etic 5-HT4 Agonists; Receptor Selectivit y and B enefit-           Gut Health-Promoting Adhesion Of Enteropathogens To Dietar y
to-Risk Profile                                                                             Polysaccharid es

BEATTIE DT, SMITH, JAM                                                                      BECKER PM 1, GALLETTI S1,2
                                                                                            1                                                                 2
Theravance, Inc ., South San Francisco, CA, USA                                                 Wageni ngen UR, Lel ystad, T he N etherlands; Uni versity of Milan, Milan, Ital y

Background: Selec tive 5-HT4 receptor agonists (e.g. TD-5108 and prucalopride)              Background: Bacterial adherence to host tissues is regarded as an important
appear to produc e more robust gastrointestinal (GI) prokinetic ac tivity i n patients      initial step for col onisation and infection. Hence, agents that interfere with the
with chronic constipation than non-s electi ve agonists (e.g. tegaserod or cisapride).      ability of pathogens to adher e to gut cells are promising antidotes. Different plant
Interac tion with non-5-HT4 receptors may also res ult in adverse effec ts; cisapride       produc ts and food stabilizers were tested in terms of their binding capacity for
and tegaserod are associated with cardi ac arrhythmias (via hERG potassium                  enteropathogenic bacteria using a mi niaturised adhesion test.
channel bloc kade) and isc hemic events, r espec tivel y. In this study, the                Method: Bacterial strains were allowed to adhere to fibrous s ubstanc es (Table 1)
phar mac ological profiles of s everal 5-HT4 receptor agonists are compared.                supplied as well coatings in micropl ates (Bec ker et al., 2007). Adhering bacteria
Methods: Recombi nant rec eptor binding affi nities were measured by radioligand            were provided with liquid medium and allowed to grow during incubation in a
techniques. Smooth muscle mec hanical acti vity was studied using guinea pig                microplate reader at 37°C. Bacterial growth was recorded as optical density (OD)
colon mounted in tissue baths. Colonic transit was measured in consci ous guinea            at 650 nm over time. The more cells were retai ned, the s horter the detecti on time
pigs (excretion time of dye injected into the colon) as was GI c ontrac tility in           of their subsequent growth. D etecti on ti mes of growth were deter mined at an OD
conscious dogs (implanted with s train gauges).                                             of 0.05 (tOD=0.05 ) in triplicate in two independent assays eac h. Means were
Results: TD-5108, TD-8954, tegas erod and cisapride had high affinity for the               compared by Fis her's unprotected least significant difference test.
human 5-HT4(c) receptor (mean pKi = 7.7, 9.4, 8.4 and 7.1, res pecti vel y). TD-5108        Results: Tabl e 1 s hows the perfor mance of various substances in terms of their
and TD-8954 had high s electi vity for the 5-HT4(c) receptor over all other 5-HT            binding capacity for different E. c oli and Sal monella enterica.
receptor types (>500- and >2,000-fold, res pec tivel y). Tegas erod and cisapride had       Table 1.      Detection ti mes of growth [h] of different E. c oli and Sal monella
affinity for other 5-HT receptors (mean pK i = 7.5, 8.4, 7.0, 7.2 and 7.2 for                             enterica. Substanc es with the shortest detection ti me bound most
tegaserod at human 5-HT2A, 5-HT2B, 5-HT2C , 5-HT7 and bovi ne 5-HT1D receptors,
                                                                                                          bacterial cells (Bec ker and Galletti, 2008).
and 6.2 and 7.4 for cisapride at 5-HT2A and 5-HT2B r eceptors, res pecti vely). TD-
5108, TD-8954, tegaserod and cisapride contracted the guinea pig col on ( pEC 50 =
                                                                                                                                     E. coli K88



                                                                                                                                                    K99




                                                                                                                                                                             S. enterica sv.


                                                                                                                                                                                               S. enterica sv.


                                                                                                                                                                                                                 S. enterica sv.
                                                                                                                                                                  ATCC



                                                                                                                                                                                       RIVM


                                                                                                                                                                                                        ATCC


                                                                                                                                                                                                                          ATCC




7.9, 8.6, 7.7 and 7.0, respecti vely); mean intrinsic acti vities (relative to 5-HT) were
                                                                                                                                     CIDC 1000




81%, 50% , 47% and 75%, respecti vel y). TD-5108, TD-8954, tegas erod and
                                                                                                                                                    CIDC 10
                                                                                                                                                    E. coli



                                                                                                                                                                  E. coli




cisapride (0.03-3 mg/kg sc), increased guinea pig colonic tr ansit; TD-5108 and TD-
                                                                                                                                                                  25922




                                                                                                                                                                                               13311


                                                                                                                                                                                                                 13076
                                                                                                                                                                             Thyp.


                                                                                                                                                                                               Thyp.
                                                                                                                                                                             1287




                                                                                                                                                                                                                 Ent.




8954 had the highes t potenc y. In dogs, po dosing with TD-5108 and TD-8954                              Substances
increased antral, duodenal and jej unal contractility more potentl y than tegaserod.                     Artichoke pomace            9.51
                                                                                                                                              c
                                                                                                                                                   7.49
                                                                                                                                                          a
                                                                                                                                                              2.86
                                                                                                                                                                         b
                                                                                                                                                                             3.68
                                                                                                                                                                                      b
                                                                                                                                                                                               3.97
                                                                                                                                                                                                        b
                                                                                                                                                                                                                  4.64
                                                                                                                                                                                                                           b-d

Conclusions: Ther e remains an unmet medic al need for agents to treat patients                          BSA (reference)            10.38
                                                                                                                                         d,e
                                                                                                                                                   8.70
                                                                                                                                                       c
                                                                                                                                                              5.14
                                                                                                                                                                  g
                                                                                                                                                                             3.95
                                                                                                                                                                                 d
                                                                                                                                                                                               5.33
                                                                                                                                                                                                   f
                                                                                                                                                                                                                  4.58
                                                                                                                                                                                                                      b

with GI disor ders suc h as chr onic constipation and constipation-predomi nant                          Carrot pomace              10.43
                                                                                                                                         d,e
                                                                                                                                                   9.13
                                                                                                                                                       d
                                                                                                                                                              3.23
                                                                                                                                                                  d
                                                                                                                                                                             4.13
                                                                                                                                                                                 e
                                                                                                                                                                                               4.48
                                                                                                                                                                                                   e
                                                                                                                                                                                                                  4.82
                                                                                                                                                                                                                      c-e
                                                                                                                                         e             d          c              a                 a                  a
irritable bowel s yndrome. F uture clinical evaluation with s electi ve 5-HT4 receptor                   Konjac gum                 10.81
                                                                                                                                         c,d
                                                                                                                                                   9.19
                                                                                                                                                       c
                                                                                                                                                              2.90
                                                                                                                                                                  f
                                                                                                                                                                             3.41
                                                                                                                                                                                 f
                                                                                                                                                                                               3.56
                                                                                                                                                                                                   f
                                                                                                                                                                                                                  3.81
                                                                                                                                                                                                                      e-f
                                                                                                         Locust bean gum             9.93          8.63       3.51           4.92              5.40               5.00
agonists such as TD-5108 and TD-8954 should deter mine whether they are                                                                  b             b          e              a                 b                  b,c
                                                                                                         Palm kernel meal            8.72          8.31       3.38           3.34              3.79               4.63
associated with a more acceptable benefit-to-risk pr ofile than ol der, less selec tive                  Pumpkin pulp and peel       7.48
                                                                                                                                         a
                                                                                                                                                   8.45
                                                                                                                                                       c
                                                                                                                                                              2.82
                                                                                                                                                                  a,b
                                                                                                                                                                             3.63
                                                                                                                                                                                 b
                                                                                                                                                                                               4.24
                                                                                                                                                                                                   c
                                                                                                                                                                                                                  5.13
                                                                                                                                                                                                                      f-g

agents.                                                                                                  Sesame seed meal            7.74
                                                                                                                                         a
                                                                                                                                                   8.19
                                                                                                                                                       b
                                                                                                                                                              2.91
                                                                                                                                                                  c
                                                                                                                                                                             3.40
                                                                                                                                                                                 a
                                                                                                                                                                                               3.80
                                                                                                                                                                                                   b
                                                                                                                                                                                                                  4.57
                                                                                                                                                                                                                      b
                                                                                                                                         a             c          a              b                 c                  d-e
                                                                                                         Tempeh (ferm. soya bean)    7.20          8.56       2.76           3.57              4.18               4.88
                                                                                                                                         a             b          a,b            b                 c                  g
                                                                                                         Tomato fruit                7.79          8.25       2.78           3.68              4.24               5.28

                                                                                            Conclusion: With growth as measuring variable for adhesion, a simpl e high-
                                                                                            throughput method was developed and applied for the screening of l arge numbers
                                                                                            of different food and feed components and bacteria.
                                                                                            References: Bec ker PM et al. (2007) J . Appl. Microbi ol. 103, 2686-2696.
                                                                                            Bec ker PM, Galletti S (2008) J. Sci. Food Agricult. 88, 2026-2035.
                                                                                            Authors‘ ackno wledgements: This study was funded by the Dutc h Product
                                                                                            Board of Ani mal Feed (PDV) and by SAFEWASTES (EU projec t no. 513949) in
                                                                                            equal shares.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                                             Page A-30
                                                         EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                              Celebrating the 100th Anni versar y of the
                                                                Nobel Prize Award to Paul Ehrlich
                                                                   Nürnberg, October 3-5, 2008
Effect of Inoculum Size on the Antibiotic Susceptibilities of                       Synthesis and QSAR Studies of CAD A Analog s with CD4 Do wn-modulating
Enterobacter iaceae Producing Shv and Ctx-M Ext ended-Spectrum Beta-                and anti-HIV Activities
Lactamases
                                                                                    BELL TW 1, SCHOLS D 2, VERMEIRE K2
BEDENIC B 1,2 , VRANES J1,3 , PLECKO V1,2 , MATULIC-BEDENIC I4, KALENIC
 1,2                                                                                1                                  2
S                                                                                     Univ. of N evada, R eno, NV, USA; Katholieke Uni v. Leuven, Belgium.
1
 School of Medicine, Uni versity of Z agreb, Zagreb; 2 Department of Clinical and            Background: HIV attac hment vi a the CD4             Me                               Me
Molecular Microbiolog y, Clinical H ospital C enter Zagreb 3Zagreb Institute of Public       receptor is an important target for                               O
               4                                                                                                                                                         O
Health, Zagreb; Trg Bana Jelaţiša 3 (retired)                                                devel oping novel appr oaches to HIV                              S         S
                                                                                             chemotherapy.          C yclotriaz adisulfonami de             O    N    N    O
Background and aim: Many extended-spectrum β-lactamas es (ESBL) producing                    (CADA, shown at right) inhibits HIV at
isolates of E. coli and K. pneumoniae are s usceptible in vitro to amoxycillin-              submicromolar levels by specific ally down-                            N
clavulanate (AMC), ceftazidime-clavul anate (CAZ/cl), and pi peracillin-taz obac tam         modulati ng cell-surface and intracellular
(TZP) but MICs incr ease substantiall y when higher inoculum is applied. The ai m of         CD4. Ai ms: 1) To s ynthesize many CADA
this study was to determine the effec t of inocul um size on the s usceptibility of E.       anal ogs having various CD4 down-
coli and K. pneumoniae isolates with well characterized ESBLs to amoxycillin                 modulati ng acti vities. 2) T o devel op
(AMX), AMC, ceftazidime (CAZ), CAZ/cl, piperacillin (PIP), TZP, imipenem (IMI)               computational models that c an be us ed to predict acti vities of novel analogs. 3) T o
and meropenem (MEM).                                                                         prepar e fluorescent analogs for studies in c ells.
Material and m ethods: Minimum inhibitory c onc entrations (MIC) were deter mined            Methods: The fi ve-step s ynthesis of CAD A was modified to produce more than 50
by broth microdiluti on method using inocula that differ ed 100 fold in density              anal ogs having various groups on the 3 ni trogen atoms, i ncludi ng some bearing
according to CLSI. The inoc ula contained 105 CFU/ml                and 107CFU/ml            the dans yl fluorophore. Testing 30 of these analogs in MT-4 cells showed a wide
approxi matel y. The study was perfor med on the set of K. pneumoniae and E. coli            range of CD4 down-modulating potencies.                Thr ee-di mensional quantitative
strains produci ng SHV-2, SHV-5, SHV-12, CTX-M-3 and CTX-M-15 β-lactamas es.                 structure-acti vity relationshi p (3D-QSAR) models were c onstr ucted using the
Results: Inoculum effec t for CAZ/cl was detected in 52% of SHV-2 producing K.               comparati ve mol ecular field anal ysis (CoMFA) and comparati ve molec ular
pneumoni ae strains followed by AMC (43%) and TZP (38%). SH V-5 producing K.                 similarity i ndices anal ysis (CoMSIA) approac hes . To generate these c omputer
pneumoni ae str ains showed the most pronounced inoc ulum effect with CAZ/cl                 models, the s olid-state structure of CADA was energy minimiz ed and us ed to
(57%) and AMC (55%) and to lesser extent with TZP (44%). Inoc ulum effect was                generate the str uctures of the other 29 analogs.
obser ved for AMC, CAZ/cl and TZP in 71% of SHV-12 producers. E. coli                        Results: A CoMFA model was generated having strong statistical significanc e ( r 2
produci ng SHV-5 β-lactamas e showed the most pronounc ed inoc ulum effect with              = 0.80, P < 0.001) and internal predicti ve ability (q2 = 0.41). Potencies of all 30
AMC (61%) followed by CAZ/cl (51%) and TZP (22%). Strains producing CTX-M                    compounds ar e predicted well by this model (within 10-fold of the experimental
β-lactamas es had a mar ked inoculum effec t with CAZ/cl (71%), AMC (57%) and                val ues). The steric and elec trosataic contributions to the model are 59% and 41%,
TZP (50%). AMC and CAZ /cl were associ ated with inoc ulum effec t against all type          respecti vel y, indicating that the sizes of groups attached to the nitrogens are more
of ESBL pr oduc ers: SHV-2, SHV-5, SHV-12 and CTX-M. TZP was less affected                   important than their polarities. The CoMSIA model also showed the strongest
by the inoc ulum size then AMC, and CAZ/cl particular y with CTX-M produc ers.               correlation between potenc y and steric bul k. One of the CADA analogs bearing a
The acti vity of TZP was mostl y c ompromised in the pres enc e of high density of           dans yl fl uorophore is as potent as CADA and has proven to be useful for
SHV-5 produci ng K. pneumoni ae.                                                             monitoring uptake and distributi on of the drug in T-cells.
Conclusions: Carbapenems were the most stable compounds to inoc ulum effect                  Conclusions: 1) Effecti ve approac hes have been developed for the s ynthesis of
regardless of the type of ESBL.                                                              many CADA analogs with a wi de range of potencies. 2) Computer models have
                                                                                             been developed for predicting potencies of novel anal ogs. 3) Fluoresc ent, potent
                                                                                             CADA anal ogs have been developed that may be useful for eluci dating the
                                                                                             mec hanis m by which CADA compounds down-modul ate CD4.




Pharmacokin etics and Pharm acod yn amics of Amphotericin B and its Lipid                    Mitochondrial Respirator y Ch ain (MRC) and Mitochondrial Permeabilit y
Formulations                                                                                 Transition (MPT) Effectors again st Heav y Met al-induced C ytotoxicit y: St ate-
                                                                                             of-the-art and Perspective
BELLMANN R
                                                                                             BELYAEVA EA
Department of Internal Medicine I, Innsbruc k Medic al Uni versity, Inns bruc k,
Austria                                                                                      Sechenov Institute of Evolutionar y Physiol ogy and Biochemistry, St.-Petersburg,
                                                                                             Russia
Background: Bec ause of its broad s pectrum, the pol yene amphotericin B (AMB)
has still an important role in treatment of invasive fungal infec tions . It is eliminated   Background: Heavy metals, suc h as cadmi um, mercur y, etc. are strong
unchanged via the bile and the urine. However, its us e is limited by infusion               carcinogenic, genotoxic and c ytotoxic agents of high environmental and
related advers e events and by its nephro-toxicity. Thr ee less toxic lipid                  occupational haz ard. Mitochondria are important targets for these pollutants. MRC
formul ations ar e available, displaying different c ompositi on of their lipid moieties     disturbance and acti vation of different ionic channels i n inner mitoc hondrial
and thus different particle siz e and s hape. Lipos omal AMB (LAMB) comprises                membrane, mainl y nonsel ecti ve MPT pore, are considered to be the key events in
small uni-laminar vesicles. Its volume of distributions is small, its half-life is short     different types of cell death. Ai ms: 1) To find effecti ve protectors agains t the heavy
and high plasma c onc entrations are achi eved. AMB lipid c omplex (ABLC) for ms             metal-induc ed c ytotoxity.
large ribbon-like structures resulti ng in a large volume of distribution a half-life of     Methods: To further underscore mechanis m(s) and role of mitoc hondrial
about 1 week wher eas onl y low plas ma level are reac hed. The 3rd lipid                    dys function in toxic action of heavy metals we us ed rat neuronal cell line PC12, r at
formul ation, AMB colloi dal dispersion (ABCD) displays i nter mediate pl asma levels,       ascites hepatoma AS-30D cells and is olated rat li ver mitochondria as a model
half-life and volume of distribution.                                                        system, as well as flow c ytometr y, different fluoresc ent probes and i on-selec tive
Methods: AMB pharmac okinetics was anal yzed in 18 critically ill patients on                electrodes, and s welling tec hnique.
treatment with lipid-formulated AMB (13 requiring hemofiltration). Li pid-associ ated        Results: We found that agai nst C d2+-induc ed injur y not onl y well- known
and liber ated AMB were separated by solid phas e extrac tion and quantified by              antioxi dants, s uch as N-acetylc ysteine, vitamin E, butylhydroxytoluene, and MPT
high performanc e liquid chromatog raphy. AMB tissue distribution was assess ed in           inhibitors – c ycl osporine A, bongkrekic acid, ADP, Mg 2+ and dithiothr eitol are
autops y sampl e obtained fr om 32 patients who had died during therapy with lipid           effec tive but also inhi bitors of different MRC components, namel y of c omplex I
formul ated AMB.                                                                             (rotenone), and of complex III (stigmatellin and antimyci n A). Interestingl y,
Results: In critically pati ents, mean peak plas ma levels (Cmax) of liberated AMB           stigmatellin was s hown to be one of the s trongest protec tors that exhibited its
amounted to 0.5 µg/ml after infusion of LAMB or ABCD, whereas C max of total                 action not onl y on isolated mitochondria but on both types of the cells.
AMB was 3.4 µg/ml and 0.8 µg after ABCD. The clearanc e (CL) of liberated AMB                Conclusions: 1) Our data pointi ng to the direct invol vement of the MRC, and
was 0.2 l/kg/h (CL of LAMB [lipid-formul ated] = 0.08 l/kg/h, CL of ABCD = 3.0               especi ally reac tive oxygen s pecies (ROS) production on res pirator y complex III in
                                                                                                 2+
l/kg/h). AMB-pharmac okinetics on and off hemofiltration were si milar.                      Cd -induc ed mitochondrial membr ane permeabilization and cell death. 2) On the
Tissue concentrati ons of 100 µg/g were reached in the liver, 70µg/g in the spleen.          basis of own findings and data in literature, we proposed a hypothetical model of
Lung conc entr ations were significantly higher after ABCD than after LAMB                   MPT pore structure and/or r egulation with the invol vement of a mitoc hondrial
treatment (33 vs.12 µg/g). Levels in myocardi um and brain were 3 µg/g and 1                 supercomplex for med by res pirator y compl ex I (P-site) and III (S-site) whic h may
                                                                                                                             2+
µg/g, respectively.                                                                          constitute not onl y critical Me -bi nding sites but also main loci for ROS generation
Conclusions: 1) T he lipi d-associated moieti es of AMB lipid formul ations dis play         that was instrumental in oxi dati on of thiol groups crucial for MPT pore induction;
considerable differences in their pharmacokinetics whereas pharmac okinetics of              moreover, depending on conditions and cell type, either one or both res pirator y
the liberated AMB fracti on is i ndependent from the preparation administered. 2) No         complexes c oul d participate in triggering of the MPT pore ass embl y and c ell death.
dose adjustment is required during hemofiltration. 3) AMB acc umulates in liver and          3) The data obtai ned are important for searchi ng new drugs which could bec ome
spleen, tissue concentrations in l ung and kidneys are intermediate and l ow in              real ―magic bullets‖ in the nearest future.
myoc ardium and brain.                                                                       Authors‘ disclo sure statem ent: The author thanks the Russi an F oundation for
                                                                                             Basic Research for financial s upport (grant N o. 07-04-00722).




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                               Page A-31
                                                               EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                    Celebrating the 100th Anni versar y of the
                                                                      Nobel Prize Award to Paul Ehrlich
                                                                         Nürnberg, October 3-5, 2008
Susceptibilities of rickettsiae to antimicrobials.                                        Immunogenicit y of Biopharmaceuticals - An Inconvenient Truth.

BENABDELLAH A, BENSSADOUN F-Z, BENSSAD M, KOUIDAD-BELKADI S-A                               BENDTZEN K

Servic e des maladies i nfecti eus es et tropic ales, CHU.Oran, Or an 31000, Algeria        Institute for Inflammation Res earch, Rigshospital et, and BioM onitor Ltd .,
                                                                                            Copenhagen, D enmar k.
Abstract:
Rickettsi ae grow onl y intracell ularly. T he antibiotic susceptibility is ass essed by    Background: Today, rec ombinant gene technolog y per mits the us e of drugs
plaque,dye uptake or IF assays . Rickettsiae are susc eptible to doxyc ycline,              which are al mos t identic al with natural human proteins, i ncludi ng antibodies (Abs).
thiamphenic ol and fluroquinolones. Betalactams, ami noglyc osides and                      Many assume that these drugs pose little or no risk of triggering specific immune
cotrimoxazole are not acti ve. T yphus group ric kettsi ae ar e susceptible to all          respons es, bec aus e patients accordi ng to dogma are tolerant towards their own
macrolides,whereas the s potted fever are mor e resistant to rifampicin than the            proteins . Unfortunately, this is not the case, and even so-called 100% human
other rickettsi ae. Ric kettsiae felis is not sus ceptible to gentamicin, erythromycin e,   biologicals may be immunogenic. I s hall foc us on the immunogenicity of anti -TNF
amoxicilline or c otrimoxazole. we present an overview of s usceptibility of                Ab constructs plus the l argely ignored problem of indi vidual variations i n drug
rickettsi ae to anti microbials.                                                            bioavailability (BA) and pharmacokinetics (PK).
                                                                                            Methods: Several methods have been used to ass ess circulati ng levels of anti-
                                                                                            TNF biologicals as well as Abs to thes e drugs. Most are based on solid-phas e
                                                                                            technolog y, e.g. ELISA, with their inherent problems of low sensiti vity, false
                                                                                            positi vity and susc eptibility to nons pecific i nterferenc e, fx by rheumatoid factors.
                                                                                            We have devel oped fluid-phase radi oimmunoassays (RIAs) for monitoring patients
                                                                                            on anti-TNF biologicals, one for functional blood levels of the drugs, and one for
                                                                                            anti-drug Abs.
                                                                                            Results: We measured BA/PK and anti-drug Abs developed in 'anti-TNF-
                                                                                            immunized' patients with rheumatoid arthritis; most of thes e were treated with
                                                                                            infliximab. The mos t sensiti ve anti-drug Ab ass ay invol ved bi nding to s olubl e and
                                                                                            intact inflixi mab r ather than to plastic-immobilized drug. Indeed, data obtained by
                                                                                            solid-phas e ass ay using cross-binding of plas tic-fixed and sol uble inflixi mab were
                                                                                            inconsistent with results obtai ned with flui d-phase RIA. Despite intravenous
                                                                                            administration, there were sizable interindi vidual variations in serum trough levels
                                                                                            of the drugs even at time-poi nts where anti-drug Abs had not yet developed; thes e
                                                                                            levels di minished or dis appeare d in parallel with Ab induction (30% and 44% of
                                                                                            patients on infli ximab were Ab- positi ve at 3 and 6 months, respecti vel y). Abs were
                                                                                            'neutralizing' in that their levels wer e positi vel y associated with i nhibition of TNF
                                                                                            binding to the drugs. There were highl y significant correlati ons between high l evels
                                                                                            of anti-drug Abs and l ater dose increas es, si de-effects and c essation of therapy.
                                                                                            Conclusions: To prevent prolonged use of inadequate anti-TNF biotherapi es,
                                                                                            indivi dualized assess ments of BA/PK and Abs s eem ess ential (personalized
                                                                                            medicine). In our hands, fl uid-phase assays are s uperior to s olid-phase assays.




The Path from Colles‘ L aw to the ―Mag ic Bullet‖                                           W ater Channel Proteins (Aquaporin s): From their Discover y in 1985 in Cluj -
                                                                                            Napoca, Rom ania (B y the use of a Doping Nmr Method and Specific
BENEDEK TG                                                                                  Labeling) to the use of their Inhib itors as Magic Bullet s

University of Pittsburgh, Pitts burgh, Penns yl vania                                       BENGA G

                The hypothesis that s yphilis might be c ounterac ted immunologicall y      Dept. of Cell and Molec ular Biol ogy, " Iuliu Hatieganu" Uni versity of Medicine and
can be trac ed to the Irish surgeon, Abraham Colles, who noted in 1837 that a               Phar mac y, Cluj-Napoc a, Romania
congenitally s yphilitic infant may be born of a mother who showed no signs of
syphilis. He interpreted this to indicate that the i nfant, i nfected by a diseased         Water channels or water channel proteins (WCPs) are transmembrane proteins
fertilizing sperm, i mmunizes its mother against s yphilis, even though it can infect       that have a s pecific three- dimensional str ucture with a pore that c an be per meated
other caretakers. This tal k will sketc h the disappointing investigations that led from    by water molecul es. T he first WCP was discovered in the human red bl ood cell
―Colles‘ law‖ to the rec epti on of Ehrlich‘s ―magic bullet.‖                               membrane in Cluj-Napoca, Romania, in 1985 by Benga‘s group. We have
                The first line of serologic investigation, initiated by Joseph Auziaz-      measured the water permeability of human red blood cells (RBCs) by a doping
Turenne in the 1840s was c alled ―s yphilization.‖ In this c ontroversial therapy the       NMR method. We s howed for the first time by NMR that the parameters
patient was ―saturated‖ over several months with hundreds of subcutaneous                   characterizing diffusional water permeability are the same i n RBCs and res ealed
injections of ―syphilitic toxi n,‖ based on the hypothesis that when s aturation was        ghosts and reported the largest series of deter minati ons of water diffusional
reached new manifestations of the disease were prevented, after which c ure might           permeability of RBCs available in literature.
be achieved. Saturation had been ac hieved when i njections no l onger elicited a           The first water channel protein (WCP), later called aquaporin 1 (AQP1) was
local inflammatory reaction. Syphilis and c hancroid had not been differentiated at         discovered in the RBC membrane by my group in 1985 in Cluj-Napoc a, Romani a,
this time. Thus the misinterpretation of ―saturation‖ of s yphilitic patients was           reported in publications in 1986 (Benga et al., Bioc hemistry, 25, 1535-1538, 1986;
frequentl y compounded by the i njection of c hancroidal rather than s yphilitic serum.     Benga et al., Eur. J. Cell Biol., 41, 252-262, 1986) and revi ewed in the following
Most responsi ble for perpetuating this tec hnique until about 1870 was the                 years. This discover y was achi eved by s 203pecific labelling of the RBC membranes
Norwegian venereol ogist, Caes ar Boec k.                                                   with the known water transport inhibitor        Hg - p-chloromercuribenzenes ulfonate
                With the advent of bacteriology it bec ame of interest why c ertai n        (PCMBS). In parallel, the water permeability was measured by the doping NMR
species were resistant to a pathogen that was lethal to another. Coul d this                technique and the inhibition induc ed by PCMBS was calcul ated. The priority of
―resistance factor‖ be transferred, either as prophylaxis or ther apy? The first            Benga in the discover y of the first WCP was ac knowledged by many outstanding
injections of serum from ani mals that could not be made s yphilitic wer e undertaken       scientists.
in 1890 on patients with sec ondar y s yphilis and wer e recognized to be ineffecti ve.     We also have a world priority in the discovery of the implications of water channel
Further trials were sti mulated by favorable reports by an Italian venereologist who        proteins in epileps y (Benga and Morariu, N ature , 265, 636-638, 1977) and
in 1892 gave subc utaneous inj ections of lambs‘ blood. Various inves tigators used         Duchenne muscular dys trophy (Serbu et al., Muscle & N erve, 9, 243-247, 1986).
serum from dogs, rabbits, sheep, and horses. When the use of ser um from                    These fi ndings were interpreted as an expression of generalized membrane
untreated ani mals bec ame rec ognized as usel ess, donor animals were pre-treated          defects affecti ng water permeability in epileps y and Duc henne musc ular
with s yphilitic ―toxi n‖ or mercur y. The greatest sti mul us to experimentation with      dys trophy. In rec ent years this idea was confirmed.
serum therapy c ame in 1893 with the announcement of succ essful serum                      Since the discover y of WCPs tremendous progress in understanding their role in
treatment of diphtheria and tetanus . The lac k of a proven pathogen of s yphilis           physiolog y and patholog y. Based on these advanc es it bec ame clear that inhbitors
added c onfusion to the s earch for an anti-s yphilitic serum.                              of AQPs c an be us ed as magic bullets in a variety of diseases, incl uding canc er.
                The immedi ate acc eptanc e of the discovery in 1905 of the                 The dopi ng NMR method should be us ed to c ompare the effec ts of inhibitors of
Spirochaeta pallida as the pathogen, rapidl y followed by discover y of a pr actical        WCPs as magic bullets . Examples of this approach will be given.
diagnostic method, may explain why, in the midst of the belief that a curative
serum for all microbial dis eas es was about to be discovered, Ehrlich‘s
announcement in 1910 of the therapeutic efficac y of medicinal ―Salvarsan‖ was
received so enthusiastic ally. Ehrlich was more circums pec t regarding the
usefulness of Sal vars an than many of its early advoca tes.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                              Page A-32
                                                          EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                               Celebrating the 100th Anni versar y of the
                                                                  Nobel Prize Award to Paul Ehrlich
                                                                     Nürnberg, October 3-5, 2008
A Study of Capecitabine and Cisplatin in the Treatment of Recurrent Carcinoma        Girolline, a new antiplasmodial leader extr acted from the sponge
of the Uterine Cervix                                                                Cymbastela canthar ella

BENJAPIBAL M, THIRAPAKAWONG C, LEE LAPHAT ANADIT C,                                     BENOIT-VICAL F1,2 , SALERY M 2, NJOMNANG SOH P1,2, AHOND
THERASAKVICHAYA S, INTHASORN P                                                          A3, POUPAT C 3
Mahidol Uni versity, Bang kok, T hailand.                                               1
                                                                                          - Laboratoire de Chimi e de Coordination du CNRS, UPR8241, 31077 T oulouse 4,
                                                                                        France.
Background: Plati num is the mainstay of treatment in advanc ed or recurre nt           2
                                                                                          - Servic e de Parasitologie-M ycologie, C entre Hospitalier Uni versitaire de
cervical carcinoma, however, the duration of response is short lived as well as the
                                                                                        Rangueil, 31059 Toul ouse 9, France.
median s urvi val. Fluorouracil (5-FU) has been shown to be acti ve i n cer vical       3
                                                                                          - Institut de Chimi e des Subs tanc es Naturelles du CNRS, 91198 Gif-sur-Yvette
carcinoma. Capecitabine, an or al fluoropyri midine carbamate, is sequentiall y
                                                                                        cedex, Franc e
converted to 5-FU by thymidine phos phoryl ase (TP) which is found at higher
concentrati ons in cer vical carcinoma than normal tiss ue. In addition, cisplatin
                                                                                        Background: Mal aria is the most prevalent parasitic disease i n the world today. In
further upregulates TP. Capecitabine plus cisplatin has the potential to be an
                                                                                        this context, there is clear demand to search for new anti mal arial agents and
active treatment, which is more conveni ent than 5-FU-cisplatin.
                                                                                        research into new antimalarial drug candi dates originating from natural sources
Methods: This study combi nes capecitabine and platinum in patients with
                                                                                        has been acti vel y pursued. Girolline (Figure1), a 2- aminoimi dazol e deri vative
recurrent cer vical carcinoma with no potenti ally cur ati ve standard treatments.      extracted from Cy m     bastel a cantharella (a New-Caledoni an Sponge) already
Sixteen patients (14 squamous c ell carcinoma, and 2 adenocarcinoma) with a
                                                                                        known for its antitumor acti vity, was tested against Plas modi um.
median disease-free interval of 11 months (range, 2-96) rec ei ved cisplati n 50        Methods: We evaluated the effects of girolline and some of its anal ogues in vitro
mg/m2 i ntravenously on day 1 and oral c apecitabine 1000 mg/m2 twice daily for         and in viv o against P. falciparum and P. vinck ei petteri, res pecti vel y. We then
two weeks with a one week res t period.                                                 evaluated its toxicity in vitr o and in vivo. We have als o determined the point of
Results: Medi an age was 50 years (range, 31-74). A total of 89 cycl es were
                                                                                        action of girolline in the er ythroc ytic life c ycle of the mal arial par asite and its
administered with a mean of 5.5 c ycles (range, 3-6) per patient. F our of the
                                                                                        synergistic action with c hloroquine.
sixteen patients had complete response (25%), 4 had partial respons e (25%), and
                                                                                        Results: We have demonstr ated that girolline presents a ver y promising acti vity
5 had diseas e stabilization (31%). Ten patients (63%) had rec urrent diseas e          against malaria both in vitro against four P. falciparum strai ns and in vivo on
outside the radiation field. The overall response rate in pati ents with recurrent
                                                                                        murine mdel. Girolline also s howed a specific mode of ac tion by inhibiting
diseas e within the pr evious irradiated field was 33% and 60% in patients with         Plas modium protei n s ynthesis. Moreover, between girolline and chloroqui ne, a
tumor outside the irradiated fiel d. The medi an follow-up ti me was 29 months
                                                                                        high synergistic effect was reported.
(range, 11-39). The median time to progression was 9 months (range, 5-37), with
                                                                                        Conclusions: Girolline is of a real i nteres t as r esearch basis for a new class of
a median overall survi val of 23 months (range, 5-37). The majority of adverse
                                                                                        anti malarials. With suc h a biological profile, girolline could be consider ed as a
events were mild and there were no grade 4 adverse events . Hematological
                                                                                        model chemical structure for new c andidates in the arsenal of new drugs and in
toxicity was the most frequent advers e event with grade 3 neutr openia in 19% of
                                                                                        particular of dr ugs able to fight malaria.
patients. Grade 2 and 3 hand-foot syndrome occ urred in 38% and 6% of patients,
respecti vel y. Ther e were no chemotherapy-related deaths.
Conclusions: This acti ve yet c onvenient c ombination of capecitabine and                                                        OH                                    OH                                   OH
cisplatin s hows a high res ponse rate, l ong time to progression and sur vi val with                                                                              5                                 5
acceptabl e toxicities for patients with rec urrent c arcinoma of uterine c ervi x.                                      N                NH2                N                    NH2           N
                                                                                                                              NH    Cl                             S     Cl                          S          Cl
                                                                                                                       H 2N        2HCl                     H2N     erythro                   H 2N           threo

                                                                                                                             1 girolline
                                                                                                                  Figure 1: Structur e of Girolline                 2        5-deazathiogirollines       3

                                                                                                                                  OBn                                                                        OBn
                                                                                                                                                                        OBn

                                                                                                                         O                                                                      S
                                                                                                                              O                              O                O                      N         OH
                                                                                                                                                                   O
                                                                                                                                                                                           BocHN
Natural Products (NP) - Microbial Metabolites (MM) - Antibiotics ( AB):                 Possible Consequences of Tr ansplacental Tran sfer of Viruses in H ealth y
                                                                                                                        4                                  5                                             6
Histor y, Facts and Problems, W here Now ?                                              Pregnants (Rewiev)

BÉRDY J                                                                                 BERENCSI GY, ÖRDÖG K, CSIRE M, KAPUSINSZKY B, YOUNES SA OBn
                                                                                                                       OBn                                                                                   OH
                                                                                                                                                                                                     4
Budapest, H ungar y, e- mail: jberdy@t-online.hu                                        Division of Virol ogy, National Center for Epidemiol ogy, Gyáli Str. 2-6, H-1097                        S
                                                                                                                          S              N3                   N                   N3                 N          Cl
                                                                                        Budapest, H ungar y                    N    OH                             S    Cl
Histor y: Great benefit and unpredictable negati ve consequenc es. The NP
                                                                                                                      BocHN                                 BocHN                        H 2N      2HCl
research and es peciall y the discover y of new MM declined in the pas t years. Few     Background: Virus es, which may c aus e illness es of the fetus were s hown to be
new drugs were discovered. T he reas ons are: resistance-problem, l ess succ ess        transfer ed frequentl y thr ough the plac enta into fetal tissues without any clinical
with HTS and c ombinatorial chemistr y, but the reasons are mai nly economic and                                                 7
                                                                                        consequenc es. Rubella and rubella vaccine, measles and measles vaccine,     8                           9 4-de
regulator y. In the last years the total s ynthetic efforts and the discover y of       Togavirus es, Flavi viruses, hepaci virus (HCV), Hepadnavirus es (HBV), , human                 NH2
compounds from higher plants and marine organis ms increasing, but s ometimes           cytomegalovirus (HCMV), human her pes virus types 1 (HSV), 6 (HHV6), 7 (HHV7),
no evident proof to the real produc er species (endophytes , symbi onts - taxol,        and 8 (HHV8), human par vovirus B19 (HPV-B19), dependovirus (AAV), human                           N
                                                                                                                                                          NH2                       HN
patellamide).                                                                           adenovirus, Eps tein-Barr virus (EBV), human papillomaviruses (HPaV), human
Facts: Close to half a million NP, including ~100 000 MM, ~20 000 microbi al AB                                                                    HN
                                                                                        pol yomavirus es (HPyV), lentivirus es and the Anellovirus TTV were detec ted in the
                                                                                                                                             Br
                                                                                                                                                          N                                 O
(~350 marketed), there are known. High perc entage, (~40 %) of all drugs and            umbilical blood, amniotic fluid or fetal tissues at the end of h ealthy pregnanci es.
about 70- 80 % of all known AB drugs wer e derived from NP, as direct drugs,            Mechanisms: IgG trans port mec hanis ms, transported maternal cells and the lipid      Br
derivati ves (semis ynthetic and modified products), and other NP mimics                rafts were s hown to be vehicles of this virus
                                                                                                                                        Brtrans port.
                                                                                                                                               N                                  N      NH
(synthesized as NP analogue). The NP libr aries have some advantages over               Results:                                               H                                  H
random s ynthetic or combinatorial chemic al libraries in sever al respects (e.g.                                                                     O
                                                                                        1.) The first consequence of the contact of the developpi ng fetal organism with              O
complex structures hardl y accessible by c hemical methods). "Nature is the best        latent viruses c an be the life-long carriage of thes e viruses upon birth.
                                                                                                                                      10                                      11   hymenialdisine
                                                                                                                                               dibromocantharelline
combinati orial chemist". T hey also meet the green chemistr y. T he bioproducts        2.) The developpi ng fetal immune s ystem might create immunotolerance to certain
have i nher ent - but perhaps undisc overed - biological functions and drug-like        viral antigens dependi ng on the fact, whether the virus es replicate or are onl y
structures, c ompatible to the host. The real func tion of MM is the communication                                                                      Figure 1 may impair compounds
                                                                                        latently pr esent in the fetal cells. This partial immune toleranc e: structures of the
with other microbes , higher organisms and the environment. They ar e the interfac e    post partum immune respons e facilitati ng tumour formation in the affected
between microbes and the rest of world. The bios ynthetic pathway of microbes,          indivi duals.
evol ved in the millions of years under evolutionar y press ure, ares mainl y           3.) Herpes viruses may acti vate endogeneous retroviral genes i n fetal cells,
undisc overed (silent genes). Onl y ~1% of the existi ng bac teria are c ultivabl e.    modifying their differentiation and s urface properties. Alternativel y the
Unlimited number of possibl e new struc tures exists in c oded for m in the             pathogenesis of autoi mmune dis eas es might be initiated by thes e modifications.
metagenom.                                                                              4.) The viruses are coding for micro RNA molec ules possi bl y infl uencing the
Where No w: Pres ently everything seems to be out of balanc e. Bac k to the pre-        replication and differentiation of the virus carrier cells. The genes of DNA virus es
antibi otic era ? What c an we do ?                                                     interfering with apoptotic mechanis ms may als o disturb the normal differentiation
1. Diversificati on of NP libraries. New ways to discover new leads to medicinal        processes in the organs of the fetus.
chemistry (ins piration for s ynthetic chemists). 2. N ew bioc hemical/genetic          5.) Finally the respons e to the mandatory anti viral vaccinations might be i mpaired
methods: mini ng and engineering of the bios ynthesis (genom). 3. Discover new          by the transpl acentall y transc ytos ed virus es.
sources (marine species , endophytes , unc ultivabl e, rare microbes). 4. New target-   Conclusion: The s ystematic testing of umbilical blood and urine of newborn
oriented effecti ve screenings. 5.Chemic al and bios ynthetic post-evolution by         babi es woul d be of ess ential importance using molec ular technics for the presence
combinatorial methods. 6. Better understanding and sol ving the probl ems of the        of the virus es in order to be prepared for yet unknown ris ks for the future life span.
drug-target-host interacti ons. T he key of our co-evol ution with microbes is the
exact understandi ng of the life-cycl e of microbes .




