ANG1005, anew therapeutic drug able to cross the Blood-Brain Barrier - PDF

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ANG1005, anew therapeutic drug able to cross the Blood-Brain Barrier - PDF Powered By Docstoc
					   ANG1005, a new therapeutic drug able to cross the Blood-Brain Barrier
   for the treatment of brain cancers.
   Michel Demeule1, Anthony Régina2, Christian Ché1, Paul Lockman3, Fancy Thomas3, Julie Gaasch3, Helen Thorsheim3, Abedelnasser Abulrob4,
   Quentin R. Smith3, Danica Stanimirovic4, Richard Béliveau2, Reinhard Gabathuler1, and Jean-Paul Castaigne1 1Angiochem Inc., 201 President Kennedy Avenue, PK-R220,
   Montréal, Québec, Canada H2X 3Y7, 2Laboratoire de Médecine Moléculaire, Centre d’Hémato-Oncologie, Hôpital Ste-Justine - Université du Québec à Montréal, Montréal, Québec, Canada H3C 3P8, 3Department of Pharmaceutical Sciences,
   School of Pharmacy, Texas Tech University HSC, Amarillo, TX 79106 and 4National Research Council of Canada, NRC Institute for Biological Sciences, Neurobiology Program, Ottawa, Ontario, Canada K1A 0R6

                                    ABSTRACT                                                                                                                                       PEPTIDE VECTOR                                                                                            IN-VIVO EFFICACY, DISTRIBUTION AND TOLERABILITY OF ANG1005
                                                                                                                                                Peptides                                      Amino acid sequence                                              Net charge                                                                                                        Efficacy of ANG1005 in the treatment of Rats
  In the present study, we have investigated the utilization of a new peptide based
                                                                                                                                                                                                                                                                                                                                                                                implanted intracranially with U87 glioblastomas
  drug delivery technology that provides a non-invasive and flexible platform for
  transporting drugs into the brain. We have demonstrated that this family of                                                               AngioPep-2                                  TFFYGGSRGKRNNFKTEEY                                                               +2                                                                                                              (Oncodesign Technologies)
  peptides (Angiopeps) is able to physiologically cross the blood-brain barrier
  (BBB) as seen by in-vivo imaging in mice using a fluorescent marker cy 5.5                                                                AngioPep-7                                  TFFYGGSRGRRNNFRTEEY                                                               +2                  Extraction and quantification of ANG1005 in
                                                                                                                                                                                                                                                                                                       mice brain by HPLC analysis




                                                                                                                                                                                                                                                                                                                                                                     Vehicle
  conjugated to an Angiopep peptide, and by in-situ brain perfusion. Based on
  this discovery, we have created several new chemical entities (NCEs), the most                                                         Visualization of AngioPep-2 vector in tumor
  advanced of which is ANG1005 formed by chemical conjugation of the peptide                                                            implanted brain using in-vivo imaging in mice                                                                                                           Tissue analyzed    Brain Tissue      Concentration
  vector (Angiopep-2) to three molecules of paclitaxel. Paclitaxel, which is                                                                                                                                                                                                                                        ANG1005           of ANG1005
                                                                                                                                                                       B            Head imaging (24h post-injection)
  normally restricted from brain, is transported very efficiently across the BBB                                                                                                                                                                                                                 In nu/nu mice
  after conjugation to Angiopep-2. Its rate of transport is 50 to 80 times higher                                                                                                                                                                                                                Mouse (n=3)      3.62 ± 0.47 μg/g      700 nM
                                                                                                                            A




                                                                                                                                                                                                                                                                                                                                                                   Paclitaxel
  than that of free paclitaxel measured using in-situ brain perfusion in rats.
  ANG1005 is also detected by HPLC analysis in normal brain and brain tumors                                                                                                                                                                                                                   ANG1005 can deliver a therapeutic dose of
  in mice 30 minutes after IV injection at level of 700 nM which is above the                                                                                               AngioPep7
                                                                                                                                                                                                                                                                                              paclitaxel to brain tissue (700nM of ANG1005
  therapeutic level of paclitaxel. ANG1005 inhibits cancer cell proliferation of
  various cell lines in vitro; its cytotoxicity is comparable to paclitaxel with an IC50
                                                                                                                                                                                                                                                                                               corresponds to 2.1 μM Paclitaxel). IC50 for
  of 10 nM. ANG1005 is 3.5 times better tolerated than paclitaxel in single-dose                                                                                                                                                                                                                            paclitaxel is 20 nM.
  rodent toxicity studies. In addition, the conjugate of paclitaxel to Angiopep




                                                                                                                                                                                                                                                                                                                                                                     ANG1005
  (ANG1005) bypasses the drug efflux pump P-glycoprotein, which is highly                                                                                               AngioPep2
                                                                                                                                AngioPep2
  expressed at the BBB. Based upon the higher distribution of ANG1005 in brain
  tumors, the effect of ANG1005 was evaluated on glioblastoma (U87) xenograft
  tumor growth in immune deficient mice. In this model, administration of                                                                                                        Angiopep2-cy5.5                       Angiopep7-cy5.5
                                                                                                                                                                                                                                                                                                                                                                                   Day 10             Day 17                    Day 24
  ANG1005 reduced tumor growth in a dose-dependent manner.
  ANG1005 is currently under evaluation in two phase 1/2 clinical trials for the                                            Angiopep2-cy5.5 (red) is detected in the brain and in the brain tumor 24 hr                                                                                                                                                                     ANG1005 is active and is inhibiting the growth of
  treatment of primary and secondary brain tumors in humans.                                                                after tail vein injection in mice. Angiopep2 concentrates in the brain tumor                                                                                                                                                                     human U87 glioblastomas implanted IC in rats.
                                                                                                                                                contrary to the control Angiopep7-cy5.5
                               INTRODUCTION
                                                                                                                                                                                                                                                                                             A direct comparison of the toxicity of Paclitaxel versus ANG1005 on beagle dogs, using 2 h IV infusion of
 The BBB is a unique, selective barrier                                                                                                 ANG1005, an AngioPep-2 conjugated with 3                                                                                                                       2.5 mg/kg of Paclitaxel vs 5 mg/kg of ANG1005. Four dogs were treated in each cohort.
                                                                                                  Tight Juntions
 formed by the endothelial cells that
 line the cerebral capillaries. These
                                                      Astrocyte foot
                                                      process                                                Pericyte
                                                                                                                                                molecules of Paclitaxel
 properties of the BBB are important                                                                                                                                        AcO     O                                                                MW =5109 daltons                                                                            White Blood Cells                  Reticulocytes                Platelets
                                                                                                                                                                                            OH
 as they provide an insulated                                                                                                               O

