1. Pharmacodynamic Paradigms in Early-Phase Cancer Clinical Trials Workshop Phase 0 Trials In Oncologic Drug Development Hilary Calvert Northern Institute for Cancer Research

2. Methodology for Phase I and Phase 0 (translational) Trials Develop trial methodology designed for targeted agents in trials with pharmacodynamic endpoints The use of pharmacodynamic or toxic endpoints present similar problems – magnitude, reproducibility, variability Endpoints –To develop methods that utilise continuously variable (scalar) endpoints rather than yes/no (Boolean) endpoints –To extend these techniques to combination Phase I trials

3. 1.Traditional –Starting dose –Modified Fibonacci escalation –Maximum Tolerated Dose (MTD) as an endpoint –Disadvantages Patient inefficient Many patients at ineffective doses Safety risk as MTD is approached No built-in confidence intervals 2.Pharmacokinetically-guided (Collins) –Establish Target Area Under the Curve (AUC) from preclinical studies –Monitor Pharmacokinetics at starting dose –Escalate in large increments to achieve target AUC in patients Inter-patient variability in PKs –Disadvantages Assumes linearity Metabolites May not be feasible 3.Continual Reassessment (O’Quigley) –Stochastic model to predict probability of DLT vs dose –Starting dose –Dose doubling –Add data to model –Predict dose with desired probability of DLT –Disadvantages Methodologically complex Needs constraints for safety May take time to converge 4.Accelerated Phase I Design (Simon) –Starting dose –Single patient dose doubling –Increase patients per cohort and reduce dose increments when mild (Grade II) toxicity is seen –Disadvantages Could be hazardous with a steep dose/toxicity relationship Little data at lower dose levels Classical Methodology for Phase I and Translational Trials

4. 1.Traditional –Starting dose –Modified Fibonacci escalation –Maximum Tolerated Dose (MTD) as an endpoint –Disadvantages Patient inefficient Many patients at ineffective doses Safety risk as MTD is approached No built-in confidence intervals 2.Pharmacokinetically-guided (Collins) –Establish Target Area Under the Curve (AUC) from preclinical studies –Monitor Pharmacokinetics at starting dose –Escalate in large increments to achieve target AUC in patients –Disadvantages Inter-patient variability in PKs Assumes linearity Metabolites May not be feasible 3.Continual Reassessment (O’Quigley) –Stochastic model to predict probability of DLT vs. dose –Starting dose –Dose doubling –Add data to model –Predict dose with desired probability of DLT –Disadvantages Methodologically complex Needs constraints for safety May take time to converge 4.Accelerated Phase I Design (Simon) –Starting dose –Single patient dose doubling –Increase patients per cohort and reduce dose increments when mild (Grade II) toxicity is seen –Disadvantages Could be hazardous with a steep dose/toxicity relationship Little data at lower dose levels

5. CI-941 – DMP-941 - Losoxantrone Similar to mitoxantrone Animal models –Activity equal to or better than doxorubicin –No or little cardiotoxicity One of 3 analogues submitted for clinical development by Warner Lambert Candidate for AUC-based dose escalation –Preclinical pharmacology established “target” AUC and linearity up to 45 mg/m 2

6. Foster et al, Br J Cancer 28(213):463-469, 1992 Recommended Phase II dose Target AUC

7. Classical Methodology for Phase I and Translational Trials 1.Traditional –Starting dose –Modified Fibonacci escalation –Maximum Tolerated Dose (MTD) as an endpoint –Disadvantages Patient inefficient Many patients at ineffective doses Safety risk as MTD is approached No built-in confidence intervals 2.Pharmacokinetically-guided (Collins) –Establish Target Area Under the Curve (AUC) from preclinical studies –Monitor Pharmacokinetics at starting dose –Escalate in large increments to achieve target AUC in patients –Disadvantages Inter-patient variability in PKs Assumes linearity Metabolites May not be feasible 3.Continual Reassessment –Stochastic model to predict probability of DLT vs. dose –Starting dose –Dose doubling –Add data to model –Predict dose with desired probability of DLT –Disadvantages Methodologically complex Needs constraints for safety May take time to converge 4.Accelerated Phase I Design (Simon) –Starting dose –Single patient dose doubling –Increase patients per cohort and reduce dose increments when mild (Grade II) toxicity is seen –Disadvantages Could be hazardous with a steep dose/toxicity relationship Little data at lower dose levels O'Quigley J et al: Biometrics, 46, 33-48, 1990