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                         Page A-33
                                                      EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                            Celebrating the 100th Anni versar y of the
                                                              Nobel Prize Award to Paul Ehrlich
                                                                 Nürnberg, October 3-5, 2008
Lacosamide is a Novel Antinociceptive and Antiepileptic Drug with a Du al         PK/PD Relationship of Antibiotics in Local Treatment of Prostethic Infection s
Mode of Action
                                                                                  BERTAZZONI MINELLI E and BENINI A
BERKELS R 1, BEYREUTHER B2 ,KREBSF AENGER N 2, FREITAG J2 , LEES G3,
              3         2
ERRINGTON A , STÖHR T                                                             University of Verona, Verona, Italy
1
 UCB GmbH, Monhei m, Germany; 2 SCHWARZ Bi oSciences GmbH, Monhei m,                         Background. Infection is the most serious complication following orthopaedic
Germany; 3U niversity of Otago, Dunedi n, New Z ealand; 4UCB, Inc., Smyrna, GA,              surgery, and delivery of antibiotics to the s urgical area is a way of reducing the
US                                                                                           infection frequenc y.
                                                                                             Pol ymethyl methacr ylate (PMMA) cements impregnated with antibiotics are
Background: Lacos ami de is an investigational drug that has demons trated                   currentl y utilised as l ocal antibiotic carrier in orthopaedic surgical-site infecti on to
positi ve res ults in Phas e III trials of neuropathic pain and epileps y. Preclinical       treat prosthetic infections (hip, knee, etc) and are an adjunct to current therapy
studies have shown neuroprotecti ve effec ts of lac osamide both in ani mal models           (surgical debridment and s ys temic antibiotic therapy).
and i n-vitro. Elec trophysiology and pr oteomics experiments have identified two            Cement intrinsic characteristics and capacity to release drug are essential for the
likel y modes of action for l acosamide.                                                     final clinical outcome as well as antimicrobial drug pharmacodynamics at the site
                                                                                             of infection.
Methods and Results: Electrophysiol ogy experiments performed in mous e                      Methods. Several experimental models in vitro and in vivo have been developed
neuroblastoma cells indicate that lacos ami de reduc es sodi um-channel availability         to better understand the rel eas e kinetic of different antibiotic from cement and to
by s electi vely enhanci ng slow-inac tivation. Enhancing slow-inacti vation is thought      opti mise their us e in clinic al practic e.
to raise c hannel-acti vati on thresholds, reducing pathophysiological neuronal              Results. Aminoglycosides (A) and vancomycin (V) show good positive characteristics:
hyperexcitability. This mec hanism is different from that of anes thetic and other           bactericidal activity, adequate release, compatibility, mechanical resistance, and
antiepileptic agents , which non-selecti vely bloc k the sodium channel pore and/or          excellent tolerability.
enhanc e fast- and sl ow-channel inacti vation.                                              The release of A and V from PMMA c ement s eems prompt and effecti ve,
                                                                                             determining high local c onc entrations. The drug el ution shows a bimodal profile,
A second mechanis m of acti on may occ ur via the bi nding of lac osamide to                 consisting of an initial high rapi d release of drug followed by a much sl ower but
collapsin-respons e medi ator protei n 2 (CRMP-2), a phosphopr otein that is                 sustained releas e. Initial drug concentration, cement s urface area and porosity are
involved in neuronal differentiation and axonal out-growth (proc esses that are              important factors in determini ng the amount of drug release.
maladapti ve in the pathophysiolog y of pain and epileps y). The interac tion of             A and V in combination show s ynergistic antimicrobial acti vity both i n vitro and in
lacosamide with CRMP-2 may underlie the apparent neuroprotecti ve effects of                 vivo against multiresistant clinical is olates .
lacosamide, since CRMP-2 appears to be important for medi ating neuroprotection              A and V concentrations in drainage fluid following spac er implant are higher than
from excitotoxic ins ult and apoptosis.                                                      thos e obtained with s ystemic admi nistration; their kinetics is superi mpos abl e.
                                                                                             These high local conc entr ations of the combi nati on are effecti ve agai nst
Conclusions: The dual mode of acti on of l acosamide repres ents two novel                   multiresistant pathogens responsi ble for prosthetic infections (high inhibitor y
mec hanis ms for the treatment of neuropathic pain and epileps y. Bas ed on current          bactericidal titre of drainage fluids).
studies , it is propos ed that selec tive enhanc ement of sl ow-inacti vati on of sodium     Moreover, the pres enc e of A and V in PMMA speci mens reduc es the growth and
channels may underlie the immediate effects of lacos ami de.                      Further    bacterial adhesi on of s usceptible and i nter mediate-resistant Staphylococci. Their
characterization of the interaction with CRMP-2 may hel p to explai n its role in            anti-adhesi ve effect depends on the characteristics of the microorganism and its
lacosamide‘s s ymptomatic and diseas e-modifying effec ts.                                   capacity of adhering to antibiotic-loaded s urfac es.
                                                                                             Conclusions. PMMA antibiotic l oaded cements improve local drug deli ver y at
                                                                                             infection site and enhance pharmac od ynamics of antibiotics for treatment of
                                                                                             prosthetic orthopaedic infections.




Development of Unique Cisplatin An alogs for Site-Specific Treatm ent of                     Mediterranean spotted fever(MSF) in Or an (Algeria)
Hormone-Dependent Female Can cer s
                                                                                             BESTAOUI L, BENABDELLAH A, BENSADOUN F-Z, KOUIDED-BELKADI S-A,
BÉRUBÉ G                                                                                     BENSAAD M

Univ. du Québec à Trois-Rivières, Trois-Rivi ères, Canada.                                   Servic e des maladies i nfecti eus es et tropic ales, CHU.Oran, OR AN 31000, Algeria

Background: Chemotherapy remains, to this day, an effec tive treatment for                   Abstract
several types of cancer. H owever, the s evere si de effects c aus ed by the treatment       MSF due to ric kettsia c onori was thought ,for many years,to be the onl y tic k-borne
limits its full potential for a cure. Thus, the development of site-specific anticancer      rickettsi al disease pr evalent in Algeria. H owever ,in rec ent years,other species
therapy is a subject of intenc e researc h. Several strategies can be used to target         within the spotted fever group of the genus Ric kettsia have been described as
cancer c ells. For ins tanc e, the us e of a carrier molecul e being able to rec ongnise a   emrging pathogens . Tick- borne agents include:Ric kettsia aesc hlimanii and
specific receptor in the cell is a tactic of c hoice used by sever al research groups.       rickettsi a massiliae. Many rickettsia of unknown pathogenicity have als o been
We have developed several es tradiol-platinum(II) (E2-Pt(II)) hybrid molec ules              detected from tic ks and could repres ent potential emerging pathogens to be
using the followi ng guiding principles a) potential for affinity towards the estrogen       discovered in the future. F urthermor e,a new spotted fever rickettsia,Ric kettsia felis
receptor b) potential for in vitro and in viv o selec tivity on hormone-dependent            ,was found to be associated with c at flees and is an emerging human pathogen.
female cancers c) eas e of s ynthesis and, d) potential for large scale industrial           Rickettsi a felis is s usceptible to doxyc yclin,thiamphenicol,and fl uoroquinolons but
produc tion.                                                                                 not to gentamicin,erythromycin ,amoxicillin or trimethoprim-sulfamethoxaz ole. The
Methods: The development of the E2-Pt(II) hybrid molec ules will be initiall y               resistance of this new s peci es to erythromycin is consistent with taxonomic
discussed. Then, the most promising hybrid deri vati ve, VP-128, is s elected to             position withi n the spotted fever group. We pr esent an overvi ew of thes e
examine its bi ological acti vity towar ds breast (MCF-7, ZR-75- 1, MDA-MB-468,              rickettsi odes ,foc using on emerging diseases.
MDA-MB-231 and HS578-T) and ovarian (OVCAR-3, SKOV-3, A2780 and A2780-
cp) cancer cells, in vitro (MTT assays) and in vivo ( xenografts model) using ER
positi ve or negati ve c ells.
Results: MTT assays revealed that VP-128 decreased the viability of breas t and
ovarian c anc er cells more efficientl y than cisplatin itself in vitro. Moreover, i n the
case of breast cancer the expr ession of ER sensitized the c ells to the growth-
suppressi ve effec t of VP-128. H oescht nuclear stai ning revealed an improved
efficienc y of VP-128 compared to cisplatin to i nduce apoptosis of breast cancer
cells, which was enhanc ed in ER-positi ve cells. In cisplatin resistant A2780-cp
cells, VP-128 was able to induce cell death i ndicating that the new drug might also
be efficient to kill cisplatin resistant cancers. Finally, using human breas t and
ovarian c anc er cell xenografts in nude mice, we found that VP-128 had s tronger
antitumour acti vity compared to cisplatin in vivo, and was mor e specific and
selecti ve towards hormone-dependent cancer c ells.
Conclusions: Experimental data show that VP-128 possess es enhanced
anticancer acti vity c ompared to cispl atin and is able to specifically target hor mone-
dependent tumours in an in vivo model. T hus ulti mately, VP-128 could provide
new and/or alternative treatment modalities for breast and ovarian c anc ers.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                Page A-34
                                                                EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                       Celebrating the 100th Anni versar y of the
                                                                          Nobel Prize Award to Paul Ehrlich
                                                                             Nürnberg, October 3-5, 2008
New Appro aches for the U se of Old Drugs – Preclinical and Clinical                         Novel Diaster eoselective Synthetic Routes to Tamo xifen, Tor emifen e, and
Pharmacological Evaluation of the Pyrimid ine Anti-neoplastics: 5-                           Droloxifene, Anti-Breast Cancer Agents via Org anoboranes
fluorouracil, z ebular ine, 5-fluoro-2‘-deoxycytidin e, gemcitabin e, and THU.
                                                                                             BHAT NG, CAVAZOS E, ORTIZ O, ELIZONDO D
BEUMER JH 1 , EISEMAN JL1 , NEWMAN EM 2, DOROSHOW JH 3, SYNOLD T W2,
         1                   1               3             1
ZEH HJ , BARTLETT DL , KUMMAR S , EGORIN MJ                                                  Department of Chemistr y,T he Uni versity of T exas-Pan American, 1201 West
                                                                                             University Dri ve, Edi nburg, Texas 78539-2999 USA;
1                                          2
  Univ. of Pittsburgh, Pitts burgh, USA; City of Hope N ational Medical Center,
Duarte, U SA; 3 N ational Cancer Institute, Bethes da, USA.                                  Background: Tamoxifen., tor emifene, and drroloxifene ar e us eful drugs us ed in
                                                                                             the treatment of breast c anc er. Several synthetic methods are known in literature
Background: Pyrimi dines include some of the oldes t anti-neoplastic agents . They           to s ynthesize them. Some of the methods give a mi xture of di astereomers.
are still an i mportant part of many anticancer dr ug regimens, and continue to              Organoborane reagents are known to ac hieve high s electi vity in organic s ynthesis.
generate interest, as evidenced by recent studi es of zebularine and 5-fluoro-2‘-            Consequentl y, highl y dias tereoselecti ve synthesis of tamoxifen, toremifene, and
deoxyc ytidine (FdC), two DNA hypomethylating agents . Our aim is to develop                 droloxifene is achieved usi ng organoborane reagents . Our current methods are
innovati ve pyrimidine anticancer drugs and improve the us e of existi ng ones.              compared with the literature methods to prepare these drug s.
Methods: We developed chromatographic ass ays for z ebularine and 3                          Methods: This study incl uded the reacti ons of al kynylborates with an el ectrophile
metabolites (HPLC-radiodetection), FdC and 4 metabolites, gemcitabine (dFdC)                 such as trimethylchl orostannane followed by stepwise Stille c oupling and Suzuki
and metabolite, 5-fluorouracil (5-FU), and tetrahydrouridine (THU) (all LC-MS/MS),           coupling. Sinc e the chemicals used are highly air-sensiti ve, all of the reactions are
and applied these to preclinical and clinical phar mac ological studies.                     carried out under nitrogen atmos phere. In the c ase of tamoxifen s ynthesis,
Results: Preclinical studies revealed that, i n vivo, [14C]-zebularine is quickl y           phenylethynyllithium is reacted with triethyl borane followed by Stille coupling with
converted to uridine, producing hydrogen peroxi de as a by-product. In mice, THU             (N-{2-(4-bromophenoxy)ethyl}N,N-dimethyl amine and Suzuki c oupling with
decreas ed metabolic degradation of FdC (>50-fold) and dFdC (5-fold) and                     bromobenzene in the pres ence of tetrakis(triphenylphosphine)palladi um. In the
substantiall y increas ed their oral bioavailability (25-fol d and 4-fold, respecti vel y).  case of toremifene, 4-chl oro-1-butynyllithium is reacted with triphenylborane
Clinical pharmacokinetics of intravenous F dC and THU were determi ned, and the              followed by reacti on with trimethylchlorostannane. T he resul ting intermediate is
oral route is being expl ored. Preclinical studies show that oral dFdC and THU is            then subjected to palladium catalyz ed Stille coupling with bromobenzene and
efficacious in a CFPAC-1 xenograft pancreatic tumor model (Treated/C ontr ol =               palladi um catal yzed Suz uki coupling with (N-{2-(4-bromophenoxy)ethyl}N,N-
30%). T he 5-FU LC-MS/MS ass ay was shown to c orrelate well with a ne wl y                  dimethylamine In a similar manner, droloxifene is synthesiz ed by the reac tion of
devel oped 5-FU immunoassay, which will allow on- demand therapeutic drug                    4-[2-(dimethylamino)ethoxy}-phenylethynyllithi um with triphenylborane             and
monitoring.                                                                                  chlorotrimethylstannane. T he r esulting intermediate is s ubjec ted to palladium
Conclusions: Pyrimidi nes are an important cl ass of anticanc er drugs, both in                                                                                -
                                                                                             catal yzed Stille and Suz uki coupling reac tions with m bromophenol and
established r egimens , and as agents i n future treatments. Their metabolism is             ethylbromide res pecti vely.
complex, but can be modulated to allow oral dosing, and different dosing                     Results: Tamoxifen is obtained in 55% isolated yi eld. Toremifene and droloxifene
schedules. New applications and targets of pyrimidine drugs are being discovered             are obtained in 48% and 50% isolated yi elds respecti vel y. These compounds are
and should maintai n interest i n this cl ass of compounds.                                  purified by column chr omatography.. The structures of thes e drugs are confirmed
                                                                                             by nuclear magnetic resonance (NMR) spectrosc opic methods.
                                                                                             Conclusions: In summation, highl y diastr eosel ecti ve s ynthesis of tamoxifen,
                                                                                             toremifene, and droloxifene is achieved using organoborane reagents, Stille, and
                                                                                             Suzuki coupling reactions. The optimiz ation of reacti on yields of thes e drugs is
                                                                                             currentl y in progress in our laborator y.




Mechanisms that Explain the Lo wer Incidence of Breast Can cer in                            Nanodevices for targeted d eliver y: An evaluation of toxicological models
Postmenopausal W omen Treated with Conjugated Estrogens (CEE). Role of
Estrogen Receptor  and  .                                                                  BHOGAL N
                1,2
BHAVNANI BR                                                                                  FRAME, Russell & Burch Hous e, 96-98 North Sherwood Street, Notti ngham, UK
1,2
  Univ. of Toronto and T he LKSKI of St. Michael Hospital,                                   Background: Engineered nanoparticles and liposomal deli ver y s ys tems
Toronto, Ontario, Canada.                                                                    (nanodevic es) are i ncreasingl y utilised and inves tigated as a means of overcoming
                                                                                             problems with the bioavailability, stability and toxicity of therapeutics. Suc h
                                                                                             systems pr omise to revoluti onise clinical management and to obviate the need for
Background: Recent findings fr om the Women‘s Health Es trogen Alone Trial                   needle-based self-admi nistration.
(JAMA 2008), showed that l ong ter m tr eatment of hysterectomized                           Methods: Some of the major challenges are described and the pros pects for
postmenopausal women with CEE not onl y did not increase the incidenc e of                   simplifying and stratifying toxicol ogical assess ment of nanodevices is consi dered.
breast c anc er, but more i mportantly, may have reduc ed the risk in thes e women           The importance of sel ecting the most appropriate toxicological models for
for this disease. These results raise s ome i mportant questions as to the                   biocompatibility testi ng is highlighted by the surge of new nanodevice-medicine
mec hanis ms invol ved and whether all types and of estrogen can impart this                 combinati ons c urrentl y i n the pipeline.
protecti on. In the pr esent study, the relati ve bi nding affiniti es (RBAs) of 11 equine   Results: Nanomaterials dis play unique reac tiviti es that are dependent on
estrogens and their functional acti vities mediated via ER and ER were                     physicoc hemical properties , surface c hemistr y and biophysical and biological
compared.                                                                                    interactions. Thes e reacti vities are often ver y differ ent from thos e of bul k materials
Methods: The RBAs were estimated by competiti ve bi nding assays using 3H-17                and can be unpredictabl e owing to material heterogeneity. T he estimation of the
estradiol and unlabelled equine estrogens. The functional acti vity of the estrogens         rate of drug releas e from nanodeliver y devices, issues pertaining to bi oretention
was meas ured i n HepG2 c ells trans fected with human ER or ER or both and                are problematic.
secreted-al kaline phos phalase (SEAP) gene and analyz ed by a chemiluminescent              Differences in anatomical featur es between test species and human, incl uding
assay.                                                                                       accessibility via natural barriers, targeting dependent on physical characteristics of
Results: In comparison to 17-estradiol (17-E2 ), the RBA‘s of mos t ring B                 tissue vasc ulature, immune c ell density and lymphatic sys tem characteristics
unsaturated estrogens were 2-8 fold lower for ER and ER, however, thes e                   present interes ting challenges. For instance, it is clear that transdermal delivery is
unique estrogens had 2-4 times greater affinity for ER than for ER. The                    likel y to depend on follicular trans port. Yet animal models, with their vastl y different
                                                                                             follicle diameters are unli kel y to gi ve an indic ation of absorption thr ough human
transcriptional acti vity of thes e 11 es trogens showed that all estrogens were
                                                                                             skin. Si milarly, it is clear that human s kin is much mor e dens ely inner vated and
functi onall y acti ve. 17-estradiol induced the acti vity of SEAP by ER to a higher
                                                                                             supplied with immune cells that most s peci es. T hus, the relevanc e of ani mal
level than any other estrogen. Acti vity of other estr ogens was 12 to 17% that of
                                                                                             models to ass ess the bioc ompati bility and as well bioavailability via this route of
17-E2 . In c ontrast, 17-E2 stimulated the acti vity of ER to a 5 fold lower level
                                                                                             deliver y is highly questionable. Whether human s ki n equi valents or cadaveric
than with ER. The acti vities of other estrogens medi ated vi a ER were 66-290%            human s kin can succ essfully replac e ani mal s tudies is dubious, particul ar given
that of 17-E2, with equilenin being the most acti ve. Exc ept for 17-E2, no                that most organotypic models lac k follicles, paracrine function and full barrier
correlation was obs er ved between functional ac tiviti es and the RBA‘s for ERs.            functi on.
The acti vity of the ring B unsaturated estrogen c omponents of CEE appear to be             Conclusions: The applicati on of existing animal and in silico approaches to
exerted predominantly thr ough ER. To our knowl edge, these are first suc h                 resolvi ng species differ ences and informing bioc ompati bility tes ting strategies of
obser vations . Moreover, depending on the estr ogen, ER can ac t as a domi nant            nanoparticles is questionabl e, calling for a review of methods for nanomaterial
repressor or dominant acti vator of ER‘s transcriptional acti vity.                         testing.
Conclusions: Taken together, thes e data indic ate that all estrogens are not the
same and have different pharmacolog y. Some ring B uns aturated estrogen
                                                                                             Authors‘ disclosur e statement: FRAME is a scientific charity that conducts
components of CEE c an via ER  inhibit the prolifer ati ve effec ts of 17-estradiol
                                                                                             research into scientificall y s ound alternati ves to animal testing that afford the
mediated vi a ER, thereby reduce the risk of breast c anc er in women who are j ust
                                                                                             highest standards of human health pr otec tion.
taking equine es trogens alone.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                  Page A-35
                                                      EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                              Celebrating the 100th Anni versar y of the
                                                                 Nobel Prize Award to Paul Ehrlich
                                                                    Nürnberg, October 3-5, 2008
New derivatives of BM 212 with improved antim ycob acter ial activit y.             The Insulin-Stem Cell Connection: W hat Insulin Does To St em Cells And
                                                                                    How W e Get It From Them
BIAVA M 1*, PORRETTA GC 1 , POCE G1, POMPEI R 2, DE LOGU A2, MANETTI F3,
         3
BOTTA M                                                                             BIEBERICH E1
1                                         2                                                 1
  Università "La Sapienza", R ome, Ital y. U niversità degli Studi di Cagliari, Cagliari,       Medical College of Georgia, GA, U.S.A.
Ital y; 3Uni versità degli Studi di Si ena, Siena, Italy.
                                                                                            Background: Replac ement of pancreatic β-cells by stem c ell-derived ins ulin
Background: According to the report 2007, compiled by the World Health                      produci ng cells (IPCs) is a promising strategy to treat diabetes . Therefor e,
Organization (WHO), the total number of new c ases of T uberc ulosis (TB)                   generating IPCs has been a major goal of ac ademic and indus trial efforts to
worldwide in 2005 had risen to approximately 8.8 million and there were                     harness the differentiation potential of embryonic or adult stem ( ES or AS) cells.
approxi matel y 1.6 million T B deaths during the s ame year. Increas ed i nfec tion with   To date, most differentiation protocols yield onl y a small proportion (<5%) of cells
the M. avium compl ex (MAC) is also contributing to the morbi dity and mortality in         that genuinel y produc e sufficient amount of insulin in response to high glucose
AIDS patients. The most urgent goal of chemotherapy of tuberc ulosis infections             levels. A major obstacle in identifying true IPCs is that serum-free culture
shoul d be the development of highly acti ve and low-cost drugs, which s hould be           conditi ons require suppl ements with insulin to ens ure survi val of the differentiating
used not only in industrialized countries but als o in devel oping ones in whic h both      stem c ells. This exogenous ins ulin is taken up by the c ells and confounds the
thes e infections are now rapidl y increasi ng. As acti ve molec ules already               determination of cell-deri ved insulin using antibody-based techniques.
introduc ed in therapy very soon generate resistance, scientists have foc used their        Methods: Our laborator y has developed, for the first time, a di fferentiati on protocol
attention on the devel opment of new antimyc obacterial compounds acting with a             replacing insulin with a recombi nant peptide analog of i nsulin-like growth fac tor-1
mode of action without cross-resistanc e.                                                   (IGF-1). This IGF-1 anal og-supplemented, s erum-free medium (NeoIT S medium)
Methods: Many pyrrole deri vatives, analogues of BM 212, have been s ynthesized             allows for the i dentification of genuine IPCs.
on the basis of previ ous results and molec ular modelling considerations bas ed on         Results:       Using      chemicall y defined      NeoIT S     medium      instead      of
the pharmac ophore model previousl y i dentified for them. All the derivati ves were        insulin/transferrin/s elenite (ITS) or serum supplement, we derived nestin(+)/C -
tested for their c ytotoxicity and in vitro acti vity against many strai ns of M.           peptide(+) and nestin(-)/C-peptide(+) cells at proportions of 40% or 25%,
tuberculosis, atypical mycobacteria, drug-resistant myc obacteria of clinical origin        respecti vel y. T hes e IPCs increased insulin s ecretion by 20-fold when expos ed to
and intr acellular myc obacteria. Protecti on Index (PI) was calculated and for the         20 mM glucose. The determination of secr eted insulin was not possible when ES
most acti ve of them the bioavailability was also evaluated. The in vi vo tests and         cells wer e differentiated i n the presence of c onventional ITS or s erum.
the s tudy of the mode of action are c urrentl y under study.                               Conclusions: Ins ulin or IGF-1 are ess enti al cell survi val factors for differentiating
Results: Some of the synthesized c ompounds revealed mor e acti ve than BM 212              stem cells. A recombi nant peptide analog of IGF-1 in NeoITS can efficientl y
against mycobacteria. In particular the PI for many of them was c ompar able to that        replace IGF-1 or ins ulin and allows for the deter mination of secreted ins ulin. Using
of reference c ompounds, Isoniazid, Streptomycin and Rifampi n. Many of the                 NeoITS will tremendousl y facilitate the identification of earl y IPCs when
synthesized compounds revealed also to be ac tive against intrac ellular                    differentiated from ES or AS cells (including induced pluripotent stem cells or iPS
mycobacteria and they showed to inhibit drug -resistant mycobacteria of clinical            cells). We will now determi ne the potential of thes e IPCs to normaliz e glucose
origin.                                                                                     levels in hyperglyc emic mice.
Conclusions: On the basis of our previous studi es and mol ecular modelling
considerations, many new derivati ves of BM 212 were i dentified. In particular
some of them revealed ver y ac tive and l ow toxic so that they can be considered
ver y promising for future studies.




Liqiud Chromatograph y and C apillar y Electrophoresis as a Tools to Stud y                 Treatment Oucom e And Manag ement Of Acromegaly Befor e And After The
Ligand-Receptor Inter actions                                                               Introduction Of Somatostatin Analog s
                                                                                            A long term follow-up s tudy
BIELEJEW SKA A
                                                                                            BIERMASZ NR, PEREIRA AM, ROMIJN JA
Institute of Physical Chemistry PAS , Kasprzaka 44/52, Wars aw 01- 224, Poland
                                                                                            Leiden Uni versity Medical C enter, Dept. of Endocrinolog y and Metabolis m, Leiden,
The phenomenon responsibl e for enantios eparation in chromatographic and                   The Netherlands.
electrophoretic methods is the same; it is the enantios electi ve interacti on between
the enantiomers and a chiral selector. The principal differ ence between thes e two         Background: Acromegal y is a rare dis eas e resulting from GH overproduction.
techniques arises from different separati on process mechanisms and s ometimes              The available treatments for acromegal y are surgery, radiotherapy and medical
different environments of c omplexation                                                     treatment. Somatostatin analogs (SMS) were the first effecti ve drugs available
The remar kable c apacity of c yclodextrin (CD) for enantios eparation has been us ed       sinds 1980s, first as three dail y injec tion and from 1998 as monthl y
to advantage in many chromatographic and electrophoretic applicati ons                      depotpr eparation. This drug changed the treatment strategy of acr omegaly, and is
The chr omatographic and electrophoretic methods which are ver y sensitive to               now us ed as the first opti on after unsucc essful s urgery, but also as primar y in stad
structure, size, s hape and dynamics of the anal ytes have been us ed not only in           of surger y in s ome patients.
separation scienc e but also in the study of mol ecular rec ognition proc eses.             Methods: Long-term bi ochemical and clinic al data are available of 164 patients
The enantiomeric separation of basic c hiral pharmaceuticals s uch as pheniramine,          diagnosed with acromegaly in the Leiden Uni versity Medic al Center from 1978
brompheniramine, metoxyphenamine, c yclopentolate, doxylamine, and ketamine                 onwards. We evaluated the long -term outcome of s urgery, irradiation and SMS
was investigated in c apillary electrophoresis (CE) and liquid chr omatography              treatment in this cohort. Bi ochemical remission and clinical outcome, i.e. mortalit y,
(HPLC) using negatively c harged sulfated--cyclodextrin (s--CD) and neutral               co-morbi dity and quality of life par ameters were eval uated. We also considered
cyclodextrins (CDs). The apparent stability constants for the model compounds               the effect of different treatment on detailed hormone secretion profile. We
with c ycl odextrins in both techniques were esti mated.                                    reviewed the effect of the introduction of SMS on the us ed treatment modalities in
Both methods seem to be c omplementary for the s tudy of complexation                       our patients, and in literature.
phenomena. It can be seen that bromphenirami ne for ms stronger complexes with              Results: The surgical remission rate is 66 % in s hort term, but ther e was a
-CD than pheniramine and doxylamine. Complexation of pheniramine and                       recurrence rate of 15 % resulti ng in a final remission rate by surgery onl y of 54%
doxylamine by -CD is ver y similar. T he weakest c omplexes -CD forms with                after a mean of 12 years. Radiotherapy was associ ated with a l ong -term remission
metoxyphenamine. For the studied compounds, TM--CD forms very weak                         rate of 75 %, however, the duration until normalization of GH excess was long and
complexes. T he stability cons tant for DM--CD is very si milar to that obtai ned for      there was a high incidenc e of hypopituitarism. Pos toperati ve SMS treatment was
-CD. From the nati ve CDs the best c hiral selec tors for the studi ed c ompounds          well-tolerated and resulted in a remission rate of 60 %. An i v dos e of octreotide
are -CD and HP--CD.                                                                       well-predicted the l ong-term outcome of medic al therapy. Detailed growth
                                                                                            hormone secr etion was nor malized following succ essful s urgery, but not after
For the studied c ompounds the bes t rec ognition between enantiomers was
                                                                                            radiotherapy and during SMS. Mortality near-nor malized, but quality of life
obtained for c yclopentolate (K1/K2=1.32, K1 /K2 =1.45 and K1/K2 =1.26 for -CD,for
                                                                                            remained impaired.
HP--CD and TM--CD, respecti vel y)
                                                                                            Conclusions: The introducti on of SMS analogs has changed the treatment
As the CE is the more efficient method, c hiral recognition is better visible in this       algorithm of acr omegaly. It is the preferred tr eatment in cas e of uns uccess ful
method than in HPLC.                                                                        surgery and in this cohort of patients treated by surgery, radiotherapy and SMS
Conclusions: The obt ained res ults shows that chromatographic and                          anal ogs about 95% of pati ents ac hieve remissio n. At pres ent equal bioc hemical
electrophoretic methods may be used as additional tools for studyi ng weak
                                                                                            remission rates are ac hieved with s urgery and medical therapy onl y. F urther study
interactions res ponsible for molec ular recogniti on between ligand and receptor           is required to assess the clinical outc ome of this new treatment regimen.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                               Page A-36
                                                      EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                             Celebrating the 100th Anni versar y of the
                                                                Nobel Prize Award to Paul Ehrlich
                                                                   Nürnberg, October 3-5, 2008
Mannitol, a Key Prob e Molecule in the Assessment of Sm all Intestin al            Safe and Effective Deliver y of Paclitaxel through Amphiphilic Beta-
Permeabilit y?                                                                     Cyclodextrin Nanoparticles

BIJLSMA PB 1 , TAMINIAU J AJM 1, JODAL M 2                                                      BILENSOY E1
1                                                                2                              1
 Academic Medical Center, Amsterdam, The Netherlands; Uni versity of Göteborg,                   Univ. Hacettepe, Fac. of Pharmac y, Dept. Pharmaceutical T echnolog y, Ankar a,
Göteborg, Sweden.                                                                               Turkey

Background: The urinar y rec over y of orally ingested sol utions contai ning Mannitol          Background: Paclitaxel is a potent anticanc er drug ass ociated with severe side
(mol.radi us 4 Å) and Lac tulos e or Cr-EDTA (mol. radii 5 Å) is a wi del y used clinical       effec ts due to the us e of c o-sol vent Cremophor in its commercial injec table
test for s mall intesti nal barrier function. In humans, recovery of Lactul ose is  0.5        formul ation. It is known that nanoparticul ate drug deli ver y s ystems can provide an
%, of Mannitol  20 %, giving a L/M recover y ratio of 0.025, which is very low                 alternative for the effecti ve delivery of anticancer agents due to the EPR effect
compared to theorethicall y expected L/M diffusion ratios of 0.8 through aqueous                facilitating the targeting of the enc apsul ated antic anc er drug to tumor cells instead
pores Aims: T o investigate the underlying mechanis ms of this discrepanc y by                  of healthy tissues. Objecti ve of this study was to develop a nanoparticulate carrier
comparison of i n vivo and in vitr o probe per meability.                                       system for paclitaxel with high encapsulation efficienc y, controlled drug releas e
Methods: 1) Small intestinal sheets from rodents (rats, guinea pigs, rabbits) and               property which may be more advantageous than cremophor vehicle in ter ms of
human intestinal bi opsies were mounted in Ussing chambers and mucos a-to-                      safety and efficac y.
serosa fluxes of L/M were determi ned (n = 4-8). Urinary recovery of orally applied             Methods: Nanoparticles of amphiphiphilic ß-cyclodextrins loaded with paclitaxel
probes was meas ured in rodents , cats and humans (n = 2-5). 2) Absorption of Cr-               were prepared using nanopreci pitati on technique. Particle size distribution and
EDTA, Mannitol and water was studied in in situ perfused jej unal l oops in                     zeta potenti al of the nanoparticles were meas ured by QELS technique.
anaestetized cats (n = 8), using four isotonic perfusion solutions with var ying                Encapsulation efficienc y of amphiphilic c yclodextrin nanoparticles were measured
contents of N aCl and glucos e.                                                                 as well in vitro releas e profiles for paclitaxel with HPLC assay. Safety of the
Results: L/M flux ratios in vitro were about 0.8 in all tested species ( 0.68 to 0.89).         nanoparticles (nanocaps ules and nanos pheres) were ass essed in ter ms of both
Urinary recover y L/M ratios in rodents ranged from 0.60 to 0.28. L/M ratios in cats            hemol ysis and c ytotoxicity to L929 cells in comparison to commercial injectable
and humans were 0.03 and 0.02, due to high mannitol recovery, resp. 29 and 22                   formul ation. Anticanc er effic ac y of paclitaxel loaded nanos pher es and
%. In in situ perfused cat jejunum there was a strong positive correlation between              nanocapsules were evaluated by MTT assay against human breas t cancer c ell line
water absorpti on and mannitol clearanc e (r = 0.99, p < 0.003), no c orrelation                MCF7 with MTT assay.
between water absorpti on and Cr-EDTA clearance (r = 0.05, p = 0.95). Li kewis e,               Results: Size for nanoc apsul es and nanos pheres wer e found to be around 350
there was a str ong negati ve correlati on between water absor ption and Cr -                   nm and 180 nm res pecti vel y and was found to be stable for a storage period of 12-
EDTA/Mannitol rati os (r = 0.98, p < 0.02).                                                     months as seen in Figure 1. Encapsulation efficienc y was increas ed b y 2 to 3 fold
Conclusions: Interspecies variati on in urinar y rec over y of mannitol is caus ed by           by inc orpor ating paclitaxel into c yclodextrin nanoparticles. The drug was released
differences s pecific for the intact s mall intestine in viv o. H yperos molality of villus     within 6 hours. Hemol ytic order was found to be Cremophor
tips in viv o varies, being highest in humans and c ats as a res ult of efficient               vehicle>nanoc aps ules>nanos pheres. Erythroc ytes were i maged by SEM after
vasc ular c ountercurrent multiplication bec aus e of their villus vasc ular anatomical         treatment with paclitaxel loaded formulations. C ytotoxicity of blank nanoparticles
structure. Thus we hypothesize that the high mannitol recover y in both s pecies is             were signficantl y lower than cremophor commercial vehicle as paclitaxel
caused by sol vent drag through pores that allow the pass age of Mannitol but not               demons trated equal anticancer efficac y i n nanoparticles.
of Lactulose. The positi ve correlation between water absorption and Mannitol                                                         0
                                                                                                                                           0   1   2   3   4 µm   µm
                                                                                                                                                                  0.8
                                                                                                                                     0.5
clearanc e in cat jejunum perfused with var ying solutions which differentially affect                                                1
                                                                                                                                                                  0.7

                                                                                                                                                                  0.6
                                                                                                                                     1.5
the c apability of the c ountercurrent multiplier mec hanism c onfirms this hypothesis.                                               2                           0.5
                                                                                                                                     2.5                          0.4
                                                                                                                                      3
                                                                                                                                                                  0.3
                                                                                                                                     3.5
                                                                                                                                                                  0.2
                                                                                                                                      4
                                                                                                                                     4.5                          0.1
                                                                                                                                      5                           0
                                                                                                                                     µm

                                                                                                Figure 1. AFM photomicrograph of paclitaxel loaded amphiphilic cyclodextrin nanoparticles

                                                                                                Conclusions: Amphiphilic c yclodextrin nanoparticles can be c onsidered as an
                                                                                                alternative dos age for m for injectable paclitaxel in ter ms of safety and efficac y.
                                                                                                Authors‘ disclo sure statement: Author would like to ac knowledge H acettepe
                                                                                                University R esearc h Fund Grant 0202301005 for financial s upport of this study.

Two Passeng ers on the Cancer Road: Clomipr amine and Lithium Chlorid e                         Analysis of drug-receptor interaction at equilibrium

BİLİR A 1 , ERGUVEN M 2, YAZIHAN N 3                                                            BINDSLEV N

                                                                                                 Synagics lab  Panum Institute  U niversity of Copenhagen  Denmark
                                        1                                 2
Istanbul Uni versity, Istanbul, T urkey Istanbul Fac ulty of Medicine, Istanbul
                                                 3
Faculty of Medici ne and Fac ulty of Pharmac y; Ankara Uni versity, Fac ulty of
Medicine, Ankara, T urkey.                                                                      Aim: We want to anal yz e our dose-res ponse data at equilibrium by models. We
                                                                                                may have an i dea about a possible physic al interpretation of obtai ned respons e
Background: The ai m was to investigate whether a tricyclic anti-depressant                     curves and use either mathematic al or mechanistic descripti ons.
clomipramine (CIM) and an anti-ps ychorotic lithi um c hloride (LiCl) potentiate the            Background: From 1901 to 1910, receptors as separate entities were recognized
cytotoxicity of vinor elbine (VNR) at SHSH-5Y human neurobl astoma c ells in vitro              by Elliott (1904) and Langley (1905) in Cambridge and Ehrlich (Frankfurt 1907).
besides their conventional mode of ac tion.                                                     Deviation fr om a simpl e Langmuirian hyperbol e (1918) was doc umented by C.
Methods: The IC 50 values of VNR, CIM and LiCl were determi ned as 8 , 14.23                    Bohr (Copenhagen 1904) for oxygen-hemoglobin bindi ng.
and 200 μM. Four sets of experiments wer e performed for 96 hrs both for                        Mono-ligand system s: For mono-ligand s ystems with dose-respons es deviating
monolayer and thr ee dimensi onal (spheroid) cultures of SHSH-5Y cells. These                   from a simple hyperbole, Hill=s equation c an gi ve you a quantitative meas ure of
were i) Control group, c ells treated with ii) Singly applied VNR, CIM and LiCl, iii)           co-oper ati vity; but without ass essing possible i nterac tions between binding sites.
VNR with CIM, and iv) VNR with LiCl.Their effects on monolayer cultures were                    Therefore, co-operative val ues from a Hill scheme mos t likely has no physical
determined by the evaluation of cell proliferation, the perc entage of cells in S-              correlate.
phas e by BrDU-LI, apoptosis by Annexin V-FITC/PI staini ng, and cAMP levels by                 For a physical descripti on of s ystems deviating from simple hyperbolis m, I suggest
RIA; on spheroi d cultures by evaluation of the p ercentage of cells in S phas e,               the homotropic two-state model, HOTSM (2004). This model can handle both
spheroi d size, and the ultrastruc ture by TEM.                                                 positi ve and negati ve co-oper ati vity as well as bell-shaped and reverse bell-
Results: In comparison to the control group, single and combination drug                        shaped r elationships .
medications significantl y reduced the proliferation index (PI) for 96 hrs. The most            Two-ligand system s: Models for two ligands may again be based on either a
potent reducti on of PI was obs er ved at VNR with CIM and LiCl for all time                    Anon-interaction@ scheme suc h as the non-competiti ve inhi bition sc heme without
inter vals. VNR with CIM and LiCl seemed to be i neffecti ve to reduce BrDU -LI of              two-states of the un-liganded receptor or on an extended Monod- Wyman-
both monolayer cell and s pheroid c ultures , s pher oids siz e, c AMP levels. VNR with         Changeux sc heme (Monod et al 1965; Rubin & Changeux 1966) with two-states.
LiCl increased apoptosis potently at 24 hrs, however VNR with CIM increased                     Both thes e approac hes assume no interaction between bi nding sites, i.e., upon
apoptosis at 96 hrs. In ultr astructural evaluation of spheroids, increased                     binding of a ligand there is no change i n the binding c onstant for a sec ond drug.
presumably autophagic vac uoles , mitoc hondria damage and debris of lytic cells in             Instead, for combinatorial drug effec ts with assumed site i nterac tion, anal ysis of
extracellular area were obs erved at VNR with CIM. Striking data were catc hed at               data may be performed by the ternar y compl ex model, TCM, a one-state scheme
VNR with LiCl applied s pheroids. The VNR applied s pheroids revealed intact                    for two ligands with site i nterac tion (Ehlert 1988), or alternati vel y by the allosteric
nuclear and c ellular membranes. LiCl led to nuclear membrane breakdown at                      two-state model, AT SM, with site interaction (Hall 2000).
nearl y all cells of spheroids. When VRL was us ed with LiCl, in addition to nucl ear           ATSM implem entation: As one of the first, Jäger et al ha ve i mplemented the
membrane breakdown, the cellul ar membranes inside spheroids dis appeared and                   ATSM (2007). Thus, physic al effec ts are quantified for naphmethoni um on
the c ellular membranes were appear ed as an unique membrane constructions                      acetylcholine bindi ng and on pilocarpine ac tivation of an M2 rec eptor subtype.
onl y in s pheroid s urfac e. As a res ult of this, one s pheroid res embled one giant c ell.   Lately, Ehlert-Griffin (2008) have extended the AT SM with an additi onal ligand. A
Conclusions: 1) Both CIM and LiCl seemed to potentiate VNR-induced                              tri-ligand two-state model.
cytotoxicity with few exc epti ons. 2) Interes tingly, VNR with LiCl led to selec tive          Conclusion: Depending on the s ynagic sys tem to be analyz ed, i.e., the actual
nuclear and cellular membranes des truction. This effect could be due to the                    dose-res pons e relations hips at equilibrium, we may choos e between Hill, Hall or
premature acti vation of c yclin dependent kinase 2.                                            other models. A critical choic e is crucial for our extrac tion and use of relevant
                                                                                                mec hanistic par ameters.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                         Page A-37
                                                              EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                    Celebrating the 100th Anni versar y of the
                                                                       Nobel Prize Award to Paul Ehrlich
                                                                          Nürnberg, October 3-5, 2008
The Pharm aco kinetics (PK) of Voriconazole in Childr en                                  Modification of the Infarct Size Limiting Effects of Statins b y Antiplatelet
                                                                                          Drugs
BINER ORHANER B
                                                                                          BIRNBAUM Y1,2 , YE Y1 ,2 , PEREZ-POLO J.R 2.
Univ. Trakya, Pediatric Hematology, Edirne, T urkey.
                                                                                          1                               2
                                                                                            The Di vision of Cardi ology, The Department of Bioc hemistr y and Mol ecular
Background: Invasi ve fungal infections are a major caus e of morbi dity and              Biology, The Uni versity of Texas Medical Branch, Gal veston, Texas.
mortality in immuncomprimised patients. T he most c ommon fungi responsible for
severe infecti ons are As pergillus. Voriconazol e is a broad spectrum sec ond            Background: Therapy for acute coronar y syndromes includes statins and anti-
generation triazole antifungal agent. It is indicated for the treatment of invasive       platel et agents. Stati ns limit infarct size (IS) in ani mal models by acti vati on of
aspergillosis. Pedi atric dos age finding and s afety evaluations have not been           phos phoinositi de-3- kinase (PI3K) with subsequent acti vation of ec to-5-
completed. Aims: 1)The ai m of this study was to review the literature about s tudies     nucleotidas e (that gener ates adenosi ne) and Akt/endothelial nitric oxide s ynthas e
done on the PK of voriconaz ole in childr en. 2) The clinical results of our 5 cas es     (eNOS) with downs tream acti vati on of inducible NOS (iNOS) and COX2. Inhibition
with As pergillus infec tion (4 pulmonar y, 1 bloods trea m) with voriconaz ole           of the PI3K, adenosine receptors, eNOS, iNOS and COX2 abrogates the IS-
treatment were shown.                                                                     limiting effec ts of statins.
Methods: Literature was searched with the key wor ds ― voriconazol e,                     Methods: Rats recei ved 3-day oral ator vas tatin (ATV) or vehicle with or without
phar mac okinetic , childr en, invasi ve fungal i nfecti on‖ and the studies were         dipyridamole (DIP, 6mg/kg/d), cilostazol (CIL, 20mg/kg/d), or as pirin (ASA, 5, 10 or
evaluated.                                                                                20mg/kg at reperfusion). Rats underwent 30-minute coronar y arter y occlusion and
Results: Children require higher doses of voriconaz ole than adults to attain similar     4-hour reperfusion.
serum concentrations over ti me because the drug exhibits non-linear                      Results: ATV (10mg/kg/d) limited IS. Intravenous ASA befor e reperfusion
phar mac okinetics in adults, but exhibits linearity in children. A significant           attenuated this effect. DIP al one and AT V (2mg/kg/d) alone had no effect on IS;
relations hip between disease progression and drug conc entr ation was described          however, IS was significantl y reduc ed in the ATV+DIP combination. M yoc ardial
in adults (Antimicrob Agents Chemother 2006;50.1570). Bas ed upon studies in              adenosine levels were higher in the ATV+DIP group than in the ATV alone, DIP
children, it appears that a pediatric dos age of 11 mg/kg admi nistered ever y 12 h is    alone and the c ontrol group. The protecti ve effec t was abolished with theophylline,
approxi matel y bioequi valent to an adult dos age of 4 mg/kg given 12 h. Plasma          indicating that it is mediated by adenosine receptor ac tivation. CIL alone, and
samples for voriconazole HPLC ass ay from 14 subjects reveal ed that i n children         especi ally when combined with ATV (2 mg/kg/d) limited IS. CIL increased
receivi ng dos ages of ≥4 mg/kg iv bid was lower than that of adult vol unteers           myoc ardial levels of adenosine and Akt and eNOS phosphor ylati on. In addition, by
receivi ng 4 and 5 mg/kg bid (Wals h TJ, et al). In another study done in 5 children      increasing tissue cAMP levels, CIL acti vated protei n kinase- A that phosphor ylates
with ages ranged from 2 to 10 years ol d voriconazole was administered at                 eNOS. CIL inhibited PT EN, thus leading to augmentation of Akt and subsequentl y
dosages varied from 3.4 mg/kg every 12 h to 8.1 mg/kg ever y 8 h and pl asma              eNOS phos phoryl ation.
voriconaz ole conc entrations were found to be unpredictabl e for thes e paediatric       Conclusions: As pirin bloc ks the IS-limiting effects of statins, wher eas both
patients (Arc h Dis Child 2008;93:578). Therefor e, using voriconazol e at                dipyridamole and cilos taz ol have s ynergistic effects with statins. It might be that
recommended dos es for adults may l ead to clinical failures in c hildren. Als o,         the anti-pl atel et regimens s houl d be modified for patients rec ei ving stati ns.
recent obs ervations suggest that hepatic toxicity and visual disturbance might be
dose rel ated. Five of our cases tr eated succ essfully without any major side effect
with voriconozole (14 mg/kg/d) will be presented.
Conclusions: 1) Children might require higher dos es of voriconaz ole than adults
to attai n si milar serum c onc entrations over time bec aus e of its linear PKs in
children. Studies about the opti mum dosage of voriconoaz ole in children s houl d be
conducted in a l arge number of children.




