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VIEWS: 10 PAGES: 47

									The NCI Approach To
 Drug Development
    Edward A. Sausville, M.D., Ph.D.
  Developmental Therapeutics Program
        National Cancer Institute
Goals Of Preclinical Drug Studies

                      Scientific framework

   • Discovery of “lead structures”
   •   “Refinement”: chemistry, pharmacology, efficacy
       =“Early” development

   •   Late development: formulation, dose form, toxicology
Goals Of Preclinical Drug Studies

                    Regulatory framework

     • IND = “Investigational New Drug” application
           = approval by FDA to conduct human studies;
           main criterion : SAFETY AND LIKELY
           REVERSIBLE TOXICITY = allows start of
           Phase I trials

     •   NDA = “New Drug Application”
          = basis for sale to public;
          main criteria: SAFETY AND SOME MEASURE
          OF EFFICACY = result of Phase II/III trials
Cancer Drugs:
How Do We Know We Have A Winner?
                                       Treatment A
 - PHASE III CLINICAL TRIAL
                              %
    = WINNER               Alive     Treatment B or no Rx

                                     Time
 - PHASE II
    = POTENTIAL
      WINNER                                       ; Time?

                               Rx

 - PRECLINICAL MODEL           Rx           Untreated
   (e.g., mouse or rat)                      Cytostatic
                      Tumor
                        Size               Cytotoxic

                                    Time
Cancer Drugs: How To Pick A Winner?

       “VALIDATED” CANCER TARGETS
           DNA
              Alkylators

              Antimetabolites

              Topo I / II


           Tubulin

           Receptors
              Nuclear

              Cell Surface (Immune?)


           Oncogene Proteins (2001: AT LAST!)
“Empirical” Drug Discovery
      SCREEN                     PHARMACOLOGY

   + ANTI-TUMOR
     ACTIVITY!!                     CHEMISTRY
    (in vitro/in vivo)

                 OPTIMIZED SCHEDULE (in vivo)

             IND-DIRECTED TOX/FORMULATION

    PHASE I: DOSE/SCHEDULE HUMAN PHARM/TOX

              PHASE II: ACTIVITY = SHRINKAGE

          PHASE III: COMPARE WITH STANDARD
Problems With Empirical Models


  •   Lack of predictive power in vivo

  •   Poor correlation of non-human with human pharmacology

  •   Divorced from biology

  •   Inefficient: many compounds screened;
           developed, but have “late” = clinical trials outcome
           at Phase III to define “validation” of compound action
KRN5500
                                                                                    N        NH


                                                                                N
                                                                                             N
                                                              OH
                                              HO                        O           NH
                    Me( CH2)8CH=CHCH=CHC(O)NHCH2C(O)NH
                                                               HO
                                                                            OH

                                                               N            NH


                                                          N
                                                                            N
                                             OH
                                  HO                 O         NH
        Me( CH2)8CH=CHCH=CHC(O)NHCH2C(O)NH
                                              HO
                                                         OH

                                                                            N
            Deacylation             SAN-Gly                                             NH


                                                                    N
                                                                                        N
                                                     OH
    H3C(CH2)8CH=CHCH=CHCO2H              HO                    O            NH
                                    H2 NCH2C(O)NH
                                                         HO
                                                                   OH



                                                  Protein Synthesis
Effect Of KRN5500 On Colo-205 Athymic
Mouse Xenografts

                           5000
Median Tumor Weight (mg)




                           4500         vehicle
                           4000       + 13.5 qdx5
                           3500         33.5 q4dx3
                                        22.4 q4dx3
                           3000
                                        20 qdx5
                           2500         50 q4dx3
                           2000       X 30 qdx5
                           1500
                           1000
                            500
                              0
                                  8    12   15    18    22    26      29   33   36   41
                                              Day Posttumor Implantation
KRN5500 Plasma Concentrations On
Effective Schedule(20 mg/kg/d) In Mice
                              10
  Plasma Concentration (M)




