Henri Caplain 2 nd Joint Annual Meeting 2007 Bad Homburg v.d.H., Germany

1. Tolerability and safety: Predictability of QT prolongation in healthy subjects from preclinical experiments Henri Caplain 2nd Joint Annual Meeting 2007 Bad Homburg v.d.H., Germany

2. Sanofi-Aventis (SA) Evaluation of Cardiac Repolarization Data for 15 Drugs

3. SCOPE OF EVALUATION • 16 completed TES: • 2003-2007; • 15 compounds. • Degree of concurrence between preclinical and clinical data: • Comparable methodology for preclinical and clinical assessments.

4. Regulatory Environment • Guidance ICH S7B: Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals; • Guidance ICH E14: Clinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential for Non-Antiarrhythmic Drugs.

5. ICH S7B • One preclinical assay not sufficient; • In vitro and in vivo complementary; • Potential to delay ventricular repolarization, related to the extent to concentration (parent + metabolites); • Estimate human risk; • Integrated risk assessment.

6. ICHS7B: NonClinical Testing Strategy Chemical/ Pharmacological Class Ion channel QT QT In vivo In vivo In vitro IKr assay * assay assay assay Relevant Non- clinical and Clinical Information Follow-up Studies Integrated Risk Assessment - up studies if Integrated risk assessment necessary Signal of risk Evidence of Risk None None Weak Weak Strong Strong Impact on Clinical Investigations Quantitative SCORING?

7. ICH E14: Scope • Recommendations for clinical studies to assess the potential of a drug to delay cardiac repolarization; • Determine whether a drug has a threshold pharmacological effect on cardiac repolarization, as detected by QT/QTc prolongation;

8. ICH E14: Positive Study • Prolongation QT/QTc: • Central tendency analysis: • 5 msec maximal mean ↑ QTc (no TdP to date); • Difference of 10 msec on UB 95% confidence interval around the mean effect on QTc. • Categorial analysis: • Absolute QTc prolongation: > 500 msec; • Change from baseline in QTc: > 60 msec. • Documented arrhythmias;

9. ICH E14: Potential Impact • Determine whether or not the effect of a drug on the QT/QTc in target patient populations should be studied intensively during later stages of development; • Basis for nonapproval of a drug or discontinuation of its clinical development.

10. SA Preclinical Experiments for these 15 Compounds

11. SA Core Battery Studies • In vitro hERG/Ikr assay: • Human ether-a-go-go-related gene (hERG) channel in CHO cells; • APD: Papillary muscle (discovery) /Rabbit Purkinje Fiber (development). • In vivo QT assay: • Conscious telemetered Dog; • Toxicology Study Non-Rodent (at least 4 weeks).

12. Criteria of evaluation • hERG: IC50 µM (concentration ng/mL); • Effect on APD: • Global (concentration ng/mL); • Increase of APD: concentration ng/mL. • Telemetered dog: ↑QTc (concentration ng/mL); • Toxicology Non-Rodent: ↑QTc (concentration ng/mL). Parent Compound + Relevant Metabolites

13. SA Thorough ECG Studies(TES)

14. TES: Design • Population: • Healthy subjects (18/65 years): 16/16 [=Pharmacological model]; • Males AND Females: 16/16 (50/50) [Gender effect = +4 msec]; • Mostly Caucasian. • Repeated administration: 15/16 (SS of unchanged compound + metabolites).

15. TES: Study Drug Doses 2 doses: 16/16 (1 with 3) • Expected maximal therapeutic dose; • + One supratherapeutic dose: DDI + concentration/effect: • 2 to 8x the expected maximal therapeutic dose; • 2 to 12x the expected maximal concentration at the maximal therapeutic dose.

16. TES: Design • Cross-over/Parallel: • Cross-over: 6/16 – 1 to 7 days; • Parallel: 10/16 studies – 3 to 28 days. • Sample size: • Cross-over: 16 to 45; • Parallel: 64 to 114. • Randomized, Double Blind: 16/16; • Placebo controlled: 16/16.

17. TES: Design • Positive control (establish assay sensitivity): Moxifloxacin 16/16; • Active control (same chemical /pharmacological class): 2/16; • ECG: • Manual ECG reading + central lab: 16/16; • Tangent method: 16/16; • Multiple ECGs at baseline + during study: intrinsic variability;

18. TES Design • 0-12 h + window around the potential Cmax; • Timepoints in analysis window: 5 to 8. • Central tendency analysis: • Primary endpoint: average effect around the median Cmax; • Cross-over: raw QTcF; • Parallel: QTcF change from baseline, with baseline as covariate.