Structure and Function of the Ubiquitous TRPC channels: Targ ets in N eed of              Starvation and Oxidative Str ess as an Inductor of Ciprofloxacin Resistan ce
Magical Bullets.
                                                                                          BIROŠOVÁ L, MIKU LÁŃOVÁ M
BIRNBAUMER L
                                                                                          Slovak Uni versity of Technolog y, Bratislava, Slovakia
Laboratory.of N eurobiolog y, Nati onal Institute of Environmental Health Sciences,
NIH, DHHS, Researc h Triangle Park, North Carolina 27708 USA                              Background: Mutation rate of bacteria is often affected by environmental
                                                                                          conditi ons. Various str ess such star vati on, oxidati ve or radiating stress can res ult
The canonic al transient receptor potential (TRPC) channels were discovered in            in increas ed frequenc y of mutations leading to antibiotic resistanc e. T he aim of this
our laboratory i n 1995- 96. T hey are homolgues of the Drosophila light-acti vated       wor k was to determi ne the mutation frequenc y leading to ciprofloxacin resistanc e
channels Trp and Trp-like and were cloned to test the hypothesis that in                  induc ed by depleted medi a and hydrogen peroxide and to find molec ular
mammalian c ells they might be at the root of not only Gq -activated non-selec tive       determinants of ci profloxacin resistanc e in s elected mutants.
cation currents but also store-operated C a2+ entr y (SOCE). In i nitially 6, now 7,      Methods: Oxidati ve stress was evoked by 3h culti vati on of S. typhi murium with
TRPCs have distant homolog y to voltage-gated c ation channels and s pan the              hydrogen peroxide in 3 conc entrations (0.4, 0.8, 2, 4 mM). Starvation was studied
membrane 6 times. Between their discover y and now, the participation of TRPCs            after incubation (3h) of bacteria in contr ol Luria-Bertani medium (LB) and in
as members of SOCE c hannels has been c ontroversial and i ndirect. An alternative        nutritionally depleted medi a 10%LB and N utrient broth No. 1. Resistant str ains
hypothesis emerged after the discovery in 2005 of STIM, a 1- pass Ca2 +-sensing           were counted on agar pl ates supplemented with ciprofl oxacin (0.06 mg/ml) after
membrane protein loc ated in the ER that organizes pl asma membrane SOCE                  72 h of inc ubation at 37°C. The frequenc y of resistant mutants (resistanc e index
channels, and i n 2006 of Orai, a 4- pass pl asma membrane protein whose loss is          RI) represents mean number of resistant c ells di vided by the total number of viable
responsi ble for a familiar form of s evere combi ned immunodefficienc y and whos e       cells per c ulture. Data repres ent the mean of three independent experiments; eac h
expression together with STIM engenders i n TRPC expressi ng cells ver y large            experiment was made in fi ve parallels and statisticall y evaluated by Student‘s t-
SOCE acti vity. This alternative hypothesis postulates Orai as the SOCE channel           test. Mutati ons in gyrA wer e deter mined usi ng AS-PCR-RFLP method. Levels of
activated by STIM and does not i nclude a role for TRPCs. However, as we                  outer membrane porin F were detected with SDS- PAGE.
published in J an 2007, Orai and TRPCs interact functionall y, seen as a TRPC-            Results: Short-term culti vation in 10% LB caused 430 fold increase of RI while in
dependent enhancement of SOCE upon expression of l ow levels of Orai . We                 Nutrient broth No.1 it was onl y 57 fold rise. In ciprofloxaci n-resistant str ains
propos ed that instead of forming channels, Orai appear to be regulatory proteins         generated by long-term s tar vati on were detected decreased l evels of OmpF
that confer C a2+-selecti ve SOC E channel properties to the otherwise non-selec tive     protein. With rising dose of H 2O2 was RI incr easing up to 33-fold of spontaneous
cation channels formed by TRPCs without Orai. In s upport, SOCE channels were             mutation frequenc y to ciprofloxaci n resistanc e. In nutritionall y depleted medium
shown in other labor atories to be dynamic ally assembled in lipid rafts from which       with H 2O2 has mutation frequenc y increased more than 103-ti me. 80% of resistant
TRPCs and Orai can be co-immunoprecipitated. A sur vey of the liter ature s hows          strains had mutation in gyrA. 37% of them had mutati on in c odon Ser-83 and 63%
that physiologic roles for TRPCs have been proven in the central ner vous system,         in codon Asp-87.
in the c ardiovasc ular s ystem, in the kidney, in epithelia, endothelia and blood-       Conclusions: 1) Star vation increases mutagenesis leading to ci profloxacin
borne cells, and in various muscle types. TRPCs act in thes e tissues either by           resistance. 2) Short-ter m treatment of S.typhi murium i n nutritionally depl eted
depolarizing their membrane or through the C a2+ they allow to enter upon                 media c aused higher increas e of ciprofloxaci n RI than long -term inc ubation.3)
associati on with Orai. The latter includes cell pr oliferation. TRPCs pl ay roles in     Long-term s tar vati on is leading to decreased levels of OmpF. 4) Oxi dati ve stress
human diseas es, including canc er. TRPCs and Orai are important targets waiting          induc ed by H 2O2 in c onjunction with lac k of nutrients in environment increas es
for the development of Magic Bullets that will help i n better understanding their        mutation frequenc y to cipr ofloxaci n resistance. 5) Maj ority of ci profloxacin-resistant
role and in ameliorating diseases invol ving altered TRPC and/or Or ai funcitons.         strains generated by H 2O2 has mutation in gyrA gene.

                                                                                          This work was supported by the Slovak Grant Agenc y VEGA (Projects
                                                                                          no.1/4305/07)




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                              Page A-38
                                                               EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                      Celebrating the 100th Anni versar y of the
                                                                         Nobel Prize Award to Paul Ehrlich
                                                                            Nürnberg, October 3-5, 2008
Identification of the anti-inflammator y targ ets of inter activ e constituents of          LC-MS for Label-Free Biom arker Discover y
Hypericum perforatum (Hp).
                                                                                            HORVATOVICH P1, GOVORUKHINA N 1, SOBCZAK-ELBOURNE I1 ,
BIRT DF and HAMMER KDP                                                                      KEMPERMAN R 1, 4, CHRISTIN C 1, HOEKMAN B1, VAN DER Z EE A2, SUIT S F3,
                                                                                            MUSKIET F2, HOEF SLOOT H 4 , SMILDE A4, BISCHOFF R 1
Dept. of F ood Sci. & Human Nutr., Iowa State Uni v., Ames, IA.
                                                                                            1
                                                                                              University of Groningen, Gr oningen, The Netherlands
                                                                                            2
Background: Hp is used as a botanical therapy for infec tive disorders. The l evel            University Medical C enter Groni ngen, Groningen, T he Netherlands
                                                                                            3
of cons tituents in H p extrac ts were lower than the c onc entration of pure                 IBM TJ Watson Res earch Center, Yor ktown Heights, NY, USA
                                                                                            4
constituents needed to reduc e lipopolys accharide (LPS)-induc ed pros taglandin E2           University of Ams terdam, Amster dam, T he Netherlands
(PGE2) produc tion in RAW 264.7 macrophages; s uggesting an interac tion of
compounds for the acti vity. The goal of this study was to identify key constituents        Background: The discover y and validati on of biomarkers is an i mportant goal in
and investigate the gene targets for the anti-inflammatory effect.                          many areas of biomedical research and drug development. Changes in indi vidual
Methods: A flavonoi d-rich bioacti vity guided fractionati on was us ed with screening      proteins or peptides as well as in protein and peptide profiles in bodily fluids or
in the LPS-induced RAW 264.7 macrophage s ystem to identify ac tive fractions               tissue bi opsies have been implicated in diagnosis and prognosis of various
from eac h round and liquid chromatography- mass spec trometr y (LC-MS) identified          diseas ed states. Rec ent developments in anal ytic al chemistry allow to obtain
constituents pres ent. CellTiter96T M Aqueous solution revealed no significant              complex, quantitati ve data from hundreds to thous ands of compounds ( protei ns,
cytotoxicity with fractions or constituents at the doses studied. Microarray anal ysis      peptides, metabolites) that r equire novel data processing and statistical
was performed with Hp fracti on, the 4 constituents combined at levels detected in          approaches to arri ve at biomar ker candidates.
the H p fracti on, and s olvent control with/without LPS.                                   Methods: Serum was prepared for pr oteomics analysis by Liquid-Chromatography
Results: A third round fracti on (3A) (10 µg/ml) significantly reduced PGE 2.               Mass Spectrometry (LC-MS) [1]. The generated data were process ed using in-
Combi ning four cons tituents at c oncentrati ons detected in the LC-MS anal ysis           hous e devel oped algorithms [2,3] and anal yzed by multi-variate statistics in
(0.17 µM chlorogenic acid, 0.08 µM amentoflavone, 0.08 µM querceti n, and 0.03              combinati on with variable s election algorithms.
µM pseudohypericin (PHCN) explained the anti-inflammatory ac tivity of the                  Results: LC-MS anal ysis of ser a from c ervical c anc er patients were anal yzed prior
fraction in light-acti vated c onditions. T he amount of each pur e constituent needed      to and approxi matel y 6 months after therapy. While differenc es in protein pr ofiles
to obs erve a significant reducti on in PGE2 was > 50 ti mes more than was found in         were mi nor for early-stage disease differenc es became significant for more
fraction 3A. Of the 4 interacting compounds, onl y PHCN was required. With LPS,             advanced stage tumors. Follow up of pati ents over multiple years showed that
the 4 component system affected 162 genes and the fraction affected 780 genes;              certain biomar ker c andidates changed in agreement with the r ecurrenc e of
40 genes were differenti ally express ed under both treatments. Important                   diseas e. Some of thes e candidates were rel ated to the gl ycos ylati on of serum
pathways for both treatments were the J anus kinase-signal tr ansducer and                  proteins .
activator of transcripti on and eicos anoid metabolism pathways.                            Conclusions: 1) Biomarker candidates have been discover ed that c orrelate with
Conclusions: 1) Interactions of c onstituents explai ned the anti-inflammator y             the res pons e to therapy for cer vical cancer. 2) Dedic ated data pr ocessing and
activity of fracti on 3A. 2) PHCN was required for the anti-inflammatory acti vity in       statistical anal ysis reduced the number of variabl es to a number that is in-line with
combinati on with one or more other constituents . 3) The gene targets identified for       the number of anal yzed sampl es.
the 4 c omponent s ystem were c onsistent with the r educ tion of LPS induc ed PGE2.        References:
This work supports the rol e of interacti ons of compounds in Hp targeting genes            1.     Govorukhina, N. I.; Reijmers, T. H.; Nyangoma, S. O.; van der Zee, A. G. J.;
that ar e important in inflammation. Supported by grants ES012020 from                             Jansen, R. C.; Bisc hoff, R. J. Chromatogr. A 2006, 1120, 142-150.
NIEHS/ODS and 9P50AT004155- 06 fr om NCCAM/ODS, NIH.                                        2.     Christin, C.; Smilde, A. K.; Hoefsl oot, H. C.; Suits , F.; Bisc hoff, R.;
                                                                                                   Horvatovich, P. Anal Chem. 2008, in press.
                                                                                            3.     Suits, F.; Lepre, J.; Du, P.; Bischoff, R.; Hor vatovich, P. Anal. Chem. 2008,
                                                                                                   80, 3095-3104.




Reactive Oxyg en Species Have a Cau sal role in the Development of Insulin                 Synthesis of Unnatural C eram ide Analog s and Th eir Antiprolifer ative
Resistan ce in Diab etic, H yper cortisolemic and Chronic Inflamm ator y States:           Properties Against a Panel of Cancer Cells
Amelioration with the Antioxidants Alph a-lipoic acid
                                                                                           BITTMAN R 1 AND ARTHUR G2
BITAR SM 1, AL-ALI W2 , AL-MULLA F2
                                                                                           1
                                                                                             Department of Chemistr y and Bioc hemistr y, Queens College and T he Gr aduate
1
 Kuwait Uni versity. Fac ulty of Medicine, Department of Phar mac ology, Safat,            Center of CUNY, Flus hing, NY 11367, USA
Kuwait; 2 Kuwait Uni versity. F aculty of M edicine, Department of Pathology, Safat,       2
                                                                                             Department of Bioc hemistr y and Medical Genetics, U niversity of Manitoba,
Kuwait.                                                                                    Winnipeg, Manitoba, CANAD A

Background: Ins ulin resistanc e, featured by an inexorable decline in s keletal           Abstract: 2S,3R-Cerami de 1 occ upies the ―hub‖ of sphingolipid metabolism and
muscle glucos e utilizati on and/or an excessi ve hepatic glucose production,              serves as a c oordi nator of eukar yotic stress res pons es and other biological
represents a major pathogenic importanc e in a cl uster of clinic al dis orders            activiti es suc h as cell growth and differentiation. Ceramide 1 plays a key role in
including diabetes mellitus, hypercortisolemia, inflammati on, and coronar y arter y       programmed cell death (apoptosis). However, sinc e 1 is a naturall y occ urring lipid,
diseas e. A novel concept suggests that exc essive generation of reac tive oxygen          it is recognized by endogenous enz ymes and can be converted into anti-apoptotic
species (ROS) contribute to the development of ins ulin resistanc e i n most if not all    lipids via phos phoryl ation and glycos ylati on. We sought to prepare unnatural
the afor ementi oned diseas e states .                                                     ceramide analogs that may be longer lived in c ells becaus e they are unrecognized
Methods: Euglycemic-hyperinsulinemic cl amp studies with an i nsulin infusion              by enz ymes. We found that some of the unnatural ceramide anal ogs have greater
index of 5 mU/kg bw/mi n were us ed to meas ure endogenous glucose production              antiproliferati ve acti vity than 1 agains t human breast cancer cell lines in vitro. One
(EGP), glucose infusion rate (GIR), glucose disposal rate (GDR) and skeletal               of the unnatural ceramide analogs we s ynthesiz ed has an exoc yclic double bond
muscle glucose utilization index (GUI). Moreover, the status of oxidati ve stress as       in the sphingoid bas e (compound 2), wher eas another has a disulfide linkage in
reflected by urinar y levels of isopr ostane and tissue c ontents of protein-bound         the N-ac yl chai n (compound 3). Their antiproliferati ve acti vities agai nst three
carbonyls and thi obarbituric acid reacti ve subs trates (TBARS) wer e also                human breast c ancer cell lines (BT549, MDA-MB-231, MCF-7), a lung cancer cell
assessed as a func tion of diabetes, hypercortisolemia and c hronic low grade              line (A549), a pros tate canc er cell line (DU145), and a cervic al cell line (HeLa)
inflammation.                                                                              were anal yz ed in vitro and c ompared with the acti vity of (2 S,3R)-N-
Results: Post-absorptive basal EGP and circulating levels of insulin, glucos e and         octanoylceramide (1). The s ulfur-containing-ceramide analog 3 and the exo-
free fatty acid wer e elevated in GK and to a lesser extent in Dex and TNF alpha-          ceramide analog 2 exhibi ted a higher antipr oliferati ve acti vity than natural
treated rats,c ompared to their corresponding control values . In contrast, steady         ceramide 1. Cas pases in cells treated with compound 3 were acti vated, indicating
state GIR and GDR of the hyperglycemic/hyperinsulinemic animals were                       that the c ells under went apoptosis. A ceramide anal og containi ng a
reduced,c onc omitantl y with impaired insulin‗s ability to suppress EGP. The              tetrahydrofuranyl ring (compound 4) and a phytoc eramide analog (compound 5)
suppressi on of s keletal muscle glucose utilizati on i n thes e animals was associ ated   exhibited a much higher antiproliferative acti vity than natural N-palmitoyl-D-
with a decrease in ins ulin‗s ability to promote the phos phoryl ation of tyrosine         erythro-ceramide against T47D, MCF7, and MDA-MB-468 cells. The s ynthes es,
residues of i nsulin r eceptor substrate-1. Similarl y, the transl ocati on of glucos e    antiproliferati ve acti vity, and mec hanistic studies of the apoptotic properties of
transporter-4 from intrac ellular compartment to pl asma membrane in response to           thes e ceramide anal ogs will be pres ented.
insulin was also reduced in these ani mals. Oxidati ve s tress-bas ed markers (e.g.
urinary is oprostane, car bonyl-bound pr otei ns, TBARS) wer e elevated in respons e
to diabetes, hyperc otisolemia and c hronic low grade inflammati on. Nullificati on of
the heightened state of oxidati ve stress in the aforementioned animal models
with antioxidants s uch as alpha lipoic acid ameliorated hepatic and s keletal muscle
insulin resistance.
Conclusions: Collec tivel y, the above data s uggest that excessi ve generation of
ROS in c onnection with their detrimental effects on lipid and protein
molec ules have a caus al role in multipl e forms of i nsulin resistanc e.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                             Page A-39
                                                       EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                            Celebrating the 100th Anni versar y of the
                                                               Nobel Prize Award to Paul Ehrlich
                                                                 Nürnberg, October 3-5, 2008
Peltophorum africanum Sond (Fab aceae) h as a role in disease control             Brain cholesterol? Long secret life behind a barrier

BIZIMENYERA ES* A, OGUTTU JW A AND KOMA LM                          B
                                                                                                            BJÖRKHEM I
a
  Department of Agriculture, Ani mal Health and H uman Ecol ogy, U niversity of                             Karolins ka Instit utet, Karolins ka Uni versity Hos pital H uddinge, Di vision of Clinical
South Africa, Private Bag X6, Florida 1710, South Africa                                                    Chemistr y, 14186 Huddinge, Sweden
b
  Department of Surgery and Theriogenol ogy, Faculty of Veterinary Medicine,
Makerere University, P O Box 7062, Kampala, Uganda                                                          The bl ood-brain barrier is almost c ompletel y imper meable for cholesterol in both
                                                                                                            directions. All choles terol pres ent in the brai n is thus a product a local s ynthesis.
Background. About 80% of people in the developing world, particularly those from rural                      Since there is a low but significant s ynthesis of chol esterol in the adult mammalian
communities where modern drugs are unaffordable, inaccessible or, unavai lable, depend on                   brain we hypothesized that there may be a compensator y flux of a c holesterol
phytomedicine for primary healthcare. However, most medical and veterinary professionals distrust
                                                                                                            metabolite from the brain across the blood-brai n barrier. About 14 years ago we
herbal medicines due to lack of scientific evidence of efficacy and safety. Hence, there is need for
their validation, before herbal medicines gain wider acceptance and use. T raditional healers,              identifi ed this metabolite as 24S-hydroxycholesterol (24OHC) and showed that in
pastoralists and rural farmers use extracts of Peltophorum africanum (a medicinal plant widely              contrast to cholesterol its elf, this oxysterol can cross the blood-brain barrier. By
spread in southern Africa and other tropical regions), to treat diarrhea, dy  sentery, pain, infertility,   measuring the concentrati on differ ence between the inter nal jugular vein and an
HIV-AIDS and to promote well-being and resistance to disease. T he extracts of the plant inhibit            artery in human volunteers, we demonstrated the production of 24OHC by the
HIV-type 1 reverse transcriptase and protease.
                                                                                                            brain to be about 6 mg/24h. Si nce the uptake of 24OHC by the liver was fund to be
Methodology. Dried leaves bark and root from mature P. africanum trees were extracted with
acetone. Chromatograms were made on silica gel plates. Minimum inhibitory concentrations (MIC)              about the same, it is evi dent that al most all 24OHC i n the circulation originates
were determined for five bacteria (Gram positive and Gram negative), and five fungal pathogens.             from the brain.
Qualitative screening for antioxidants was done by spray      ing chromatograms with 0.2% 2 , 2-            In s pite of the fact that cholesterol does not pass the bl ood-brain barrier,
diphenyl-1-picryl hydrazyl (DPPH) , and quantification done in comparison with L-ascorbic acid and          hypercholesterolemia is a risk fac tor for Alzheimer Disease (AD). We hypothesized
T rolox (6-hydroxy-2, 5, 7, 8-tetranethylchromane-2-carboxylic acid). Anthelmintic activ was  ity
                                                                                                            that there may be a metabolite of cholester ol fluxi ng in the opposite direc tion from
evaluated by effects of extracts on the egg hatching and l arval development of parasitic nematodes
Haemonchus contortus and Trichostrongylus colubriformis.                                                    the circul ation into t he brain. This metabolite was identifi ed as 27-
Results. The extracts showed substantial activity against both Gram-positive and Gram-negative              hydroxycholesterol (27OHC), also using the catheterization appr oach. The uptake
bacteria, with Minimum Inhibitory Concentration (MIC) values of 0.08 mg ml -1 for Staphylococcus            of this oxysterol by the human brain was found to be about 5 mg/24h. Si nce there
aureus and 0.16 mg ml -1 for Pseudomonas aeruginosa. T he extracts showed higher antifungal                 is a good c orrelation between levels of cholesterol and 27OHC in the circulati on, it
activity than amphoterin B. T he acetone extracts of the bark, and root of P. africanum showed
                                                                                                            seems likel y that the uptake of 27OHC by the br ain is related to the levels of
higher antioxidant activity than L-ascorbic acid (Vitamin-C) and T rolox (6-hydroxy-2, 5, 7, 8-
tetramethylchromane-2-carboxylic acid), a synthetic vitamin-E analogue, and much higher than                cholesterol in the circulation.
Ginkgo biloba extract (EGb 761). T he respective EC 50 for the P. africanum root and bark extracts,         In spite of the relati vely high influx, levels of 27OHC in the brain are ver y low,
L-ascorbic acid, and EGb761 were 3.82µg/ml, 4.37µg/ml, 5.04µg/ml, and 40.72 µg/mL. T he                     indicating an efficient metabolism. T he major metabolite was i dentified as 7a-
                                              b
standardised extract of Ginkgo biloba (EG 761) is widely employed for its significant benefit in            hydroxy-3-oxo-4-chol estenoic acid. This acid ver y efficientl y passed a model for
neurological disorders. The extracts inhibited egg hatchability and larval development (from L1 to
                                                                                                            the blood-br ain barrier and we found a net flux of it from the human brain into the
infective stage L 3) of both Haemoncus contortus and Trichostrongylus colubriformis (both parasitic
nematodes of ruminants) at concentrations of 0.1-1.0 mg ml -1. The plant extracts, at the                   circulation. T he c onversion of 27OHC into the steroid acid can be regarded as a
concentration of 5-25 mg ml -1 completely lysed larval forms (L 1) and eggs of the nematodes.               regulated detoxification. 27OHC is an efficient suppress or of choles terol synthesis
Conclusion. P. africanum extracts have therefore, potential for treatment of inf7ction-related              and we have shown that the compound is abl e to increase amyl oid formation in
diseases by either directly inhibiting bacterial growth or by stimulating the immune system of the          neuroblastoma c ells.
host. The traditional use of P. africanum concoctions against diarrhea, dy   sentery and unthriftness,
may be also due to anthelmintic activity as these signs are consistent with parasitic gastroenteritis.
Gastrointestinal nematodes exasperate diarrhea in HIV -AIDS patients, as well as disease-related            Different pathogenetic aspects of the oxysterol crosstalk over the blood-br ain
production losses arising from stock mortality, severe weight loss and poor production in                   barrier will be discuss ed in the lecture and it is suggested that the flux of 27OHC
ruminants. Antiox  idants are also important in boosting the immunity, critical in the management of        from the circulation into the brai n is the missing link between hypercholesterolemia
helminthosis. T here is ample scientific and empirical evidence supporting the use of plant-derived         and AD.
antioxidants in the control of neurological diseases, as antioxidants have neuro -protective
(preventing apoptosis), as well as neuro-regenerative roles. Due to the high antioxidant activity of
its extracts, P. africanum has prospects in the management or control of neurodegenerative
diseases. T hus there is great potential of P. africanum extracts in disease control.




Therapeutic Differentiation of Insu linoma C ell Lin es Tr eated with                                       Development of a new 3D-Human Air way Epithelium / W hole-blood Co-
Streptozotocin                                                                                              culture Model Combined with Multi-Analyte Profile (M AP) Analyses for
                                                                                                            Assessing Drug Effects
BLOCH K, VARDI P.
                                                                                                            BLUM M 1, STEIN GM 1, CONSTANT S2, WISZNIEWSKI L2, HUANG S2, MAPES
                                                                                                             3         3            1
Tel Aviv Uni versity, Felsenstei n Medic al Research Center, Petac h Tikva, Israel                          J , SPAIN M , SCHMOLZ M
                                                                                                            1
Background: Streptoz otoci n (STZ) is a member of a group of al kylating                                     EDI GmbH, Reutlingen, Ger many, 2Epithelix Sàrl, Plan-Les-Ouates , Genève
antineoplastic drugs, and is clinically acti ve agai nst insulinomas. STZ toxicity                          Suisse, 3R ules-Bas ed M edicine Inc ., Austin, TX, U SA
depends on glucos e trans porter protein- 2 (GLUT-2) expression and generation of
free radicals. As with many other chemotherapeutic drugs, repeated treatment                                Background: The dialogue between cells of the i mmune s ystem and cells of
with STZ, may induc e a selection of resistant cell populations. Differentiation                            various tissues controls immune reac tions and is in part mediated by a variety of
therapy has been proposed as a promising approach to sel ecti vel y engage the                              cytokines , chemokines etc. T his network may be strongl y influenc ed by the
process of tumor c ell differ enti ation during chemother apy. Accordi ng to this                           application of drugs. Aim of our investigations was to develop an innovative
approach, c ytotoxic agents c an induc e drug resistance, but in c ertain conditions,                       organo-typical human air way epithelial co-culture model for the anal ysis of
can also lead to rec over y of normal c ell homeostasis. Ai m: To esti mate toxin                           immunopharmacological ac tiviti es of drugs.
resistance and differ entiation of i nsulinoma cell s urvi val following expos ure to STZ.                  Methods: A differenti ated airway epithelium, M ucilAir, was c ombined with whole-
Methods: A parental highl y differentiated mouse insulinoma c ell line (BTC-tet) and                        blood cultures in a two-c hamber system to study the effec ts of betamethas one
low differenti ated rat insulinoma cell line (RINm) were repeatedl y exposed to STZ.                        applied onto the epithelium sitting on acti vated immune cells from a healthy donor
The cell populations (RIN-S and BTC-S) survivi ng such treatment were examined                              mimic king an inflamed tissue environment. 92 mediators and other parameters
as to their resistance to STZ, hydr ogen peroxide, nitric oxide and c ytokines.                             were tes ted in the supernatants of the c ell cultures by multiplexed bead ass ays
Western blot anal ysis was applied to estimate GLUT-2 and bcl-2 expression in                               (RBM MAP analysis).
parental and STZ selected cells. Ins ulin content and s ecretion, c ell proliferation,                      Results: Betamethas one exhibited its typic al, strong pharmacological effec t pr ofile
morphological and chromos omal characteristics wer e studied.                                               on both, the immune and the epithelial cells: It dose-dependently inhibited a
Results: Repeated STZ treatment of parental ins ulinoma cell lines resulted in the                          variety of pr o-inflammator y mediators, being either T hel per cell type 1- (Th1),
selection of cell sub-populations with multiple resistance to different toxins. The                         Th2-, or macrophage-ass ociated, s uch as interferon (IFN)-               IL)-5 and
enhanc ed toxin toleranc e may be explai ned by a low level of GLUT-2 and high                              tumor necrosis fac tor (TNF)-, respectivel y. In contrast, IL-10 as a mediator of
expression of bcl-2 in s elected c ells. In addition, STZ selec ted cells displayed a                       regulator y T cells was up-regulated after 24h of co-cultur e. Furthermore, epithelial
lower r ate of cell pr oliferation when compared to untreated cells. M oreover, BTC -S                      cells were cultured for another 6 days showi ng a dos e-dependent effect on e.g.
and RIN-S cells showed 2- 5 times higher levels of intrac ellular insulin content and                       the monoc yte c hemotactic protei n-1 (MCP-1).
improved insulin res ponse to glucose than their parental cells. STZ-based                                  Conclusions: From the data we will pres ent in this poster, it is evi dent that the
selection changed cell morphol ogy and increased frequenc y of pol yploidy i n RIN-S                        highly complex, organo-typical co-cultur e model provi des an excellent, in vivo-like
(2.2% ) compared to RINm c ells (0.7%).                                                                     tool to study in vitro not onl y the phar mac okinetics and pharmacodynamics of
Conclusions: Repeated STZ treatment of ins ulinoma cell lines res ults in the                               inhaled drugs, but also the harmful effects of toxicants that get access to the
selection of cell subpopulations poss essing multipl e toxin resistance, a low rate of                      human lung.
proliferation and enhanced functional acti vity. Further characterization of STZ
selected beta cells could provi de useful lessons for opti mization of differentiation
therapy of cancer.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                  Page A-40
                                                                EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                       Celebrating the 100th Anni versar y of the
                                                                          Nobel Prize Award to Paul Ehrlich
                                                                            Nürnberg, October 3-5, 2008
Drug Potential of Nigerian MedicinE R elated Plants: A SURVEY                                Purposeful Drug-Excipients Ph ysico-Chemical Inter actions – W hat does that
                                                                                             mean for Optimiz ation of Drug Deliver y and Safet y?
BOBOYE B AND AKHARAIYI F
                                                                                             BOGDANOVA S1 , PAJEVA I2, AVRAMOVA N 3, HRISTOVA Y2
Department Of Microbiolog y, Federal U niversity Of T echnolog y, P. M. B. 704,
                                                                                             1                                                              2
Akure, Ondo State, Nigeria.                                                                    Faculty of Pharmac y, Medical Uni versity-Sofia, Bulgaria; Bulgarian Ac ademy of
                                                                                             Sciences , Bulgaria; 3 F aculty of Chemistr y, Sofia Uni versity, Bulgaria
Medicinal plants are plants used whole or parts to pr event and cure health
problems, promote and rehabilitate natur e to the living population at primar y,             Background: Drug-excipients inter actions attract res earch interest as a
secondar y and tertiary health care deliveries. In Nigeria, the use of plants at             biopharmaceutical tool to infl uenc e the onset, intensity and the duration of drug
various levels of health c are deli ver y has been in practice for many centuries pas t;     perfor manc e In vivo. Aim: to improve the dissolution charac teristics and opti mize
as old as the histor y of human bei ngs. These plants bel ong to several families            In vitro drug release profile of some non-steroidal anti-inflammator y drugs -
including Legumi nosae, M alvaceae, Mimos aceae, Euphorbiaceae, Compositae,                  indomethaci n (IND), ibuprofen (IBP), naproxen (NAP) by In vitro interacti ons with
Acanthac eae, C annaraceae, Passiflorac eae, Rutaceae, Zingiberac eae,                       hydrophilic pol ymers - pol yvinyl(pyrrolidone) (PVP), pol yethyleneglyc ol (PEG),
Bombac aceae, Ol acac eae, Apoc ynaceae, Guttiferae, Liliaceae, Sapindac eae and             hydroxyethyl c ellulose (HEC), sodium alginate, dextran and silica-PVP- or silica-
Combretac eae. Specific examples of the phyto-organisms are Pericopsis,                      methylmetacr ylate nanohybrids.
Phaulopsis, Myr obal an, Pleioc arpa, Picralima, Fig, Bhadram, Akerbia, Millefoil,           Methods: Different phar maceutical techniques – s olid dis persions-, ads orbates
Bear?s breac h, Copper leaf, Acal ypha, Acaci a, African mallow and Baobab.                  formation, sol-gel reacti ons and appropriate moder n physico-chemical (FT-IR, X-
sThese pl ants ar e used for differ ent medicinal purposes suc h as stimulant and            ray, DSC, s olid state C13 and Si29 NMR, AFM, TEM) - and in silico methods for
carminative, avert fever, coughs, asthma, flatulence, helmi nthic and microbial              characterization have been applied.
problems, c anc er, hypertension, l epros y, vitamin deficienc y, lac k of homeostasis,      Results: Changes in physico-chemical properties of dr ugs have been ac hieved -
pox and ulcer.                                                                               pol ymorphic transitions (IND), inhibition of cr ystallization and amorphization (IND,
                                                                                             IBP), complex for mati on (IND,IBP), particle size reduction. All changes are related
                                                                                             to significant incr ease of In vitro drug dissol ution. Special emphasis has been
                                                                                             put upon the eluci dation of the character of occurring interactions on molec ular
                                                                                             level. The modern s ol-gel technique has been applied to develop Silica-PVP-IBP
                                                                                             model nanohybrids of pr olonged, pH-independent IBP releas e.The main
                                                                                             advantage of this r eaction is that it takes plac e at mild experimental c onditi ons and
                                                                                             enables drug immobilization i nto the inorganic-organic networ k.
                                                                                             Conclusions: Physico-chemical tr ansformations of s ome non-steroidal anti-
                                                                                             inflammator y drugs by means of purposeful i nterac tions with pol ymers and
                                                                                             nanohybrids have been proven. Significant impr ovement of In vitro drug
                                                                                             dissolution and modified drug release profiles wer e registered. The res ults give
                                                                                             reasons to ass ume an increase of In vivo drug ac tivity and s afety.




Escit alopram-the fir st ASRI. A Magic Bullet in the treatment of depression                   The influence of cyt arabin e and m yristic acid on aspirin binding with serum
and anxiet y.                                                                                  albumin. Sp ectroscopic stud y

BØGESØ KP                                                                                      BOJKO B 1, SUŁKOWSKA A1, MACIĄŻEK-JURCZYK M 1, RÓWNICKA J1, ZUBIK-
                                                                                               SKUPIEŃ I1 AND SUŁKOWSKI WW2
Lundbec k Res earch D K, Ottiliavej/9, DK 2500 Valby, Denmar k
                                                                                               1
                                                                                                 Department of Physical Phar mac y, F aculty of Pharmac y, Medic al Uni versity of
Selecti ve Ser otonin Uptake Inhibitors (SSRI‘s) are drugs (bullets) that are targeting        Silesia, Sosnowiec, Poland
                                                                                               2
the serotonin trans porter in order to bl oc k the reuptake of the neurotransmitter              Department of Environmental C hemistry and Tec hnology, Institute of Chemistr y,
serotonin. Escitalopram has , in contras t to other SSRI drugs been sho wn to                  University of Silesia, Katowice, Poland
interact with the s erotonin trans porter at two different sites: the primar y bindi ng site
shared with other SSRI‘s and a separate allosteric binding site. A consequenc e of             Background: In the body drugs are transported mos tly vi a circulator y s ystem as
this unique allos teric interacti on is a prolonged diss ociation half-life from the           the c omplexes with albumin. This is an i mportant part of the drug metabolism
primar y bindi ng site resulting in ver y efficient inhi bition of the ser otonin uptake.      since the bound fraction of a drug has no phar macol ogical effect. The
Escitalopr am is therefore due to this s elf-enhancing effec t a Magic Bullet which            simultaneous bi nding of other exo- and endogenous ligands (such as drugs or
has been termed Allosteric Serotonin U ptake Inhibitor (ASRI).                                 fatty acids) can alter protei n affinity towards drug and change the conc entration of
                                                                                               its free frac tion. In our study the influenc e of c ytarabine (araC) and myristic acid
Escitalopram is the active enantiomer of the racemic drug citalopram. While the R-             (MYR) on aspirin (ASA) bi nding with defatted bovine serum albumin (dBSA) was
enantiomer originally was believed to be inactive, recent investigations have                  investigated.
surprisingly show that the R-enantiomer inhibit the effect of the S-enantiomer.                Methods: NMR spectra were recorded on Bruker Avance 400 spec trometer using
Pharmacological and clinical studies have shown a clear tendenc y for faster onset of          5 mm tubes . For water signal suppression the pres aturation method was us ed. All
antidepressant action and more than two-fold higher potency of escitalopram compared           solutions were prepared i n D2O. The chemical s hifts of proton resonances were
with citalopram.                                                                               calculated in relati on to DSS signal (0.05 ppm).
                                                                                               Fluorescence emission s pec tra were recor ded on Kontron SFM 25
The pres entation will foc us on c omparing the profile of escital opram/ASRI with             spectrofl uorimeter at excitation wavelength of 280 nm. Binding constants Ka were
SSRI‘s and discuss the mec hanism behind and the cons equences of the inhibition               calculated with the us e of Sc atchard method. All meas urements were obtained at
of S-cital opram by its R-enantiomer. A molec ular model of the serotonin                      310K.
transporter will be presented as well as possibl e mechanisms i nvol ved in the                Results: The association constants calculated for the first (subdomain IIA) and
allosteric interaction.                                                                        second (subdomain IIIA) class of ASA bindi ng site is 84,2x103M -1 and 1,81x103M -
                                                                                               1
                                                                                                 , respec tivel y.
                                                                                               1
                                                                                                 HNMR spectra show that dBSA and araC i nduce c hanges in c hemical s hifts of
                                                                                               ASA proton resonances. The effect is more evident for the aromatic ring of ASA
                                                                                               participati ng in the for mati on of the c omplex than for its methyl group. C hanging of
                                                                                               chemical shifts of ASA proton resonances are not enough significant to obs erve
                                                                                               differences i n drug binding in the pres enc e of MYR.
                                                                                               Spectr ofluori metric anal ysis indic ate that above [MYR]/[dBSA] mol ar ratio 4:1 KaI
                                                                                               and KaII of ASA- dBSA compl ex decreas e approxi matel y by 50 and 35% .
                                                                                               Conclusions: 1. ASA is displac ed by araC in both high and l ow affi nity bi nding
                                                                                               sites. 2. Bindi ng of MYR with albumi n infl uences formation of ASA compl exes with
                                                                                               albumin. This effec t depends on MYR:dBSA molar r atio. 3. In cas e of multidrug
                                                                                               therapy or hyperlipidemia the monitoring therapy may be nec essar y.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                 Page A-41
                                                              EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                    Celebrating the 100th Anni versar y of the
                                                                       Nobel Prize Award to Paul Ehrlich
                                                                           Nürnberg, October 3-5, 2008
Current syst emic and lo cal therapies of intra- abdominal sep sis                        A gen eric, multidim ension al SPE-platform for undisturbed LC-MS/MS
                                                                                          analysis of basic drug s in native b iofluids
BONDAR VM
                                                                                          BOOS K-S, MORELLO R
Department of Surgery, C offee Regional Medical Center, Medical College of
Georgia, Douglas, Georgia, USA                                                            Laboratory of BioSeparation, Institute of Clinical C hemistry, Medic al Center of the
                                                                                          University of Munich, Munich, Ger many.
Abstract
Intra-abdominal sepsis (IAS) or infection is defined as inflammation of the               Background: In bioanal ytical LC-MS electros pray ionizati on is ver y susceptibl e to
peritoneum due to pathogenic microorganis ms and their products . IAS is not a            so called matri x effects that are ca used by coeluting low (LMW) and high (HMW)
local disease, affecti ng the entire body, its organs and s ystems, producing             molec ular weight sampl e components. Due to the natural variati on of endogenous
systemic inflammatory respons e s yndrome (SIRS). A favorable outc ome for IAS            compound concentrations in biofluids s uch as urine and blood pl asma withi n an
depends on the ability of host defenses to overcome pathogenic microorganis ms.           indivi dual, between i ndi viduals and between species, matrix effects are hardl y
The c urrent treatment of IAS consists of: 1. s urgical control of the source of          predictable. In order to mi nimize matrix effects we tr eated the native bi ofluids by
infection, 2. reduction of intra-abdominal bacterial i noc ulum and 3. supportive         on-line multidi mensional Solid Phase Extracti on (MD-SPE) prior to on-line LC-
measures. T he pharmacological and mec hanical means for combating intra-                 MS/MS.
abdomi nal bacteria and the c ombination of the abo ve measures will be reviewed.         Methods: The MD-SPE platform (High SPEed, CTC Anal ytics) relies on the
The sys temic and loc al strategies to treat primar y, secondary and tertiar y for ms of  combinati on of two SPE columns . The first SPE column (25 x 2 mm ID) is packed
IAS will be discuss ed.                                                                   with Res tricted Acc ess Material (RAM; LiChrospher ADS RP 4, dp 25 µm, Merc k
                                                                                          KGaA) which allows size-exclusion (SEC: 1st dimensi on) and reversed phas e
                                                                                          chromatography (RPC: 2nd dimension). The sec ond SPE column (20 x 2.1 mm
                                                                                          ID) contai ns a mi xed-mode phase (MMP; Oasis WCX, dp 30 µm, Waters) which
                                                                                          allows weak cation exchange (IEX: 3rd di mensi on) and hydr ophobic interaction
                                                                                          chromatography (HIC: 4th dimension). Anal ytic al separation of the model anal ytes
                                                                                          (antidepressants) was achieved on a Zorbax RX C18 column (75 x 4.6 mm ID, dp
                                                                                          3.5 µ m, Agilent). A Quattro Micro MS ( Waters) was us ed in ESI(+) MRM mode.
                                                                                          Matrix effec ts were monitor ed in post-column i nfusi on experiments.
                                                                                          Results: The RAM-SPE column repeatedl y and ver y efficientl y depl eted pl asma
                                                                                          samples (20 µL) with regard to HMW matrix components suc h as proteins and
                                                                                          quantitativel y extracted the target anal ytes (pka value higher than 6.5) in less than
                                                                                          60 sec. After trans fer onto the MMP-SPE column residual LMW matri x
                                                                                          components (< 15 KDa) were eluted to waste within 60 s ec. This extensi ve and
                                                                                          highly s electi ve sampl e clean-up res ulted in almost no detectable matri x effec ts. In
                                                                                          additi on, MS-scans (m/z range from 100 to 1000) of blank plas ma and urine from
                                                                                          different indi viduals and species revealed no masses , i.e. no residual matri x
                                                                                          components [1].
                                                                                          Conclusion: A c ombination of orthogonal chromatographic separati on modes , i.e.
                                                                                          SEC-RPC-IEX-HIC, for on-line SPE of native bi ofluids s uch as plas ma and urine
                                                                                          completel y eliminates matri x effects in LC-ESI-MS/MS detection of basic drugs.