                               1




                              0.1
                                    0   1   2        3    4   5

                                            Time (days)
Summary Of KRN5500 Phase I


     •   26 patients as IV once per day over 5 days

     •   Dose limiting toxicity = interstitial pneumonitis

     •   MTD = 2.9 mg/M2/d x 5

     •   Achieve only 0.75 - 1 M at 3.7 mg/M2/d x 5

     •   4/6 patients with >25% incr Cmax have
              grade 4 toxicity

                                                 Data of J. P. Eder, DFCI
In Vivo i.e., Intact Animal Tumor Models

 •   The information received depends on the question asked:
      not all models are appropriate for all questions

 •   Drugs need different types of models at different times
      in their “discovery / development” life cycle
         • “Pharmacology” models to qualify compound
         • “Efficacy” models to define potential for biologic effect
              Target driven
              Target unselected
         • “Biology” models to confirm ONLY the target
             or molecular events related to target are affected
In Vivo Activity vs Clinical Activity (39 Agents)

                         a. 20% or greater response                          b. 20% or greater response
                             in any phase II trial                                 in >1 disease
                    25

                            p=0.14                                                  p=0.04
 Number of Agents




                    20


                    15


                    10


                     5


                     0
                             <33%           >=33%                                <33%          >=33%


                              Percentage of Xenograft Models That Were Active
                                                (T/C<=40%)
                                              Clinically Active   Clinically Inactive
“Rational” Drug Discovery

 MOLECULAR TARGET SCREEN             PHARMACOLOGY
    Biochemical                          (to affect target)
    Engineered cell
    Animal (yeast/worm/fish)
                                         CHEMISTRY
          TARGET-DEPENDENT IN VIVO MODEL

                 IND DIRECTED TOX/FORM

   PHASE I: DOSE/SCHEDULE: HUMAN PHARM/TOX;
                ? AFFECT TARGET

        PHASE II: ACTIVITY = ? AFFECT TARGET

     PHASE III: SURVIVAL/TIME TO PROGRESSION
How To “Build” A Cancer Cell
       Cancer cells possess defined “families” of lesions
                      with common outcomes
         • “Misfiring” or Abnormality of Cell Cycle
                     S              - “Oncogenes” turned on
                                    - “Suppressor genes” turned off
  G0         G1             G2
                                    - Mimic Growth Regulatory
                                           “Signal Transduction”
                     M
         •   Imbalance of Genes Regulating Cell Death

                      Cell Proliferation          Cell Death
                                                                Tumor Size


         •   Immortality / Telomerase
         •   Angiogenesis / Invasion Phenotype
        (After Varmus, Bishop, Weinberg, Croce, Folkman, Hanahan … etc.)
Molecular Target Definition - How To?

 •   BIOLOGY:
       * Cytogenetics            Breakpoints            Molecules (bcr-abl)
       * “Positive” selection from tumor DNA            Active oncogenes
            (signal transduction)
       * Tumor gene expression profiling (CGAP)


 •   “ RETROFIT” ACTIVE MOLECULES:
       * Binding partners (geldanamycin, rapamycin, fumagillin)
       * Computational algorithm (molecule       target)
             - COMPARE
             - Cluster analysis

 •   “CLASSICAL:”
       * Cell metabolism / Cell cycle effects
       * Suggest single targets           Inefficient

 •   CHEMICAL GENETICS:
       * Libraries of molecules and precisely defined organisms
bcr-abl As Target: Rationale


    • Apparently pathogenetic in t9:Q22 (Ph+) CML/ALL
    •   Absence in normal tissues

    •   Modulate signal transduction events downstream
           Maintenance of chronic phase
           Adjunct to bone marrow transplantation
bcr-abl Fusion Protein


    bcr   SH2    SH2 V SH2/SH3 kinase         NT   DNA Actin




           bcr



                 autophosphorylation


                                        Phosphorylation of
                                        other substances
                                       McWhirter JR, EMBO 12:1533, 1993
Example Of “Rational” Approach:
bcr-abl directed agents
                                                              CO2H
                                                                                                       OH
                     OH                                              OH
                                             OH
                               NHCHO