8. Comparison of mCRM 1 Method with Traditional Method - Pemetrexed Proc ASCO 1997, Abs no 733 ScheduleQ21D 2 WQ4x6W 3 Dx5Q21D 4 Escalation MethodmCRM Traditional Doses mg/m250-70010-400.2-5.2 No. Dose levels7410 MTD600304 Months to MTD91229 Pts near Phase II dose20/3716/2411/38 1.Rinaldi DA et al: Cancer Chemotherapy and Pharmacology 44 (5): 372-380, 1999 2.Rinaldi DA et al: Journal of Clinical Oncology 13 (11): 2842-2850, 1995 3.McDonald AC et al: Clinical Cancer Research 4 (3): 605-610, 1998 4.Faries D: J Biopharm Stat 4:147-164, 1994

9. Classical Methodology for Phase I and Translational Trials 1.Traditional –Starting dose –Modified Fibonacci escalation –Maximum Tolerated Dose (MTD) as an endpoint –Disadvantages Patient inefficient Many patients at ineffective doses Safety risk as MTD is approached No built-in confidence intervals 2.Pharmacokinetically-guided (Collins) –Establish Target Area Under the Curve (AUC) from preclinical studies –Monitor Pharmacokinetics at starting dose –Escalate in large increments to achieve target AUC in patients –Disadvantages Inter-patient variability in PKs Assumes linearity Metabolites May not be feasible 3.Continual Reassessment (O’Quigley) –Stochastic model to predict probability of DLT vs. dose –Starting dose –Dose doubling –Add data to model –Predict dose with desired probability of DLT –Disadvantages Methodologically complex Needs constraints for safety May take time to converge 4.Accelerated Phase I Design (Simon) –Starting dose –Single patient dose doubling –Increase patients per cohort and reduce dose increments when mild (Grade II) toxicity is seen –Disadvantages Could be hazardous with a steep dose/toxicity relationship Little data at lower dose levels Simon R et al: Journal of the National Cancer Institute 89 (15): 1138-1147, 1997

10. Methodology for Phase I and Translational Trials Traditional –Starting dose –Modified Fibonacci escalation –Maximum Tolerated Dose (MTD) as an endpoint –Disadvantages Patient inefficient Many patients at ineffective doses Safety risk as MTD is approached No built-in confidence intervals Pharmacokinetically-guided (Collins) –Establish Target Area Under the Curve (AUC) from preclinical studies –Monitor Pharmacokinetics at starting dose –Escalate in large increments to achieve target AUC in patients –Disadvantages Inter-patient variability in PKs Assumes linearity Metabolites May not be feasible Continual Reassessment (O’Quigley) –Stochastic model to predict probability of DLT vs dose –Starting dose –Dose doubling –Add data to model –Predict dose with desired probability of DLT –Disadvantages Methodologically complex Needs constraints for safety May take time to converge Accelerated Phase I Design (Simon) –Starting dose –Single patient dose doubling –Increase patients per cohort and reduce dose increments when mild (Grade II) toxicity is seen –Disadvantages Could be hazardous with a steep dose/toxicity relationship Little data at lower dose levels SLOW AND STEADYHARD TO GET FAST AND LOOSE CHEAP AND CHEERFUL

11. Use of Pharmacodynamic Endpoints Almost always useful as a secondary endpoint –Clinical “proof of principle” of an effect on the target May be useful as a primary endpoint if –Target is known, is single and is known to mediate the therapeutic effect –Level of target suppression needed is known (50%, 90%, 99%?) –Required duration of target effect is known –It is possible to measure all of the above Methodology required for trials with a Pharmacodynamic endpoint –Requires definition of a dose where an effect of sufficient magnitude is present for sufficiently long in a sufficiently high proportion of the patients –Endpoint is scalar (e.g., 95%) rather than Boolean (e.g., DLT present or not) –Interpatient variability and confidence intervals –Prediction of duration of effect Use of a scalar (continuously variable) methodology will also be of value where toxicity is used as an endpoint