                                                                                            [1] K.Georgi, K-S Boos, C hromatographia 63 (2006) 523-531




Control of ceramid e levels b y ceram ide kinase: evidence from knockout                    Discover y of novel non-cyclam polyn itrogenated CXCR4 cor eceptor
animals and u se of a novel potent inhibitor                                                inhibitors

GRAF C, BOATH A, ROVINA P, SCHANZER A, NIKOLAY R, REUSCHEL, R,                              PETTERSSON S1, PÉREZ-NUENO V1 , ROS-BLANCO L1, C LOTET B2, CLOT ET-
NIWA, S, BORNANCIN F.                                                                       CODINA I2, ARMAND-UGÓN M 2, EST É J2, TEIXIDÓ J1 , BORRELL JI1
                                                                                            1
Novartis Institutes for BioMedical Research, Brunnerstrass e 59, A-1235 Vienna               IQS, Uni versitat Ramon Llull, Barcelona, Spain; 2R etrovirology Laborator y
(Austria)                                                                                   IrsiCaixa, Badal ona, Spain.

Background: The s phingolipi d ceramide is an i mportant regulator of c ell biolog y        Backgro un d: CXCR4 and CCR 5, chemokine coreceptors of the primary recepto r (CD4 )
(e.g. apoptosis, differentiation) and its l evels therefore mus t be tightl y controlled.   for the HIV cell fusion and entry, have been validated as targets fo r therapeu tic
Ceramide ki nas e (CerK) is a unique enz yme that s pecificall y phosphor ylates            intervention against AIDS. Bicyclams were the first non peptidic low molecular weight
ceramide into the bi oacti ve lipid c eramide-1- phos phate (C1P). Rec ent data from        compounds with specific interaction with CXCR4, the most potent bicyclam being
our laborator y have provided compelling evi dence that CerK is a key regulator of          AMD31 00 (IC50 = 1-10 ng/mL ). Howe ver, it showed poor o ral absorption and toxicity
both C1P and c erami de l evels.                                                            related to its high positive charge at physiological pH.
                                                                                            T o overcome such problem, we designed a combinatorial library of tetraamines 1, which
Methods: CerK-deficient (Cerk–/–) Balb/C mic e were generated as described in
                                                                                            preserve the main features of AMD31 00: a) at least two nitrogen atoms on each side of
Graf et al. J Immunol 2008, 180:3457- 66, and c ompar ed to control liter mates in all      the p-phen ylene moiet y, one in the ben zylic position and the other(s ) in a hete rocyclic
studies . The diamino-benz othi azole deri vati ve N VP-231 was s ynthetized as             system and b) similar distances between suc h nitrogen atoms with those present in
decribed in Graf et al. Mol Pharmacol 2008 74(4), in press. CerK acti vity ass ays          cyclam.
using either fluoresc entl y label ed cer amides or 32P-AT P were described in Boath        Me th ods: 19 compounds we re initially selected by evaluating a series of molecular 2D
et al, J Biol Chem 2008 283:8517- 26 and Rovina et al, Bi ochem J 2006 400:255-             and 3D descriptors. A PCA reduced the initial set of descriptors to 5 components which
65, res pecti vel y. Growth and c ulture of COS-1 c ells and bone marrow deri ved           were used fo r the di versity selection. Anti-HIV activity (EC50 ) and cyt otoxicity (CC50 )
macrophages as well as Liquid C hromatography/Mass Spectrometr y (LCMS)                     measurements were carried out in MT -4 cells infected with HIV-1.
anal ysis was performed as decribed in Graf et al. J Immunol 2008, 180:3457- 66.            Results: T he first subset of compounds showed EC 50 in the range 0.9-18  g/mL. A
Results: First, using a fluor escently labeled ceramide molecul e to trace CerK             second subset of 17 compounds afforded 1 2 compounds presenti ng EC50 in the range
                                                                                            0.2-2.7  g/mL. T he third and final subset, covering up the total of 53 synth esized, was
activity we found that C1P is short-lived compar ed to other ceramide metabolites,
                                                                                            selected using QSAR techniques and ligand and structure -based drug design (using our
thus fulfilling an essential criteria for signaling function. This suggested that CerK
activity might be useful to dis pos e of exc ess ceramide. Then we pr ofiled C erk–/–       CXCR4 an d C CR5 m odeled corecepto rs). Among the m, 1 {8,8} showed an EC50 value of
                                                                                            0.008  g/ mL and a CC50 > 25  g/mL, presenting nearly the same activity as AMD3100
mice and we found indeed that ablation of CerK not onl y decreas es C1P levels but          but showing no cell toxicity at tested concentrations.
also leads to an increase in c erami de levels. Finall y, we identifi ed and                Concl usio ns: 1) A diversity oriented selection has allowed the syn thesis of tetraamines 1
characterized NVP-231, the first potent, specific and reversible CerK inhibitor.            covering a broad ran ge of activity values, useful fo r QSAR calculations. 2) T his approach
Consistent with the obser vati ons i n Cerk–/– mice, prelimi nar y experiments with         afforded compoun d 1{8 ,8}, with an E C50 value of 0.008  g/mL and a C C50 > 25  g/mL. 3 )
NVP-231 s uggest that C erK inhibition may have potential to regulate C1P and               Studies on the mode of action of compounds 1 showed specific inhibition of the CXCR4
ceramide levels.                                                                            coreceptor.
Conclusions: Thes e results establish CerK as a novel, key regulator of ceramide                                                                  N   nN
                                                                                                                                                       H             H
levels and s upport further exploration of CerK inhibiti on as a r ationale for the                                          NH HN                                   N       N
treatment of pr oliferati ve disorders.                                                                   NH   N                                              1          n
                                                                                                                             N   HN
                                                                                                          NH HN                               N        N
                                                                                                                                                       H             H
                                                                                                                                                                     N           N
                                                                                                                   AMD3100
                                                                                                                                                            1{8,8}

                                                                                            Reference s
                                                                                            - S. Pettersson, V. Pérez-Nueno, L. Ros-Blanco, R. Puig de La Bellacasa, M. O. Rabal, X.
                                                                                            Batllori, B. Clotet, I. Clotet-Codina, M. Armand-Ugón, J. Esté, J. I. Borrell, J. Teixidó, Chem.
                                                                                            Med. Chem. 2008, DOI 10.1002/cmdc.200800145 (July 31)
                                                                                            - S. Pettersson, L. Ros-Blanco, J. Teixidó, X. Batllori, R. Puig de La Bellacasa, J. I. Borrell, S.
                                                                                            Nonell, M. O. Rabal, V. Pérez -Nueno, J. Esté, I. Clotet-Codina, M. Armand-Ugón, WO
                                                                                            2008/049950 (02.05.2008)




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                            Page A-42
                                                        EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                               Celebrating the 100th Anni versar y of the
                                                                  Nobel Prize Award to Paul Ehrlich
                                                                    Nürnberg, October 3-5, 2008
Heparin as an inhibitor of cancer progression; the role of selectins                 According to registration: BASU Chinm ay

BORSIG L1, VARKI A2                                                                             Alter ations in F emale Reprodu ctive Organ s of C yclic R ats Treated with
                                                                                                Aqueous Extract of Moringa Oleifer a Lam: Indication of Po ssib le Role in
1                                        2
    Univ. of Z ürich, Zürich, Switzerland; Univ. of Californi a, San Diego, U.S.A.              Epithelial Ovarian Cancer

Background: Heparin and low molecul ar weight heparin ( LMWH) are widel y used                  BOSE CK
in canc er patients at ris k of venous thromboembolis m, whic h is a recognized
complication of malignant disease. Rec ent clinical trials with LM WH and meta-                 Cancer Clinic, Cancer Nirnaya O C hikits a Kendra, Kol kata, India.
anal ysis of earlier clinic al trials with unfracti onated heparin indicate that heparin
affec ts also c anc er progression. Meanwhile, heparin and LMWH were r epeatedl y               Background: A hormonal etiology of epithelial ovarian c ancer has long been
shown to reduce metas tasis i n a variety of ani mal models. Heparin is a natural               suspec ted seeing its incidence menopausal age, and now the role of FSHR has
produc t, c omprising a pol ydisperse mi xture of highl y sulfated glyc osaminoglycan           also been demons trated. Many ovarian cancer cell line express FSHR in them. In
chains, onl y a fr action of which bind antithrombi n.                                          studies of the antic anc er potential of plants us ed in fol k medicine of Bengal,
Methods: Experimental metastasis study was performed in wild type as well as in                 extracts of plants suc h as Oroxylum indicum, Moringa oleifera lam, Aegles
P- and/or L-selec tin deficient mice. Treatment with heparin or their derivati ves was          marmelos c ould be c onsidered as potenti al sources of antic ancer c ompounds.
perfor med onl y shortl y before or after the application of tumor cells. The extend of         Amongst them onl y Moringa oleifera lam has unique anticancer as well as
metastasis was evaluted after 28 days.                                                          hormonal property, whic h may or may not be attributable to is othi oc yanate or
Results: We provided evi dence that heparin has an additional biol ogical acti vity             glucosinolate that it contai ns. An ani mal experi ment was planned to see effect of
which inhi bits binding of P- and L-selecti n to their natural ligands . Sinc e also            Moringa root extract i n female reproducti ve s ys tem of mice.
modified heparins without any anticoagulant acti vity were found to attenuate                   Methods: 5 adult female mice of s wiss strain of 30 gm each 2-control, 3-treated
efficiently metas tasis, the heparin effect on c anc er progression may not be                  kept on stoc k diet, pellet, havi ng nutritional value of 7 daysAn aqueous extract of
principall y due to inhibition of coagulation. In fact, we have demons trated that the          the root was prepared accordi ng to a traditional method. In brief, 500 g of the root
selectins play critical roles during the hematogeneous phas e of carcinoma                      were plac ed i n a contai ner with 750 ml of water and boiled for 30 mi n. The
metastasis in ani mal models. Natural selecti n ligands on carcinoma cells were                 prepar ation was left standing to cool and was then filtered. T he filtrate, containing
identifi ed as mucins carryi ng sialyl ated, fucos yl ated and sulfated c arbohydrate           66.7 mg root in 1 mL, was plac ed i n s mall vials and kept in 4 degree C refrigerator
structures. H eparin treatment i n wild type mic e resulted in attenuati on of                  until us e. 1 ml of extrac t was used orall y dail y for 45 days .
metastasis si milar to the one obs erved either P- and/or L-sel ectin i n mic e,                Results: Attenuation of ovar y and uterus was seen while mice tolerated the herb
indicating that inhi bition of s electi ns significantl y c ontributes to the anti -cancer      extrct well. There was reversal to pre estrus phase of adult mice as was revealed
effec t. Inhibition of selecti n-mediated i nterac tions with carcinoma c ells was found        by PAP s mear from vagina. In histolog y there was abs enc e of follicles in
to be critical for earl y stages of a metas tatic c ascade. Most i mportantl y,                 comparison to c ontrol ovary. There was l esser amount of fibrosis i n treated ovary.
attenuation of metastasis can be achi eved at clinicall y acc eptabl e dosages. In              Conclusion: Isothioc yanate of Moringa may inhibit proliferation of ovarian
additi on, heparin contains als o other biological acti vities incl uding inhibition of         granulosa and other cells as it induc es apoptosis vi a caspase-9 and -3 pathways,
heparanase, modulation of chemokines and growth fac tors ac tiviti es.                          a family of calcium- dependent c ystei ne proteases. It may also act by inhibiting
Conclusions: The availabl e evidence from preclinic al anal ys es, together with the            ERK1/2 and Akt sur vi val signaling while simultaneousl y acti vating pro-apoptotic
promising obser vations from clinical trials, merits further inves tigation of heparin as       p38 and JNK1/2. There are reports to show that Moringa induc ed decreas e in
a potential anti-metastatic therapy.                                                            cerebral dopamine and norepi nephrine which may i nfluence and lower NGF and
                                                                                                FSHR through central mechanis ms. There is strong possibilty of using this agent
                                                                                                in epithelial ovarian cancer and, as such, a cell line experiment is urgentl y
                                                                                                necess ary.




Serum and Alveolar Concentrations of Antibiotics during the Treatment of                        Therapeutic effects of Crocus sativu s (saffron) on respir ator y diseases
Ventilator-Asso ciated Pneumonia
                                                                                                BOSKABADY MH, ASLANI MR, AHMADZADEH H, GHASEMZADEH RAHBARDAR
BOSELLI E1 ,2, BR EILH D 3,4 , ALLAOUCHICHE B1,2                                                M, NEMATI A
1                                            2                              3
 Hosp. Édouard Herriot, Lyon, Franc e; Uni v. of Lyon, Lyon, France; Hosp. Haut-                Dept. of Physiol ogy and Pharmacological Res earch Centre, Medical Sc hool,
Lévêque, Pessac , Franc e, 4Uni v. of Bordeaux II, Bordeaux, Franc e.                           Mashhad Uni versity of M edical Scienc es, Mas hhad, Iran

                                                                                                As indicated in ancient Iranian medical books Crocus sativus (Iridaceae) or saffron has therapeutic
Background: We ass essed the ser um and al veolar c oncentrations of anti biotics               effects on respiratory diseases. T herefore in a series of experiments, the follwing effects of
                                                                                                cumulative concentrations of saffron and its constituent, safranal was examined on tracheal chains
frequentl y us ed during the treatment of ventilator-ass ociated pneumonia (VAP) in             of guinea pigs: relaxant, stimulatory effect on -adrenoceptors, inhibitory effect on histamine (H1)
critically ill patients in order to optimiz e anti microbial treatment in this particular       receptors and the inhibitory effects on muscarinic receptors
population.                                                                                              In precontracted trachea by 10 µM methacholine (roup 1) all concentrations of
Methods: Various antibi otics (β-lactams, tobramycin, levofloxaci n or linezolid)               theophylline, extract and safranal showed significant relaxant effects compared to that of saline
were admi nistered in intermittent or continuous infusion to critically ill patients with       (p<0.05 to p<0.001). In precontracted trachea by 60 mM KCl (group 2) also theophylline, extract
                                                                                                and safranal showed concentration dependent relaxant effects compared to that of saline (p<0.05
VAP. At steady-state (after two days of therapy), blood s amples were withdr awn                to p<0.001 for different concentrations except two low concentrations of safranal). However, in
from each patient and serum c oncentrati ons were meas ured by high-performanc e                group 3 (precontracted incubated tissues with propranolol, chlorpheniramine and atropine by 60
liquid chromatography. Simultaneously to blood s ampling, antibi otic concentrations            mM KCl) the extracts of Crocus sativus showed a weak relaxant effect (p<0.05 only for highest
were deter mined in epithelial lining fl uid (ELF) obtained from standardiz ed mini -           concentration).
bronchoal veolar lavage, which is a reliabl e method for the measurement of                              T here were clear leftward shifts in isoprenaline curves obtained in the presence of only
                                                                                                higher concentration of the extract in group 1(non incubated tissues) and it‘s both concentrations in
alveolar drug conc entrations.                                                                  group 2 (incubated tissues with chlorpheniramine) compared with that of saline. T he EC 50 (the
Results: The 122 patients were of similar age (50-70 yrs), weight (60-80 kg) and                effective concentration of isoprenaline, causing 50% of maximum response) obtained in the
creatinine clearanc e (60- 100 mL/min). The anti biotic daily dose for each patient,            presence of both concentrations of the extract and safranal in group 1 and only in the presence of
administered in i nter mittent or c onti nuous infusion, the s erum and ELF                     two concentrations of the extract in group 2 was significantly lower compared to saline (p<0.05 to
concentrati ons and the ELF/s erum conc entration ratios appear in T able 1.                    p<0.001). The max    imum responses obtained in the presence of both concentrations of the extract
                                                                                                and safranal in group 1 were significantly lower than that of saline (p<0.005 for all cases).
Conclusions: Ser um and al veol ar conc entrations and al veolar diffusion of                            EC50 histamine obtained in the presence of chlorpheniramine, all concentrations of the
antibi otics from both different and similar class es exhibit wi de variations in criticall y   extract and safranal in all three groups (incubated trachea with: 1) indomethacin, 2) indomethacin,
ill patients with VAP. Indi vidual antibiotic dos ages may be helpful to optimiz e the          propranolol, and atropine and 3) indomethacin and propranolol) were significantly greater than
administration regimen of antibi otics and therefore PK/PD parameters ( T>MIC ,                 those of saline (p<0.05 to p<0.001) except medium concentration of the extract in group 2 and its
Cmax /MIC and AUC/MIC) during the treatment of VAP.                                             low concentration in group 3 (0.5 and 0.25 mg/mL respectively). The EC50 obtained in the
                                                                                                presence of all concentrations of extract and safranal in group 2 were greater than group 1 and 3
                                                                                                (p<0.05 to p<0.001). Maximum response obtained in the presence of all concentrations of extract
Table 1. Mean steady-state s erum and ELF antibi otic concentrations                            and safranal in group 2 were greater than those of group 1 and group 3 (p<0.05 to p<0.001).
                                                                                                         T he results of Inhibitory effects on muscarinic receptors showed clear parallel rightward
                                          Serum concentration (mg/L)      ELF
                                                                                  ELF/serum     shifts in methacholine-response curves obtained in the presence of atropine, 2 low concentrations
        Antibiotic (dose/day)      n      Intermittent     Continuous    conc.                  of safranal and extract compared with the curves obtained in the presence of saline. The EC 50
                                                                                  conc. ratio
                                         Cmax       Cmin       Css      (mg/L)                  obtained in the presence of atropine, two lower concentrations of safrana l and all concentrations
           Cefepime 4 g            20     -           -       13.5       14.1        1.04       of the extract was significantly higher than that of saline         (p<0.01 to p<0.001). T he maximum
                                   15                                                           responses obtained in the presence of all concentrations of the extract was significantly lower than
          Ceftazidime 4 g                 -           -       39.6       8.2         0.21
                                                                                                that of saline (p<0.01 to p<0.001).
          Ertapenem 1 g            15    30.3        0.8       -         9.4         0.32
                                                                                                         T hese results showed a potent relaxant effect of saffron, a relatively potent stimulatory
    Levofloxacin 500 mg qd         12    12.6        3.0       -         11.9        1.18       effect of the extract from Crocus sativus on ß 2-adrenoceptors, an inhibitory effect at histamine H 1
    Levofloxacin 500 mg bid        12    19.7        7.7       -         17.8        1.27       receptors and a possible inhibitory effect at muscarinicreceptors on tracheal chains of guinea pigs.
     Linezolid 600 mg bid          16    17.7        2.4       -         14.4        1.05       T he results also indicated that the safranal is, at least in part, responsible for the relaxant effect of
Piperacillin/tazobactam 12/1.5 g   10     -           -       25.4       12.7        0.46
                                                                                                Crocus sativus.
 Piperacillin/tazobactam 16/2 g    10     -           -       38.9       19.1        0.43
    Tobramycin 7-10 mg/kg          12    22.4         -        -         2.6         0.12




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                Page A-43
                                                             EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                  Celebrating the 100th Anni versar y of the
                                                                    Nobel Prize Award to Paul Ehrlich
                                                                       Nürnberg, October 3-5, 2008
Different therapeutic effects of Nigella sativa                                         Rediscovering antibiotics of altern ative medicine: case of apitherap y

BOSKABAD Y MH, MOHSENPOOR N, TAKALOO L, JAVAN H, FARHADI J                               BOUKRAA L; H AMMOUDI SM; BENB AREK H; BENHANIFIA MB; AISSAT S;
                                                                                         AHMED M; MESLEM A
Dept. of Physiol ogy, M edical School, Az adi Square, Mashhad Uni versity of
Medical Sciences , Mashhad, IR Iran                                                      Laboratory of Microbi ology, Department of Veterinary Scienc es, Tiar et Uni versity,
                                                                                         Algeria
In a series of studies the following effec ts of Nigella sativa on guinea pig tracheal
chains and human airways were studied.                                                   Background: Antibiotic-resistant bacteria continue to be of major health c onc ern
The res ults s howed: (1) Aqueous and mac erated extracts from Nigella sativa            world- wide. Sinc e the us e of anti biotics bec ame wides pread, bac teria have
showed significant relaxant effec t compared to s aline ( p<0.001). The extr acts        progressivel y developed resistance. Consequentl y, scientific efforts have been
caused a non parallel rightwar d shift in methac holine conc entration respons e         made to devel op new compounds to be us ed beyond conventional anti biotic
curve and EC 50 methacholine obtained i n the presence of extracts were                  therapy. It has been propos ed that the healing effect of bee pr oducts could be due
significantly greater than that of s aline (p<0.05 to p< 0.001). (2) plant extr acts     to various physical and chemical properties. H ydrogen peroxide, volatiles, organic
caused parallel right ward s hifts in histamine c onc entr ation res ponse c urves       acids, flavonoids, bees wax, nectar, pollen and propolis are, among others, the
obtained c ompared to s aline. The EC 50 histamine i n the pres ence of extracts were    chemical fac tors that provide antibacterial properties.
significantly greater than slain (p<0.05 to p<0.001). (3) There was a l eftward s hift   Methods: Bacteria of medic al conc ern s uch as Ps eudomonas aeruginos a
in isoprenaline c onc entration res ponse cur ve in the pres ence of aqueous extrac t.   Escherichia coli and Methicillin Resistant Staphyloc occus aureus were subj ected
(4) Different extr acts showed significant relaxant effec t on tracheal chains           to the action of bee products; namel y honey, propolis, royal jelly and bee venom.
incubated with Chlor phenirami ne and propranolol, contr acted by methac holine          In the majority of cas es, the mi nimum inhibitor y concentrations (MICs) were
(p<0.01 to p<0.001) but did not s how any relaxant effect on trac heal contracted by     obtained using the incorporation method. The MIC was determined by finding the
KCl. (5) Aqueous extract caused a rightwar d shift in the CaCl 2 res pons e c urves      plates with the l owest c onc entration of honey on which the strain would not grow.
and EC 50 CaCl 2 hin the pres enc e of axtrac t was significantly greater than the       Results: all bee produc ts have shown antibac terial acti vity. Sensiti vity of bacteria
presenc e of saline (p<0.05). (6) However, thymoquinon, main c ons tituent of the        to bee products varies considerabl y within the product and the varieties of the
plant di d not s how any significant relaxant effect on trac heal chains . (7) Boiled    same product. Propolis has been found to have the strongest action agai nst
extract of this pl ant c aus ed significant increas es in all meas ured pul monar y      bacteria. This is probabl y due to its richness i n flavonoids. Anti biotic-resistant
functi on tests (PFTs), (p<0.05-p<0.001) comparabl e to the effect of thophylline. (8)   bacteria have been found to be sensiti ve to the action of hi ve products .
Two months administration of boiled extract also c aused significant impr ovement        Conclusion:
in PFT val ues, res pirator y s ymptoms , ches t wheeze and dr ug usage in asthmatic             The frequent use of antibi otics has lead to the emergenc e of antimicrobi al
patients. (9) Concentrations of extracts of the plant showed significant reduction of             resistance.
cough number (p<0.001 for all cases) whic h wer e significantl y greater than that of            Honey and other bee produc ts were subjected to laborator y and clinic al
codei ne (p< 0.05-P<.001).
Results showed a potent relaxant effec t of Nigella sativa on trac heal chains , a                investigations during the past few dec ades and the mos t remarkable
relativel y potent bronchodilator y effect on asthmatic airways and a potent                      discover y was their anti bacterial acti vity.
antitussive effec t.                                                                            The emergenc e of antibiotic resistant bacteria, particularl y methicillin-
                                                                                                 resistant Staphyloc occus aureus (MRSA) has pos ed problems in the
                                                                                                 management of c hronic wound infections. Many studi es have shown that
                                                                                                 application of hi ve products to severel y infected c utaneous wounds is
                                                                                                 capable of clearing infec tion from the wound and i mprovi ng healing.
                                                                                                Bee products are natur al products; without adverse effects on tiss ues,
                                                                                                 they can be s afel y used on burns and ins erted into c avities and sinuses to
                                                                                                 clear infec tion.




Neurotensin Agonists: Novel Analg esics with Syn erg y to Morphine                       Current Medical Countermeasures (Vaccines- Antibodies- Antibiotics) to
                                                                                         Protect Humans from the Anthrax Bioter rorism .
BOULES M 1, SHAW A1, BARBUT D 2, RICHELSON E1
                                                                                         BOUZIANAS DG
1
 Mayo Clinic, Jac ks onville, F L, USA ; 2Sarentis T herapeutics, New Yor k City, NY,
USA                                                                                      Technological Educational Ins titute of Thessaloni ki, Thessaloniki-Macedonia,
                                                                                         Greece.
Background: Neurotensin (NT) is a widely distributed neuropeptide in the central
ner vous s ystem that modul ates nociception at several different levels, but is         Background: B. anthracis spore attac ks through the US mail s ystem have
associated with hypotensi on and hypother mia. NT exerts its effec t through NT          demons trated their feasi bility as a bioterrorism weapon. Vacci nati on appears to be
receptors, of which there are thr ee known subtypes (NTS1, NTS2 and NTS3).               the most effecti ve and ec onomical form of mass protecti on, however, the current
Morphine is a µ-opioi d receptor agonist that is commonly us ed for the treatment of     vacci nes have drawbac ks that jus tify the immense efforts for the development of
many types of pain, but it is us uall y ass ociated with side effects that can be        improved treatment modalities. T his review summarizes the current human
serious. We hypothesize that sel ecti ve NT rec eptor agonists may represent a           approaches devel oped mai nly since the 2001 events against inhalati onal anthrax.
novel class of analgesics and their use in conjunc tion with morphine will have          Methods – Results: The increased research acti vity has led to a huge expansion
synergistic properties with morphine which may reduc e the dos e administer ed and       in the existing literature. The pr esent work is bas ed on an extensive review of the
its side effec ts.                                                                       international literature. Combination of postattac k pr ophylactic vaccination with
Methods: Studies wer e done to test for the use of a novel peptide analog of NT          antibi otic therapy is the most effecti ve strategy. C urrent appr oaches agai nst
(NT69L) as a new class of analgesic drugs, ac ting through the NT rec eptors, alone      anthrax toxins are focus ed on agents that affect cr ucial steps of the intoxication
and in combination with morphine. The anti nocicepti ve acti vity of NT69L and           process. High-affinity toxi n-specific monoclonal antibodi es have a significant
morphine was studied in rats using the hot plate test to deter mine if there is          clinical effec t, if they are administrated rapidl y. While effec tive antibi otics,
synergism between the two drugs in reducing pain. The NTS2 r eceptor                     antitoxins and vacci nes are available, concerns over their safety and effec tiveness
antagonist, levocabastine was used to deter mine the receptor subtype invol ved in       have driven the development of 2nd and 3rd generation products that act rapidl y
the s ynergistic effect of NT69L and morphine.                                           and with mini mal adverse effec ts. Protec tive antigen (PA) is the princi pal
Results: The administr ation of both NT69L and morphi ne res ulted in a dose-            immunogen of the 1st generation licens ed vaccines . A 2nd generati on vacci ne is
dependent analgesic effect. Isobolographic anal ysis was us ed to study the              based on highl y purified r ecombinant PA (rPA) and is likel y to recei ve licensing
antinociceptive inter actions between the two drugs. The isobolographic anal ysis        approval. T he 3rd generation vaccines aim to enhance the effic ac y of the previous
demons trated that the combination of sub-analgesic dos es of NT69L and                  vacci nes. T hey would ideally be given via the oral, nasal or dermal routes for
morphine was s ynergistic in the hot plate test. Pretreatment with the NTS2              deliver y of rPA in a single dose facilitating stoc kpiling and mass vaccination
receptor antagonist, levoc abastine, attenuated the synergistic effect of NT69L and      programs. DNA vaccination coul d form the basis for multiagent vaccine
morphine in the hot plate test.                                                          devel opment. The development of novel agents is hamper ed by the difficulty in
Conclusion: The res ults pr ovide preliminary data s upporting the hypothesis that       demons trating effec tiveness in humans.
the s ynergistic combi nati on of NT69L and morphine would i mprove the                  Conclusions: Treatment r esponse to a deliberate release of B. anthracis spores
phar mac ological treatment of pain while minimizing specific adverse effects of         includes the pr ompt admi nistration of antibiotics. There ar e conc erns for the
each of the drugs at a higher dose. Both NT receptors NTS1 and NTS2 are                  availabl e antitoxins and vaccines over their effec tiveness and toxicity. Des pite the
important for the s ynergisitic effect of NT69L and morphine.                            intensi ve anthrax res earch, there has been as yet no real progress. The huge
                                                                                         efforts are expected to provide an array of novel protecti ve age nts and their
                                                                                         balloni ng list reflects the emergenc y of the global community to combat the anthax
                                                                                         threat.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                          Page A-44
                                                 EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                        Celebrating the 100th Anni versar y of the
                                                          Nobel Prize Award to Paul Ehrlich
                                                             Nürnberg, October 3-5, 2008
Cefepime Neurotoxicit y in Persp ective                                       Poor permeabilit y of blood brain b arrier for cr eatin e: Autonomous brain
                                                                              synthesis of cr eatin e, and consequences for creatine d eficiency syndrom es
BRAGATTI JA1, SILVA D 1, CASTILHOS RM 1, AZAMBUJA M 1, GARCÉS EO1 ,
           2             2              2
BARROS EG , THOMÉ FS , BIANCHIN MM                                            BRAISSANT O
1
    Hospital de Clínic as de Porto Alegre, Brazil                                                                                Inborn Errors of Metabolism, Clinical Chemistry Laborator y, Centre H ospitalier
2
    Universidade Federal do Rio Grande do Sul, Porto Al egre, Brazil                                                             Universitaire Vaudois and U niversity of Lausanne, 1011 - Lausanne, Switzerland.

                                                                                                                                 Abstract :
Background: Cefepi me, a widel y us ed antibiotic, is mor e effecti ve than other
cephalosporins against many Gram positive and negati ve microorganisms , and                                                     In mammals, creati ne (Cr) is taken up from the diet, or can be s ynthesized
has a main rol e in the treatment of febrile neutropenia. Its safety profile was                                                 endogenousl y by a two-step mec hanism invol ving L-arginine:glycine
considered adequate, but increased mortality was recentl y found with its use. M ost                                             amidi notransfer ase (AGAT), whic h yiel ds the i nter mediate guanidinoacetate
common neurol ogical side effects are s omnolence, dis orientation, hallucinations,                                              (GAA), and guanidinoac etate methyltransferas e (GAMT), which converts GAA to
and epileptic seizures . Jall on et al. firstly r eported on 19 patients who devel oped a                                        Cr. Cr is distributed through the bl ood and is taken up by cells with high energy
confusional state that reverted after cefepime disc onti nuation. Cefepi me?s                                                    demands through a specific Cr trans porter, SLC6A8. It was thought for a long time
neurotoxicity cl earl y res ults from its accumulation in the central ner vous s ys tem                                          that mos t, if not all, of the Cr needed by the brai n comes from the peripher y
(CNS). Excessi ve dosage and impaired renal clearanc e are important risk factors,                                               through blood brain barrier (BBB). However, our rec ent work has shown that
as well as ur emia, extreme ages, and meningitis. Our group employed three                                                       AGAT and GAMT are express ed i n CNS and that brain cells in vitro s ynthesize
studies regardi ng clinical, EEG, and phar mac ological issues of c efepime                                                      their own Cr. SLC6A8 is also expressed in CNS, but not in as troc ytes , particularl y
encephalopathy                                                                                                                   in their feet sheathing microcapillaries at BBB. Thes e data suggested that BBB
Methods: We previousl y published a series of seven patients with cefepime                                                       has a limited permeability for Cr, and that CNS might depend more on its own
encephalopathy. Thereafter, we s tudyi ed the r enal failure as a ris k factor for this                                          autonomous Cr s ynthesis than on Cr s uppl y from the blood.
clinical entity, appl ying the C oc kcroft-Gault formula to measure glomer ular filtration
rates (GFR). The third study, now o ngoing, looks for detecting the incidenc e of                                                The brain is the mai n organ affected in pati ents suffering from Cr deficienc y
cefepime encephalopathy in different grades of renal failure. When neurological                                                  syndromes c aus ed by either AGAT, GAMT or SLC6A8 deficiencies, which all three
manifestations during cefepime treatment are detec ted, we record an EEG and                                                     are charac terized by an abs enc e, or a s evere decrease, of Cr in CNS, as
measure c efepime blood l evels as s oon as possibl e. Events are classifyied                                                    measured by magnetic r esonanc e s pectrosc opy. Because SLC6A8 is pres ent in
according to Naranjo?s algorhythm, and renal impairment as abs ent, slight,                                                      microcapillar y endothelial c ells, AGAT and GAMT deficient patients c an be treated
moderate, s evere, and termi nal.                                                                                                with oral Cr supplementation. However, due to the absence of SLC6A8 in the
Results: In the first study, our patients used cefepime doses between 4 to 8 g/day.                                              surrounding astroc ytes, ver y high dos es of Cr mus t be used, and the
Somnol ence, agitation, or myoclonic jer ks developed after 2 to 9 days after                                                    replenishment of cerebral Cr takes months and results onl y in the partial
starting treatment. Al most all patients had renal failure, and onl y one had c efepime                                          restoration of the c erebral Cr pool. Cr suppl ementation of SLC6A8 deficient
dosage adjusted for renal func tion. The EEG pattern described by Jallon et al.                                                  patients is inefficient to res tore c erebral Cr levels.
were pres ent in all pati ents . In the s econd study, we included 498 patients, 111
with renal failure. Five patients (1%) had enc ephal opathy, all in the renal
impairment group. We als o found that encephalopathy?s incidence increases with
decreasi ng clearance r ates. Indeed, in our present study, we hope to s how that
cefepime?s dosage is s o important than renal function to the pathogenesis of
cefepime encephal opathy.




Low-dose steroid s in critically ill patients: W ho should be treated and when?                                                  The design of drugs which do not create r esistance: folding inhibitors

BRIEGEL J                                                                                                                        BROGLIA R A1,2 ,3 AND TIANA G1,2
                                                                                                                                 1
Munich, Germany                                                                                                                      Department of Physics, Uni versity of Milano, Milano, Ital y
                                                                                                                                 2
                                                                                                                                     INFN, sez. di Milano, Ital y
Ongoin g an d sever e systemic i nflammati on affect in g critica lly i ll p atients may ca use a dren al i nsufficie ncy an d   3
                                                                                                                                     The Niels Bohr Institute, Uni versity of Copenhagen. Copenhagen, D enmar k
steroid res istance. As th is cli nica l entity is difficu lt to dia gnos e, many stu dies on l ow-d ose cortic osteroi d
therapy us ed i ncl usio n criteri a bas ed o n the und erlyi ng d iseas e, clin ical sym ptoms, time w ind ows after o nset
of the dise ase, a nd bioc hemic al testing. S eptic s hock is witho ut do ubt th e b est kno wn dis ease with sever e           Conventional protein inhibitors cap the active site of their targets, preventing the binding of the
systemic infl ammati on and rel ated adr ena l i nsuffici ency. In m any studi es, pers istin g se ptic sh ock after flui d      substrate. An alternative approach has been recently developed to block the activity of proteins
and v asopr essor resusc itation bec ame the m ain inc lusi on criteri a. Other targ et gr oups in lo w-dos e                    and consists in preventing their folding to the native, biologically-active conformation. It is based on
corticosteroi d trials wer e pati ents with ARDS, ac ute l ung inj ury (ALI) d ue to p neum oni a, card iac sur gery w ith       recent understandings on the overall folding mechanism of proteins, and in particular on the
cardio pulm onary by pass, acute pa ncreatitis, or trauma.                                                                       observation that some protein segments display residual native structure already in the denatured
                                                                                                                                 state of most proteins, and that the same segments often participate to the formation of the
Target group septic shock                                                                                                        transition state, as shown by protein-engineering experiments. T he theoretical picture which
Septic sh ock an d l ow-d ose c orticostero id ther apy have be en exte nsive ly i nvestig ated duri ng the last d ecad e
[1-6]. The ma in fi ndi ng sup ported by al l stud ies is th at hydr ocortiso ne 20 0 to 30 0 mg per d ay a ccele rate s         emerges is that folding is guided by Local Elementary Structures (LES) and stabilized by few,
shock reve rsal, i.e. reduces the time on vas opress or therapy. Des pite differe nt inclus ion criter ia of septic              highly conserved (―hot‖) amino acids. Docking of the LES give rise to the transition state and to the
shock (i.e. early vs. l ate septic sh ock, hypote nsio n and poor ly resp onsiv e to fluid and vas opress ors                    postcritical folding nucleus (FN), which inevitably grows into the native state. Intervening a folding
resuscitatio n vs. the broad er defi nitio n give n by the ACCP and the SC CM), this findi ng is co nsistent an d                reaction can, in principle, be achieved by interacting the polypeptide chain with peptides (called p -
supporte d by the m ost recent tria l, the larg e Euro pea n CORTICUS study [1- 6]. In the latter cli nica l trial,              LES) whose sequence is identic al to those of the LES that define the FN of the target protein. As
improvem ent in c ardi ovascu lar p hysio logy di d not trans late i n impr oved s urviva l as it h as be en d emonstrate d      the concentration of p-LES increases, the protein may nucleate by the assembly of the protein
earli er i n the Ann ane study [1,2]. In ad ditio n, ad justment of the ap propr iate tar get gr oup by m eans of                chain with peptide LES, leading to a nonproductive folding. T his can be viewed as changing the
corticotropi n tests did not make a differenc e in the CORTICUS tria l [1]. This is in marked contrast to the                    folding from a unimolecular reaction to a bimolecular reaction.
results of the Anna ne study, in whic h the be nefit of hydroc ortison e and fl udroc ortison e was fou nd in th e target        T hese are two important advantages of these non-conventional (folding) inhibitors with respect to
group with a bl unted resp onse to c orticotrop in as defi ned by p ost -corticotrop in cortis ol incre ase of ¡ Ü9 µg/dl.
The reaso ns for the differenc es foun d in the tw o cli nica l trials rem ain to b e disc ussed. First ana lyses reve ale d
                                                                                                                                 conventional (active-site centered) ones. First, their molecular structure is suggested direc tly by the
that patients in the A nna ne study wer e more sev erely i ll (SAPS II: 59 vs. 49 pts.), had early septic sh ock (time           target protein. One needs not to design or optimize anything, just find the LES of the protein to be
wind ow of i nclus ion: 8 vs. 7 2 hrs.), had more sever e arteria l hy potens ion an d hig her v asopr essor doses at            inhibited, because the design has been performed by evolution through a myriad of generations of
inclus ion, did not r espo nd to vol ume th erapy for at least one ho ur, an d h ad m ore pne umon ia as u nder lyin g           the organism that expresses the protein. Moreover, the probability that the protein can develop
infection. The most importa nt difference was the 2 8-day morta lity of the placeb o grou p whic h was 61 % in                   resistance through mutations is much smaller than in the case of conventional drugs. In fact, a
Annan e study an d 3 1.5% in th e CORTICUS tria l. A p ost -hoc ana lysis of pati ents i n CORTICUS wh o h ad a                  folding inhibitor binds to a LES, and a protein cannot mutate the amino acids of a LES under risk of
systolic blo od press ure that pers isted be low 9 0 mm Hg at 1 day after fluid a nd vaso pressor res uscitatio n                denaturation.
(n=126) sho wed a rate of d eath of 56.1% i n the pl aceb o grou p and a n abso lute re ductio n in mortal ity of 11.2%                                                                                              s
                                                                                                                                 T he above strategy has been applied to HIV -1 Protease, an enzyme which play an essential role
in the hy drocortis on e gro up. These r esults that are very s imil ar to those rep orted by A nna ne [2]. CORTICUS                           cle
                                                                                                                                 in the lifecy of the v     irus. Consequently, its inhibition can control AIDS. T he HIV -1 PR is an
patients w ho rece ived th e study dru g withi n 12 h ours after b asel ine d id n ot show a ny sign ificant d ifferences i n
outcome w hen com pare d with p atients w ho h ave rece ived th e study dru g later. The res ults of the CORTICUS                homodimer, each monomer containing 99 amino acids. It reaches the native conformation following
trial led to the ne w recommen dati on of the survivin g sepsis cam pai gn that only p atients with se ptic shock                a three-state mechanism in which each monomer folds independently of each other and afterwards
poorly r espo nsive to fl uid res uscitatio n an d hi gh-d ose vas opress or thera py sho uld rec eiv e hydroc ortison e         they dimerize.
therapy, but n ot all p atients fulfi lli ng the broa der defi niti on of septic s hock g iven by th e ACCP and th e SCCM.       We have identified the segments containing the residues 23-33 and 83-92 as those associated with
The optimal time w ind ow for initi ation of hydr ocortiso ne in se ptic shock rem ains u ncertai n.                             the LES of the monomer, the segment 83-92 being the one that becomes more structured in the
                                                                                                                                 early folding events. Experiments in silico 1,2, in vitro3 (enzyme) and in infected cells 4 (virus, also
Target group ALI-ARDS                                                                                                            multi-drug resistant v irus) have shown (see also 5) that peptides with identical sequence to the
Ongoin g systemic i nflammati on associ ated w ith excess ive fibr opro liferati on in p ersistent AR DS has bee n               segment 83-92 (p-LES(83-92)) are able to inhibit enzymatic activity by inhibiting folding, as testified
propos ed as an other in dicati on for corticoster oid ther apy. Methylpr edn isol one treatme nt exceed ing the d ose of
                                                                                                                                 by circular dichroism experiments 3. Furthermore, ―long-term‖ studies of pharmacological pressure
corticosteroi d rep lacem ent therapy in the stud ies on s eptic shock by a factor of two improv ed pu lmon ary
function an d reduc ed multi ple org an dysfuncti ons by profo und imm unom odu latio n of the persistent                        conducted by passing in PBMC of a wild-type virus in the presence of p-LES(83-92) or Atazanavir
inflammatory proc ess [7, 8]. The larg e-scal e tri al of the ARDS netw ork, h owev er, di d not su pport th is                  (ATV) have shown the very high genetic barrier to mutations displayed by the LES peptide. In fact,
interventi on [9]. Des pite s ign ificant a nd s ubstanti al improv ements i n p ulmo nary physi olo gy assoc iated with a       genotipic sequenceing 6 showed that p-LES (83-92) did not select for any mutation leading to
high er num ber of venti lator-free d ays an d ICU-free d ays at day 2 8, the use of corticoster oi d did not result i n a       resistance, since the pattern of mutations at baseline and after 9 months of in -vitro
lower mortal ity at day 60 after ra ndom isatio n. An incre ased rate of retur n to assista nt bre athin g ass ociate d          pharmacological pressure were just alike. On the other hand, ATV selected for primary and/or
with muscle weak ness has b een d iscuss ed as mai n reaso n why im prove d physi olo gy di d not translate i n                  secondary mutations. T he transferibility of the folding-inhibition strategy to other proteins testify to
improve d o utcome. A post-h oc a nalys is rev eal ed th at pati ents with A RDS of 14 to 2 8 days durati on ( n=48)             the universality of the folding-inhibition scenario for the design of leads of drugs which are unlikely
had ev en i ncreas ed 6 0-day morta lity wh en they were assig ned to the c orticostero id gr oup wher eas a tre nd to
                                                                                                                                 to generate resistance 7,8.
improve d surviv al was s een i n the gro up recr uited betwe en day 7 and 13 after ons et of ARDS [9]. It has been              1. Brogli a RA et a l (20 05) Protei n Sci. 14:2 668- 268 1 2. Brog lia RA et al. (2 007) La riv ista de l Nuov o Cim ento
criticized th at substa ntial imba lanc es at bas eli ne i n the latter post -h oc an alysis gro up w ere pr esent favo urin g   29:1-11 9 3. Brogli a RA et al (200 6) Proteins 6 2:928 4. Rusc oni S et al (20 07) Procs. Of the Internationa l
the place bo gro up.                                                                                                             School of Physics ―E. Fermi‖, Course C LXV on Prote in Fold ing a nd Dru g Desi gn, IOS Press, Amsterdam, 293-
                                                                                                                                 299 5. Bro gli a et al ( 20 08) Curr. Opi n. Struct. Bio l. 18:6 0-66 6. Ferr amosca S et al., XVII Internati ona l HIV
[Regrettably n ot enou gh spac e on this pa ge.]                                                                                 drug res istance w orksho p, basic princ ipl es and c lin ical impl icatio ns, June 1 0-14, 20 08 Sitg es, Spai n (see
                                                                                                                                 http://merlino.mi.infn.it/pub licati ons/articl es2/asbtract_ 200 8.pdf) 7. Bro gli a RA et al. (2 007) Pr otei ns 67:4 69 8.
                                                                                                                                 Cald arin i M et al (2008) Prote ins
                                                                                                                                 (in press; see http://merlino.mi.i nfn.it/publ icatio ns/articles 2/lysozyme 12fi g.pdf)