  Natural                                            N
                                                                          HO
  product                                                                                                         OH


  empiric lead       OH
                                             OH
                                                                          HO
                                                    HO

                   erbstatin                 lavendustin                       piceatannol
                                                  CO2Me
                          OH
                                                                                        HN        N
  1st generation                                                                                            NH2
                                   NH
  synthetic
                                                                                   CN             CN
                                   AG957                             HN
                                                                                                       AG1112
                          OH

                                                                               N

  2nd generation               N        NH           NH                             N
                                                                                             Me
  synthetic;                        N                     O
                                        Me
  in clinic
                                                          CGP 57148B = STI571
                                    N
STI571:
An Oral In Vivo Bcr-abl Kinase Inhibitor
                           N           NH                NH           O                                            100




                                                                                        % Tumor Free Survival
                                                                                                                    90
                               N                                                                                    80
                                       Me                                                                                                                     KU812 control mice
                                                                                                                          70
                                                                                                                                                              U937 control mice
                                                                                                                          60
                                                                                                                                                              KU812 3x50 mg/kg i.p.
                                                                                                                          50
                                                                                                                                                              U937 3x50 mg/kg i.p.
                               N                                                                                          40
                                                         N                                                                                                    KU812 3x160 mg/kg oral
                                                                                                                          30
                                                 N                                                                        20
                                                                                                                                                              U937 3x160 mg/kg oral
                                            Me                                                                            10
                     120                                                                                                   0                                                                 (days)
 % Phosphorylation




                                                                                                                                    0        10       20     30     40        50        60
                     100
                                        Intraperitoneal           Oral                                                             1.8




                                                                                                                Tumor Weight (g)
                     80                                                                                                            1.6
                                                                                                                                              control mice    3x160 mg/kg oral
                                                                                                                                   1.4
                     60                                                                                                            1.2
                                                                                                                                    1
                     40
                                                                                                                                   0.8
                     20                                                                                                            0.6
                                                                                                                                   0.4
                      0                                                                                                            0.2
                           0       1        2    3   4       5    6       7   8   9 (hrs)                                           0                                                   (days)
                                                                                                                                         0        5          10          15        20
                               Tyr phosphorylation in vivo
                                                                                                                                             Antitumor activity in vivo


                                                                 le Coutre et al, JNCI 91:163, 1999
 Efficacy And Safety Of A Specific Inhibitor Of The
 Bcr-abl Tyrosine Kinase In Chronic Myeloid Leukemia
         BRIAN J.DRUKER,M.D.,MOSHE TALPAZ,M.D.,DEBRA J.RESTA,R.N.,BIN PENG,PH.D.,
LISABETH BUCHDUNGER,PH.D.,JOHN M.FORD,M.D.,NICHOLAS B.LYDON,PH.D.,HAGOP KANTARJIAN,M.D
       RENAUD CAPDEVILLE,M.D.,SAYURI OHNO-JONES,B.S.,AND CHARLES L.SAWYERS,M.D.


                                   White Cell Count                                 Ph Chromosome + Cells
                                                                             100
  (cells x 10-3 / mm3)




                                                                   % in Metaphase
                         100                                                        80

                                                                                    60
                         10
                                                                                    40

                          1                                                         20
                               0   30   60      90    120   150
                                                                                    0
                                                                                         0   100   200   300   400
                                             Duration of Treatment with STI571 (Days)



                                                      NEJM 344: 1031, 2001
NCI Drug Discovery

     A Marriage Of Empirical And Rational Opportunities
    • Aids to find targets / link targets with drugs
        -Cancer Genome Anatomy Project (CGAP)
        -Developmental Therapeutics Program In Vitro Drug Screen