12. PARP Inhibitor Phase 1 (0.5?) Trial: AG014699 Potent inhibitor, IV administration Not expected to be active as a single agent (BRCA data not known at the time of design) Expected to potentiate monomethylating agents and Topoisomerase I active compounds Tumour biopsies required for PD endpoint Desire for single agent data on PARP inhibitor Combination study with temozolomide undertaken –PARP inhibitors potentiate temozolomide –Temozolomide active in melanoma –Melanoma patients have multiple lesions, biopsies relatively easy –Single dose of AG14699 scheduled 1 week before combo

13. PARP Inhibitor – Clinical Plan Single agent PARP Inhibitor PARP Inhibitor + temozolomide 50% PD Assays - surrogate Stage 1 – Phase 1 patients – dose escalation of PARP inhibitor Single agent PARP Inhibitor PARP Inhibitor + temozolomide PD Assays - surrogate PD Assays - tumour PARP Inhibition achieved: Stage 2 – Melanoma - dose escalation of temozolomide

14. PD End point PARP Inhibitory Dose (PID) Dose of AG-014699 causing ≥50% inhibition on PARP-1 ex vivo in peripheral blood lymphocytes 24 hours after 1 st dose, with a plateau in the degree of inhibition between dose levels. Validated quantified immunoblot using monoclonal antibody against PAR

15. (SW620 xenografts)

17. PARP immunoblot assay with grateful thanks and credit to Alex Bürkle and Ruth Plummer permeabilised cell suspension expose to NAD+ and oligonucleotide for 6 min stop reaction with ice-cold 12.5µM 699 blot known number of cells on to nylon membrane probe with 1° anti-PAR antibody probe with 2° HRP-conjugated antibody expose to ECL and measure luminescence PAR formed

18. PARP Assay Validation Minimise / explain variability –Enzyme stable with freezing?? –Inhibition stable with freezing –Can inhibition be measured in PBMCs? Establish procedures for handling samples –Does sampling and transport affect result? –Consistency of assay reagents Provide standards for acceptability of results –Control samples –Intra- and inter-assay variability Thanks to Ruth Plummer Probably 1-2 person years

19. Schedule: 28 day cycle -10 to -41 48152228 ↑↑ ↑ PKPKPK PDPDPDPK Comet :Temozolomide :AG014699 Day : Biopsy Biopsy in Part 2 (melanoma patients) only PK (plasma) and PD (lymphocytes) in Part 1 (any tumour) and 2 (melanoma) First cycle only

20. Part 1Part 2 Number1715 Male:female3:48:7 Mean age (range)56 (31-72)48 (32-68) Performance status 0:1:2 7:10:09:6:0 Tumour typeSarcoma 3Melanoma 15 Melanoma 3(13 cutaneous, 1 ocular, 1clear cell sarcoma of soft tissue) Colorectal 3 Others 8 Previous treatmentPretreated but no DTIC/Temozolomide Chemonaive Patient Demographics

21. Dosing and toxicity Cohort 699 dose (mg/m 2 ) TMZ dose (mg/m2) nDLT Part 1 (n=18) 111003None 221004None 341004None 481004None 5121003None Part 2 (n=15) 6121353None 7121703None 8122003None 9182006 1/6 plus 3 C2 dose delays

24. Mean tumour PARP activity at 6 hours after a single dose of AG014699

25. AG14699 Phase 0/1 Trial Interpretation 12 mg/m 2 AG14699 causes profound inhibition of PARP in PBMCs and ~90% inhibition in melanoma 12 mg/m 2 AG14699 can be given with a “full” dose of temozolomide Protocol criteria have been met, but: –Might 18 mg/m 2 with a dose-reduction for temozolomide work better? –What would be the variability of the level and duration of tumour inhibition? –Do we need a longer period of inhibition for single agent treatment of BRCA tumours? –What might the effects on PARP homologues be?