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                                                                 Page A-45
                                                                  EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                         Celebrating the 100th Anni versar y of the
                                                                            Nobel Prize Award to Paul Ehrlich
                                                                               Nürnberg, October 3-5, 2008
Novel targets and magic bullet s for treating cardiovascular disease                           A Ther apeutic Conundrum: Low Molecular W eight Heparins in Patients with
                                                                                               Kidney D ysfunction.
BROOKS G
                                                                                               BROPHY DF
University of Readi ng, Ber kshire, UK
                                                                                               Virginia Commonwealth Uni versity, Rich mond, VA, U.S.A.
Cardiovascul ar diseas e repr esents a major clinical problem affecting a significant
proporti on of the world‘s popul ation and it r emains the major c aus e of death in the       Background: Patients with c hronic kidney diseas e (CKD) are frequentl y viewed
EU and the res t of the Western world. Furthermore, the burden on healthc are                  as being at high risk for bleeding. In point-of-fac t however, CKD pati ents not onl y
systems is increasingly high; the overall cost of cardiovascular dis ease to the EU            maintain a bleedi ng tendenc y but also exhi bit a high frequenc y of thromboembolic
economy is esti mated to be in excess of 192 billion Euros per year. The majority of           diseas e. Indeed, cardiovascular disease is the leading caus e of death in CKD
therapies currently availabl e for the treatment of cardiovascular disease do not              patients. Therefore, clinicians ar e frequently c onfronted with choosing the most
cure the problem but merel y treat the symptoms. Furthermore, many c ardioac tive              appropriate antithrombotic therapy in CKD patients undergoing percutaneous
drugs have serious side effects and have narrow therapeutic wi ndows that can                  coronar y i nter vention. Clinical trials have shown that low molec ular weight
limit their us efulness i n the clinic. Thus, the development of more sel ecti ve and          heparins (LMWH) have greater effic ac y and less adverse effects c ompared to
highly effec tive therapeutic strategies that c ould cure s pecific c ardiovasc ular           unfracti onated heparin (UFH). However, CKD patients pres ent a special
diseas es would be of enor mous benefit both to the patient and to thos e countries            circumstance suc h that LMWHs carr y an incr eased risk of adverse bleeding in
where health care s ystems are res ponsible for an incr easing number of patients.             such patients compared to those with normal renal func tion. This presentati on will
There is increasing evidenc e to suggest that targeting the cell c ycle machi ner y in         provide perspecti ve on the clinical use of LMWH in CKD patients.
cardiovasc ular cells (e.g. c ardiac myoc ytes, vasc ular smooth muscl e cells                 Methods: A Medline literature review was conducted to identify primar y literature
(VSMCs), endothelial cells) provi des a novel approac h for the treatment of certain           describing the efficac y and ris k of adverse bleeding when UFH and LMWH drugs
cardiovasc ular diseases , including pos t-infarct heart failure, restenosis, in-stent         were used at therapeutic doses i n CKD patients.
stenosis and bypass graft failure. It has been demonstrated that certain cell c ycle           Results: In major clinical trials, enoxaparin has been associ ated with increased
molec ules that are i mportant for regulati ng terminal differentiation in car diac            bleeding rates. A recent meta-anal ysis carefully described the available data for
myoc ytes (e.g. c yclins, c yclin- dependent ki nas es [CDKs], CDK i nhibitors, E2F            all LMWH products i n non-di alysis-dependent CKD patients with r espect to
transcription factors) can be targeted to reinitiate cell division and repair in the           bleeding. n the primar y anal ysis, 4971 patients with a creatinine cl earance < 30
myoc ardium post-infarction. Furthermore, cell cycle molec ules that control                   mL/min had an increas ed ris k of bleeding compared to thos e without renal
exc essi ve VSMC proliferation in disorders such as restenosis, in-stent stenosis              insufficienc y (5.0% vers us 2.4%; odds ratio 2.3, [95% CI 1.2 to 4.3], p=0.01). In a
and bypass graft failure have als o been targeted effec tivel y in recent laborator y          secondar y product-based anal ysis of these same data, enoxaparin us e increased
and clinical s tudi es. T he results of these studies illustrate the exciting possibility of   the rate of major bleeding to 6.0% in CKD patients compared to 2.4% in non-CKD
targeting components of the cell c ycle machinery to develop magic bullets to                  patients (odds rati o 2.6, [95% CI 1.2 to 4.3]). T he li kelihood of a major bleeding
improve cardi ac functi on and prognosis for patients with heart failure and for               event increased further when the enoxaparin dosing regimen was c onsi dered.
patients with atheroscl erosis.                                                                Safety data for other LMWH is difficult to ascertai n bec ause of the relative few
                                                                                               availabl e data.
                                                                                               Conclusions: These data suggest that non-adjusted enoxaparin dosing may lead
                                                                                               to serious bl eeding in CKD patients. The appropriate enoxaparin dos e reduction
                                                                                               has yet to be confirmed. The us e of UFH is pr udent in patients with advanced
                                                                                               CKD.




Epinephrine Vasoconstrictor Drug-Drug Interactions R evisited                               Magic bullets for the treatment of inflamm ator y bo wel disease – yet to
                                                                                            come?
BROW N RS
                                                                                            BRUNNER M
Howar d Uni versity College of D entistr y, Was hington, DC, USA
                                                                                            Medical Uni versity of Vi enna, D epartment of Clinical Pharmac olog y, Vi enna,
                                                                                            Austria
Drug-Drug interactions with regard to epinephrine vasoc ons trictor local anesthetic
formul ations have been vastl y overstated in the past. Suc h purported drug                Background: Infl ammator y bowel dis eas e (IBD), an immune- mediated chr onic
interactions include tricyclic antidepress ants, non-specific beta bl oc kers and           intestinal conditi on, encompasses two idiopathic inflammator y diseases of the
cocaine. These supposed but mistaken interacti ons are widel y published in many            intestinal trac t: Crohn‘s disease and ulcerati ve colitis. Currentl y, medical treatment
Dental Phar mac ology Texts and have established problematic clinical                       for IBD aims at induction and maintenanc e of remission. Established therapi es,
considerations negati vel y influenci ng pharmacologic patient therapy. A portion of        such as 5-ami nosalic ylic aci d c ompounds , cortic osteroi ds, i mmunomodulators or
the rati onal e for suc h purported drug interac tions incl ude poorly designed studies     calcineurin inhi bitors, however, lac k specificity or effecti veness and might c aus e
and inapplicable c ase reports.          The major misconceptions are due to                significant long term si de effects.
misunderstandi ngs of adrenergic pharmacol ogy. Lac k of understanding with                 Recent advanc es in the knowledge of pathogenesis and immunolog y of IBD has
regard to epinephrine‘s beta two rec eptor‘s influence upon blood press ure                 led to the development of novel therapi es directl y targeting specific aspects of the
dynamics and the difference between alpha adrenergic local versus s ystem effects           inflammator y process, such as c ytokines and receptors invol ved in T-cell
are probl ematic. A misunderstanding of the positive attributes of loc al anesthetic        activation, selecti ve adhesion molec ule bloc kers, anti-inflamator y c ytokines,
vas oconstrictor acti on and limited knowledge of s ympathetic acti vation and the          modulators of the intes tinal flora or monoclonal anti bodies. T he first monoclonal
actions of endogenous nor epinephrine among dental clinicians has contributed to            TNFα antibody, has been succ essfull y i ntroduced into clinical practic e for the
this problem and resulted in the mis use of phar mac otherapeutics. The lack of             treatment of IBD approximatel y 10 years ago. Most other novel therapies are
toxicity is further influenc ed due to epi nephrine‘s exc eedi ngly s hort half-life. T o   undergoing differ ent s tages of clinical eval uation.
add to the above, the mec hanism of tric yclic antidepr essant pharmacolog y is not         Conclusions: Although during the last years progress has been made in both
common knowledge among dental clinicians and even s uch simpl e drug -drug                  defini ng the mechanisms underl ying the devel opment of infl ammator y bowel
interactions as the additi ve dr ug-drug interaction between coc aine and other local       diseas e and expanding the spectrum of effecti ve therapies , it might be too earl y to
anes thetics is overlooked. F urthermore, many recent human studies which                   know, whether the currently tested c ompo unds will be routinely employed in IBD
demons trate the relati ve safety of epinephrine vasoc onstriction will also be             treatment. It seems rather unlikel y, however, that one single drug will pr ove to be a
discussed. In conclusion, an evaluation of potenti al drug-drug epinephrine                 magic bullet.
vas oconstrictor interactions is important with regard to clinical dental care and
appears to limit fears regarding several noted but highly s uspect previ ousl y
accepted drug-drug interactions.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                              Page A-46
                                                               EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                       Celebrating the 100th Anni versar y of the
                                                                          Nobel Prize Award to Paul Ehrlich
                                                                             Nürnberg, October 3-5, 2008
Transcr iptional Changes Induced b y Imatinib and Nilotinib in the Chronic                   according to r egistr ation: Bugelli Cainelli Geb ara!
Myelogenous L eukemia (CML) Cell Line K562
                                                                                             Immunogenic and Adjuvant Activities of Bordetella pertussis protein s
BRUENNERT D, KOCH A, GATT ERMANN N, KRONENWETT R, HAAS R,
NEUMANN F                                                                                    CAINELLI GEBAR A VCB, LOPES APY, WOLF PS, FERREIRA VRF, QUINTILIO
                                                                                             W, RAW I
Heinrich-Heine-Uni versity, Duessel dorf, Germany
                                                                                             Instituto Butantan, S ão Paul o, SP, Brasil
Background: Nilotinib is a selec tive bcr-abl tyrosi ne ki nas e inhibitor that is 30-fold
more potent than Imatinib in vitro. To examine the molec ular and functional effects         Background: Adjuvants are essential c omponents to enhance the
of Nilotinib and Imatinib we performed gene expression and functional anal yses in           immunogenicity of vaccine antigens. Extrac ts of bacterial origin are well known
K562 c ells following in vitro treatment with the two tyrosine kinase inhibitors.            and widel y used immunomodul ator y substances . B. pertussis is a promising
Particular emphasis was put on 1539 genes which we found to be differentiall y               candi date since it produc es several components acti ng on the immune s ystem of
expressed in primar y CD34+ cells from pati ents with CML in c hronic phas e in              the host.
comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al.,                       Methods: The proteins were purified by anion exchange chr omatography (Hitrap
Leukemi a 2006).                                                                             Q Sepharose HP) followed by extracti on from a preparati ve gel (SDS-PAGE), and
Methods: Affymetrix U 133A 2.0 microarrays c overing 21.722 probe sets wer e                 were further anal ysed by mass sp ectrometr y and tested in mice. Female BALB/c
                                                        7
used to anal yse the gene expression profile of 5x10 K562 cells after 24h in vitro           mice, 4-6 weeks old were subcutaneously injected 2 times at weekl y i nter vals with
treatment with Imatinib (0.5 µM) or Nilotinib (0.05 µM) (half maxi mal inhi bitor y          the pr oteins (1µg/0.1ml/mouse) al one or mi xed with DPT (Diphtheria-Pertussis-
concentrati on). FISH anal ysis confirmed the K562 cell line to be BCR -ABL                  Tetanus) vaccine formulated without alumi num hydr oxide (NA-DPT), (2µl/mous e).
positi ve. Gene expression data of the treated c ells were c ompared with the data of        DPT vaccine c ontaining alumi num hydroxide (DPTBut) was us ed as control
untreated c ells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell           (2µl/mous e). Non-immunized mice (saline sol ution injected) were used as control.
Death D etec tion ELISAPLUS, Roc he) and c ell cycle (FITC BrdU Flow Kit, BD                 Blood was collected 1 day before and 20 days after the first immunization, and
Phar mingen) assays wer e performed.                                                         mice were challenged by i ntracerebral route with live B. pertussis. Sera were
Results: Looki ng at those 1539 differentially expressed genes in K562 cells which           assayed for antibodies and isotypes. ELISA was us ed for all evaluations.
distinguish patients with CML from healthy donors, we found that Imatinib led to a           Statistical anal yses i ncluded one-way ANOVA followed by D unnet‘s multi ple
significant downregul ation of 187 and upregulati on of 45 genes. In general, the            comparison tests considering p<0.05, and 2 test, with Yates correction.
effec t of Nilotinib with regard to the number of genes affec ted and degree of              Results: We have purified two B. pertussis proteins , and identifi ed by mass
suppressi on was mor e pronounced resulti ng in the downr egulation of 418 and               spectrometry as the 73 kDa N-ter minal -domain of Br kA autotrans porter protein
upregulati on of 41 genes . Of note, genes affected by Nilotinib incl uded all genes         and the Cpn60/60 kDa c haperonin.
altered by Imatinib s uch as those related to bcr-abl signalling. Downregulati on of         Immuniz ations with 73k+60kD a+NA-DPT elicited a significant increase (p<0.01) of
genes involved i n cell c ycle was onl y obser ved following Nilotini b expos ure. The       the antibody respons e against pertussis, and of pertussis specific IgG1 and IgG2a,
stronger effect of Niloti nib is in line with the results of cell c ycle experiments         when c ompared to DPTbut immunization. The former group have als o showed a
showing that Nilotinib exposed cells had the lowest proportion of acti vel y c ycling        better protection rate (42%) against the ch allenge than the latter (27%).
cells. The proportion of apoptotic K562 cells was 5.5 fold greater following                 Conclusions: Our results s uggest that mouse immunizati on with both proteins
treatment with Nilotinib in comparison to Imatinib after 24 hours.                           mixed with NA-DPT c ould s timulate Th1 and T h2 c ells, induce significant higher
Conclusion: Nilotinib is apparentl y more potent than Imatini b with r egard to the          levels of IgG1 and IgG2a antibodies , and medi ate a partial pr otecti on agai nst the
number of genes downregul ated and the degree of their suppression. Many of the              challenge. T herefore, these protei ns are good c andidates to be exploited as
suppress ed genes are associ ated with bcr-abl signalling and c ell cycl e.                  adjuvants for incl usion i n vacci nation protoc ols.
                                                                                             Financial Support: FAPESP, Fundaç ão Butantan, CNPq
Authors‘ disclosure statement: Imatini b and Nilotinib were provided by Novartis.
The wor k was supported by Leukaemieliga e. V.




Online m ethod for measuring of the activities of antibiotic efflux pump s in              Red Blood Cell Membr ane F att y Acid Analysis in Never-Medicated Fir st-
Esch erichia coli and Pseudomonas aeruginosa                                               Episode and Chronic Med icat ed-Schizophrenic Patients

BUIVYDAS A1 ,2 , SENŢILO A1,2 , STUKAITĖ S1 , BAMFORD D 2, DAUGELAVIŢIU S                  BURETIĆ-TOMLJANOVIĆ A1 , GIACOMETTI J1, NAD ALIN S1, RUBEŃA G2 ,
R1                                                                                         VULIN M 3, TOMLJANOVIŠ D 4
1                                                                                          1
  Ţiurlionio 21/27, Department of Bi ochemistr y and Biophysics, Vilnius Uni versity,       School of Medicine, Uni versity of Rijeka, Rijeka, Croatia; 2Clinical Medic al Centre,
Vilnius, Lithuani a LT-03101;                                                              Rijeka, Croatia; 3 Ps yc hiatric Hos pital Lopaţa, Rijeka, Croatia; 5Private ps yc hiatric
2
  Viikinkaari 5 (PO Box 56), Institute of Bi otec hnology, Uni versity of Helsinki,        practice, Rijeka, Croatia
Helsinki, Fi nland.
                                                                                           Background: Reduced n-3 and n-6 pol yunsaturated fatty acids (PUFAs) content
Background: Multidrug resistance (MDR) pumps, decreasing intrac ellular                    in red blood cell (RBC) membranes and abnormal membr ane phos pholipid
concentrati ons of antibi otics are major causes of resistanc e in opportunistic           metabolism were repeatedl y i mplicated in the etiol ogy of sc hizophrenia.
pathogens suc h as Ps eudomonas aeruginosa. This phenomenon ensures the                    Methods: Gas-chromatography anal ysis of fatty acids content in RBC was
viability of bacteria under high c onc entr ations of antibi otics and complicates the     perfor med in the group of never-medicated first-episode schiz ophrenic patients,
treatment of infected pati ents . Fast methods are needed to determi ne the                chronic medicated patients and healthy controls. Differenc es between group
efficienc y of drugs inac tivating MDR pumps for the success ful treatment of              means were i nvestigated using ANCOVA. Group, sex and s moki ng status were
patients.                                                                                  used as predictors, while covariates were body mass index (BMI) and age. The
Methods: Aerated sus pensions of Esc herichia coli and P. aeruginosa c ells were           level of statistic al significanc e was s et to 0.01.
studied in thermostated reaction vessels, and changes in the extrac ellular                Results: Three groups of subjects significantl y differed in total n-3 fatty aci d,
concentrati on of indicator lipophilic cati on tetraphenylphos phonium ( TPP+) was         22:6n-3, 22:5n-3 and 22:0 RBC content, delta-9-des atur ation (D9C18) index,
monitored using selecti ve electrodes. Tetrac ycline was us ed as the model                peroxidizability index (PI), double bond index (DBI), and DBI/PI rati o. BMI was
antibi otic, and, phenylalanyl arginyl β-naphthyl ami de (PAβN), reserpine and             significantly correlated to average chain length (ACL), D9C18 index and
chlorpromazine were used as inhi bitors of the differ ent MDR pumps.                       PUFA/monouns aturated fatty acid ratio. First-episode patients had significantl y
Results: Depending on the outer membrane per meability, membr ane voltage and              higher total s aturated fatty acid (SFA) c ontent, particularl y 18:0, l ower total PUFAs
activity of the MDR pumps , wt c ells and the mai n MDR pump mutants of P.                 content, particularly 18:2n-6 and 22:5n-3, lower D9C18 index, PUFA/SFA and
                                                                         +
aerugi nos a and E. c oli accumul ated different amounts of TPP . Addition of              DBI/PI ratios c ompared to controls. T he differ ences between c hronic medicated-
tetrac ycline and the pump inhibitors c aus ed detectable alterations of the amount of     patients and controls showed similar pattern. Onl y mi nor but highly significant
      +
TPP acc umulating. Using the outer membrane-permeabilizing and the pl asma                 differences were found between two groups of schiz ophrenic pati ents ( 22:0, 22:5n-
membrane depolarizing compounds, acti vity of the pumps, affinity of the                   3).
substrates and efficienc y of the inhibitors were eval uated.                              Conclusions: 1) The results confirmed a disturbance of lipid homeostasis in RBC
Conclusion: Online monitoring of T PP+ fluxes across the bacterial envelope can            membranes as intrinsic feature of schiz ophr enia. 2) Reduced total n-3 FAs and
be applied for studies of the acti vity of MDR pumps in E. c oli and P. aerugi nos a,      total PUFAs content, reduc ed PI, DBI and D9C18 index, and higher total SFA and
for deter mination of the pump s electi vity to s ubs trates and for eval uati on of the   18:0 content were found regardl ess of illness duration and anti ps ychotic treatment.
efficienc y of pump inhi bitors.                                                           3) Nicotine usage was not a significant predictor of the F As composition in RBC
                                                                                           membranes of investigated subjects . 4) Drugs affecting phos pholipid metabolism
                                                                                           and providing recovery of lipid homeostasis in c ellular membranes might have
                                                                                           beneficial effec ts in sc hizophreni a.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                              Page A-47
                                                        EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                               Celebrating the 100th Anni versar y of the
                                                                  Nobel Prize Award to Paul Ehrlich
                                                                    Nürnberg, October 3-5, 2008
Conceptual basis for can cer tr eatment: from single drugs to kits with serial       Pro-Atherogenic and Pro-Inflamm ator y Alter ations in Mononuclear Cell
programmed actions of multiple substances-drugs                                      Populations Induced b y Oxid ized Lo w D ensit y L ipoprotein (oxLDL) and High
                                                                                     Glucose Levels in T ype II Diab etic Patients. Anti-Inflammator y Drugs, an
BURLAKA D                                                                            Altern ative?
                                                                                                                1          1              1              1                     1
Department of Bi otec hni cal Pr obl ems of Di agnostic s, Insti tute of Cr yobiol og y      BUSTAMANTE, M , DÍAZ, F , MUÑOZ, M , GUZMÁN, C , LLANCAQUEO, A ,
and Cr yomedici ne N ati onal Ac ademy of Sci enc es of U kr ai ne, Kyi v, Ukrai ne.         NÚÑEZ, L1, CAMPOS, L1 , RIVAS, CI2 , VER A, JC 2, GROSS, H-C3 & BACHEM, M 3.
                                                                                             1
Background: Well-known paradoxical effect that both high-dose estrogens and                   Laboratory of Mol ecular Diagnosis, Catholic Uni versity of Concepción,
                                                                                                                 2
anti-estrogens cause tumor regressions. In this cas e important is an                        Concepción, Chile; D epartment of Phatophysiol ogy, U ni versity of Conc epci ón,
understanding of the properties of a tumor. Aims: 1) To study of the infl uence of           Concepción, Chile; 3 Einrichtung Klinische Chemie, Uni-Klini kum, Ul m, Germany
the same hor monal environment on tumor growth and charac ter of c hange of the
receptors level i n tumors with different hormone sensiti vity.                              Background: T ype II diabetes mellitus is a ris k factor in the devel opment of
Methods: Conc entr ati on femtomol es/mg pr otein ( fM/mg) of es trog en                     atherosclerosis. T wo factors are c entr al to the effect of diabetes, oxidative
rec eptor (ER) was deter mined in trans planted mic e mammar y tumors with                   modification of LDL and high glucos e concentrations. We hypothesized that
intensi ve reproducti on mode (2-6 parturition without lactating) (n=20) and virgin          mononuclear cells from diabetic pati ents in the presence of oxLDL and high
(n=20). The levels of ER were deter mined by means of the dextran-coated                     glucose levels undergo pro-infl ammator y alterations, whic h woul d enhanc e the
charc oal tec hniq ue. Mice of line С3H/Sn (n=69) contained in cages on 5 female             devel opment of atheroscl erotic plaques. Therefore, our aims were: 1) To meas ure
+2 mal e (nor mal reproduction) in a room with daylight. Virgin female mic e (n=47).         the i nduction of necrosis and apoptosis i n mononuclear cells from diabetic
Results: The data pr ovides both estrogen-positi ve and –negati ve tumors that               patients, 2) to measure the r eleas e of pro-inflammator y interleukins in s timul ated
actually are evoked from the s ame tissu e, so that one can deter mine at what               cells, and 3) to analyz e the possi ble us e of meloxic am as an alternati ve treatment.
level(s) the hor mone s ensiti vity or dependenc y exists. The widel y accepted              Methods: This study i ncluded c ells from 140 diabetic pati ents and 105 controls.
hypothesis that the interacti on of estr ogen with its cellular rec eptors determines        The indi viduals were grouped by age, sex and obesity for the meas urement of
the hormone dependenc y of mammar y tumors should now be challenged on the                   necrosis and apoptosis; 15 di abetic and 15 controls for quantification of
basis of the following obser vations : a) tumors occuring in experimental ani mals           interleukins ; and 20 patients and 20 c ontrols to study the effect of meloxic am (7,5
with per manent high estrogen levels are rec eptor-positi ve(~20-100fM/mg) and with          mg/day/30days). The res ults ar e presented as the mean ± SD of at least three
low estr ogen levels (<2fM/mg) are receptor-negative; b) tumors regrowi ng after             independent experiments (a value of p < 0,05 was considered as significant, t-
complete or partial regression as a result of endocrine abl ation or hormone                 test).
administration are no c onsidered to be autonomous or hormone dependenc e but                Results: Cell necrosis and apoptosis was increased in mononuclear cells from
environment dependenc e; c) growth of a tumor at cyclicall y changing hormonal               obes e, di abetic patients older than 50, without significant sex-related differenc es.
level leads to heterogeneity of mammar y tumors that complicates hormone                     Moreover, the release of pr o-inflammator y interleukins , in particular those rel ated
therapy.                                                                                     with the devel opment of atherosclerosis, was also increased in diabetic cells.
Conclusions: 1) Series of parameters of a tumor suc h as the invasi veness,                  Interes tingly, meloxicam treatment decreas ed necr osis, apoptosis and release of
heterogeneity and others are consequence of adaptic properties of tumoral cells.             pro-inflammator y interleukins from diabetic mononuclear cells.
2) Ability of tumoral cells to adapt for change of a surrounding microenvironment            Conclusions: Some of the pro-inflammatory effects obser ved when diabetic cells
ans wers on a question on a paradoxicality of a hormone therapy. 3) High                     are subjec ted to the synergistic action of oxLD L and high glucose conc entrations,
sensiti vity of tumors to change of steroids conc entration (10-9M) discovers an             such as necrosis, apoptosis and interleukin releas e, are reduc ed by treatment with
epigenetic path of redifferenti ation a tumor onc ogenome i.e. multipl e-stage               meloxic am. Ac knowlegment: FONDECYT 1040977, DIP-UCSC, Prof. Max
therapy.                                                                                     Bachem.




Tacrolimu s: a highly effective new therap y for chronic glomeru lar diseases?

BUTANI L

Profess or and Chi ef, Pedi atric Nephrology, Co-Instructor of R ecord, 3rd year
Pediatric Clerkshi p, UC Davis Medical Center, 2516 Stoc kton Boulevard,
Sacramento, CA 95817, USA


Abstract:

Tacrolimus is a potent immunos uppressive agent that has been demonstrated to
be superior to c yclos porine after renal trans plantati on as an effecti ve and safe
anti-rejection drug. Bas ed on the exc ellent outc omes in the trans plant popul ation,
the us e of tacrolimus has been extended to patients with a variety of different
chronic glomerular diseases that are immune- mediated, s uch as steroi d resistant
nephroses and lupus nephritis, to name a few. T he aim of this presentation is to
critically appraise data related to the safety and efficac y of tacroli mus i n adults and
children with immune medi ated renal diseas es, outside the transpl ant setting. New
evi dence discussing the potenti al benefits and side-effects of long-term tacrolimus
use (suc h as subclinical nephrotoxicity) will be discussed, s o as to allow the
audi ence to make an evidence- based assess ment on whether tacr olimus is a
reasonabl e strategy to treat patients with c hronic glomerul onephritis.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                              Page A-48
                                                         EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                 Celebrating the 100th Anni versar y of the
                                                                    Nobel Prize Award to Paul Ehrlich
                                                                      Nürnberg, October 3-5, 2008
The two main problems in evaluating resist ance to antiparasitic drugs in              Botulinum Toxin in restrictive strabismus
populations of naturally infected hosts: efficacy variabilit y and cut -off value
for resistance                                                                         CÁCERES TOLEDO M

CABARET J, ANTOINE T                                                                           Clinical-Surgical Hospital. Her manos Ameijeiras. Department of Ophthal molog y,
                                                                                               Havana-Cuba
INRA, IASP 1232, 37380 Nouzilly, France
                                                                                               SUMMARY
Background: Variability of parasite/host res ponse to antipar asitic drugs is l arge in
field conditions depending on drug and galenic formul ation. Resistance to                     The restricti ve s trabis mus is the most frequent sequel in T hyroid Orbitopathy
anthelmi ntics has been well doc umented i n sheep and goats , partl y in c attle and
less frequentl y in man. We dis pos e of different evaluati ons of efficac y for               Objective. We want to determi nate the effecti veness of botulinumToxin (botox) in
anthelmi ntics, none of them being a golden standard. We do not really know up to              correct restrictive strabismus and eliminate the di plopi a in primar y positi on of look
what l evel of effic ac y we consider that we ar e facing a resistance phenomenon. In          and reading.
that res pect it has been consi dered that less than 90% (horses) or 95%                       Methods. we c arried out a descripti ve, prospecti ve and l ongitudi nal s tudy to 10
(ruminants) efficac y was the limit for stating on resistanc e. We are in great need           patients (17 eyes) with restrictive strabismus of March to J une of the 2008, using
of evaluation of efficac y of dr ugs and cut- off value for resistanc e.                       botulinum Toxin with direct method, without any elec trical control, we have
Methods: Since distributi on of effic ac y val ues is not known and is cl earl y not a         registered the effect and stability in strabis mus and i nfluence in the s ystemic
Gaussian distributi on, we propose a bootstr ap c onfidence evaluati on, using a               diseas e.
freeware we c ons tructed (Bootstreat availabl e on demand or on i nternet). The               Results. BotulinumToxin´injec tion i n the affec ted muscle was useful to c orrect
bootstrap evaluation is bas ed on different eval uation for mulas for effic ac y (before       restrictive strabismus and offers excellent results in the eli minati on of the diplopia
/after treatment with and without untreated control, after treatment in treated and            and i n the patient's immediate c omfort; we don't obs erve any influenc e in thyr oid
controls, either using arithmetic or geometric means). The evaluation of cut-off for           systemic diseases. The treatment can be to c orrect strabismus c ompletely or
resistance is a completel y open problem. We propose a two steps‘ method:                      waiting to go to surger y in a better moment. The botulinumToxin´s effec tiveness
transfor mati on of indivi dual efficac y data that res ults in a Gaussian distribution        could be for 4 to 6 months, but i n some patients is for ever.
and then when efficac y is not real we suppos e that Gaussi an distribution is                 We c oncludes that the therapeutic is a ver y good c hoice to treat strabismus not
acceptabl e and progressivel y with increasing dos es and /or efficac y, there is a            onl y for the efficac y but also for the s afe and i nnocuous of the direct method of
departure from suc h a distribution. We use data from anthelmi ntic treatments                 application
(tetramis ole, macroc yclic lactones, benzimi dazol es) in c attle or sheep.
Results: Boots trap r esults indicate that us ual procedure for estimating efficac y are
not accur ate since confidence intervals reach s ometi mes more than 20% of the
mean. The cut-off values are highl y dependent on drug/dose (example in
tetramisole) and galenic for m used (injectable or per os i vermectin). We propose a
strategy for evaluating resistanc e in the field.




H pylori: Treatment for the patient only or the who le fam ily?                                Antican cer Activit y and L ack of Toxicit y of CZ48 Admin ister ed Or ally To
                                                                                               Nude Mice Carr ying Xenografts of 22 Hum an Tumors
SARI YS, C AN D, TUNALI V, SAHIN O, KOC O , BENDER O.
                                                                                               CAO, Z, KOZIELSKI, T, VARDEMAN, GIOVANELLA BC
SB Istanbul Traini ng Hospital, Istanbul, Tur key. didemcan73@hotmail.com
                                                                                               CHRISTUS Stehlin Foundation for Canc er Research, 1918 C henevert
Aim: T o compare the effects of treatment of H pylori-infected i ndi viduals with the          Street,Houston, TX 77003 USA
effec ts of treatment of indi viduals as well as all H pyl ori-infected famil y members.
Methods: H pylori-positi ve patients with si milar demographic s pecifications were
prospec tivel y randomized with respect to treatment, with a triple regimen of either          Background: Camptothecin, an al kaloid extrac ted from the tree Camptothec a
patients and all H pylori-positi ve family members livi ng togetherI (group I) or                    i
                                                                                               acum nata and s ome of its derivati ves were found to have potent anticancer
patients onl y (group II). Nine months after treatment, all patients were ass essed            activity against human tumors growing as xenografts i n nude mice. However,
for H pylori positi vity.                                                                      thes e compounds were much l ess acti ve against tumors of cancer patients.
Results: T here were 70 H pylori-positi ve patients in each group; patients in groups          Further studies established that all the camptothecins were converted to their
I and II lived with 175 and 190 H pylori-positi ve relati ves, res pecti vel y. Age, s ex      inacti ve form in the human blood by the opening of their lactone ring caused by the
and H pylori positi vity rate were si milar in both groups of relati ves . Nine months         presenc e of human serum albumin.
after 14 d standard tripl e therapy, H pylori positi vity was 7.1% in group I patients         Methods and Results: In order to prevent s uch an opening, our labor ator y has
and 38.6% in group II pati ents [P < 0.01, OR = 8.61 95% confidence interval (CI):             devel oped CZ48 (Camptothecin-20(S)-propionate hydrate). We have tested it for
2.91-22.84].                                                                                   anticancer acti vity admi nistering it orally to nude mice carryi ng xenotr anspl ants of
Conclusion: The present res ults indicate bad environmental hygienic conditions                22 human c ancers ( 4 col on, 2 l ung, 5 pancreas, 5 breast, 2 melanomas, and 2
and close intra-familial relati ons hips ar e important i n H pyl ori contaminati on. Thes e   sarcomas, 1 prostate and 1 bl adder) at doses of 50 mg/kg/day to 2000 mg/kg/day
findings indicate all family members of H pylori-positi ve indi viduals should be              daily, 5 to 7 days/week. The tumors were growing subcutaneously on the bac k of
assessed for H pyl ori positivity, particularl y in developi ng countries where H pylori       3 month old mice. The treatment started when tumors were measur able by c aliper
prevalence is high; they also s uggest pati ents, their spous es and all H pyl ori-            (200-300 mm3). CZ48 was administer ed s uspended in cottonseed oil.
positi ve famil y members of H pylori-positi ve indi viduals should be treated for H           Conclusion: Two concl usions clearly emerge from this study: 1) CZ48 is totall y
pyl ori infecti on.                                                                            devoi d of toxicity at the maximum doses used which were administered for more
                                                                                               than 300 days; and 2) CZ48 is a potent anticancer drug against human cancer
                                                                                               xenografts wit h a wide spectrum of acti on which c aused c omplete growth inhibition
                                                                                               in 20 out of 22 human cancers treated.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                 Page A-49
                                                            EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                 Celebrating the 100th Anni versar y of the
                                                                   Nobel Prize Award to Paul Ehrlich
                                                                      Nürnberg, October 3-5, 2008
SAPPHIRE: A Structural-energ etic Approach to B-cell Epitope Pr ediction               Molecular strateg ies to improve the anti-tumour activit y of Zoledronic acid in
                                                                                       prostate cancer cells
CAOILI SE
                                                                                       CARAGLIA M 1* , MARRA M 1, ZAPPAVIGNA S 1, LOMBARDI A 1, SANTINI D 2,
University of the Philippines Manila, Manila, Philippines .                            ABBRUZZESE A 1

Background: B-cell epitope prediction facilitates the design of antibody-bi nding          Department of Bi ochemistr y and Biophysics, Sec ond Uni versity of Naples, Via
constructs for the development of novel vaccines and i mmunodiagnostics. This              Costantinopoli 16, 80138, Naples; 2C ampus Biomedic o Uni versity, Sec tion of
wor k ai med to gain insights into the problem of B-cell epitope prediction using          Oncolog y, Rome, Ital y.
structural energetics.                                                                     * Corresponding author
Methods: Structural-energetic analysis was applied to pepti de and protein
antigens. A possibl e rate-limiting process of local epitope unfolding was                 Background: Zoledronic acid (ZOL) is an aminobisphosphonate able to inhibit the prenylation of
considered for the cross-reacti on of antipeptide anti body with protein antigen.          intracellular proteins through the inhibition of farnesylpirophosphate synthase. Prenylation is
                                                                                           essential for the maintenance of the activation of components of signal transduction pathways
Immunodomi nance was treated as a thermodynamicall y determined hi erarchical              regulating apoptosis and proliferation such as ras and ras -related proteins. ZOL has demonstrated
steric-exclusi on phenomenon. The algorithm thus developed was implemented as              a direct anti-tumour effect in vitro and in preclinical models and its ability in preventing skeletal
the c omputer program SAPPHIRE (Str uctural-energetic Anal ysis Program for                related events is proven in patients with bone metastases from different origi ns. Clinical evidence
Predicting Humoral Immune Res pons e Epitopes), with the estimated affinity for                                                                                 ity
                                                                                           on its direct anti-proliferative effects is emerging, but its activ is limited by the excessive
antibody as the main criterion for epitope predicti on. Predictions were r endered on      accumulation in the bone. Methods: We describe several strategies in order to improve the anti -
                                                                                           tumour activity of ZOL based on preclinical and biological rationales. Results: T he first strategy is
the cross-reacti vities of pol yclonal anti bodies to 38 peptides with 15 globular         to combine ZOL with either cytotoxic drugs or other biological agents such as the farnesyl -
proteins of known structure and eval uated against published experimental data             transferase inhibitor tipifarnib focusing also on the importance of the sequence of administration of
comprising 18 positi ve and 20 negati ve binding interactions.                             these drugs. T he synergistic interaction with other agents could lower the active concentrations of
Results: Structural-energetic parameters c ould not be unambiguously assigned to           ZOL allowing the achievement of anti -tumour concentrations also in extra-bone sites. The second
cysteine in view of its capacity for disulfide bond formation. The energetic               strategy is to find new molecular targets of ZOL through the use of technological platforms such as
                                                                                                             s.
                                                                                           DNA microarray We have analysed the gene modulation induced by ZOL in androgen -resistant
contribution of histidine c ould not be determined i n view of the unc ertainty of its     prostate cancer PC3 cells with cDNA microarray platform to identify new molecular targets of ZOL
protonati on state at physiologic pH. The binding contexts defi ned by the types of        in prostate cancer. The gene coding for cysteine-rich, angiogenic inducer, 61 (CYR61), often over-
participati ng antibodies (antipeptide or antiprotein) and antigens (pepti de or           expressed in tumour cells, resulted highly down-regulated with a fold-change of 5.58. T herefore,
protein) were all fundamentally different from one another. Predictions on genuine         we have studied the effects of different concentrations of ZOL on CYR61 protein product an d we
antibody-antigen cross-reacti vity could be evaluated against empirical data onl y         have found that CYR61 protein expression was significantly decreased after exposure to ZOL on
                                                                                           both PC3 and DU145 cell lines. T he effect of ZOL on CYR61 expression was dose and time-
with regard to i nterac tion between antipeptide antibody and protein antigen.                                                                          ity
                                                                                           dependent and was due to a reduced transcriptional activ of CYR61 promoter as demonstrated
Maxi mum areas under the recei ver operator characteristic curves were                     by transfection with a plasmid encoding for luc -CYR61 promoter. Interestingly, other signal
approxi matel y 0.71 either with or without c onsi derati on of i mmunodominanc e.         transduction inhibitors did not induce or induced less effect on CYR61 modulation if compared to
Conclusions: 1) B-cell epitope predicti on is potentiall y complicated by the              ZOL. Moreover, ZOL reduced CYR61 expression through decreased activation of ras -raf-1-
presenc e of c ysteine and histidine. 2) The evaluation of B-cell epitope predictions      dependent pathway that was dependent from isoprenylation inhibition since they were antagonized
                                                                                           by the addition of either farnesol or geranylgeraniol. Finally, we have investigated the role of
against empirical data is meaningful onl y if the two pertai n to exactl y the s ame       CYR61 in the regulation of growth inhibition and invasion/motility of PC3 cells using a shRNA for
types of anti body (antipeptide or anti protei n) and antigen (peptide or protei n). 3)    CYR61 in order to down-regulate the expression of CYR61 protein. We have found that shCYR61
Predictions on genuine antibody-antigen cross-reacti vity can be evaluated agai nst        enhanced inhibition of proliferation induced by ZOL. Since CYR61 was reported to be involved in
empirical data in the cas e of interaction between antipeptide anti body and protein       the resistance to taxanes we have evaluated if ZOL could sensitize PC3 cells to Docetaxel (DT X).
antigen.                                                                                   We have found a sequence-dependent synergism induced by the combination between ZOL and
                                                                                           DTX on PC3 cell growth inhibition and similar results were recorded after transfection of PC3 cells
                                                                                           with shCYR61.
                                                                                                                                     Fa-CI plot

                                                                                                          3.0                                                                       Effect
                                                                                                                                                                                50%     75%
                                                                                                          2.0                                                       DTX  ZOL   0.475 0.623
                                                                                                                                                                    DTX + ZOL   0.760 1.135   CI
                                                                                                     CI




                                                                                                          1.0                                                       ZOL  DTX   0.873 0.822

                                                                                                          0
                                                                                                                0              0.2   0.4          0.6   0.8   1.0
                                                                                                                                       Effect
                                                                                                                    dtx72z48                CI = 1.0


                                                                                           Conclusions: In conclusion, it is possible to design new molecular rationale-based therapeutic
                                                                                           strategies in androgen-independent prostate cancer based on the use of ZOL.