    • Where the target is known: build its assessment into
     the selection and development of a compound: e.g.,
     17-allylamino 17-demethoxy geldanamycin (17-AAG)

    • Where the target is unknown, define it or a proximal
     indicator of effect in parallel with conventional
     development path: e.g., UCN-01 protein kinase
     inhibitor
Cancer Genome Anatomy Project: PROCESS



     •   Tumor material (archival)

     •   “Laser capture microdissection” of tumor cells
            from defined sections

     • Creation of tumor-derived cDNA libraries
     •   Sequence to establish uniqueness

     • Deposit in public domain
Gene Expression: The Cell’s Fingerprint


                                                                  Normal Cell
                                                                  Cancer Cell




    Establishing for a cell the repertoire of genes expressed, together
    with the amount of gene products produced for each, yields a
    powerful "fingerprint". Comparing the fingerprints of a normal versus
    a cancer cell will highlight genes that by their suspicious absence or
    presence (such as Gene H ) deserve further scientific scrutiny to
    determine whether such suspects play a role in cancer, or can be
    exploited in a test for early detection.


                          http://cgap.nci.nih.gov
NCI In Vitro Drug Screen

    1985 Hypothesis:
      • Cell type specific agents
      • Activity in solid tumors
    Emerging Realities:
      • Unique patterns of activity, cut across cell types
                          AND
        Cell type selective patterns found
       •   Correlations of compound activity
           - relate to molecular “target” expression
           - generate hypothesis re: molecular target
                    National Cancer Institute Developmental Therapeutics Program
                                       Dose Response Curves
                    NSC: 643248-Q/2 (a rapamycin)       Exp. ID: 9503SC35-46

                                             All Cell Lines
                100


                    50
Percentage Growth




                     0


                    -50



          -100
                          -9       -8          -7        -6           -5       -4
                                    Log10 of Sample Concentration (Molar)
Pattern Recognition Algorithm:
COMPARE
   • Goal: COMPARE degree of similarity of a new
            compound to standard agents
   •    Calculate mean GI50, TGI or LC50
   •    Display behavior of particular cell line as deflection
            from mean




       resistant                mean                    sensitive

   •    Calculate Pearson correlation coefficient:
                   1 = identity ; 0 = no correlation
Agents With Similar Mechanisms Have
Similar Mean Graphs
        Leukemia

          NSCLC

   Small Cell Lung

            Colon


             CNS

        Melanoma


          Ovarian

            Renal

                     Taxol        Halichondrin B    Daunorubicin

                             Tubulin               Topoisomerase II
Drug Target Clusterings
Reveal Clues To Mechanism




    5FU/DPYD                                      L-Asparaginase
                                                  /ASNS
       Nature Genetics 24: 236, 2000; http://dtp.nci.nih.gov
Geldanamycin Structure
                        benzoquinone
                             O
                        R
                                           O

                                      NH
                             O
                                 OH        MeO
     ansa ring
                                                       carbamate
                                                  OCONH2
                 Me O


                              NSC                 R
     Geldanamycin            122750              OMe
     17-AAG                  330507              NHCH2CH=CH2
Benzoquinoid Ansamycins
Initial Cell Pharmacology
                     Reduce levels or inhibit transformation by a large number of PTKs:
                      src, yes, fps, erbB1, lck
                     e.g., 17AAG decrease erbB2 under conditions where overall
                      transcription/translation little affected

                                                             Effect of 6 hr, 0.35 M
            150                                           herbimycin A on SKBr3 cells
                              MDA MB 453
                                                             Parameter       % of control
            100                          p185 Protein       p185 protein           35
% Control




                                         p185 PY            p185 Y-P                5
                                                            erbB2 RNA             130
             50
                                                            Prot syn               84
                                                            RNA syn                90
              0                                             ATP                    99
                  0       2    4    6       8               ATP/ADP               108
                              Hours

                                (Miller et al, Cancer Res 54: 2724, 1994)
DTP, NCI In Vivo Evaluation
Of Geldanamycin In PC3 Prostate CA
                Early Stage, Athymic Mouse Xenograft
    Dose                                 Drug    % Opt     Growth
   (mg/kg)        #        Schedule     Deaths   T/C (D)   Delay

     0            20       qd x 5 (9)     0        ---        ---
     3.4          6        qd x 5 (9)     3       Toxic       ---
     2.3          6        qd x 5 (9)     1      33(22)       50
     1.5          6        qd x 5 (9)     0      90(15)        3

 Route of administration – i.p.