 environment for stable neuronal                                                                                                                                                                                                                                                                                                                  X109 cells / L                    X1012 cells / L            X109 cells / L
                                                                                                                                                NH           O
 function.                                                                               Blood                                                       2'                                 H         O
                                                                                                                                                                                                        O             Paclitaxel               O             Paclitaxel

                                                                                         Vessel                                                                  O                          OAc
                                                                                                                                                                                 OH O

  Endothelial cells forming the BBB:                                                                                                                 O           O           O
                                                                                                                                                                                                              O        NH                            O        NH
                                                                                                                                                                                                                                                                                                                                              Taxol®     ANG1005                 Taxol®     ANG1005   Taxol®         ANG1005
  •  Express tight junctions
  •  Lack fenestra
  •  Lack transendothelial channels
                                                                                                             P-gp efflux
                                                                                                               pump
                                                                                                                                                         O            NH2                                                                                                                                 Pre-treatment                           9.7       10.6                  0.031      0.044       250             275
                                                                                                                                                                            Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly                        Arg-Asn-Asn-Phe                        Thr-Glu-Glu-Tyr
  •  Lack pinocytic vesicles                                                                                                                                                                                      N
                                                                                                                                                                                                                  H
                                                                                                                                                                                                                                                         N
                                                                                                                                                                                                                                                         H
  •  High levels of the active efflux                                          Endothelial cell                                                                  OH     O                                                   O                                      O
                                                                                                                                                                                                                                                                                                          4-days post-treatment                   4.3       9.7                   0.011      0.065       200             275
                                                      BRAIN
     pump (P-gp)

                                                                                                                            Detection and Transport of ANG1005 across the Blood-Brain Barrier Measured                                                                                                    8-days post treatment                   8.7       10.4                  0.11        0.10       340             290
                                                                                                                                by in situ Brain Perfusion in Rats. The Kin ( BBB Transfert Constant) is
                 EXPERIMENTAL MODELS                                                                                                determined (A) and Regional Analysis of Uptake of ANG1005 (B)                                                                                              During initial infusion, Paclitaxel was not well tolerated, as opposed to ANG1005 which was very well
1. Brain Tumor Distribution after iv inj 5. In situ Brain Perfusion in Rats                                                                                                                                                                                                                      tolerated (Cremophor). These biological observations demonstrate that at an equivalent molecular
   of fluorescent conjugates :               • Perfusion in rats                                                            A                    Transport Rate                                                       B                                                                           dose of Taxol®, ANG1005 does not trigger bone marrow toxicity as opposed to Paclitaxel alone.
   •   Injected animals were viewed in the near-              •        Regional distribution
       infrared mode (Red) (a 660- to 680-nm
       excitation and a 700-nm longpass emission
       filter) using Zeiss Axiovert 200 fluorescent
       microscope (Carl Zeiss).                                                                                                                                                                                                                                                             CONCLUSIONS :
   •   IC implantation of 70,000 U87 cells
   •   IV injection after 10 days of fluorescent
       conjugates
                                                                                                                                                                                                                                                                                            • Angiopep2-cy5.5 shows higher distribution in brain tumors (mice with IC implanted U87
2. Brain uptake of ANG1005 and                                                                                                                                                                                                                                                                glioblastoma cells)
   plasma PK after iv injection in mice                                                                                                                                                                                                                                                     • 50 to 80 times more ANG1005 is transported into brain parenchyma as compared to Paclitaxel
   •   Measure of radioactivity
   •   HPLC analysis after tissue extraction                                                                                                                                                                                                                                                  (in-situ rats)
   •   LC-MS-MS analysis after tissue extraction
3. Tumor model for efficacy in rats                                                                                                                                                                                                                                                         • Homogenous distribution of ANG1005 in brain regions (in-situ rats)
   •   Intracranial implantation of human                                                                                  • ANG1005 has high brain uptake with parenchymal localization.                                                                                                   • Therapeutical amounts of Paclitaxel is delivered in the brain using ANG1005.
       U87 glioblastoma cells followed by
       evaluation of the efficacy by following
                                                                                                                           • In comparison to ANG1005, Paclitaxel had 50-80 fold lower brain uptake.                                                                                        • Inhibits intracranial tumor growth as measured by MRI in rats
       the tumor size by MRI (Oncodesign)                                                                                  • Uptake was comparable across all measured regions except those with low
                                                                                                                                                                                                                                                                                            • Higher tolerability of ANG1005 during infusion without Cremophor in formulation and for bone
4. Toxicity study done by ITR Lab                                                                                            perfusion fluid flow.
                                                                                                                                                                                                                                                                                              marrow at equivalent dose of Paclitaxel