The d ynamic h ypothesis of latent tubercu losis infection offer s a n ew r ational        Vascular disruption: an old mech anism rediscover ed for targeting tumor
to develop future therapeutic strategies.                                                  blood supply

CARDONA P- J                                                                               CARLSON RO, PLEIMAN CP, BAICHWAL V, MATHER G, SWABB E, YU M

Unitat de T uberculosi Experimental. Institut Ger mans Trias i Pujol. Uni versitat         Myriad Phar mac eutic als, Inc., Salt Lake City, UT, USA
Autònoma de Barcelona. Badalona. Catalonia. Spain. www.ute.galenic om.c om; e-
mail: pjcardona.igtp.germanstrias@genc at.c at                                             Background: Vascular disrupting agents (VDAs) are cancer drugs that act
                                                                                           through destruc tion of tumor neovasc ulature. The majority of known VDAs act
Background: It has been pos tulated that once infected by Mycobacterium                    through tubulin bindi ng which leads to des tabilization o f microtubules in
tuberculosis, its latent form can be retained for the whol e life, dor mant in old         neovascular endothelial cells, increased vascular permeability, loss of perfusion
lesions. With the aid of r esuscitation fac tors thes e bacilli can reac tivate towards    and tumor necrosis. Many VDAs are currently in all phas es of clinical
active tuberculosis. These assumptions raise at leas t thr ee rel evant questi ons to      devel opment. T he preclinical and clinic al history of MPC-6827, a quinaz oline-
ans wer: (1) how can dormant bacilli remain in the lungs for the whole life?               based tubulin targeting agent under development by M yriad Pharmac eutic als, is
Considering the cons tant cellular tur nover and healing of injured tiss ues; (2) How      presented as a c ase s tudy for discover y and development of a VDA.
are the resuscitation factors provi ded to the dormant bacilli, while immersed in old      Methods: MPC-6827 was identified through a drug discover y proc ess that began
lesions?; (3) why can a 9-month treatment with isoniazi d eliminate the dor mant           with a screen for apoptosis induction in a cancer cell line and continued through
bacilli? As isoniazid is acti ve onl y against growing bacilli, this treatment should be   biochemical, c ellular and animal model charac terization of acti vity. Subs equentl y,
provided for life, to avoid the reac tivation of dormant bacilli.                          safety and efficac y of MPC-6827 was s tudi ed i n two Phase I clinical trials.
Methods: Using experi mental models of latent tuberculosis infection (LTBI) in             Results: MPC-6827 was found to compete with colchicine for tubulin bi nding and
mice, we have demonstrated that the granulomas are c harac terized by:                     to des tabilize tubulin assembly using biochemic al and cellular ass ays. MPC -6827
Results: ( 1) the dr ainage of nonreplicating bacilli by the foamy macr ophages            was also obser ved to potentl y kill tumor and endothelial cells i n c ulture and rapidl y
towards the alveol ar spac es; (2) the constant formation of new foamy                     damage tumor neovasculature, induc e tumor necrosis and inhi bit tumor g rowth in
macrophages; (3) the presence of l ocal immunodepr ession, c haracterized by a             xenograft models. Further more, MPC-6827 did not show decreased potenc y in
high apoptosis, lac k of lymphoc ytic proliferation and anergy; ( 4) the reduction of      tumor cells overexpressi ng the ABC trans porters, P-gp (MDR-1), MRP-1 and
the immunol ogical response and foamy macrophages accumulation after a s hort-             BCRP-1, whic h mediate multidr ug resistance and maintai n the bl ood- brain barrier.
term chemotherapy, and the bacilli reactivation once is finished; (5) administration       MPC-6827 demonstrated high brain availability in mice, with expos ure i n exc ess of
of a vaccine based on frag ments of M. tuberc ulosis (RUTI) allows the control of          14-times that of pl asma. A maximum-tolerated dos e (MTD) of 3.3 mg/m2 was
this reacti vation by inducing a pol yantigenic res ponse against secreted and             elucidated in a Phas e I clinical trial.
structural antigens .                                                                      Conclusions: MPC-6827 is a potent c ytotoxic agent that inhibits tumor growth
Conclusions: The ―dynamic hypothesis‖ suggests that LTBI would be caused by the            primarily through vasc ular disruption. MPC-6827 is also not a substrate for ABC
constant endogenous reinfection of nonreplicating bacilli. This hypothesis is the onl y    transporters, which likely explai ns the extensive brain expos ure in mice. Based
one that may explain the efficacy of the 9-month isoniazid treatment, and supports a       upon the preclinic al data and upon identification of an MTD in humans, MPC -6827
therapy based in the elimination of the local immunosuppression by a short-term            has been advanced t o Phas e Ib/II trials in glioblastoma and melanoma.
chemotherapy, followed by a therapeutic vaccination able to generate immunity against
structural antigens to detect the resting nonreplicating bacilli and to avoid its
reactivation.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                          Page A-50
                                                       EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                              Celebrating the 100th Anni versar y of the
                                                                 Nobel Prize Award to Paul Ehrlich
                                                                   Nürnberg, October 3-5, 2008
Comparison of oral pharm acokinetics of nifedipine in different population s        Cross-kingdom Vaccines : dogma and her esy

CARRASCO-PORTUGAL MC 1, FLOR ES-MURRIETA FJ1,2                                               CASSON E A* & T OROSANTUCCI A
1                                                                            2
 Unidad de Investigación en Farmacol ogía, INER, Mexico City, Mexico; Escuela                Department of Infec tious, Par asitic and Immuno-medi ated Diseas es
Superior de Medicina del IPN, Mexico City, Mexic o                                           Istituto Superior e di Sanità
                                                                                             ROME (Ital y)
Background: Nifedi pine is a calcium c hannel bloc ker that is wi del y used in the
treatment of hypertension and angina pectoris. It has been described that this drug          *Presenting Author
is importantl y metabolized by CYP3A4 and differences in its pharmacokinetics
have been described. In fact, it has been suggested that pl asma levels are higher
in Nigerians, South Asians and Mexicans when compared with Caucasi ans. In
order to extend this information, the purpos e of this study was t o evaluate the oral       A vaccine made up by an algal β-glucan (laminarin; β-1-3 glucan with occ asional
phar mac okinetics of nifedipine in Mexic ans and to compare the pharmacokinetic             β-1-6 single glucose side c hai ns), conj ugated with diphtheria toxoid as a c arrier
parameters reported in different populations by means of a meta-anal ysis.                   protein component , protec ts agains t infections by different fungi and induc es
Methods: Twenty male healthy volunteers were enrolled in this study that was                 antibodies c apable of inhibiting fungal growth .T his is a sort of ―cross-kingdom‖
approved by the Ins tituti onal R esearch and Ethics Committees . All were fit               vacci ne because the immunizing antigen and the vaccination target belong to two
according to medical history, clinic al examination and suitable laborator y tes ts.         different kingdoms, and this is certainl y the first cas e in the field of human
After an overnight fast, subjects recei ved an oral dose of 10 mg nifedipi ne and            vacci nes, which are generall y bas ed on the dogma ― one or more specific antigens
blood sampl es were collect ed during 8 hours. Plas ma was anal ys ed by a validated         against one disease‖. T hus, it is ―heretic ally‖possible to c onvey in a single
HPLC method. Phar mac okinetic parameters were obtai ned by non-compartmental                immunol ogical tool the potenti al to protect against multiple infections, in our case
approach and compared by meta-analysis with thos e reported in different                     all those c aused by β- glucan -expressing fungi or bacteria. The generation of
populations.                                                                                 antibodies with the potential of directl y inhibiting the growth of, or killing the fungal
Results: Nifedi pine was rapidl y absorbed r eachi ng the maximum between 30 to              cells also opens an exciting perspecti ve for both ac tive and passi ve vacci nati on in
60 minutes , then dec ayed with a half-life of about 5 hours. When pharmacokinetic           immunoc ompromized subjects.
parameters obtained in this study were compared with thos e reported in other                The above approach c ould be theoreticall y extended to non-fungal infec tions by
populations by meta-anal ysis, it was obser ved that AUC reached in non-caucasian            selecting the appr opriate molec ular patter n shared by a given microbi al group (
(Asian, African and Mexican) populations was al most twice the r eported in                  e.g. peptidoglycan for Gram positive bacteria). Noteworthy , the molec ular
Caucasians, indicating interethnic differenc es in the oral pharmacokinetics of this         patterns are those microbial molec ules which foster natural i mmunity through their
drug.                                                                                        binding to the pattern-recognition structures on host c ells. Thus, single-
                                                                                             component, molec ular pattern- based vacci nes would merge the broad target
Conclusions: It is c onfirmed the existence of interethnic differenc es in t he oral
                                                                                             range typic al of innate immunity with t he highly foc ussed specificity of the adaptive
phar mac okinetics of nifedi pine, non-caucasians reac hing higher levels than
                                                                                             immunity.
Caucasians and ther efore, blindy extrapol ation of dos age regimens between
populations is not adequate for this drug.




Heparin as anti-inflammator y and anti-met astatic drug- new potentials of an                Electro chemical Behavior of Flavonoids in the Pr esence of Metal Ion s
old player
                                                                                             CERDÁ MF1, KREMER C 2, TORRES J2, HEINZEN H 2, BERTUCCI A2,
CELIE JW 1, BEELEN RH 1                                                                      DOMINGUEZ S3
1                                                                                            1                                                                                  2
 Dept. Mol ecular Cell Biolog y & Immunolog y, VU Uni versity Medical Center,                 Facultad de Cienci as, UdelaR, Montevideo, Uruguay; Fac ultad de Químic a,
Amsterdam, the Netherlands.                                                                  UdelaR, Montevi deo, Uruguay; 3Uni versidad de La Laguna, España
                                                                                             Background: Flavon oids ar e a lar ge gro up of phytoc hemic als ub iqu itously foun d i n ma ny fo od prod ucts of
Background: Heparin is a well- known compound bas ed on its antic oagulant                   vegetab le or igi n. They h ave b een r eporte d to hav e a br oad spectrum of pharm acol ogic al activ ity. In additi on,
activiti es. This mol ecule is a highly sulfated carbohydrate glycos ami noglyc an-          due to the prese nce of hy droxyl grou ps in th eir mo lecu lar structure, flavo noi ds exh ibit stron g anti oxid ant
                                                                                             properti es. Previo us works in this fie ld sh ow that the ir anti oxid ant activity is mo difie d in th e pres ence of meta l
chain, bel onging to the famil y of heparan s ulfates. H eparan s ulfates are                ions by the form ation of so lub le com plex sp ecies whic h cha nge the abi lity to scaven ge free ra dica ls. The goa l
ubiquitousl y expressed throughout the body, and are es peciall y known for their            of this work was to (i) m ake a comp arative stu dy o n the e lectroch emica l be havi or (cycl ic voltamm etry) of the
roles in cell adhesion and migration, angiogenesis and wound healing. Bas ed on              flavono ids qu ercetin (qrc), rutin (rut), rhamn etin (rha) an d isorh amneti n (irh) on g old e lectrod es, and (ii) to
                                                                                             study the influ ence of th e re leva nt metal io ns C u(II) and Fe(III) on th e e lect roch emica l ox idati on of the fo ur
the roles of heparan sulfates in physi ological processes , the use of heparin as
                                                                                             flavono ids.
anti-inflammatory and anti-metastatic drug has gained increasi ng research                   Methods: Qrc and rut were reage nt grade, purc hase d from commerci al sourc es and us ed with out further
interest.                                                                                    purificati on. Rh a and irh w ere iso lated from B. trimera. The is olate d comp oun ds wer e further char acterize d by
                                                                                             HPLC-DA D ana lysis an d HPLC co-c hromato grap hy with pur e stand ards purc hase d from Indofine a nd Fluka.
Methods: Sever al studies have exami ned the effecti vity of heparin, low molec ular         For cyclic voltamm etry exper ime nts, a polycrysta lli ne Au- pc disc ( 99.99 9 %, 3 mm diam eter) shro ude d into a
weight heparin and synthetic heparin mimetics in various experimental                        Teflon body an d a Pt sheet, 3.0 cm 2 geometric area, were use d as workin g and co unter electro des,
inflammator y models and tumor growth and metastasis.                                        respective ly. The refer ence was th e Ag/Ag Cl e lectrod e (3 M KCl, E = 0.2 30 V vs. S HE). The so lutio ns wer e
Results: Heparin and its derivates have been shown to have broad anti-                       prepar ed by m ixin g the flav ono id and/or the metal i on in bas ic me dia (p H ar oun d 9) to reac h 1 mM
                                                                                             concentrati on in water. The io nic strength was ad justed up to 0.1 5 M with NaC lO 4 ass upp orting el ectrolyte,
inflammator y properties in models of delayed type hypersensiti vity, peritonitis,           and ad justed to the des ired p H with di lute N aOH or HClO 4. Potentia l scan rates w ithin the ra nge 0.0 05 Vs -1 ≤ v
meningitis, allergic encephalomyelitis, airway (allergic) and cutaneous                      ≤ 0.05 Vs-1 wer e empl oyed.
                                                                                             Results: Evalu ated free flavo noi ds prese nt simil ar features, incl udi ng many a nod ic contrib utions l ocated i n the
inflammation, myocar ditis, as well as ischemia/reperfusi on-induced infl ammation in        positive p otentia l rang e. Some of them can be c ertain ly attributed to the red ox be havi or of the system Au-pc in
the heart, li ver and kidney. The mec hanism i nvol ved is likel y based on the ability of   the supporti ng el ectrolyte an d the others aris e f rom the electro active moi eties of the flavon oids.
heparins to bloc k leukoc yte adhesion and migration through bi nding of the selec tin       Table 1. Anodic co ntributi ons ascr ibe d to re dox pr ocesses invo lvin g functio nal grou ps of the flav ono ids. E(V)
                                                                                             vs. SHE
famil y of adhesi on mol ecules (es peci ally L-selectin and P-sel ectin), as well as        Anodic pe aks (Ean1) at ca. 0.3 V and 0.5 V c oul d rise                          qrc         rut           rha       irh
chemokines and c ytokines. In addition, there is increasing evidence that heparins           from the ox idati on of the OH m oiety from chatec ol to                        0.43        0.38           0.35      0.32
can inhibit tumor growth and metas tasis by affecting angiogenesis, cell                     quin one foll owi ng a two on e-el ectron ste ps p ath.              Ean1
                                                                                                                                                                             0.56        0.45           0.53
                                                                                             Contrib utions (E an2) at 0.6 8 V in rh a an d 0.87 V in qrc
proliferation, as well as cell-matrix interactions. We will shortl y discuss the several                                                                          Ean2       0.87                       0.68
                                                                                             could b e assig ned to oxi datio n of the OH located in
lines of evidenc e.                                                                          the same rin g than th e qui non e. This proc ess was n ot           Ean3       0.97                       1.13      1.07
Conclusions: Although heparin has been known and used for decades, it is                     detected in rut, caus ed by to the block ing of this positio n with the su gar resi due. In the case of irh this si gna l is
                                                                                             also abse nt, may be du e to difficu lties to reac h th e e lectrod e s urface or th e formati on of a n i ntramol ecul ar
becoming increasi ngly clear that this highl y potent compound may be effecti ve in          hydrog en b ond w ith the O of the qui non e. Final ly, contrib utions at ca.1 V (Ea n3) can be attribute d to the
several other clinically i mportant s ettings such as (chronic) inflammation and             oxidati on of some of the rem ain ing OH gr oups. Whe n copp er is ad ded, a n ew co ntributi on (not pre sent for the
cancer. Efforts are bei ng made to generate heparin-mi metics with targeted                  free flavon oids) is detecte d at ca. -0.2 V, ass ign ed to the c op per re dox b ehav ior itse lf. The strong i nteractio n
specificities to enhanc e their potential use i n clinic.                                    Cu(II)-flavono id mak es co pper r educti on l ess favora ble. As a cons equ ence, a po orer p articip ation of the
                                                                                             copper i on i n the formatio n and pr opa gatio n of the free rad icals is expecte d. After additio n of Cu, only the E a n1
                                                                                             is mainly affected and a ppe ared at less p ositive potenti als than i n the case of the free flavo noi ds, indic ating a n
                                                                                             increas ed r educti on pow er of th e co pper c ompl exes in aqu eous sol ution. Wh en Fe(III) is add ed to th e
                                                                                             flavono id conta ini ng sol utions, features ar e quite sim ilar to thos e observ ed for the copp er compl exes.
                                                                                             Conclusions: 1) The study of the electrochem ical b eh avior of the flavo noi ds in a que ous sol ution is a p owerfu l
                                                                                             tool to und erstand the r edox proc esses r elate d to th e Anti oxid ant Activity of these natur al antiox ida nts. The
                                                                                             oxidati on of the catecho l moi ety produc ing a q uin one ca n be spl it into two in dep end ent sign als an d ind ivid ual ly
                                                                                             analyz ed. 2) Further ox idati on of the rema ini ng OH gro ups of the q uin one ca n als o be stud ied i n this med ium,
                                                                                             as we ll as th e re ductio n of th e fl avon oids. 3) U pon comp lex ation with Cu(II) or Fe(III), the spec ies with 1:1
                                                                                             stoichiom etry prese nt a sim ilar v oltammetric pattern c ompar ed to th e free flav ono ids. H owev er, the oxi datio n
                                                                                             of the flavonoi ds is thermo dynam ical ly favored, esp ecia lly i n the case of the Cu(II) compl exes. 4) The compl ex
                                                                                             species als o prese nt cha nges i n the a bil ity of th e meta l i on to b e r educ ed, i.e., th e meta l r educti on is l ess
                                                                                             favorabl e in th e pres ence of the flavo noi ds. The flavo noi ds cou ld se quester Cu(II) and Fe(III) preventi ng the ir
                                                                                             reductio n a nd s ubse que ntly the formatio n of free rad icals. 5) The infl uenc e of C u(II) on th e AA of the
                                                                                             flavono ids, at l east from a therm odyn amic p oi nt of view, is m ore notori ous tha n in th e cas e of Fe(III). On the
                                                                                             other ha nd, the i nteractio n with fl avon oids re gar din g the poss ibi lity to red uce the m etal i on is also mor e
                                                                                             favorabl e for Cu(II). This work was ac cepted for publication in Inorganic Biochemistry: Re sea rch
                                                                                             Progress, NOVA Publishers, Ed. by J.G. Hughes and A.J. Robinson.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                               Page A-51
                                                                EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                        Celebrating the 100th Anni versar y of the
                                                                           Nobel Prize Award to Paul Ehrlich
                                                                             Nürnberg, October 3-5, 2008
Treatment of Hepatic Abscess: Magic Bullets and Beyond                                        Anti-Angiogenic and Anti-Neoplastic Potential of Prostate-Sp ecific Antigen
                                                                                              (PSA)
CERW ENKA H
                                                                                              CHADHA KC
Department of Surgery, M edical Uni versity of Graz, Austria.
                                                                                              Roswell Par k Cancer Institute, Buffalo, NY, USA
Background: Constant improvements of anti biotics have had crucial influenc e on
clinical management of pyogenic liver abscess (PLA). Advanc es in microbiol ogical            Background: In pr ostate cancer, disease progression and prognosis are rel ated
diagnosis as well as i n inter ventional radiolog y have als o contributed to better          to angiogenesis and the degree of tumor vascularizati on correlates with
outc ome, but s uccess ful treatment of PLA s till remai ns a consi derable clinical          progression and the development of metastatic diseas e. PSA protei n present
challenge.                                                                                    within the prostate tissue microenvironment repres ents the pool most critical to the
Methods: Clinical data of a s eries of 76 patients with PLA were anal yzed. Initiall y,       pathogenesis of pros tate cancer. Tissue-PSA (T-PSA) levels correlate with
broad-s pectrum anti biotics wer e given, and treatment was modified according to             prognosis in prostate canc er, as well as in breast cancer. Hypothesis: PSA down
sensiti vity testi ng as soon as possible. When indicated, additi onal therapy with           regulates pro-angiogenic growth fac tors and up-regulates anti-angiogenic growth
percutaneous puncture/drainage, endoscopic papillotomy/s tenting or s urgical                 factors.
inter ventions was used.                                                                      Methods: In vitro and in vivo studies wer e carried out usi ng prostate tumor (PC-
Results: The diseas e was confined to the right hepatic lobe i n 76% of the patients          3M) cells in cultur e and in nude mice. We anal yz ed modulation of protein
and to the left lobe in 7%; in 17%, both l obes were affected. Fifty-eight patients           expression in PC-3M cells by PSA using 2D-DIGE anal ysis coupl ed with HPLC-
(76%) had a single PLA and 18 patients had multiple abscess es. Etiolog y was                 MS/MS and SEQUEST data mini ng. Biological networ k analysis was carried out
biliary in 38%, hematogenous in 11%, posttr aumatic in 9% and cr yptogenic or                 using MetaCore integrated software designed for functional anal ysis of
attributabl e to rare r easons in the remaining patients . Microbiological cultur e was       experimental data. Gene expression data for several regulated proteins were
sterile in 24%, whic h was at least partly due to antibiotic pre-treatment.                   confirmed by real-time, quantitati ve PCR. Anti-angiogenic potential of PSA was
Staphyloc occi, Streptococci and E.coli were most often identified. Anaerobes were            also obs erved using human umbilical vein endothelial cells (HUVEC) in an in vitro
found in 15%. Factors associated with the need for surgery included empyema of                anti-angiogenic assay.
the gallbladder, underl ying malignanc y, perforation, vascul ar complications                Results:
(hepatic arter y thrombosis) and foreign bodies (e.g., toothpick, infected ventriculo-        a. Inc ubation of PC-3M cells with purified PSA res ulted in a significant down-
peritoneal shunt). In patients with biliary fistul ae it was important to ensure prompt       regulation of expression of 147 genes includi ng genes like VEGF, IL-8, EphA2,
bile flow (for i nstance, by s phinc terotomy/stenting)                                       CYR61, Bcl 2, Pi m-1 onc ogene, and uPA, that are associated with
Conclusions: In s urgical departments, we usually treat a s elected group of                  angiogenesis/tumor progression i n different c ancers and up-regulation of
patients with very severe forms of PLA. ―Magic bullets‖, adapted due to the res ults          expression of 137 genes including IFN and IFN-related genes and peptide
of microbiol ogical testing, c ons titute the mainstay for trea tment. However,               inhibitors of angiogenesis.
additi onal therapy with inter ventional radiolog y and/or surgery was us uall y              b. Forty one proteins were significantl y (p<0.05) changed in abundance in PC -3M
required in our pati ents and success ful management of underl ying diseas es                 cells treated with PSA. Many down regulated protei ns including N8 gene product
played a decisi ve role for positive outc ome.                                                long isoform, laminin rec eptor, Vimentin, DJ-1 and Hs p60 are known to be
                                                                                              involved in tumor progression.
                                                                                              c. PSA inhi bited significantl y, in a dose dependent manner, the
                                                                                              migration/c hemotaxis and attac hment functi ons required for tube formation by
                                                                                              HUVEC.
                                                                                              d. PSA delivered to PC 3M xenografts engrafted to nude mice resulted in inhibition
                                                                                              of tumor growth.
                                                                                              Conclusions:
                                                                                              1. In PC-3M cells, PSA down regulates pro-angiogenic growth factors and up
                                                                                              regulates anti-angiogenic growth factors.
                                                                                              2. Enz ymatically acti ve and inacti ve forms of PSA have anti-angiogenic acti vity in
                                                                                              vitro.
                                                                                              3. PSA has antitumor ac tivity against PC-3M xenografts in nude mice.




Magic Bullet s Versu s Shotgun Drugs in Can cer Th erap y and Prevent ion                    Capitalizing on CYP450 pol ymorphism in HIV tr eatment optimiz ation

CHAKRABARTY AM 1, FIALHO AM               2
                                                                                             TO KW1, LEE SS2,3, Chan DPC 3, LIU ST4, LEE KCK1 , CHEUNG SW2, CHAN CY2 .
1
    University of Illinois, Chic ago, USA, 2 Instituto Superior Tec nico, Lisbon, Portugal   1
                                                                                              Department of Medicine and Therapeutics, 2D epartment of Microbi ology, 3Stanley
                                                                                             Ho Centre for Emerging Infecti ous Diseases, The Chinese Uni ver sity of H ong
Background: The pioneering ideas developed over hundred years ago by Paul                    Kong, Hong Kong; 4Eas t Lake H ospital, Shenz hen, PRC
Ehrlich led not only to his Nobel Prize but also to the modern day way of rational
drug design. The c onc ept of pr omiscuous drugs, where a single drug can target             Background: The use of highl y ac tive antiretroviral therapy (HAART) is now the
not onl y multiple steps i n cancer progression and prevention but als o infections          standard in the clinical management of HIV infection. HIV patients are nor mall y
caused by viruses and parasites, is more recent and contrasts with the s hotgun              prescribed with 2 nucleoside re verse transcriptase inhibitors (NRTI) plus one of
approach of one target – one dr ug conc ept.                                                 the two other major cl asses of c ompounds – protease inhi bitors (PI) or non-
Methods: Bacterial proteins s uch as azurin or Laz are pr oduc ed as weapons by              nucleoside reverse transcriptase inhi bitors (NNRTI). As C ytochrome P450 is the
pathogenic bacteria to prevent other intruders to invade their habitats during               major enz yme s ystem for the metabolis m of the l atter two antiretrovirals, treatment
colonization in human tissues. Assays invol ving induc tion of apoptosis and cancer          respons e may var y from one ethnic group to another. Treatment opti mization
cell death as well as protei n-protein interacti ons have shown the ability of az urin or    could be effected by exploiting the phar mac okinetic and phar mac oegnomic
Laz to inhibit the growth of various cancers, HIV/AID S and malaria/toxoplas mosis-          patterns.
causing parasites , thus acting as a magic bull et.                                          Methods: NNRTI is commonl y prescribed as a c omponent of first-line regimen in
Results: The ability of az urin or Laz to induce apoptosis through complex                   treatment-naïve patients. The pharmac okinetics of efavirenze, an NNRTI is
formation with tumor s uppr essor pr otein p53 or inhibit growth of cancer cells by          correlated with the CYP450 2B6 pol ymorphis m in a C hines e population.
interfering in receptor tyrosine kinas e-mediated c ell signaling or preventing              Results: We have s tudi ed the prevalence of G516T of CYP450 2B6 gene in
angiogenesis has been s hown. In vivo c anc er regression by azurin has als o been           healthy Hong Kong Chines e bl ood donors and found it to be high (TT genotype
demons trated. A chemicall y-s ynthesized 28- amino acid peptide derived from                23%; allelic frequenc y of T at 516 0.43) compared to Cauc asian popul ations.
azurin, ter med p28, is non-toxic to animals includi ng monkeys and is under                 Interes tingly t here‘s also variation among different ethnic groups in C hines e
consideration by the U.S.FDA for phase I human clinical trials as an anticancer              population. In a followup study we recr uited 68 Chi nese HIV patients from a
agent. Azurin and p28 c an als o interfere in onc ogenic transformation to prevent           hospital in Southern China. All patients have been recei ving a HAART regimen
precanc erous lesion formation i n mous e alveolar and ductal mammar y glands                comprising of efavirenz, an NNRTI metabolized largely through CYP450 2B6. The
expos ed to a carcinogen DMBA. Many patents have been issued to cover thes e                 allelic frequenc y of 516T in this cohort was 0.23. T he TT genotype was associ ated
inventi ons.                                                                                 with a higher level of efavirenz at 8-12 hours post-dos e, as c ompared to GT or
Conclusions: While Paul Ehrlich emphasized human ingenuity to design new                     GG. The phar mac okinetic pattern of TT was likewise different.
chemicals for the treatment of i nfec tions and c anc er, suc h efforts are limited to our   Conclusions: 1) The deter mination of CYP genotype and allelic pattern ca be a
ability to develop validated targets. Our understanding about c ancer is an evol ving        useful adjunct in the clinical moni yoring of HIV I infection. 2) Adjus tment of
process and will take years for a fuller grasp. A single or few targets als o allow the      efavirenz dos age in accordanc e with CYP genotypic or allelic pattern c ould be a
diseas e agents to change such targets to bec ome drug resistant. Bacteria have 3            potentiall y us eful strategy for opti mizing treatment outcome and minimizing
billion years of evolutionar y wisdom and c an design proteins as weapons that               adverse effects. 3) Clinical guidelines on the use of antiretr oviral ompounds may
target multipl e diseas e agents and multi ple steps in the disease pr ogression             need to be refi ned acc ording to the pharamcogenomic pattern of the l ocal
pathway to prevent rapi d drug resistanc e. We believe suc h multi-targeting                 population.
bacterial proteins will be our next generation drugs as envisioned by Paul Ehrlich.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                             Page A-52
                                                                  EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                         Celebrating the 100th Anni versar y of the
                                                                            Nobel Prize Award to Paul Ehrlich
                                                                              Nürnberg, October 3-5, 2008
Novel Clinically R elevant Proteasom e Inhibitors and HDAC Inhibitors                          Neom ycin, Kanam ycin and Pyranm ycin: Synthesis, Antib acterial Activity and
                                                                                               New Applications
CHANDRA J1, MILLER C 1, RIVERA N 1
                                                                                               CHANG CW T
1
  Children‘s Canc er Hospital Uni versit y of T exas M.D. Anderson Cancer Center.
Houston, T exas, U.S.A.                                                                        Department of Chemistr y and Bioc hemistr y, Utah State Uni versity, Logan, Utah
                                                                                               84322, USA
Background: Agents with specific biologic targets, that display more selec tive
killing of leukemia c ells as compar ed to nor mal lymphoc ytes require further study          Background: Anti biotic resistance represents stringent probl ems for the global
in thes e dis eas es. Here we foc us on two distinct cl asses of new agents:                   health. Development of new anti biotic is urgent. Utilizing synthetic methodologies
proteasome i nhibitors and histone deacetylase i nhibitors (HDACi) and highlight a             (glycodi versification), we have s ynthesiz ed libr aries of neomycin and kanamycin
specific agent withi n eac h class as possessing unique properties with potential              classes of aminogl ycosides. Novel aminoglycosides with prominent antibac terial
therapeutic benefit. NPI-0052 is a proteasome inhibitor distinct from bortez omi b,            activity against a panel of resistant bacteria including Pseudomonas aerugi nos a,
which is approved by the Food and Drug Administration (FDA). PCI-24781 is an                   mecilline-resistant Staphyloc occus aureus (MRSA) and vancomycin-resistant
HDACi which targets a distinct class of HDAC‘s more specificall y than does                    enterococci (VRE) have been identifi ed. In additi on, we have also disc overed new
vorinostat, the onl y FDA approved HDACi. Here we tested these c ompounds in                   applications of aminogl ycosides. T hes e include antifungal, anti viral and potential
leukemia c ells to determine the mechanism of c ytotoxicity and to c ompar e them to           therapeutic for neural dis ease.
approved counterparts.                                                                         Methods: We empl oyed two different s ynthetic s trategies: 1) library construction
Methods: Cell lines were repres entati ve of ac ute myelogenous leukemia and                   of aminoglyc osides using glycodi versification approac h, and 2) leads construction
acute lymphoc ytic leukemia. Cell death was ass essed by meas uring DNA                        using carbamate approach. Whol e-cell based assay and mol ecular modeling have
fragmentation by propidium iodide s taini ng and flow c ytometr y. Cas pas e                   been employed for revealing useful structural ac tivity relationshi p.
activation was measured by acti vity ass ays and by western blotting. Oxidative                Results: From our studies on carbohydrates and aminoglycosides, we have made
stress was quantitated using dichlorofluoresci en and dihydroethidium to meas ure              several important discoveries whic h can be divi ded into two as pects i n chemistr y
levels of intr acellular peroxides and superoxide, respecti vel y. Proteasome acti vity        and biolog y areas.
assays were c onducted using fluorogenic peptides. Histone ac etylati on was                   In chemistr y area, we have invented several s ynthetic protocols to be us ed in the
assessed by western blotting for histone H 3 ac etylation. Combination indices were            synthesis of di verse glyc oconjugates of biological interest. In addition to the librar y
based on Chou and Tal alay‘s methods.                                                          synthesis of aminogl ycosides, we have developed methods li ke dideoxygenation
Results: Dose respons e and time course studies revealed that NPI-0052 is more                 at mild condition, regiosel ecti ve Staudinger reaction, and azido-migration for
potent than bortez omi b and inhibits the catal ytic acti vities of the proteas ome more       creating new structural scaffolds on aminogl ycosides.
effec tivel y than bortezomib. Similarly, PCI-24781 exerted unique effects, c ausing           In biol ogical area, one of the newly developed aminogl ycosides, pyrank acin
histone hyper acetylation at lower doses than vorinostat. Cells lac king cas pas e-8           manifests superior acti vity than clinicall y used ami kacin and gentamicin agai nst
did not display histone acetyl ation by PCI-24781. Surpisingl y, NPI-0052 pr omoted            various resistant bacteria. Two ami noglyc osides exert impressi ve acti vity (low
histone acetylation which was dependent upon caspase-8 and oxi dati ve stress.                 micromolar) against MRSA and VRE. In the antifungal studies, we have identified
Synergy of N PI-0052 with several HDACi was stronger than seen with bortezomib.                two leads with promi nent acti vity against fungal pathogens, suc h as F usarium
Conclusions: 1) NPI-0052 is more potent alone and i n combi nati on with HDACi                 gram  inearum. In the effort of developi ng potential ther apeutics for the treatment of
than bortez omi b. This agent also acetylates histones in an oxi dant and cas pas e-8          spinal musc ular atrophy (SMA), we have notic ed a l ead with eminent acti vity in
dependent manner. 2) PCI-24781 action was also dependent upon cas pase- 8,                     mouse model.
suggesting that promoti ng caspas e-8 acti vity may complement the acti vity of both           Conclusions: Through our continuous effort, we have developed new
agents in leukemia.                                                                            aminoglycosi des with broad spectr um acti vity against resistant bacter ia.
                                                                                               Additionall y, new acti vities and applicati ons have als o been discovered.




Competing Causes of Death from a Randomized Tr ial of Extended Adjuvant                       The Antagonistic Role of Curcumin against Nicotine induced Genotoxicit y
Endocrine Th erap y for Breast Cancer: NCIC CTG MA.17                                         on different Organs of F emale Rats under Restricted Dietar y Protein

CHAPMAN JW 1, MENG D 1, SHEPHERD L1 , PARULEKAR W1, INGLE JN 2,                               BANDYOPADHYAYA G1, SINHA S1, CHATTOPADHYAY K2 ,
MUSS HB3, PALMER M 1, YU C 1, GOSS PE4                                                        CHATTOPADHYAY BD 1
1
    NCIC Clinical Trials Group, Kingston, Canada; 2Mayo Clinic, Roc hester, USA;              1
                                                                                                  Jadavpur Uni versity, Kol kata, India; 2Uni versity of Calcutta, Kol kata, India.
3
    University of Vermont, Burlington, U SA; 4H arvard Uni versity, Bos ton, USA.
                                                                                              Background: Nicotine, a major phar macol ogically acti ve substance of tobacco for
Background: Older women with earl y-stage breas t canc er experience higher                   several diseas es, has proven to be a potential genotoxic compound. It is abs orbed
rates of non-breast cancer-related death. We examined factors ass ociated with                through lungs with s moki ng and mai nly metabolized i n liver, yet its effect on li ver
cause-specific death in a large cohort of breast cancer pati ents treated with                injuries and other organs particularly in r estricted protein diet are not clear. The
extended adjuvant endocrine therapy.                                                          aim of this study was to investigate the genotoxicity of nic otine and corresponding
Methods: In the MA.17 trial c onduc ted by the National Cancer Institute of Canada            the protecti ve role of curcumin on li ver, ovar y and uterus of female populations
Clinical Trials Group, 5170 breast canc er pati ents (medi an age = 62 years; range           particularl y who us ed tobacco but depri ved of healthy di et.
= 32-94 years) who were dis eas e-free after approximatel y 5 years of adj uvant              Methods: The study was investigated by: 1) Meas urement of total DNA
tamoxifen treatment were randoml y assigned to treatment with letr ozole (2583                concentrati ons and 2) Anal ysis of DNA damage by Comet assay i n the
women) or placebo (2587 women). The median follow-up was 3.9 years (range =                   investigated tissues of female rats maintained under altered protein diets. Two
0–7.0 years). We investigated the associ ation of 11 bas eline factors on the                 groups of femal e albino rats (15 ani mals each) were maintai ned in normal protein
competing risks of death from breast cancer, other malignancies, and other                    diet (18% cas ein) and restricted protein diet (5% casein) respec tivel y. Eac h group
causes . All statistical tests wer e two-sided li kelihood ratio criterion tests.             was di vided in three subgroups (5 animals each). First subgroup was s erved as
Results: During follow-up, 256 deaths wer e reported (102 from breast cancer, 50              control and sec ond and third subgroups (experimental) was injec ted nicotine
from other malignancies, 100 from other caus es, and four from an unknown                     tartrate ( 2.5 mg/kg body weight/day) subc utaneousl y and the third s ubgroup was
cause). Non- breast cancer deaths acc ounted for 60% of the 252 known deaths                  administered c urcumi n (80 mg/ kg body weight/day) orall y. The animals were
(72% for thos e ≥70 years and 48% for thos e <70 years). Two bas eline fac tors               sacrificed after 21 days of treatment and the total DNA c ontent in the s pecified
were differentiall y associ ated with type of death: cardi ovasc ular diseas e was            tissues were meas ured. The DNA damages in the tissues were determi ned by
associated with a statistically significant increased risk of death from other caus es        Comet ass ay. R esults were analyz ed by One way Anal ysis of Variance, all pair
(P = .002) and osteoporosis was associ ated with a statistically significant                  wise Multiple Comparison Pr ocedure (Holm-Sidak) and Func tion CORREL of
increased risk of death from other malignancies (P = .05). An increased risk of               Microsoft Excel.
breast c anc er-specific death was associated with l ymph node invol vement                   Results: Total DNA contents of all investigated tissues were decreased more
(P<.001). Increas ed risk of death from all three causes was ass ociated with ol der          significantly (P< 0.001) by nicoti ne in both dietary conditi ons. Significant ( P< 0.01)
age (P<.001).                                                                                 increase of total DNA content in nor mal diet and more significant (P<0.001)
Conclusions: Non-br east c ancer-related deaths were mor e common than breast                 increase of that i n protein restricted diet was obs erved due to c urcumin
cancer-related deaths in this cohort of 5- year c ancer s urvi vors, especially among         suppl ementations. Curc umin more significantly (P< 0.001) reduc ed the DNA
older women.                                                                                  damage percentage of the liver tissues in protei n-restricted c ondition.
                                                                                              Conclusions: 1) The degree of nicoti ne-induced genotoxicity increas es in protein
                                                                                              restricted condition. 2) Curcumi n effecti vel y reduces the effect of nicotine as
                                                                                              obser ved in tissues . 3) The protecti ve role of curc umin is more pronounced under
                                                                                              protein-restricted c ondition.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                     Page A-53
                                                     EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                           Celebrating the 100th Anni versar y of the
                                                              Nobel Prize Award to Paul Ehrlich
                                                                Nürnberg, October 3-5, 2008
Dose-dependent Antiherpesvirus Activit y of an Ethnomedicinal                    Blocking Effect of an Immuno-Suppressive Ag ent, C ynarin , on CD28 of T-
Ph ytophores: Sear ch for Magic Bullet                                           Cell Receptor Found b y a Novel Pharmaceutical Method

CHATTOPADHYAY D 1*, ARUNACHALAM G1, MANDAL AB2, MAHMUD TAREQ                                      CHEN HM
HASSAN KH AN 3, AND CHAKRABARTY S1
                                                                                                  National Nano Device Labor atories, HsinChu, Tai wan, R.O.C.
1
  ICMR Virus Unit, I.D. & B. G. H ospital, GB 4, 1ST Fl oor, 57, Dr. Sures h C
Banerjee Road, Beliaghata, Kol kata 700010, Indi a;                                               Background: Resting T-cells ar e sti mul ated to initiate i mmune acti vity in
2
  Department of Biotec hnology, Central Agricultural Res earch C entre, Port Blair,               respons e to s pecific exter nal sti muli. T he ―signal 2‖ pathway is in part initiated by a
Andaman and Nicobar Islands, India; and                                                           binding interaction between CD28 (T-cell receptor) and CD80 (B-cell receptor).
3
  School of Molecul ar and Structur al Biolog y, Department of Phar mac ology,                    Recent efforts to find new i mmuno-suppressi ve drugs have foc used on cell
Institute of Medical Bi ology, Uni versity of Tromsø, 9037 Tromsø, Nor way.                       membrane rec eptor‘s antibodies. However, using anti bodies as drugs has many
                                                                                                  drawbac ks. Also, unli ke s mall molecules , whic h mimic metabolites in the body, the
Backgro un d: Ophiorrhiza nicobarica Balkr., a wild herb popularly used as an antiinfective       large antibodies might induce other immune s ystem problems and therefore
medicament by the Shompen and Nicobarese tribes of Grate Nicobar Islands, India, was              reduce their therapeutic utility.
investigated for its anti-herpes virus and antibacterial activities.                              Methods: In this study, cynarin was purified from Ec hinac ea purpurea extracts on
Me th ods: T he w hole herb was ext racted in wate r and eth anol, and fractioned in n -butanol   a silica gel open c olumn with serial el ution buffers. Based on HPLC anal ysis, the
for the isolation of bioactive compounds using T LC, HPLC, HPT LC and N MR. T he in vitro         active frac tion including cynarin was collec ted at a retention time of 32.612 mi n.
and in vivo toxicity of the extract were determined in Vero cell and in mouse model. T he
                                                                                                  The UV absor ption peak shifted from 338 nm (crude extract) to 330 nm (c ynarin
antiviral activity and its mechanism was tested by Cytopathic effect (CPE), Plaque
                                                                                                  pure fraction). The c ytotoxicity of c ynarin tr eatment of T-cells was measured by
reduction assay, Yield reduction assay, and Dose response curve using the H erpes
Simplex Virus type 1 (HSV -1) and HSV type 2 (HSV-2 ). T he EC50 (50 % protection                 MTT colorimetric assay. Molecular modeling and doc king among cynarin and
against virus induced cytopathic effect) and Selective Index (SI; ratio of 50% cellular           CD28/CD80 were done by PRODRG2 based on AutoD oc kTools.
cytotoxicity to EC50) was determined. T he antibacterial activity was tested by disc              Results: Cynarin, a potenti al immunosuppress ant that bl oc ks the interaction
diffusion and agar dilution methods on 150 bacte rial isolates of human origin, and the           between the CD28 of T-cell rec eptor and CD80 of antigen pres enting c ells, was
mechanism was tested by biochemical and transport studies.                                        found in Ec hinac ea purpur ea extr act by a new pharmaceutical screeni ng method:
Results: T he e xtract was non-to xic up to 3.6 mg ml-1, while at 300 mg ml-1 it completely       After Flowing Through Immobilized Rec eptor, (AFTIR; G.-C. Dong et al., J. Med.
inhibits plaque formation in HSV-1 and HSV-2. T he antibacterial activity was noticed at          Chem., 2006, 249: 1845-1854). This Ec hinac ea c omponent is a s mall molec ule
128-2000 µg ml-1 against 25 Gram negative and 53 Gram positive isolates. T hese data              that is able to s pecific ally bloc k ―signal 2‖ of T-cell acti vation. In this study, we
are highly significant (p<0.05) compared to the control. T he bioactive part of the e xtract      further confirm that cynarin effecti vel y bloc ked the binding between CD80 of B-
contains ursolic acid (triterpene), que rcetin (flavonoid), ?-sitosterol and harmine (? -
                                                                                                  cells and CD28 of T-cells, and pr ovide details of its mec hanis m of ac tion .
carboline indole alkaloid). T he isolated ursolic acid and quercetin in combination (100?g
                                                                                                  Conclusions: The r esults above confirm both that AFTIR is a promising method
ml-1 each) showed highly significant (P>0.001) anti -HSV activity (EC50 = 37.2 fo r HSV -1
and 45.1 for HSV-2), while harmine showed moderate anti -HSV activity at 300?gml -1               for screening selecti ve ac tive c ompounds from herbal medicine and that c ynarin
(EC50 = 74.4 for HSV-1 and 82.2 for HSV-2 ). Interestingly the eclipse phase initiation of        has great potential as an immuno-suppressi ve agent.
HSV-2 was delayed at 100 mg ml -1 of ursolic acid. T he yield reduction inhibition was 85%
with ursolic acid, 68% with harmine and 51% with quercetin respectivel y for HSV -1.
Mo reover, the ursolic acid in combination with acyclovir showed the highest activity (E C50
20.1 and 32.2, SI was 87.5 and 90.4 respectively). T he results re vealed that the ea rly
stage of multiplication was blocked by quercetin while ursolic acid and harmine block the
late stage of multiplication. While membrane destabilization in susceptible bacteria is
noticed with ursolic acid (50-100?g ml-1 ).
Concl usio n: T he O. nicobarica ext ract ha ve good anti -HSV activity, along with mode rate
antibacterial activity possibly due to its ursolic acid, quercetin and or harmine content.
Studies showed that the ext ract is acting on early and late stage of HSV multiplication and
have SI value grate r than 20. T hese results suggest that this herb ha ve the potential in the
management of HSV infections, particularly in primary health care.




