Clinical Trial Preparation for Molecularly Based Agents

1. Clinical Trial Preparation for Molecularly Based Agents Edward A. Sausville, M.D., Ph.D on behalf of Dr. Louise Grochow Chief, Investigational Drug Branch Cancer Therapy Evaluation Program National Cancer Institute

2. Changing the Paradigm: Targeted Therapy Development Agent Selection: Murine tumors Xenograft models Credentialed targets molecular models Priority: Log cell kill Growth inhibition Trial Design: Empirical Hypothesis driven Dose Endpoint: Toxicity Molecular effect When to measure? Endpoint Eval: H&P, clin. lab Complex assays Eligibililty: Any solid tumor Presence of target

3. Changing the Paradigm: Phase II Ph II Goal: Tumor shrinkage (cure) Tumor stabilization (eliminate progression) Ph II Metric: When? Anatomic imaging 8 weeks Functional probes Establish time points Dose Finding More is better Molecular effect Combinations: Empiric Pathways and preclinical proof of principle Patient Selection Histology Molecular pathology

4. What Experts Will You Need to Work With? • Basic, translational, and clinical colleagues • Preclinical experts in model design, toxicology, and activity • Clinical trials experts • Statisticians • Experts in molecular target assessment • Pathologists • Imagers • Interventional radiologists

5. So You Have a Good Idea: Now What? • Characterize the target • Screen libraries against the target OR • Synthesize based on in silico drug design • Select lead compound based on pharmacology • Screen for activity in engineered lines • Validate in animal models mimicking human disease • Develop reporters/probes to assess target effects • Design a clinical trial

6. Clinical Trial Design: Dose Finding (Phase I) Studies • Establish disposition and safety • Establish dose that produces target effect (Target Effect Dose) • Establish relationship between dose/schedule and effect • Agent may be non-toxic (endostatin) • (Expensive) agent may have effects at doses that are not toxic • Dose response relationship may not be monotonic (interferon)

7. Possible Trial Designs • PK/target concentration-guided • Accelerated Titration Design: • Minimize patients treated at inactive doses • Incorporate biological target effect • Other designs incorporating biological effect • O6BG: AGT suppression • single dose before planned tumor resection • PS 341: proteasome inhibition • circulating normal PBM • EGFR inhibitors: assess target presence • bcr/abl tyrosine kinase inhibitor: response

8. O6BG: Rationale for Development • Alkylator-resistant tumors often have increased intracellular alkylguanine-DNA alkyltransferase (AGT), DNA repair enzyme • O6BG depletes AGT • When AGT < 10 fmol, activity of DNA alkylating agents that form adducts at the O6 guanine position increases • O6BG and BCNU more active in vivo than BCNU alone Friedman et al. JCO 16:3570, 1998

9. O6-BG Phase 1 Trial Design in Malignant Glioma • Undetectable AGT (< 10 fmol/mg protein) occurs in 20% in absence of O6-BG • O6-BG administered 18 hrs before craniotomy • BCNU-induces interstrand cross-links by 18 hrs • Up to 13 patients entered at each dose • At any time, if AGT detectable in 3 patients, dose was escalated • Biologic endpoint: undetectable AGT in > 11 of 13 patients Friedman et al. JCO 16: 3570, 1998

10. Glioma AGT Activity After O6-BG AGT Undetectable Dose No. of (mg) Patients 0 9 0 40 3 0 60 3 0 80 13 8 100 11 11 Friedman et al. JCO 16:3570, 1998

11. O6-Benzylguanine: Phase I Trial • Suppression of AGT activity in peripheral blood mononuclear cells (PBMC) did not predict suppression of AGT activity in tumor tissue in phase I studies • PBMC: 36 mg/m2 • Tumor : 120 mg/m2 • Immunoreactive AGT in PBMC a poor surrogate Spiro TP, et al. Clin Cancer Res.7:2318, 2001

12. Clinical Trial Design WithBiologic Endpoints • Evaluate for target effect as active concentration is approached • Expand cohort when any biologic effect seen • reproducibility of effect • importance of well defined confidence interval • Escalate dose • until maximal expected effect is seen • until maximal effect occurs in maximal fraction of patients • Additional steps to confirm • effect is maximal • rate of effect is maximal

13. Support for Early Clinical Trials:Cancer Therapy Evaluation Program • CTEP supply agent • CTEP support for infrastructure • Cooperative agreements (Dose finding) • Contracts (Phase II) • Other support for infrastructure • Grant support • Quick trials (R21) • Clinical study section • Cancer Center Core Grants • General Clinical Research Centers

14. Applying to CTEP for Agents • Letter of intent (LOI) • Form available on CTEP Web site http://ctep.info.nih.gov • Review criteria • Strong scientific hypothesis • Supporting preliminary data • Adequate patient accrual • Innovative correlative studies • Ability to meet regulatory requirements • Not duplicative • Agent available • Industry sponsor concurs

15. Preparing a Protocol • LOI approved after reviewed by CTEP’s Protocol Review Committee (+ extramural investigators if needed) • Address critique in LOI approval letter • Use available templates (supplied by CTEP) • Include relevant details (or references) for correlative studies • Submit electronically to CTEP PIO • Review by CTEP PRC

16. What Do You Need to Measure Drug Effect on Target? • Characterized assay or probe to report effect on target relevant to therapeutic agent • Quality assurance: calibration assays, interfering processes, sample handling • Reproducible • Sensitivity • Specificity • Economically and practically feasible • Validate in engineered model

17. Desirable Information for Design of Dose-Finding Trial • Relevant models: • Optimal schedule • Optimal dose • Concentration required for effect on target • % change in target associated with efficacy • Variability in dose/target and target/efficacy relationships • Estimate of fraction of tumors that will have effect • Normal target values, variability • Target effect on tumor vs. other tissues (e.g., PBM) • Does patient’s tumor have relevant target?