Conclude:      Narrow therapeutic index on this schedule
               Solubility of agent major problem for other schedules
Geldanamycin Bead




       Bead




                                         O

                                                 R4
                                                      O

                          R2                                Me
                                                 NH

                                         O
                                    Me        H2 NC(O)O



                    MeO        R1
                                         Me      Me       OMe
Geldanamycin Beads
Identify Hsp90 As Binding Partner
                       R. Lysate



           p90


                   1     2     3      4

   1) Bead-Geld              3) Bead-Geld + Geldampicin

   2) Bead-Geld + Geld       4) Bead

                                   Neckers et al, PNAS 91:8324, 1994
Hsp 90
A.                      degradation        C.
     X-mRNA       X                                            G0 
                                                  erbB2
          ER                                      EGFR
                       folding                  lck, met,                  raf
                 X                                 etc
                 hsp
                                 X
nucleus           90

               Immature      Mature
                                                                             EIF2
                  X            X           pAKT             Hsp 90           kinase
B.
                          hsp
                           90          *         ER
                                                 PR                      Cyclin D
          nucleus                hsp             etc
                            *     90
                                                            telomerase
      *                    hsp
                            90
Three Dimensional View Of Geldanamycin
Binding Pocket In Amino Terminus Of Hsp90




               Stebbins et al, Cell 89:239, 1997
17-AAG Binds To Hsp90 & Shares Important
Biologic Activities With Geldanamycin

                                                             p185erbB2                                                           Raf-1
                         control
                           0.03




                           0.03




                                                                                                  control
                                                                                                    0.03




                                                                                                    0.03
                           0.1
                           0.3
                           0.5


                           0.1
                           0.3
                           0.5




                                                                                                    0.1
                                                                                                    0.3
                                                                                                    0.5


                                                                                                    0.1
                                                                                                    0.3
                                                                                                    0.5
                           2




                           2




                                                                                                    2




                                                                                                    2
                           17-AAG              GA     (M)                                              17-AAG         GA     (M)


                         120                                                                      120
erbB2 (% of base line)




                                                                         Raf-1 (% of base line)
                                                    17-AAG                                                                  17-AAG
                         100                          GA                                          100                         GA
                          80                                                                       80

                          60                                                                       60

                          40                                                                       40
                          20                                                                       20
                           0                                                                        0
                               0      1   10    100 1000 10000                                          0   1     10   100 1000 10000
                                          dose (nM)                                                              dose (nM)
                                   Schulte & Neckers, Cancer Chemother Pharmacol 42: 273, 1998
UCN-01
                                O     NH    OH




                               N             N
                                      O
                                             Me


                                             OMe
                                                         IC50
Potent antiproliferative agent          NHMe            37nM (DTP screen)
Protein Kinase C (PKC) inhibitor                     ~30/~600nM
Ca2+ dep/Ca2+ independent
Cell cycle arrest/CDK inhibition, unrelated to PKC   300-600 nM
DNA-damage G2 checkpoint abrogation                    ~50nM
Induces apoptosis                                    100-1000 nM
UCN-01 Abrogates Radiation-
Induced G2 Arrest In CA46 Cells
                                                  -Irradiated
                                              -Noc           +Noc   UCN-01
                         Asynchronous                                (nM)
                  2000
                               G1                                     0
                  1600
    Cell Number




                  1200
                                    G2/M
                  800
                                S                                     30
                  400