Synergistic inhibition of taxol- r eisistan ce primar y ovarian can cer cells b y                 From Mono- to Dimeric-IRFs: The Heart of the Matter in Activation of the
oridonin and wogonin                                                                              Interferon Regulator y Factor s

CHEN S1, WADES J1, JONES M 1, BUTLER-MANUEL S2                                                    CHEN W , LAM SS, SRINATH H, SCHIFFER CA, LIN K, ROYER WE
1
  Ovarian and Prostate C ancer R esearc h Trust Laborator y, Guildford, Surrey, UK,               Dept. of Biochemistr y and Mol. Phar m., U niv. of Massac hus etts M ed. School,
2
  St. Luke Cancer Centre, Guildford, Surrey, U K                                                  Worcester, MA 01605

Abstract: In researching the mol ecular principle of Chinese herbal medicine for                  Background: Members of the Interferon Regulator y Factor (IRF) family of proteins
cancer therapy, we use the composition of the original US phyto product ―PC                       play important roles in develo pment of the immune system, hos t defens e,
SPES― as a study model. Now no longer availabl e, PC SPES was reported by                         inflammation, apoptosis and tumorigenesis. Acti vation of these proteins in the
several research groups to be acti ve in suppressi ng hormone refrac tor y pros tate              cytoplas m is triggered by phosphor ylation of Ser/Thr residues in a C -termi nal
cancer. Among the isol ated phytoacti ve chemicals, baicalein, oridonin                           autoinhibitor y region. Phosphor ylati on stimulates dimerization, trans port into the
isoliquiritigenin and wogonin were found to indi viduall y or combi natorially inhibit            nucleus and assembly with the coac tivator cAMP r esponse elem ent-binding
the ovarian c anc er cell lines s ensiti ve (A2780) or resistant (PT X10) to taxol. The           binding protein (CBP)/p300 to acti vate transcription of type I i nterferons and
activity was confirmed by MTS c ell viability assay, SR B antiproliferation assay and             other target genes. However, it is unknown how the phosphor ylation of IRF
colony formation ass ay. Although the four agents shar e some common molec ular                   proteins acti vates dimerization and why di merizati on favors bindi ng of CBP/p300.
targets includi ng the i nacti vation and down regulati on of transcription factors NF-           Methods: To that end, x-ray cr ystallography,
kB and androgen r eceptor, oridonin s eems to dis play the most potent acti vity in
modulati ng canc er cell apotosis and stabilization of p53 protei n. To full y
understand the action of botanic al medicine, it is essential to investigate the                  Results: We present here that the 2.0Å r esoluti on cr ystal structure of a di meric
combinator y effect of acti ve agents. Our investigation has led to the discover y that           form of the IRF-5 transac tivation domain (residues 215-477) in which Ser 440 has
differential s ynergism is dependant on the cancer c ell type. While the strongest                been mutated to the phos phomi metic Asp. The struc ture reveals a s triking
synergistic inhibition was obser ved i n prostate canc er c ell line DU-145 (isolated             mec hanis m of di merization in whic h the C-termi nal autoinhibitor y domai n attains a
from hormone therapy refractor y pati ents and androgen r eceptor insensiti ve)                   highly extended c onformation permitting extensive c ontacts to a sec ond subunit.
induc ed by the c ombination of oridonin and baical ein, the c ombined pair of                    Based on comparison with cr ystal structures of IRF-3, these r esults provide a
oridonin and wogoni n exhibited the mos t potent antiprolifer ati ve acti vity i n ovarian        structural basis for the coupling between dimerization and CBP binding in IRF
cancer cells A2780 and PTX10. Sinc e taxol is the last treatment option in ovarian                famil y members, in which the C-terminal autoi nhibitory domain plays a dual rol e. In
cancer therapy, we further tested our finding on the primary ovarian cancer cells                 the unphosphor ylated form, the C-ter minal autoinhibitory domai n bi nds to and
directly isolated from the ascitic fluid of patients. Preliminary results reveal that the         mas ks the hydrophobic CBP/p300 bindi ng surfac e.
indivi dual and combined agents mar kedly suppress the proliferation of primar y                  Conclusion: Phos phoryl ation s timul ates the unfolding of the C-termi nal
cancer c ells. In c ontr ast, taxol failed to inhibit both primar y c anc er cell c ultures up    autoinhibitor y domain which then for ms extensi ve contacts with a second IRF-5
to a conc entration of 100 nM. In view of the different c hemical struc ture and                  subunit, leaving a hydr ophobic s urfac e free for binding CBP/p300.
biological acti vity of terpenoi ds and flavonoids , we are interested to understand the
scientific rationale of the combination. On this ground, we pres entl y are studying
the underl ying molec ular mec hanism and the s ystems bi ology networ k affected by
phytochemicals. T his report discusses our initial findings. We thank Dr. Marianne
Poruchyns ky of NIH (Roc kville, Mar yland) for the gift of PTX10 c ell line.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                      Page A-54
                                                        EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                             Celebrating the 100th Anni versar y of the
                                                               Nobel Prize Award to Paul Ehrlich
                                                                  Nürnberg, October 3-5, 2008
iTRAQ-coupled 2D LC-MS/MS Analysis of Proteomics Profile in Cel ls                 New targets for new drug discover y opportunities.
Incubated with S- or R-enantiomer s of Chiral Drugs: from Metabolic
Path ways to Biomarker Discover y                                                  CHENE P

CHING CB, SUI JJ, ZHANG JH, CHEN W N                                                         Oncolog y Res earch, Druggability-Enz ymology-Profiling Unit, Novartis, Bas el,
                                                                                             Switz erland.
School of Chemical and Biomedical Engineering, Nanyang Technological
University, Singapore                                                                        Today most phar mac eutical c ompanies use si milar strategies to target the s ame
                                                                                             proteins . As a cons equenc e ther e is a ver y high competition between the different
Background: Protein profile in cells incubated with indi vidual enantiomers of               phar ma, a reduction of the potential mar kets , a lower innovation and a limited
chiral drugs is important to understand their differential mechanis ms of action.            freedom to operate. Overall the pr oducti vity is decreasi ng. The identification of
Using propranolol, a β-blocker used for controling hypertension and myoc ardial              new drug targets is then very important for the future of the pharmaceutical
infarction, the main aims are: 1) To establish cellul ar protei n profile by LC-MS/MS        industr y.
apparoac h. 2) To identify specific proteins secreted from cells i ncubated with             During this presentation we shall examine the ATPas es as a new famil y of drug
propranolol.                                                                                 targets. From a brief structural anal ysis of the ATP-binding site of these enz ymes,
Methods: The vascul ar smooth muscle A7r5 cells were cultured and incubated                  we s hall see that some thes e enz ymes are very good drug targets for a s trateg y
with the S- or R-propranolol at a c oncentrati on which showed no significant effect         aiming for the identification of ATP c ompetiti ve inhibitors.
on the cell viability. A 4-plex multiplex strategy was us ed to simultaneously detect        Finally, we will s how that it is possible to identify l ow molec ular weight inhi bitors of
and quantitate differenc es in intrac ellular as well as extracellul ar protei ns in         thes e enz ymes and to develop them up to the clinic.
untreated vasc ular smooth muscl e cells and thos e inc ubated with S- or R-
propranolol. The anal ysis was performed on an Agilent 1200 nanoflow LC sys tem
interfac ed with a QSTAR XL mass spec trometer (Applied Bios ystems). Relative
abundanc e quantitati on and protein identific ation were perfor med using
ProteinPilotT M Software 2.0 (Applied Bios ystems).
Results: A total of 40 and 13 unique proteins were identified i n three independent
experiments in cells and c ulture medium of cells i ncubated with S- or R-
propranolol, res pecti vely. For intracellular proteins, t he higher level of enz ymes
involved in c ellular anabolis m and antioxidant acti vity in c ells incubated with the S-
propranolol was supported by Real-Time PCR and Wes tern bl ot anal yses. The
increase in the anabolic acti vity was als o supported by the higher intrac ell ular
concentrati on of the metabolic cofac tor NAD +. For extrac ellular protein, only T-
kininogen was found to be significantl y increas ed in c ells incubated with S-
propranolol by Western bl ot anal ysis.
Conclusions: 1) Metabolic effect ass ociated with propranolol treatment was
revealed. The relevant metabolic enz ymes may be us eful targets for future
phar mac eutical interventions to reduc e clinical side effects following propranolol
treatment. 2) The higher level of secreted T-ki ninogen fr om cells inc ubated with S-
propranolol may provi de a link of this vasoac tive pepti de to treatment of
cardiovasc ular dis ease. 3) Our approach may be a platfor m for drug -target cells
anal ysis and biomarker discovery.




Association bet ween Alpha-2a-Adr energic Receptor Gen e and                                 From Gene Expr ession Profile to Identification of Molecular Target Gr anulin-
Methylph enidate R esponse in Korean Childr en and Adolescents with ADHD                     Epithelin Pr ecur sor for Liver C ancer

CHEON K-A1*, CHO D-Y2, SHIM J-O1, KOO M-S1                                                   CHEUNG ST, FAN ST
1                                                2
    Univ. Kwandong, Koyang City, South Korea; Labgenomics, Seoul, South Korea.               Department of Surgery, T he U niversity of Hong Kong, Pokfulam, Hong Kong.

Background: Methylphenidate (MPH), known to be effecti ve for the attention                  Background: Primary liver cancer, hepatocellul ar carcinoma (HCC), is the third
deficit probl ems, bloc ks norepinephrine trans porters and low oral dos es of MPH           cancer killer worldwide. The maj ority of HCC patients have no effec tive therapeutic
have more effect on norepinephrine than on dopamine in s ubc ortical areas. Alpha-           option. Aims: 1) To identify novel therapeutic target from the HCC microarray
2a-adr energic receptor (ADRA2A) is a key c omponent of the noradrenergic                    genome-wi de expression profiles. 2) To develop neutralizing monoclonal antibody
system. The ai m of this study was to evaluate the ass ociati on between the                 (mAb) agai nst the therapeutic target and examine its func tional effect.
ADRA2A pol ymor phism and the clinical improvement of symptoms with MPH                      Methods: The expression profiles of more than 200 HCCs and adjacent li ver
treatment in Korean subj ects with ADHD.                                                     tissues were exami ned. Genes that demonstrated differential over expression in
Methods: This study included 114 ADHD children (mean age= 9.08±1.94 years)                   significant portions of tumors, revealed clinicall y relevant biological functions , and
who were recruited from the child ps ychi atric clinic at uni versity hos pital in South     preferabl y a secretory factor (diagnostic ther apeutic concern) were short listed for
Korea. The subjects who had an greater than or equal to 50% compar ed with the               further eval uati on. The granulin-epithelin precursor (GEP, also named progranulin,
baseline ADHD rating scale (ARS) scores and who had 1 or 2 point of Clinical                 acrogranin, or PC-deri ved growth factor) was identifi ed as potenti al therapeutic
Global Impressi on–impr ovement (CGI-I) score after 8 weeks of treatment were                target. The GEP expression on mRNA level was subsequentl y validated by real -
considered as the ‗good res ponse‘ group. After perfor ming genotyping for                   time quantitati ve RT-PCR, on protei n level by wes tern blot and
ADRA2A, we examined the c orrelation the ADRA2A pol ymorphis m with MPH                      immunohistoc hemistr y. The GEP mAb was raised against the peptide designed at
respons e and also c ompared the change of total ARS scores between genotypes                the carboxyl-terminal of GEP. Neutr alization effec t was examined on hepatoma
at ADRA2A.                                                                                   cells and normal liver cells in vitro and in viv o.
Results: e found that while 76.9% of the subjects with G/G genotype showed a                 Results: The GEP overexpression was demonstr ated in more than 70% of HCC
good respons e, 46.0% and 41.7% of the subjects with C/G and C/C genotype                    tissues. GEP controls HCC cells proliferati on, i nvasion and tumorigenicity. Thes e
showed a good res ponse to MPH treatment acc ording to AR S ass essed by parent              in vitro and in vivo data c orroborated the clinical findings that GEP over expression
(Pearson χ2 value= 11.929, df=2, p=.003). We als o found a significant differ ence of        is associated with aggressive canc er features including large tumors, venous
the change at total ARS scores between the subjects with and without G/G.                    infiltration (micrometastasis) and earl y recurrence after curati ve surgery. The GEP
(t=2.21, df=1, p=.029). In ter ms of treatment res ponse acc ording to the CGI-I,            mAb inhibited the growth of human hepatoma cells Hep3B and HepG2, but
significant correlation was found between genotypes at ADRA2A (Pearson                       revealed no significant effect on normal liver cells MIHA. In the nude mice model
  2
χ value=7.250, df=2, p= .027).                                                               transplanted with human HCC cells Hep3B, GEP mAb decreased the ser um GEP
Conclusions: Our findi ngs provide evidenc e of an associati on between the                  level and inhibited the growth of es tablished tumors in a dose-dependent manner.
ADRA2A genotype and res ponse to MPH treatment ass essed by both parents and                 The GEP mAb r educ ed tumor cell pr oliferation via the p44/42 MAPK and Akt
clinician in ADHD s ubjects .                                                                pathways, and reduc ed tumor angiogenesis with reduc ed microvessel density and
                                                                                             vasc ular endothelial growth factor (VEGF) level.
                                                                                             Conclusions: 1) GEP is a novel therapeutic target for liver canc er treatment. 2)
                                                                                             GEP mAb inhi bit tumor growth in a dosage dependent manner with anti -
                                                                                             proliferati ve and anti-angiogenic functions.

                                                                                             Ackno wledg ement: The res earch wor k was supported in part by the Sun C.Y.
                                                                                             Research Foundation for Hepatobiliar y and Pancreatic Surgery, the Seed Funding
                                                                                             Program of the Uni versity of Hong Kong, and the Hong Kong Res earch Grants
                                                                                             Council (HKU 7392/03M, HKU 7560/06M, HKU 1/06C).




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                  Page A-55
                                                              EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                     Celebrating the 100th Anni versar y of the
                                                                        Nobel Prize Award to Paul Ehrlich
                                                                           Nürnberg, October 3-5, 2008
Application of Superp aram agnetic Nanoparticles in Pur ification of Plasmid               PATZ1 gene h as a critical role in the spermatogenesis and testicular tumors
DNA and Recombinant Protein from Bact erial Cells
                                                                                           CHIEFFI P
CHIANG CL, CHEN CY
                                                                                           Department of Experimental Medicine, II Uni versity of N aples, Napl es, Ital y.
Department of Chemic al and Materials Engineering,Southern Tai wan Uni versity,
710 Yung-Kang City, Tai nan Hsien, Tai wan                                                 Background: PATZ1, als o named MAZR or ZSG, is a recentl y discovered
                                                                                           ubiquitousl y expressed transcripti onal regulator y factor gene whos e product binds
Background: The purification of plas mid DNA or recombinant protei n is                    to the RIN G finger protei n RNF4 that in turn ass ociates with a variety of
fundamental to life scienc e research, but some is olation methods c an be                 transcription regulators. Due to the pres enc e of the POZ domai n, PATZ1 acts as a
physicall y and chemicall y damaging. Magnetic separation offers a gentle                  transcriptional repressor affecting the bas al acti vity of differ ent promoters.
alternative. Targets are captur ed on magnetic particles coated with a target-             Recentl y, it has been shown that PATZ1 is an androgen rec eptor (AR) coregulator
specific surface, and s eparated from the sample using a magnetic field.                   that acts by modulating the effect of the AR c oacti vator RNF4.
Methods: Nanosized superparamagnetic nanoparticles (Fe3O4) were prepared by                Methods: To gain insights into its biological role, we gener ated mic e lac king the
                                              2+   3+
chemical coprecipitation method using Fe , F e salt, and ammonium hydroxide                PATZ1 gene (PATZ 1-/- )and utilized different technical approaches (Norther n blot,
under a nitrogen atmosphere. A quic k and reliabl e method for the isolati on and          Western blot and immunohistochemistry anal ys es) to characterize the expression
purification of transfec tion-grade plas mid DNA has been developed, using PEI-            PATZ1 gene.
modified magnetic nanobeads as a solid-phas e ads orbent. We demonstr ated a               Results: Male PATZ1-/- mice were unfertile, suggesting a crucial role of this gene
useful plasmi d, pRSET B-EGFP, encoding the green fluorescent protein with T7              in spermatogenesis. C onsistentl y, most of adult testes from these mice s howed
promoter, was amplified in DE3 strain of E. coli. A new immobilized metal ion              onl y sper matogonia and few s permatoc ytes, associated with increased apoptosis,
affinity (IMA) adsorbent contai ning superparamagnetic nanoparticles and coated            and c omplete abs enc e of spermatids and s permatoz oa, with the subs equent loss
with hydrophilic resins are also proposed to impr ove the purificati on of His-tagged      of tubular structure. The anal ysis of PATZ1 expression, by Northern blot, Western
proteins .The GMA-IDA-coated magnetic F e3O4 were employed for the direct                  blot and immunohistoc hemistry, revealed its pres enc e in Sertoli cells and, among
extraction of recombinant protei n, EGFP-(His) 6, from E. coli lysates as a model          the germ c ells, exclusi vely in the spermatogonia. Sinc e PATZ1 has been indic ated
system.                                                                                    as a potential tumor suppress or gene, we also looked at its expression i n tumors
Results: Up to appr oximatel y 819 μg of high-purity (A260/A280 ratio= 1.86) plas mid      derivi ng from testicul ar germ cells (TGCTs, carcinoma in situ, seminoma,
DNA was is olated from 100 ml of overnight bacterial c ulture. T he eluted plas mid        teratoma, and embr yonal carcinoma). Although expressi on of PATZ1 protei n was
DNA was us ed direc tly for restriction enz yme digestion, bacterial cell                  increased in thes e tumors, it was del ocalized in the c ytoplas m, s uggesting an
                                                                                   2+
transfor mati on and ani mal cell trans fection applicati ons with succ ess.T he Cu -      impaired functi on.
charged GMA-IDA-coated ads orbent had the highes t yield and purification fac tor          Conclusions:Thes e res ults indicate that PATZ1 plays a crucial role in nor mal
at 70.4% and 12.3, r espec tivel y.                                                        male gametogenesis and that its up-regulation and mis-localizati on could be
Conclusions: 1) PEI- modifi ed magnetic nanobeads deli ver significant ti me-              associated to the development of TGCTs.
savi ngs, overall higher yields and better tr ansfection efficienci es c ompared to
anion-exchange and other methods . 2) GMA-IDA-coated magnetic ads orbent
could be used as a suitabl e adsorbent for recombinant His-tagged protein from
aqueous solution. Res ults proved that this new protein purification ads orbent
provides a fast and efficient method for purifying His-tagged proteins with high
yield and low bac kground.




Pathological Studies on Th ym ic L ymphoma in M edaka Fish                                Mifepristone Acts as Progesterone Antagonist of Non-genomic R esponses
                                                                                          but Agonist of Immunosuppression in Human T Cells
CHIEN 1 C-H, CHIEN EJ1, LIN T-L2 , AND LIAO CF1,2
                                                                                          CHIEN EJ 1 , LAI JN2, CHIEN C-H1, SHIE MC 1 , LEE WF 1
1
 University, National Yang-Ming, Taipei, ROC; 2Ins tituti on Academia Sinica, Taipei,
                                                                                          1                                        2
ROC                                                                                        University, National Yang-Ming,Taipei, H ospital, Taipei Municipal Yang-Ming,
                                                                                          Taipei, Republic of China.
Background: Five orange col or strain of Japanese medaka (Or yzias latipes) fish,
age from 8 to 24 months ol d, were diagnos ed with thymic lymphoma. Grossl y, the         Background: Progesterone is an endogenous immunomodul ator to suppress T
tumors protr uded unilaterally out of the side of the head from the thymus.               cell activation during pregnanc y. The stimulati on of membrane progesterone
Methods: Specimens wer e anes thetized with MS-222. Peripheral blood smears               receptors might be the cause for rapid non-genomic respons es in elevating
and tumor cells i mprints wer e stai ned by Gi ems a–Wright stain. The bodies of          intracellular c alcium ([Ca2 +]i) and decreasing intrac ellular pH (pHi) in human T
victims were fi xed in Bouin‘s fl uid ready for histopathol ogical study. Obtained        cells. Mifepristone (RU486) exhi bits mi xed agonist/antagonist effects of
tumor cells and tissues were fi xed by 2.5% glutaral dehyde in 0.1 M cacodylate           progesterone on immune c ells. It is nec essar y to explore whether thes e
buffer for SEM and T EM studies.                                                          complicated effec ts come from RU486 being antagonist of r apid non-genomic
Results: Metas tatic l esions had infiltrated vi a both direct extension and vasc ular    respons e by proges terone.
system in all fish by histopathological exami nati on. The neoplastic mononucl ear        Methods: The respons es in pH i and [Ca2+]i changes were meas ured usi ng the
cells with trans verse splitti ng were obs erved in blood s mears. Mitotic neoplastic     fluorescent dyes, BCECF and fur a-2, res pecti vel y, i n T cells. Proliferation was
cells commonly appeared two or three nucl ei in tumor tissue imprint. No virus            determined by [3H]-thymi dine inc orpor ation i nto phytohemagglutinin (PHA) -
particles was found after the inves tigation by TEM examination. The res ults             stimulated T cells.
obtained from SEM studi es, the thymic neoplas tic cells obtained fr om victims were      Results: Equimolar amounts of the pr ogesterone antagonist RU486 bl oc ked the
identifi ed to neopl astic lymphoc ytoid and lymphoblastoid. T he surface of              progesterone- medi ated non-genomic respons es on [Ca2 +]i incr eases and pH i
lymphoblas toid      was s mooth with s ome lamellate and pits. In addition,              decreas es. RU486 did not bloc k the inhibitory effec ts of progesterone on PHA-
lymphoc ytoi d c ells had microvilli in appear ence. The res ults obtained from TEM       stimulated T cell proliferation. Rather, RU486 was inhibitory. This inhibitor y effects
studies , l ymphoblast oid poss essed nuclear poc kets and s wollen mitoc hodria.         on proliferation c aus ed by proges terone and RU486 were additi ve.
Besides , l ymphoc ytoi d possess ed few c ytoplas m and vesicles inside the cells.       Conclusions: These res ults demonstrate that RU486 has dose-dependent mi xed
Conclusions: The thymic lymphoma pres ent ed in medaka at l east origined from            progesterone antagonist/agonist effects in T c ells. RU486 is antagonistic to
two different kinds of c ells. The l ymphoblast lymphoma/leukemia type s howed            progesterone-sti mulated non-genomic respons es, but agonistic and s ynergistic
aberrance in nucl ear and edema in mitoc hondria. The l ymphoc ytic                       with progesterone to suppress PHA-stimulated T cell proliferation. Our fi ndings
lymphoma/leukemia showed aberranc e in few c ytoplas m, and nuclear cleft. The            suggest a new light on the clinical application of RU486.
victims must s uffer sever ely from anemia and neoplastic c ell proliferati on.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                          Page A-56
                                                     EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                           Celebrating the 100th Anni versar y of the
                                                              Nobel Prize Award to Paul Ehrlich
                                                                Nürnberg, October 3-5, 2008
Dual mechanistic anorexigenic and anti-adipogenic therapeutic for the            A potential molecular link for metabolic stress and car cinogen esis: AMP-
treatment of obesit y                                                            activated protein kinase

CHIN KV1,4,5, QASEM A1, SAUNDERS RA1 , SZLUDLAREK, M 1, CHIN A1, BOSIO                                                                                CHING YP
RM 2, WU Q1, SH APIRO J1 , NAJJAR S3,4
                                                                                                                                                      The Uni versity of H ong Kong, Pokfulam, Department of Anatomy, LKS medic al
Department of M edicine1, Department of Surger y2, D epartment of Physiolog y,                                                                        school H ong Kong
Phar macol ogy, Metabolism, and Cardiovasc ular Sciences 3, Center for Diabetes
and Endocrine Res earch4 , and Department of Bioc hemistr y and Canc er Biolog y5,                                                                    Background: AMP-acti vated protein ki nas e (AMPK), whic h is a s erine/theronine
The Uni versity of Toledo, College of Medicine, 3000 Arlington Avenue, Tol edo,                                                                       protein kinas e, is found to be a key regulator i n glucose and lipid metabolism in
Ohio 43614, USA.                                                                                                                                      respons e to cellul ar stress and hormones such as leptin and adiponecti n.
                                                                                                                                                      Metabolic stress es, li ke heat shoc k, hypoxia or ischemia, and glucos e deprivation,
Background: The sharp rise in obesity in the last decade is one of the most                                                                           have been demons trated to acti vate AMPK acti vity. Acti vation of AMPK ai ms to
serious public health risks worldwide. Currentl y approved therapies for obesity                                                                      switch off pathways that c ons ume AT P, li ke inhibition of car bohydrates and lipid
exhibit modest efficac y and limiting side effec ts. We show here the i dentification                                                                 synthesis; while s witch on pathways that generate AT P, li ke fatty acid oxidation,
of a novel phar mac ological agent, ECP00068, for the treatment of obesity.                                                                           glucose transport and gl ycol ysis. Rec ently, AMPK has been s hown to be the
Methods: The morbi dl y obese leptin-deficient, ob/ob, and the leptin receptor-                                                                       molec ular target of a wildl y used anti-diabetic drug, metformin. Rec ent publications
deficient db/db mice, as well as the glucos e intolerant/type II diabetes proned                                                                      suggest that AMPK acti vation res ults in s uppressing cell proliferation. AMPK lies
Ceacam-/- , and the diet-induc ed obes e (DIO) mice, were treated with ECP00068.                                                                      upstream and downstream of two tumor suppress ors, TSC2 and LKB1,
Effects of ECP00068 on the differentiation of preadipoc ytes into adipoc ytes and on                                                                  respecti vel y, i ndicating that AMPK may also involve in carcinogenesis. Li ver
differentiated adipoc ytes were exami ned. Mechanis ms of action of EC P00068                                                                         cancer ( hepatocellular c arcinoma, HCC) is one of the most common cancers
was inves tigated by whole genome DNA microarray during the differentiation of                                                                        worldwide. However, the molecular mechanism underl ying the devel opment of
preadi poc ytes into adipoc ytes .                                                                                                                    HCC is still unclear. Here, we examined if AMPK is rel ated to HCC for mation.
Results: We showed that ECP00068 caus es appetite suppression that results in
up to 50% reducti on in food intake, decreas e in visceral and subcutaneous                                                                           Methods and Results:
adipose tissues , and weight l oss in ob/ob and db/db, Ceac am- /- , and DIO mic e.                                                                   Using real-time quantitati ve PCR, we obs erved that AMPK2 was significantl y
ECP00068 inhibits the proliferation and differentiation of preadipoc ytes, and                                                                        underexpress ed in HCCs (47.6%) as compared to its corresponding nontumorous
causes either dedifferentiation or delipidation of adi poc ytes. Gene expression                                                                      liver samples. To further confirm the effec t of AMPK on hepatocarcinogenesis, we
profiling showed that i nhibiti on of differentiati on by ECP00068 was accompanied                                                                    established stabl e HCC clones expressing short-hairpin RNA (shRNA) that can
by the tr anscriptional i nhibiti on of a large cl uster of fat r egulator y genes with                                                               specificall y knoc kdown the endogenous AMPK 2 and assay for the pr oliferation
functi onal equivalenc e in C. elegans. Additionall y, ECP00068 increas es the                                                                        rate as well as anchorage-independent growth i n s oft agar. The res ults s howed
expression of a transcriptional repressor, zinc finger protein 68 (Zfp68), which                                                                      that the stabl e AMPK2 knoc kdown clones proliferated much fas ter and formed
inhibits the CCAAT /enhancer bi nding proteins (C/EBPs) and the peroxis ome                                                                           more c olonies than the vector contr ol. Since p53, an important metabolic
proliferator-activated rec eptor  (PPAR).                                                                                                           chec kpoint protei n, was rec entl y reported to be regulated by AMPK, we quer y
Conclusions: 1) The unique dual anor exigenic and anti-adipogenic                                                                                     whether AMPK-regulated HCC cell growth is p53-dependent. Using in vitro kinase
phar mac ological profile of ECP00068 suggests a first-in-class anti-obesity drug.                                                                    assay, we showed that AMPK2 catal ytic domain can directl y phosphor ylate p53.
2) Drug-induced tolerance resulting from chr onic pharmac otherapy for obesity can                                                                    Interes tingly, we found that AMPK phos phoryl ated p53 at a novel site T 150, which
be overcome by a cyclical drug holiday treatment strategy. 3) Zfp68 is a repressor                                                                    affec t the stability of p53 protein.
of C/EBP and , and PPAR, master regulators of adipogenesis.
                                                                                                                                                      Conclusions: our results s uggest that AMPK may mediate its tumor suppression
                                                                                                                                                      functi on through regulation of p53 i n HCC.




An Entr y to Clavams from Chiral Vin yl Ether s and Chloro sulfon yl Isocyanate                                                                       Bulletprooves to Abacavir-related H yp ersensitivit y R eaction in HIV-1-
                                                                                                                                                      infected Population
CHMIELEW SKI M
                                                                                                                                                      CHOLEW INSKA G
Institute of Organic C hemistry of the Polish Ac ademy of Scienc es, 01-224
Warsaw, Kas przaka 44/52, Poland, e-mail:c hmi el@icho.edu.pl                                                                                         Hospital for Infecti ous Diseases. Warsaw, Poland

                                                                                                                                                      Background: Current antiretroviral (ARV) therapy are effecti ve, allowing HIV-
[2+2]C ycloadditi on between c hiral vinyl ethers 1-4 and c hlorosulfonyl isoc yanate
                                                                                                                                                      positi ve indi viduals to live longer, but at the s ame time they have resulted in many
leading to c orresponding β-lactams 5-8 was inves tigated. Reacti ons pr oceeded
                                                                                                                                                      drug-related c omplications. One of it, is hypersensiti vity reac tion (HSR) to
with excellent di astereosel ecti vity. The special attention was foc used on the
                                                                                                                                                      abac avir (ABC)treatment. Phar mac ogenetics associations can infl uenc e on HIV-1
problem of s tereocontrol in the for mation of a desired configuration of the C -4
                                                                                                                                                      treatment. F ew recent s tudies have sh own that patients who were positi ve for HLA
carbon atom of the azeti dinone ring. Adducts 5-8 were transfor med into cl avams
                                                                                                                                                      B*5701 allel es have a high risk allergic reaction to abacavir. The indi vidual loci of
9-12.
                                                                                                                                                      alleles haplotype has been combined to susceptibility to ABC-HSR. Preval enc e of
                                             TIBSO                                                                              R
                                                                                                                                 2                    HLA B* 5701 among Caucasian ar e 5-7% .
              CH2OBn
                                                O                                 TIBSO                                                               Methods: This is retrospecti ve analysis of clinicall y diagnos ed ABC-HSR among
      BnO                 O                                   O                                O                                                      217 treated with abacavir-containi ng ARV regimen HIV-1 infec ted indi viduals,
              OBn                                                                               O                                O
                                                                                                                                                      hospitalized between 2003-2008 in the Hospital for Infection Diseases in Warsaw.
                                                                      O
                                                                                                    O                    1
                                                                                                                                             OTIBS    The aim of anal ysis was attempt to determine of * B57 allel es c arriage in 12
                                                                  O                                                     R
                                                                                                                                                      patients (M-9, F-3, av. age 39), who developed ABC-HSR incidenc e. Multi ple
                      1                                   2                                    3                                    4
                                                                                                            O                                         symptoms were obs erved i n this c ases , defined as abac avir -related allergic
                                                                                                                             2                   O
              CH2OBn                            TIBSO                                         H                             R                         reaction, acc ording to HSR c haracteristic signs and time onset between 1-6 weeks
                                                  H                                                                           H
      BnO                 O                         O                             TIBSO                 NH
                                                                                                                              H
                                                                                                                                                      after abac avir initiation. T he HLA test was performed in all 12 patients defi ned as
                                                                  O                                                                         NH
              OBn                                                                              O
                                                                                                O                            O                        ABC-HSR, using of method Sequens Bas ed T yping [Atria Genetics].
                              NH                   NH                                                                                       OTIBS
                                                                                                                                                      Results: During recent 5 years, abacavir-containing antiretroviral therapy recei ved
                                                                              O
                                         O                                                          O               R1                                217 hos pitalized patients . In 12 (5,5%) out of this group have clinicall y confirmed
                                                                          O
                  5 O                                         6                                7                                    8                 ABC-HSR and discontinued ABC treatment in consequenc e. Simply s ymptoms,
          H       H
                                               H                                          H
                                                                                                        O           R2 H H                            such as s kin and gastrointestinal reac tion, were obs erved in 4 (33%) i ndi viduals. In
                          O                          O    H                                    O                                        O
AcO                                                                   O                                                                               2 other (17%) constitutional and severe respiratory s ymptoms were reported.
                                                                                                                O                                R1
                                   OAc
                  N           H
                                               N
                                                          H                   O           N                                      N            H       Among 6 other (50%), 3 or more HSR signs including fever, paresthesis,
      O                                  O                                                          H               O
                                                                          O       O                                                                   hypotensi on, tachycar dia, li ver parameters elevation, were obs erved. The onset of
              9                                      10                                       11                                    12                HSR occurred between 6 day and 6 week afterwards. No death registered. The
                                                                                                                                                      HLA B* 5701 test performed in subs equence H SR, using the sequence typing
      R1=Ph, Furyl; R2=H, Me; TIBS = 2,4,6-triisopropylobenzenosulfonyl                                                                               method. Onl y 1 pati ent was H LA B*5701 positi ve. It concernd 36y men with AIDS-
                                                                                                                                                      C3 categor y, who rec eived 5-th ARV-drug regimen due to resistanc e and
                                                                                                                                                      intolerance previous antiretroviral agents.
The adduct 5 was subjected to the sequence of reactions, in which glycolic                                                                            Conclusions: 1. HLA B*5701 screening in HIV-1-infected indi viduals has the
cleavage and intramol ecular al kylation of the nitrogen played cruci al roles.                                                                       predicti ve val ue to ABC-HSR risk. As a result, cases of abacavir-related
Adducts 6-8 were trans formed into clavams 10-12 via intramol ecular al kylation of                                                                   hypersensiti vity reaction inc orrectly di agnosed clinicall y, as well as abacavir
the nitrogen atom.                                                                                                                                    unnecess arily discontinuation, can be reduc ed i n clinical practice.
The antibac terial and antifungal properties of all clavams obtained were
investigated to show, however, only in few cas es interesting ac tivity.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                                                        Page A-57
                                                          EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                               Celebrating the 100th Anni versar y of the
                                                                 Nobel Prize Award to Paul Ehrlich
                                                                    Nürnberg, October 3-5, 2008
The Therap eutic ―Cure‖ of Xenograft Tumors b y the Third Generation                 Deferoxam ine and Defer sirox as magic bullets ag ainst iron overload
Epothilone: Iso-oxazole-Fludelone
                                                                                     CHOUDHRY VP (M.D. FIAP, F IMSA, FISHTM, FIACM)
CHOU TC
                                                                                     Director, SUNFLAG PAHUJA CENTRE F OR BLOOD DISORDERS, Sunfl ag
Memorial Sl oan-Kettering Canc er Center, New Yor k, NY, USA                         Hospital, F aridabad, Har yana-121002, India.