18. 105 104 103 102 101 100 PS-293 PS-273 PS-341 0.1 1 10 100 1000 10000 Correlation Between 20S ProteasomeInhibitory Potency & Growth Inhibition for 13 Dipeptide Boronic Acids Correlation r2=0.92 Mean GI50 (nM) Ki (nM) Adams et al., Cancer Res. 59:2615, 1999

19. 700 600 500 400 300 200 100 0 Vehicle (n=15) PS-341 0.3 mg/kg (n=15) Treatment PS-341 1.0 mg/kg (n=10) 0 1 2 3 4 5 6 Effect of PS-341on PC-3 Tumor Growth in Mice Tumor Volume (% Vehicle) Week Adams et al., Cancer Res. 59:2615, 1999

20. 120 100 80 60 40 20 0 120 100 80 60 40 20 0 Vehicle 0.3 0.6 Vehicle 0.1 0.3 1.0 3.0 PS-341 (mg/kg) PS-341 (mg/kg) Effect of PS-341on 20S Proteasome Activity Mouse WBC PC-3 20S Activity (% Vehicle) 20S Activity (% Vehicle) Adams et al., Cancer Res.59:2615, 1999

21. 120 MDACC MSKCC Mayo 100 NYU Wisconsin UNC 80 DFCI Cortes 60 40 20 0 0.1 1 10 Ex Vivo Proteasome Activity:1 Hour Post Treatment % 20S Activity 1.96 mg/m2 PS-341 (Log dose, mg/m2)

22. MS-275 HDAC Inhibitor NSC 706995 • Collaboration between NCI and Nihon Schering • Very “differential” activity in NCI screen • High oral bioavailability • Range finding tox suggests GI/Marrow DLT • on qd x 14 schedule

23. A B C D 1 2 3 4 5 6 7 8 9 10 Effect of HDACIs on Histone Acetylation PC-3M Prostate Cancer Cells Peripheral Blood Lymphocytes Acetylated H3 Acetylated H4 A. Control B. TSA, 0.3M, 8hr C. MS-275, 0.3M, 24hr D. MS-275, 1.0M, 24hr 1. Control 2. MS-275, 0.3M, 2hr 3. MS-275, 1.0M, 2hr 4. MS-275, 0.3M, 8hr 5. MS-275, 1.0M, 8hr 6. MS-275, 0.3M, 24hr 7. MS-275, 1.0M, 24hr 8. TSA, 0.3M, 2hr 9. TSA, 0.3M, 8hr 10. TSA, 0.3M, 24hr

24. Control 0.1 M 0.3 M 1.0 M G1 S G2/M 38 22 39 61 3 36 62 2 36 61 2 37 Cell Cycle Phase Analysisof MS-275 on Prostate Cancer Cells PC-3M, 24h Treatment

25. p21 DAPI p21 DAPI Effect of HDACIs on P21waf1 Expression Control TSA 0.3 M 8 h MS-275 1 M 24 h MCF-7 Breast Carcinoma Du145 Prostate Carcinoma

26. 101 100 10-1 10-2 Control 4 8 12 16 20 24 28 32 36 40 MS-275 TSA Real-Time PCR of p21 cDNA in PC-3MAfter HDI Treatment Rn Cycle Number

27. Options for Preliminary Efficacy (Phase II) Trials • Conventional and/or molecular target eligibility? • Standard phase 2 design targeting RR, survival, PFS, clinical benefit • Standard with biologic endpoint • Standard targeting non-progressor rate • Ratio of TTP for new and previous therapy • Tumor growth before and after treatment with each patient as her own control • Multi-arm randomized selection designs • Randomized discontinuation design

28. Clinical Trial Design: Combination Studies • Combinations of novel agents, novel agents with cytotoxics • Preclinical models or plausible molecular hypothesis • Biologic target effect designs – proof of principle • Dose escalation schemes

29. Tumor Tissue Studies in Clinical Trials • Difficulty obtaining tumor tissue • Difficulty obtaining multiple samples • Which time points? • Tumor heterogeneity: results vary depending upon % tumor vs. normal vs. necrotic cells • Methodological issues: sample handling, sensitivity, reproducibility, complexity, availability • For newer targets, limited information on normal ranges, variability, magnitude of desired effect

30. Setting Priorities for Clinical Trials • Credentialed target • Evidence that agent effects target • Evidence that target effect correlates with useful therapeutic effect • Reliable way to measure presence of target • Probes that permit assessment of target effect in clinical trial

31. Selecting and Evaluating New Agents for Cancer Treatment • Test strategies for selecting agents for trials • Test hypothesis of molecular diagnosis • Evaluate novel clinical trial designs • Target effect is necessary but not necessarily sufficient (pathway crosstalk, downstream events, other proliferative advantages • If proposed target is not accurately characterized, a useful agent might be discarded • This is much simpler if the agent is non-toxic, cheap and highly selective in its effects on a critical target • This is much harder if the agent is promiscuous in its targets, toxic, and the molecular pathology of the disease is complex

32. Acknowledgements • O6BG: H. Friedman, S. Gerson, H. Spiro, E. Dolan, J. Pluda • Biopsy data: J. Haaga, A. Dowlati • PS341: J. Adams • MS275: J. Trepel