                     0
                         0 30 60 90 120 150

                           DNA Content                               100



 Wang et al, JNCI 88: 956, 1996
                                                                     300
A DNA Damage G2 Checkpoint Is
Mediated By CDKs: UCN-01 Action
                                                                    Chk 1 mediates
                                                                 the G2 checkpoint

 UCN-01
          spRad3(hATM/hATR)

          Chk1            Chk1-P
                                                     14-3-3

                 Cdc25             Cdc25-P(Ser216)            Cdc25-P(Ser216)-14-3-3
                 Active               Active?                      Inactive

 Cdc2-P(Tyr15)              Cdc2
   Inactive                 Active
                                                               Adapted from Weinert,
   G2 arrest          Enter mitosis                           Science 277:1450, 1997
                                                                                     in vitro




                                                     Chk1 kinase



                      in vivo (HeLa)
           Control                                                        Control

           UCN-01                                                         UCN-01
                                                                          UCN-02
           Staurosporine
                                                                          Staurosporine
           CGP41251                                                       CGP41251
                                                                          K252a
           K252a
                                                                          K252b
           K252b                                                          KT5823
           KT5823                                                         Rebeccamycin
                                                                          EDTA
                                                                                                UCN-01 Inhibits Chk-1 Kinase




           Rebeccamycin
b                                                                         Control
                                                                   Chk1




a Cdc25C
                                       Cdc25 motif
UCN-01 Infusional Phase I Trial
G2 Checkpoint Abrogation

                              70
 % G2 checkpoint abrogation




                                                                % G2 checkpoint abrogation
                                                                                             70
                              60
                                                                                             60
                              50
                                                                                             50
                              40                                                             40
                              30                                                             30
                                                                                             20
                              20
                                                                                             10
                              10
                                                                                              0
                              0                                                                   30      40       50       60
                                   0     10     1000   100000
                                        nM UCN-01                                                      UCN-01 (mg/m2/day)

                                       UCN-01 w/o plasma
                                       UCN-01 in plasma
Challenges In Pursuing The
Molecular Therapeutics Of Cancer

   •   Must change thinking from histologic to molecular
          diagnoses (CGAP, array technology)

   •   Develop new means (imaging, probes) to assess
          molecular pharmacodynamics

   • Must move away from cytotoxicity as sole primary
           endpoint: assess and evaluate cytostatic effect

   •   Promote patient participation in clinical trials

   •   Develop speed and efficiency in answering critical
           clinical questions
Goals For Cancer Drug Screening
In The New Millennium

    • Associate novel chemotypes with defined targets
         • may utilize purified targets at the “front end”
         • may define targets in pathway/organisms
         • may “retrofit” molecules to targets or pathways
            by statistical approaches

    •   Allows facile tools for chemical/pharmacological
             optimization

    •   Define targets of relevance to and translatable in
            early clinical trials
Summary:
Developmental Therapeutics Program, NCI

    •   Novel agents directed at molecular targets
           important to cancer pathogenesis
           and progression

    •   Interdisciplinary collaborators: academia,
            industry, intramural NCI

    •   Contribute agents and regimens for use by
           intra / extramural investigators
Acknowledgements
   NCI
   V. Narayanan, R. Schultz
   J. Johnson, S. O’Barr
   M. Hollingshead, S. Stinson         H. Piwnica-Worms
   L. Rubinstein                           Wash U
   A.Monks, N. Scudiero                V. Pollack
   K. Paull, D. Zaharevitz, S. Bates       Pfizer
   S. Holbeck, J. Weinstein            M. Roberge
   A. Senderowicz                          U. Brit. Columbia
   A. Murgo, S. Arbuck
   G. Kaur, P. Worland, Q. Wang
   P. O’Connor
   L. Neckers, L. Whitesell
   D. Newman
Our next speaker is:

Ms. Jill Johnson
Office of the Associate Director
Developmental Therapeutics Program

								
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