17-Iso-oxazole- 26-F3-9,10-dehydro- 12,13-desoxy-epothilone B (Iso-Flu or KOS-               Patients w ith an emias such as tha lassem ia, sick le ce ll dise ase, refractory anem ias and my elo dyspl astic
                                                                                             syndromes, ap lastic a nemi a, requir ing mu ltipl e bl ood transfus ions for their s urviva l deve lop h emosi deros is
1803) is designed by di verted total s ynthesis based on phar mac ological property.         and/or h emoc hromatos is. The excess iro n gets dep osited in the liv er, heart and end ocrin e or gans lea din g to
In vitr o, it has an IC 50 of 0.27nM against CCRF-CEM cells, which is 4- x more              multipl e orga n fail ure. It is a major caus e of morbi dity and mort ality in thes e pati ents. Iron overl oad is ma nifest
potent than paclitaxel (taxol). It is 638- x and 637- x mor e potent than taxol against      as elev ated l iver ir on co ncentrati on (LIC) and elev ated ser um ferritin leve ls. An incr ease d risk of iro n-in duce d
                                                                                             cardiac d iseas e has b een o bserve d whe n LIC val ues exc eed 1 5 mg of iron per gram of dry w eig ht (15
drug-resistant CCRF-CEM/taxol and CCRF-CEM/vinblas tine c ells, respecti vel y. It           mg/Fe/g/dw), and ser um ferriti n va lues a bove 25 00µg/L. L iver bio psy h as bee n the ―go ld stand ard‖ for iro n
showed microtubule-stabilization acti vity si milar to taxol. When compared with             bala nce stud ies, but the tech niq ue is i nvasiv e proc edur e, expe nsive, a nd su bject to vari abi lity with in an d
dEpoB and dehydel one, Iso-Flu is metabolicall y stable in mouse pl asma when                betwee n rese arch su bjects. Liv er iro n co ncentrati on (LIC) has b een m easur ed n on-i nvasiv ely by biom agn etic
                                                                                             susceptometry us ing a low critic al temp erature (l ow-TC) su perco nducti ng qu antum i nterferenc e devic e
delivered as either a 6 hr-i.v. infusi on or oral administration; has i mproved water        (SQUID) bioma gnetom eter, wh ich a llo ws the m easur ement of th e par amag netic susc eptib ility of the iro n
solubility; has increas ed bi oavailability and tissue penetrati on for prolonged tumor      stored in the liver as hem osid erin a nd ferritin. The results of bioma gnetic susc eptometry meas ureme nts of
tissue drug retenti on; and demonstr ates favorable pharmac okinetic and                     hepatic non- heme ir on h ave be en re ported to b e strong ly correl ated w ith those o btain ed by co nventi ona l
                                                                                             analys is of liver bi opsy. An other tec hni que use d is T2* ima gin g of heart. L ow T2 * va lues {<8 ms com pare d
phar mac odynamic properties . The critical-optimal conditions for therapy and low           with (>20ms)} are rel ated to risk of heart failur e and d eath in ir on-ov erlo ade d thalass emic pati ents.
toxicity are 6 hr-i.v. i nfusion at 25- 30mg/kg ever y 14 days, 3x dos es. In human          Bullets as Iron Chelators: Deferoxa mine: It was the first chelator dev elo ped a nd has bee n used exte nsive ly
xenograft tumors: for CCRF-CEM/taxol at optimal conditions , Iso-Flu led to 4/4              in multi ple d isord ers ne edi ng iro n che latio n. It is a hexadent ate chel ator, it binds ir on tig htly, and the ir on-DFO
                                                                                             complex is excr eted in b oth urin e an d stool. More than 4 0 years of clin ical ex peri ence w ith deferox amin e
complete remission (CR) without any relapses for over 3 months, whereas taxol                (Desferal, DFO) in ir on-ov erlo ade d patie nts it reduces iron-r elate d compl icatio ns an d thereby improv es qua lity
had no therapeutic effect, and c yclophos phami de (CTX) suppress ed tumor without           of life an d over all s urviva l, when admi nistere d in ade quate doses over l ong peri ods. The stan dard r egim en to
a CR. For CCRF-CEM xenograft, Is o-Flu, taxol and CTX all ac hieved CR, but 2/4              remove excess iro n is by subc utane ous (sc) infus ions of DFO over 8-1 2 h ours, o n 5 to 7 days each we ek
                                                                                             becaus e of short plasm a ha lf-life. The DFO-iron ch elate is ch arge d an d does not read ily enter and l eave ce lls.
of CTX treated relaps ed in one month. For the pancreatic Bx-CP-3 xenograft, Iso-            Parentera l a dmin istration with use of an infusi on p ump has bee n the m ajor limitati ons. The studi es reve ale d
Flu (25mg/kg, Q12D x3, i.v. infusion) led to suppression and shrinkage but no CR,            that the comp lia nce decre ased sig nifica ntly w ith the incre asin g a ge. D eferasir ox (Exja de ®, ICL67 0) is an N-
whereas taxol (20mg/kg, Q2Dx8, i.v.) and gemcitabine (40mg/kg, Q2D x10, i.v.)                substituted bis- hydroxy phe nyl-triaz ole w as deve lop ed un der a ratio nal dr ug dev elo pment pro gram from
                                                                                             over70 0 compo unds. It represe nts a new cl ass of tridentate ir on ch elators w ith a hi gh spec ificity for iron w ith a
suppress ed tumor growth onl y 90% and 60% , respecti vely. For hepatoma Hep,                plasma half-l ife of 8 to 16 ho urs. It is practical to admi nister the dr ug o nce a d ay oral ly and to ma intai n
Iso-Flu, (30mg/kg, Q16x2, i.v. infusion) led to growth s uppression and s hrinkage           effective plasm a leve ls. It is able to scav eng e non-tra nsferrin- bou nd ―l iab le p lasma ir on, resp onsi ble for tissu e
but no CR, whereas taxol (25mg/kg, Q2D x4, i.v.) and CTX (50mg/kg, Q2D x3, i.v.)             damag e‖. Two mol ecul es of the dr ug form a sta ble iron c ompl ex wh ich is excreted in the fec es. Iron is
                                                                                             chelate d, both from the retic ulo end othel ial cel ls (RE c ells) as w ell as var ious pare nchyma l tissu es. The
and 5-fluorouracil (40mg/kg, QD x5, i.v.), led to onl y 15% , 55%, and 30% growth            chelate d iro n is cl eare d by the liver and excreted thr oug h the b ile. ICL 670 prod uced a li near dose- dep end ent
suppressi on, respecti vel y. The superior data ar e in addition to early results for Iso-   rise in net iron excretio n, with w ide v ariati on se en at 40 m g/Kg/day w hil e vari ation w as much less i n the dos e
Flu against mammar y MX-1 (i.v. infusion or oral), neuroblastoma SK-NAS, lung                of 20 mg / Kg. It is highly selectiv e for iron an d does n ot ind uce the excreti on of zinc or cop per.
                                                                                             Clinical Trials: In patients rec eiv ing 20 m g/Kg defer asirox the iron burd en was ess enti ally unch ang ed if the
A549 and A549/taxol, ovarian SK-OV-3, mammar y MCF-7/Adr xenografts over                     basel ine LIC v alu es w ere betwe en 7 a nd ≤ 14mg Fe /g dw. Whi le th e p atients r eceiv ing 30 m g/Kg the iro n
other chemotherapeutic agents [Chou, et al. Proc. AACR 47: 115, 2006; 48: 342,               burde n was re duce d. In frequently transfuse d pati ents, define d as in divi dua ls receiv ing 2 to 4 units per mo nth
2007, and C hou, et al. PNAS (in press)]. Thus, so far, we have not been able to             (or 7-14 ml/Kg/mo nth) of packed RBCs, oral d eferas irox onc e dai ly in d ose of 20 mg/Kg l ed to mai ntena nce of
                                                                                             LIC, neutral ir on b ala nce, an d stable s erum ferriti n leve ls. In anoth er larg e ph ase 2 study, p atients wer e
find any other cancer therapeutic agent that is even approxi mate to Is o-Flu in             random ized to r eceiv e onc e-da ily d eferasir ox (10 or 20 mg / Kg; n=2 4 in b oth gro ups) or DFO (40 mg / Kg, 5
overall therapeutic efficac y in xenograft experi mental s ystems .                          days / week; n=23) for 48 weeks. Decrease i n liver iro n conce ntration (LIC) wer e compar abl e in the
                                                                                             deferasir ox 20 mg/Kg/d ay and DFO grou ps; basel ine v alu es of 8.5 and 7.9 mg Fe / g dw fell to 6.6 an d 5.9 mg
Authors‘ disclo sure statem ent:                                                             Fe / g dw, respectiv ely, by week 48. His het an d his c oll eag ues o bserv ed d aily s ing le d ose of 2 0 mg/ Kg w as
                                                                                             well tol erated a nd was effective as an ir on che lator. Most common advers e events with a n appar ent
The intellectual property rights for epothilones at Memorial Sloan-Kettering Cancer          relatio nshi p to deferas irox wer e transi ent, gastrointesti nal events wer e observ ed i n 15.2% of patients that
Center have been licens ed to Kos an/Bristol-Myers Squibb Pharmac eutic als.                 inclu ded ab domi nal pa in, n ause a a nd v omitin g, di arrhe a, an d co nstipati on. Ski n ras h w as se en in 10.8% of
                                                                                             patients. Deafn ess, ne urose nsory de afness, w ere obs erved infre que ntly. Mi ld tra nsie nt d ose dep end ent
                                                                                             increas e in serum cr eatin ine was s een in n early 1/3 of cas es. There were no epis odes of neutro pen ia,
                                                                                             agran ulocytos is or throm bocytop eni a i n any of the treatment grou ps. N o a dverse effects o n gr owth or
                                                                                             devel opme nt in pe diatric p atients w ere obs erved. It shoul d also faci litate p atient comp lia nce, a critica l factor in
                                                                                             effective pati ent ma nag ement to m ainta in low iro n b urde ns in pati ents re quir ing frequ ent b loo d transfusi ons.
                                                                                             This drug m eets al l ide al pr operti es of effective si ngl e ora l iron c hel ators with a practic al mi nima l sid e eff ects.
                                                                                             Thus it serves as a sensatio nal ma gic bu llet of the century ag ai nst hemosi deros is and h emochr omatosis.




Nanomedicin es - Self Nano emulsif ying Drug Deliver y Syst em (SNEDDS) and                  Akt Inhibitor s: A N ew Str ateg y T argeting Long-Living HIV Macrophag e
Nanosuspension for Oral and Par enteral Formulations in Cancer Th erap y                     Reservoirs
with Significant Impact s on Ph armacokin etics and Biodistributions
                                                                                             CHUGH P, RIVERA-PABON O, KIM B
CHOW DSL1, GUPTA P1 ,2, QI Y1,3 , LIANG D 4, WISNIECKI P5, SH AH JC 5
                                                                                             University of Roc hes ter, Roc hes ter, NY, USA
1
 Univ. H ouston, Hous ton, TX, USA; 2 Schering-Plough, Kenil worth, NJ, USA;
3
 BioMarin, Novato, CA, USA; 4T exas Southern Uni v., Hous ton, TX, USA; 5Pfizer              Background: The infection of CD4+ T cells with Human Immunodeficienc y Virus
Global R esearc h and D evelopment, Groton, CT, USA                                          (HIV-1) is well-studied and typicall y leads to virally-induced c ytolysis. In c ontras t,
                                                                                             infected macrophages are able to uniquel y evade the apoptotic effects of HIV-1
Background: Mebendazole (Mbz), a highl y lipophilic new anti-neoplastic agent                infection. These cells bec ome infec ted early on and persist throughout viral
(aq. Solubility: 0.7 μg/ml), is challenging for pharmaceutical formulation                   pathogenesis. In addition, brain macrophages, or microglia, are thought to
devel opment. Aims : 1) To develop SNEDDS and Nanosus pension (NS) for oral                  significantly contribute to the development of HIV-1 ass ociated neurological
and parenter al administrati ons in c anc er therapy. 2) To characterize their               problems. Since these c ells seem to play a key r ole in HIV-1 pathogenesis, the
phar mac okinetics (PK) and biodistributions (BD). 3) To establish the impac ts of           study of the interactions between macrophages and HIV-1 is vital to understanding
droplet/particle size on PK and BD of Mbz.                                                   their role as persistent viral reser voirs. Here, the pro-sur vi val effec t of HIV-1 is
Methods: For SNEDDS, s ys tematic approaches in excipient s electi ons and                   presented as a mec hanism for es tablishi ng these c ells as viral res ervoirs.
SNEDDS-based nanoemulsion (NE) region i dentifications, using ter nar y phas e               Methods: Primary human macrophages were infected with HIV-1 M-tropic YU-2
diagram enabl ed the effecti ve formulation optimizations . NS of sizes of 128-167,          virus or a ps eudotyped vector s ystem HIV-GFP and used to perform experiments
250-253, 739- 891 and 1552-1781 nm, were prepared using milling tec hnique. The              pertaini ng to cell sur vi val and the role of PI3K signaling pathway. Miltefosine
PK and BD of the nanoformulations were comparati vel y characterized in Sprague              along with related PI3K/Akt inhi bitors were us ed to antagoniz e the effects of HIV.
Dawley r at and Swiss athymic nu- mouse models.                                              Viral production and c ell death were monitor ed.
Results: The SNEDDS-based NE of 35 and 143 nm significantl y improved the                    Results: We previously found that HIV-1 i nfecti on and the expressi on of HIV-1
oral bioavailability of Mbz, 228 and 120 ti mes of that of unfor mulated s uspension,        Tat pr otei n were able to induce a pro-sur vi val effect i n primar y human
respecti vel y. With parenter al administrati ons of the NE, Mbz yielded prolonged           macrophages. Subs equent studies showed that thes e pr o-survi val effects c oul d be
half-life, 644 min vs 173 min, and s ustained expos ures in organs, especiall y in the       attributed to the acti vati on of the Akt sur vi val pathway. A series of mechanistic
lung, AUC of 12.4 vs 1.8 (hr*ug/g)/(mg/kg), from the c osol vent refer ence (CS).            studies further revealed that the modulation of the Akt pathway was key in
Droplet sizes (35 and 478 nm) impacted PK and BD, with an even higher                        establishing the extended sur vi val phenotype i n these c ells following infection.
concentrati on and a longer retenti on half-life with 35 nm than 478 nm NE. The Mbz          Specificall y, we obs er ved a reduction in PT EN protein levels and a significant
from NS exhibited prolonged half-lives , 13-30 hr vs 3 hr of CS. The Vss were                increase in Akt acti vity. Based on t hese data, the role of Akt inhibitors as an anti-
significantly larger, 1.19-1.69 L vs 0.06 L of CS, and peripheral V2 were                    HIV therapy was tested. Interes tingly, Akt inhibitors and specificall y Miltefosine
substantiall y l arger, 24- 49 L vs 0.4 L of CS. The size i mpacts were also                 were found to bloc k the c ytoprotecti ve effec t of HIV- 1 infection and Tat expression
demons trated.                                                                               in primar y human macr ophages. Furthermore, a drastic decreas e in viral
Conclusions: 1) SNEDDS of Mbz with dropl et sizes of 35 – 478 nm were                        produc tion was obs erved in HIV-1 infected macrophages followi ng treatment with
formul ated for oral and parenteral administrations. 2) Nanosus pensions of Mbz              Miltefosine and related PI3K/Akt inhibitors.
with 4 particle sizes ranging from 128 to 1781 nm were devel oped. 3) Greater                Conclusions: Collecti vel y, thes e data s uggest that Akt inhi bitors like Miltefosine
tissue distributions and slower in vi vo diss olution of NS were key parameters              may provi de a means of targeting long-lived viral reser voirs and may offer further
responsi ble for the size-dependent PK distinction of M bz. 4) Both                          insight into novel therapeutic targets for anti-HIV therapy without concerns of viral
nanofor mulations offer potential merits of sustained and targeted c anc er therapi es,      resistance.
in lung and li ver.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                                                  Page A-58
                                                       EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                             Celebrating the 100th Anni versar y of the
                                                                 Nobel Prize Award to Paul Ehrlich
                                                                    Nürnberg, October 3-5, 2008
The urokinase system is a natural inducer of cancer cell drug r esistance          Magnetic Alginate Nanospheres: a Novel Vector for Targ eted Drug Deliver y

CHUN MH, HOFFMANN MK                                                                                CIOFANI G1 , RAFFA V1 , MENCIASSI A1,2, MICER A S1, CUSCHIERI A1
                                                                                                    1
ApoT ech, Seocho gu, Banpo dong, Gu-ganpo 106-505, Seoul Korea                                          Scuola Superiore Sant‘Anna, Pis a, Ital y; 2IIT Networ k, Genova; Ital y.

Background: Cell-bound uroki nas e converts serum plas minogen to plasmi n. High                    Background: The expl osive growth of nanotec hnol ogy in the last years has
membrane uroki nase expression is associated with enhanced c anc er cell                            produc es dramatic innovations in pharmacolog y. Precise targeting of drugs to
metastasis for mati on and poor disease prognosis. Experiments were conducted to                    diseas ed cells or locations within organs is considered the ―magic bullet‖ in
explore the relati ons hip between urokinase acti vity and cancer cell drug sensiti vity.           medical therapy, but it is not yet achieved by c urrent drug deliver y methods.
Methods: MCF7 Human breast c anc er cells wer e cultured for 2 days in the                          Magnetic materials have been proposed for biomedic al purpos es to a large extent
presenc e or absence of plas min, and subsequentl y expos ed for additional two                     for several years. In this paper, the development, characterization, and in vitr o test
days to doxorubicin, paclitaxel, or tumor necrosis fac tor. Cell survi val was                      of alginate nanoparticles, embeddi ng magnetite, which responds to externall y
determined.                                                                                         applied magnetic fields , are pres ented.
Results: After culturing in the presence of plasmi nogen-c ontaining human serum,                   Methods: Magnetite powder was obtained reac ting iron(II) and iron(III) ions in an
50% MCF7 cells were killed with 15 ng/ml doxorubicin. When c ells were c ultured                    aqueous ammonia sol ution. Magnetite is then isolated from the reaction
in the presence of plasmi nogen- depleted human s erum and thus bloc ked                            suspension and completely dissol ved in an alg inate solution. Alginate magnetic
urokinas e ac tivity, as little as 5 ng/ml had an equal c ytotoxic effec t.                         particles were realized by a homogenizati on proc ess and reticulati on with calcium
Addition of exogenous pl asmi n to plas minogen-depleted c ulture restored the drug                 ions. Such microparticles were c haracterized in terms of external morphol ogy, size
resistance of cancer cells.                                                                         distribution, zeta potenti al, magnetic properties and drug releas e behaviour. In
Bloc king canc er cell‘s urokinase acti vity by anti-uPA antibody enhanced the                      vitro testing was performed with NIH/3T 3 and PC12 cells.
susceptibility to doxorubicin toxicity.                                                             Results: Concerni ng the magnetic pr operti es, magnetizati on cur ves show the
The life pres erving effect of pl asmi n is demons trated to be mediated by the i nsulin-           typical trend of superparamagnetic materials. Important parameters, such as
like growth factor (IGF). IGF is known to induc e drug resistanc e in c anc er cells.               magnetic per meability (μ r = 12.3) and magnetic momentum (μ = 2.25∙10-25 A∙m2),
Bloc kage the IGF pathway abr ogate d the ability of plasmi n to render cancer cells                were derived by employing Langevin theor y. A drug releas e of about 5-6 days was
drug resistant.                                                                                     assessed usig al bumin as protein model.
Other apoptosis-induci ng canc er drugs were tested in the presence or in the                       Nerve growth fac tor (NGF) loaded nanoparticles were tested on PC12 c ells. Cells
absence of plas min. Pl asmin- dependent drug resistance was obser ved in eac h                     showed neuronal phenotype and developed neuritis with length strictly dependent
case.                                                                                               on the distanc e from the nanospheres, fi xed in a region of the Petri dish thanks to
Conclusion:                                                                                         an external magnetic field. Finally, dynamic colture of NIH/3T3 c ells incubated with
1. Cancer cells utilize the urokinas e s ystem to acquire drug resistanc e.                         fluorescei n loaded magnetic nanoparticles defi nitel y demonstrated the possibility
2. Drug sensiti vity is r estored when the urokinase s ystem is inhibited                           to guide these nanovectors through a circuit simul ation blood circulation.
3. Insulin-like growth factor (IGF) is identified as a mediator of this pl asmin effect.            Conclusions: 1) Alginate nanoparticles with a cor e of magnetite and filled with
                                                                                                    specific drugs were r ealized and fully c harac terized. 2) Magnetic nanos pheres
                                                                                                    loaded with neurotrophic factors were able to trigger cell differentiati on strictl y
                                                                                                    depending on the distanc e of the release s ource. 3) The pr opos ed s ystem was
                                                                                                    successfully tested on dynamic cultur es simulating blood circulation.




Treatment and r elo cation of wheat flour sen sitized workers b y an allergenic                     Management of Deep Space Neck Infection. Five Year Exp erien ce.
vaccine
                                                                                                    ČIZMAREVIČ B, DEBEVC D, LEVART P
CIRLA AM, LORENZINI R A, CIRLA PE
                                                                                                    Univ. Clinical C enter Maribor, D epartment for otorhinolar yngolog y and
Italian Center Environmental Occupational H ealth (CIMAL), Milan-Cremona, Ital y                    maxillofacial surger y, Maribor, Slovenia.

Backgro un d: Proteins binding IgE are natural components of wheat flour (WF) and the               Background: Deep nec k spac e infections pres ent as deep nec k abscess or
inhalation of particles during working is a m ajor cause of W F occupational allergy (WOA )         necrotizing fasciitis.They have s evere potential for complication which can be life
for bake rs, pastry-ma kers or pizza-make rs with workplace-related respirato ry symptoms.          threateni ng. Aims: To find out predicti ve factors for mor e fulmi nant progression of
T hey develop rhinitis and subsequently asthma with unfa vou rable consequences on                  infection.
quality of life, often being forced to give u p their jobs and to claime compensation.              Methods: We retrospecti vel y anal yzed medical data for patients surgically treated
Although drugs t reatment could be useful fo r limited pe riods, attempts to realize a
                                                                                                    for deep nec k space i nfecti on between 2002 and 2006. Our group of patients
desensitization by continous administration of incremental doses of an allergen-specific
                                                                                                    consists of 79 male and 53 female. We di vided them in two groups: deep nec k
vaccine (AV) we re carried out in the clinical practice of recent years. An AV containing the
                                                                                                    abscess and necrotizing fasciitis.
most relevant fraction fo r W OA (water/salt soluble albumins and globulins) and standards
related to this subcutaneous immunotherapy (SIT ) are the remarks of the author‘s                   Results: 115 patients had deep nec k abscess and 17 necrotizing fasciitis.
experience, whose e vidence is proposed.                                                            Infec tion started with nondental origin (angina, epiglotitis, infec ted haemathoma) in
Me th ods: 43 subjects were AV-t reated (39 M, 4 F, mean age 39 yr, range 23 -67 ); ba kers         8 patients in first group and in 6 patients in s econd group. Among comorbi dities
27, pastry make rs 13, pizza make rs 4. All had a diagnosis of W OA with rhinitis and 60%           Diabetes mellitus was the mos t common, others wer e prolonged treatment with
suffered also of persistent mild asthma (prick test and specific -IgE positive, occupational        corticosteroi ds, cirrhosis and chemotherapy.
challenge confirmed). WF-AV (Lofa rma Labo ratories, Milan) was characterized by SDS -              Conclusions: Dental infections are the most common cause of deep nec k
PAGE and immunoblot; content of 14-17, 36 and 50 Kd proteins as major allergens was                 abscess es and necrotizing fasciitis. Nondental origin is more likel y to caus e
documented. T he product was a retard prepa ration in aluminium hydroxide; an in -house             fulminant infection and necrotizing fasciitis. Diabetes mellitus was the most
reference standa rd (RAST Units, RU) and quality control we re de veloped. SIT was always
                                                                                                    common comor bidity.
performed by the same single-patient man agement plan, wi thout stopping the work,
basing on a slow induction schedule of 4 months and a montly maintainance with 8000
RU for 4-7 yrs. Subjects were retrospectively interviewed after SIT by questionnaire and
data were registered about s ymptoms, SIT -compliance, job conditions, global efficacy.
Results: SIT was tolerated without ad ve rse effects; only 7 cases repo rted local reactions
during starting months of SIT . Above all 34 subjects keepped up regula rly his own job, 16
of them being at work after W F-SIT was discontinued from several yrs. Nobod y claimed
persistent asthma, while many snee zed again sometimes during wo rk in the last year, but
feeling rarely poor nasal occlusion or runny nose (see table). Only 9 subjects changed
the activity at risk, because of better en gagements elapsed or being retired fo r ageing;
nobody for worsening of symptoms. T he immunological parameth ers (reduction of cell
reactivity and of wheat-specific IgE) are reported elsewere.

 Symptoms                 Never            Sometimes            Often               Total
 (n.workers)                (n.)               (n.)               (n.)               (n.)

 Eyes                       14                 20                  0                 34
                                                                                      34
 Nose                        4                 24                  6
 Breath                     10                 20                  4                 34


Concl usio ns: 1) W OA ma y b e treat ed b y SIT with an AV. 2) A W F-AV was pro ved to be
effective by a standa rdized p rotocol, duration at least 4 yea rs. 3) Giving up a q ualified job
by ba ke rs and similar e xposed o nes ma y be pre vented, with economic and social
advantages. 4) Compliance of SIT reduces the sensitization, im proves symptoms and
promote the w ork relocation in most of cases.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                                           Page A-59
                                                               EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                    Celebrating the 100th Anni versar y of the
                                                                      Nobel Prize Award to Paul Ehrlich
                                                                         Nürnberg, October 3-5, 2008
Zolpidem after br ain Damage                                                              New Applications for Micro- and Nanoscaled Drug Deliver y System s

CLAUSS RP1, NEL H W2                                                                       COESTER C
1
  Royal Surrey C ounty Hos pital, Guildford, Surrey, UK
2
  Famil y Practic e, Springs, South Africa                                                 Ludwig Maximilians Uni versity, Munich, Germany

11 January 2009 mar ks the tenth anniversar y of the awakening of Louis from his           Our group comprises four members loc ated in the Pharmaceutical Tec hnology of
state of i mpaired c onsciousness 30 minutes after swallowing 10mg Zolpi dem. In           the Department Pharmac y, LMU-Munich, headed by Mr. Ger hard Winter. We are
the past decade, Zolpi dem has been effecti ve in a multitude of patients with br ain      concerned with the pharmaceutical devel opment of colloi dal c arriers for the
damage ranging from birth injur y to traumatic brain injur y to stroke and others.         targeted gene and tumor therapy by deli ver y of siRNA, RNA, plas mid DNA, and
SPECT and PET s tudi es in thes e pati ents s how reacti vation of brain metabolism in     classical chemotherapeutics. The idea behi nd is to use simpl e and straight forward
quiescent brai n regions after inj ury, designated functional neurodormanc y. MEG          formul ation approaches, which are eas y to i mplement on a larger scale e.g. for the
studies show that thes e suppress ed neurodormant brain areas have a                       industrial producti on. Our colloidal carriers are produced solel y out of
characteristic slow wave magnetic rhythm that nor malises after Zolpi dem, but not         biocompatible, bi odegradabl e and low toxic materials like phos pholipids, porcine
after placebo or other sl eepi ng drugs suc h as Z opiclone. Neurodor manc y arises as     gelatin and chemic ally modifi ed deri vati ves of them. Besides our focus for
a physiological thread that is pres ent in a multitude of unr elated brain pathologies,    advanced pharmaceutical quality, we c hase for a high therapeutical effic ac y in
possibl y due to a basic physi ological protection mec hanism that is initiated after      numerous clinical studies .
brain damage. It forms a target for Zolpidem whic h re- activates neurodor mant            Our research is based on proper inventi ons and is one of the worldwide leading
tissue and normalises clinic al features that occ ur because of the s uppressed            groups in the fiel d of gelatin nanoparticles. The group‘s focus here is on high-end
neurodor mant brain.                                                                       pol ymer and particle anal ysis with as ymmetrical flow field flow fr actionation (AF 4),
                                                                                           novel in-vitro si mulati on models and the use of fluoresc ent or radioacti ve labeled
                                                                                           nanoparticles for whole body in-vivo imaging. For several years the group hol ds a
                                                                                           strong knowledge in the field of immunity based tumor therapy with CpG
                                                                                           oligonucleotides delivered by nanoparticles made of gelatin and other natural
                                                                                           pol ymers. A new matter of our group is the knoc kdown of disease - related genes
                                                                                           by siRNA. This is anticipated to gain momentum with the ai d of our newl y
                                                                                           established cell culture facility.
                                                                                           A further challenge we have rec ently taken is the devel opment of acousticall y
                                                                                           active gas-filled microparticles, also called microbubbl es. Microbubbles are known
                                                                                           for over 15 years as contrast agents for the ultrasound diagnostic imaging.
                                                                                           Recentl y, they have also proven promising carriers for drug and genes. After being
                                                                                           injected, microbubbles can be vis ualized on the target site by usi ng diagnostic
                                                                                           ultrasound. Followi ng, by increasing ultrasound intensity they can be destroyed
                                                                                           and release their therapeutic load. Our research on microbubbl e carriers diverges
                                                                                           in the fields of targeted therapy of solid tumors and targeted gene therapy.




The AD AM9 Disintegrin Domain (AD AM9D) Inhibits Platelet and Tumor C ell                  In situ Photopolymerized Co atings for pH-Specific Drug Deliver y from
Adhesion to Subendothelial Matrix under D yn amic Flo w Conditions                         Pellets

COMINETTI MR 1, MARTIN ACBM 1, RIBEIRO JU 1, FAUVEL-LAF ÈVE F2, CRÉPIN                     CONCHEIRO A, MAYO-PEDROSA M
M 2, SELISTRE-DE-ARAUJO, HS1
                                                                                           Univ. Santiago de C ompostela, Santiago de Compos tela, Spain.
1
    Federal U niversity of São Carlos, SP, Brazil; 2IN SERM Unit 553, Paris, France.
                                                                                           Background: Coating of drug-containi ng pellets enables the delivery to be
Background: Members of the AD AM (A Disintegrin And Metallopeptidase) protein              sustained or site-specific. As an alter nati ve to the traditi onal coati ng procedure
famil y are c ompos ed by a s eries of c ons erved protei n domains including a            consisting of deposition of successi ve layers of preformed pol ymers, tailore d
disintegrin domai n, which interacts with c ell surface i ntegrins. Adhesi on to and       coatings can be obtained by photopolymerization/ cross-linking of acrylic
extravas ation through the endothelial lining of bl ood vess els is critical for tumor     monomers on the pellet surfac e. Particularly, we have applied this new approac h
cells to establish metastasis. The objecti ve of this work was to verify the effec ts of   for preparing hydrogel coati ngs able to provide theophylline pellets with pH -
the disintegrin domain of ADAM9 (AD AM9D) on the adhesion of tumor c ells (MDA-            dependent release rate.
MB-231) and pl atel ets to c ollagen type I, under flow to simulate shear conditions       Methods: Pellets of theophylline (20%), powdered c ellulose (60%) and
found in the circulati on.                                                                 pol y(vi nylpyrrolidone) (20%) were obtained by extrusion-spheronization and then
Methods: The recombi nant ADAM9D was produced by cloning into a pGEX-4T-1                  placed on a tray adapted to a vortex stirrer, pulverized with soluti ons of cross-
vec tor whic h was us ed transform E. c oli AD494(DE3) cells. T he s ynthesis of           linker and acrylic acid (AA) or AA:lauryl acr ylate (LA) 88:12 mol ar ratio in
GST/ADAM9D was induc ed by IPTG (0.1mM, 4h). After purification on a                       ethanol:water medi um and immedi atel y irradiated with 366 nm UV-light. The
Glutathione-Sepharose 4B resin, the ADAM9D was releas ed from GST by                       coating process i nvol ved s everal s essions of pul verization and irradi ation.
cleavage with thrombin and further purified in a Benzamidine-Sepharose 4B                  Theophylline release was tested i n 900 ml of 0.1N HCl or pH 7.4 phosphate buffer
column. MDA-MB-231 br east tumor c ells label ed with cell trac ker red were               at 37ºC.
previousl y i ncubated with ADAM9D (5µM) or PBS (contr ol) and then mixed with             Results: The new photopol ymerization process was partic ularly adequate for the
whole bl ood prepared labeled with calcei n green. T he mixture was perfused at a          coating of pellets sinc e enabled: i) an eas y and homogenous s praying of the
shear rate of 1500sec-1 in a flow c hamber on a collagen type I-coated coverslip.          monomeric sol ution on the pellets , ii) a quasi-instantaneous pol ymerization, and iii)
Adhered platelets and c ells in each field wer e differentially counted using the          a deep-thorough cure resulti ng in the formation of a well-structured networ k, of a
software Image J. The results were statistically compared with a two- way anal ysis        high consistenc y but still flexi ble. No significant i ncreas e in pellet size (ca. 1 mm)
of variance (ANOVA), followed by Bonferroni‘s significant differenc e post hoc             was obser ved after coating. Non-c oated pellets rapidl y disintegrated and released
anal ysis.                                                                                 all drug in few mi nutes. When onl y AA was used for the c oating, the releas e
Results: Rec ombinant ADAM9D was able to inhibit about 50% of breast tumor                 process was sustained for 4 hours in 0.1 M HCl and finished in 2 hours in pH 7.4
cells and platelet adhesion to c ollagen type I, under fl ow conditions.                   medium. By contrast, c oating with AA:LA mi xtures and a cross-linker notabl y
Conclusions: AD AM9D can be us ed as a tool for i nvestigating the role of ADAMs           hindered the releas e at acid pH (onl y 50% releas ed at 4 hours), without
in metastasis and c anc er progression and for the design of sel ecti ve inhi bitors       significantly compromising the fast deli ver y at neutr al pH.
against the adhesion and extravasation of c anc er cells.                                  Conclusions: Photopol ymerization/cross-linking of AA:LA on pellet surfac e
Financial s upport: F APESP, CNPq, IN SERM.                                                enables site specific deli ver y as a functi on of pH and opens a wide range of
                                                                                           possibilities for preparing tailored c oatings adapted to s pecific triggering pH
                                                                                           val ues. The coating conditions us ed in the pres ent work could be easil y adapted to
                                                                                           industrial sc ale.
                                                                                           Authors disclosur e statement:
                                                                                           Some i nfor mation described i n this abstract is the s ubjec t of patent applications
                                                                                           filed by the Uni versity of Santiago de Compostela (ES 200600757).




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                              Page A-60
                                                              EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                     Celebrating the 100th Anni versar y of the
                                                                       Nobel Prize Award to Paul Ehrlich
                                                                          Nürnberg, October 3-5, 2008
Role of Drug Metabolism in the Developm ent of Epler enone (EP): A Lesson                  Is there a m agic bullet for pro state cancer?
Learned from Spironolactone (SP).
                                                                                           COREY E1, KORECKIJ T1, MORRISSEY C 1, VESSELLA RL1,2
COOK CS1, BERRY LM 2 ZHANG L3
                                                                                           1
                                                                                             University of Was hington, Seattle, WA, USA
1                                           2                                              2
  Baxter H ealthc are, R ound Lake IL, USA, Amgen Inc Cambridge MA, USA,                     Veter an Administration, Seattle, WA, USA
3
  Pfizer Inc , Groton CT, USA
                                                                                           Background: Patients with advanc ed prostate canc er (CaP) frequentl y suffer from
Background: EP is a highly selecti ve aldos terone bloc ker appr oved in the US for        significant morbi dity secondary to bony metastases. Therapeutic modalities that
the tr eatment of hypertension and heart failur e. During development of the drug,         would eliminate or slow the growth of metastatic CaP and improve quality of life
extensive metabolism studies were nec essar y since another competitive                    are of great interest. Although current docetaxel-bas ed regimens offer superior
aldosterone antagonist, potassium canrenoate (PC), that is structurall y related to        survi val advantage over previous standard therapi es for men with castrati on-
EP, was shown to be metabolized to potent mutagenic metabolites which appear               resistant metastatic CaP, this i mprovement resulted in an over all survi val of onl y
to be ass ociated with myelogenous leukemi a. PC is a potassi um salt of canrenoic         18-19 months.        Novel ther apies with greater effic ac y, either alone or in
acid for med from SP. However, SP did not produc e these mutagenic metabolites             combinati on with docetaxel, are needed to continue to move the field forward to
because a thiol metabolite of SP inhibited for mati on of mutagenic metabolites from       benefit afflicted men. T he obj ecti ves of our studies were to c harac terize CaP
canrenoate.                                                                                metastas es and tes t new treatment modalities.
Methods: In vitro metabolism studies of EP were c arried out using human li ver            Methods: Metastases of prostate cancer were obtained from patients who had
microsomes and cDNA-express ed CYP450 isozymes. Major metabolites were                     died from advanc ed C aP and had c ons ented to be part of the rapi d autops y
isolated and identifi ed using MS and NMR. Inhi bition of metabolism i n human li ver      program. There are two prevalent ways of selec ting agents for combination
microsomes was examined in the presence of various c hemical inhi bitors at a              therapy: utilize compounds that act by cl earl y distinc t mechanisms (e.g. affecting
concentrati on of 10 M and with human CYP450 antibodies . Urinary and fecal               either the tumor or microenvironment) or select compounds that affect different
                                                                  14
metabolites were identified following oral administrati ng of [ C]EP to healthy            points i n a specific signaling pathway critical to c anc er progression. In preclinical
subjects at dos e of 100 mg/person.                                                        testing, a model of experimental bone metas tas es was used wher eby CaP tumor
Results: Metabolic pathways of EP were 6- and/or 21-hydroxylati on by                     cells wer e injected directl y in to tibiae of SCID mic e. Ani mals were treated with
CYP3A4/5 and 3- keto reduc tion. The major metabolite identified was 6-OH EP.             various agents incl uding docetaxel, zoledronic acid, RAD001, and the effec ts on
There was no evidence for any alteration of the 9,11-epoxi de ring or carboxy              tumor growth and bone remodeling were monitored.
methyl ester. In contrast to canrenoate metabolism, no 6,7-epoxy metabolite, a           Results: Our results clearl y show that metastas es of pros tate canc er are
precursor to mutagenic metabolites, was found with EP.                                     phenotypicall y heterogeneous not only between different patients but also within a
Conclusions: 1) Maj or metabolic pathway of EP was hydroxyl ation by CYP3A4/5.             single pati ent. Our preclinical studi es showed that while multiple agents are able to
2) Unlike PC, there was no 6,7-epoxide metabolite for med with EP. 3)                    slow the growth of CaP in the bone, no single agent resulted in eradic ation of the
Demonstrati on of stability of 9,11- epoxide ring and abs enc e of 6,7-epoxy             tumor. Our further efforts have demons trated that combi nations of various agents
                                                                                           inhibit the growth on CaP in the bone more than any single agent alone.
metabolite was ess ential i n the development of EP.
                                                                                           Conclusions: 1) CaP is an extremel y heterogeneous disease; 2) Significant
                                                                                           heterogeneity of CaP tumors makes treatment with a single agent unli kel y to res ult
                                                                                           in prolonged clinical res ponses; 3) Effic ac y of drugs is affected by the site of
                                                                                           metastas es; 4) None of the tested agents was capable of eradicating the dis eas e;
                                                                                           and 5) Agents used in c ombination had more pronounced inhibitor y effects.




Corticotrophin releasing factor as drug target for modif ying dopaminerg ic                 BDNF and Its Intracellular Signaling Path ways as Drug Targets in Addiction
system neuroplasticit y in cocaine addiction
                                                                                            COROMINAS M 1, RONCERO C 1, RIBASES M 1, C ASTELLS X2, CASAS M 1 .
COROMINAS M 1, RONCERO C 1, C ASTELLS X2, CASAS M 1.
                                                                                            1
                                                                                             Department of Ps ychiatr y, Vall d'Hebron Uni versity Hospital, Barcelona, Spai n;
1                                                                                           2
 Department of Ps ychiatr y, Vall d'Hebron Uni versity Hospital, Barcelona, Spai n;          Department of Clinical Pharmacol ogy, Vall d'Hebron U niversity Hospital,
2
 Department of Clinical Pharmacol ogy, Vall d'Hebron U niversity Hospital,                  Barcelona, Spain.
Barcelona, Spain.
                                                                                            Background: Craving and relapse, the most challenging aspects in the treatment
Background: Cumulati ve evidenc e has demonstrated t hat stress plays a crucial             of coc aine addiction, still lack an effecti ve pharmac ological treatment. Aims : This
role in c ocaine addicti on by enhancing the rewarding pr operti es of the drug and         review examines the evidence s upporting the invol vement of brain-deri ved
inducing relapse during withdrawal. Corticotropin R eleasing F actor (CRF) is one of        neurotrophic factor (BDNF) and its intracellular signaling pathways , mitogen-
the major effectors of stress in different structures of the rewar d circuitr y, such the   activated protein ki nase (MAPK/ERK) and phos phatidylinositol 3- kinase (PI3-K), in
ventral tegmental area (VTA) and the amygdala. Ai ms: T his paper explores the              the cerebral neuroplas tic mechanis ms in different structures of the
pattern of interacti on between CRF, dopamine (DA) and glutamate (GLU) in                   mes ocorticolimbic dopamine s ystem underlying ps ychostimulant abus e and
different structures of the mes ocorticolimbic circuitry during cocaine c ons umption.      dependence, and also explores the possibility of using pharmacological inhi bi tors
Understandi ng the neuroc hemical and cellul ar mec hanisms involved in thes e              of these intracellular pathways as a new s trateg y to prevent c ocaine craving and
processes would be useful in the development of new phar mac ological strategies            relapse during withdrawal.
for the treatment of coc aine addiction.                                                    Methods: A systematic literature review was conduc ted of ani mal and human
Methods: A systematic literature review was conduc ted of ani mal and human                 research on BDNF and its intrac ellular pathways in ac ute and repeated c ocaine
research on the effects of stress through the CRF s ystem. T he i nterac tion between       consumption and withdrawal.
CRF, dopamine and glutamate in VTA and the amygdal a during repeated cocaine                Results: Animal research demons trates the invol vement of BDNF, ERK and PI3-K
consumption and withdrawal wer e the focus of this review.                                  in cocaine addicti on. R epeated c ocaine expos ure and withdrawal induc e an
Results: In the VT A, stress-induc ed increase of CRF potentiates the acti vity of          increase of BDNF and ERK in different cerebral areas, including the ventral
GLU trans mission, whic h in turn incr eases the excitability of the DA sys tem in          tegmental area, nucl eus accumbens (NAc) core and shell, amygdala and
cocaine-experienced animals. Bl oc kade of CRF-R2 prevents stress-induced                   hippocampus. After repeated coc aine admi nistration there is also a PI3-K up-
increase of s ynaptic plas ticity in the DA s ystem and rel apse during withdrawal. In      regulation and a down-regulation in the NAc and prefrontal cortex, res pec tivel y.
the amygdal a, during early and late cocai ne withdrawal, there is a progressive            These mol ecular changes ar e associ ated with experimental meas ures of c ocaine
increase of CRF releas e that c oincides temporall y with behavioral anxiety during         addicti on, craving and relapse during withdrawal. Pharmacological bloc kade of the
cocaine withdrawal and contributes to rel apse. This increase in CRF acti vity              ERK a) prevents the acquisition of behavi oral sensitization (increase of s ome
potentiates GLU tr ans mission, long -term potentiati on, and s ynaptic pl asticity.        behavioral responses), b) abolishes the ass ociation of environmental cues to
Selecti ve antagonists of CRF-R1 are useful for preventing anxiety during                   cocaine, and c) decreas es cocai ne seeking during withdrawal. Pharmacological
withdrawal and reinstatement of c ocaine seeking.                                           PI3-K inhi bition after repeated coc aine prevents the expression of behavioral
Conclusions: 1) Both CRF-R1 and CRF-R2 inhibitors may be useful for                         sensitizati on.
preventing withdrawal s ymptoms and relaps e in coc aine addicts. 2) Bearing in             Conclusions: 1) BDNF modulates s ynaptic pl asticity in coc aine addiction,
mind the interactions between CRF, glutamate and D A in the different structures of         especi ally during withdrawal, c ontributing to the transiti on from sporadic c ocaine
the mes ocortilimbic sys tem, the use of combined pharmacol ogical strateg ies              consumption to addiction and relapse. 2) ERK and PI3-K casc ade inhibitors are
involvi ng all these neurotransmitters shoul d be consider ed i n the treatment of          potential therapeutic strategies to be further investigated in the context of
cocaine addiction.                                                                          addicti on.




All abstracts are listed in alphabetical order of the presenting author.
Abstracts                                                                                                                                                            Page A-61
                                                                EHRLICH II –2nd World Conferenc e on Magic Bullets
                                                                     Celebrating the 100th Anni versar y of the
                                                                       Nobel Prize Award to Paul Ehrlich
                                                                          Nürnberg, October 3-5, 2008
Anti-HIV And Anti-Cancer Drug-Drug Interactions                                            Docking calculations of heme d erivatives on CYP2B4

CORONA G                                                                                     ROSALES-HERNÁNDEZ MC, TRUJILLO-FERRARA J , CORREA-B ASURTO J*

Experimental and Clinical Pharmacolog y Unit, NATIONAL CANCER INSTITUTE                      Sección de Estudi os de Posgrado e Investigación y Departamento de Bioquímica,
CRO-IRCCS. Via Franc o Gallini 2, 33081 Aviano (PN) ITALY                                    Escuela Superior de Medicina, Ins tituto Politéc nico Nacional, Plan de San Luis y
                                                                                             Díaz Mirón s/n, Casc o de Santo T omás, México, D.F. 11340, México,
The challenge in the treatment of human immunod eficiency virus (HIV)-related                jcorreab@ipn.mx
malignancies is represented by the need to maintain an adequate control of HIV
infection while managing the malignant disease with anticancer therapy. Combination          Cytoc hrome P- 450 is a group of enz ymes invol ved in the biotransfor mati on of
therapy with highly active antiretroviral therapy (HAART) and anticancer chemotherapy        many substances , including drugs. T hese enz ymes possess a heme group ( 1) that
(CT) has been found to be effective and may contribute in reducing morbidity and             when it is properly modifi ed induc es several important physicochemic al changes
extend survival of HIV patients with cancer. However, an increased toxicity of the           tha