26. PARP Homologues PARP-1most prevalent most of existing data relates to PARP-1 PARP-2responsible for residual PARP activity in PARP-1 knockouts PARP-2 knockouts are also viable Double knockouts not viable PARP-3Unknown PARP-4V-PARP - drug resistance PARP-5Tankyrase 1 - involved in telomerase activity PARP-6Tankyrase 2 PARP 7..upwards ? function

27. Two Dimensional CRM Method Developed in house by James Wright New targeted agents will be used in combination with both traditional cytotoxics and other targeted agents –“Multikinase inhibitors” For every single agent Phase I there will be many combination Phase Is Toxicities may potentiate or antagonise For any two drugs, there is a range of maximum tolerated dose pairs MTD of Drug A MTD of Drug B Toxic Antagonism Toxic Additivity Toxic Synergy

28. D 1 D 2 0.000.250.40.60.80.95 0.00 0.160.220.300.400.48 0.250.160.240.300.400.510.62 0.400.220.300.370.490.600.68 0.600.300.400.490.600.700.77 0.800.400.510.600.700.790.84 0.950.480.620.680.770.840.88 Priors are constructed showing the probability of dose limiting toxicity for each pair of doses Two Dimensional CRM Method Developed by James Wright, PhD Student, 1997-2000 CRM Methodology requires that the probability of DLT at each level is estimated before the start of the trial (priors) A model relating the probability of DLT to dose is created using the estimated data points As real data accumulate during the course of the trial they are used to modify the model A problem for single agent studies is that the initial estimates may be way out For combination Phase I studies, single agent data are already available, facilitating the estimation of priors Hypothetical example: Data derived from single agent Phase I Studies Data estimated from mechanistic knowledge and experience

29. CRM Method – Illustration with Completed Trial OSI 211 in combination Phase I with carboplatin OSI211 – Liposomal Lurtotecan Carboplatin

30. Combination Continual Reassessment Method of OSI 211 + Carboplatin. Probabilities of Dose Limiting Toxicity (DLT) Based on Priors OSI 211 dose (mg/m 2 ) 1.21.62.02.42.83.23.8 40.040.080.160.290.470.650.85 50.140.270.450.650.800.900.97 Initial Estimates OSI 211 dose (mg/m 2 ) 1.21.62.02.42.83.23.8 Carbo Target AUC 40.210.270.350.420.510.590.70 50.290.370.470.570.660.740.84 OSI 211 dose (mg/m 2 ) 1.21.62.02.42.83.23.8 40.0250.050.100.190.330.510.76 50.290.370.470.570.660.740.84 After first 6 patients CarboOSI 211DLT? 41.60/3 42.00/3 After 17 patients CarboOSI 211DLT? 41.60/3 42.00/3 42.42/5 51.20/1 51.60/3 52.02/2 Decreasing Risk of DLT Increasing Risk of DLT Carboplatin AUC is expressed in µg/ml × min Confidence intervals were calculated but are not shown

31. Proposed Enhancements of 2-Dimensional Phase I Methodology Use a scalar rather than a Boolean endpoint (e.g., reduction in neutrophil count rather than MTD) Modify for use with Pharmacodynamic endpoints

32. Mean Tumour PARP Activity at 6 Hours after a Single Dose of AG-014699 0 20 40 60 80 100 0 510152025 AG014699 Dose (mg/m 2 ) Tumour PARP Activity (% pre-treatment) ▲ ▲ ▲ Predicted curve 95% Confidence intervals Instead of this We want this

33. Methodology for Phase I and Translational Trials - Needs Trial methodology designed for targeted agents in trials with pharmacodynamic endpoints methods that utilise continuously variable (scalar) endpoints rather than yes/no (Boolean) endpoints Extension of these techniques to combination Phase I trials –Models to detect trends may be more appropriate than hypothesis-testing We need to use these methods where available and develop new mathematical models where they are not Early investment in PD assay development and validation

34. Agouron/Pfizer Heidi Steinfeldt Zdenek Hostomsky Raz Dweji Gerrit Los Cancer Research UK Newcastle Patients Research nurses Ruth Plummer Nicola Curtin Herbie Newell Roger Griffin Chris Jones Alan Boddy Barbara Durkacz Bernard Golding Plus the other clinical investigators Acknowledgements