19. hERG: G = > 10 µM; Y = 1 to 10 µM ; O < 1 µM PF: G = 0; Y = ↑APD; O = ↑APD+EAD+triangulation In vivo: G = 0; Y = ↑QTc (trend); O = ↑QTc (DP)

20. QTcF/QTcN: G < 5 msec MMI; O> 5 msec MMI QTcN = Study specific correction TD = Expected Max Ther Dose; STD = Supra Ther Dose Dose = STD/TD; Conc = STDconc/TDconc HR effect: - = No effect; + = Significant effect (TD/STD)

21. TN = true negative – 7/15 FP = False positive – 2/15 FN = False negative – 4/15 TP = True positive – 2/15

22. hERG: IC50 = 0.45 nM ↑ APD: 10 µM (DP) In-vivo concious non-rodent: ↑ QTc QTcF TD = 0.04 msec QTcF STD = 2.18 msec Dose STD/TD = 5 Conc STD/TD = 7 (8.32 ng/mL) No HR effect Case Study #1: False Positive (Drug 4) POSITIVE SIGNAL NEGATIVE TES FALSE POSITIVE

23. hERG: IC50 = 0.22 µM ↑ APD + EAD In-vivo concious non-rodent: ↑ QTc (trend) QTcF TD = 0.7 msec QTcF STD = 6.98** msec Dose STD/TD = 4 Conc STD/TD = 3 (35.6 ng/mL) No HR effect Case Study #2: True Positive (Drug 12) POSITIVE SIGNAL POSITIVE TES TRUE POSITIVE

24. hERG: IC50 = 5.15 µM/M = 25.7 µM ↑ APD: No In-vivo concious non-rodent: No QTcF TD = -1.42 msec QTcF STD = -2.95 msec Dose STD/TD = 5 Conc STD/TD = 8 (2250 ng/mL) No HR effect Case Study #3: True Negative (Drug 5) NEGATIVE SIGNAL NEGATIVE TES TRUE NEGATIVE

25. hERG: IC50 > 11.1 µM (6133 ng/mL/M = 2.8 µM ↑ APD: No In-vivo concious non-rodent: No ↑ QTc QTcF TD = 0.0 msec QTcF STD = 10.0*** msec Dose STD/TD = 6 Conc STD/TD = 5 (881 ng/mL/M = 115 ng/mL) No HR effect Case Study #4: False Negative (Drug 11) NEGATIVE SIGNAL POSITIVE TES FALSE NEGATIVE

26. Summary of Evaluation of SA Cardiac Repolarization Data for 15 Drugs

27. CONCLUSIONS ON PREDICTIVITY OF PRECLINICAL MODELS • Consistent with previous data: • ILSI/HESI program; • ABPI retrospective analysis; • FDA experience on 19 drugs (2005). • No universal predictivity: • False positive and false negative for every test; • Only one result should not to be considered.

28. CONCLUSIONS ON PREDICTIVITY OF PRECLINICAL MODELS • Careful search for confounding factors (PK, metab). • Knowledge in that field is rapidly evolving (CPMP, Points to consider in 12/1997 recommended PF test; ICH S7B consider this test as only an additional test and ask for hERG. In the (near?) future, recommendations will evolve… • Need to find a more accurate scoring for preclinical assessment; • Need of innovative methods of preclinical investigation.

29. Preclinical data captured the majority but not all the drugs that prolong QT (2/15), even with a conscientious methodology consistent with ICH S7B and E14

30. Preclinical Evaluation Never ?

31. SUMMARY ON PREDICTIVITY OF CLINICAL THOROUGH ECG STUDY NEXT STEP ….. BE PATIENT AND ENJOY THIS NICE MEETING!

32. Back-up

33. The First in vitro Model: hERG Channel Whole cell Patch clamp method: hERG channel stably expressed in Chinese Hamster Ovarian cell (IKr) Used Protocol Recording Ikr Inhibition/ Cumulative Concentrations

34. hERG Channel: Sources of Variability Experimental design Experimental design Statistics Statistics Criteria of acceptation Analysis of Analysis of conc conc - - Expression system Expression system response curve response curve Number of conc conc to built to built [K [K ] in superfusion ] in superfusion + + a a conc conc - - response curve response curve Main Main ERG ERG h h medium medium Main Main ERG ERG h h patch patch - - clamp clamp patch patch - - clamp clamp Number of cells/ Number of cells/ Superfusion Superfusion issues issues issues issues concentration concentration temperature temperature Validation with a Validation with a Stimulation Stimulation reference reference protocol protocol Current reading Current reading Reversibillity Reversibillity protocols protocols Nature of blockade Rundown quantification Rundown quantification Incubation time Incubation time Limitations of in vitro models

35. The Second in vitro Model • PAPILLARY MUSCLE: GUNEA-PIG (Discovery) • PURKINJE FIBER: RABBIT (Development) • Measure the action potential of a conductive tissue (or cell); • Action potential = result of the activity of the various channelsinvolved in the depolarization and in the repolarization phases;

36. The Second in vitro Model • PAPILLARY MUSCLE: GUNEA-PIG (Discovery) • PURKINJE FIBER: RABBIT (Development) • Microelectrode technique; • Tissue pacedat fixed frequency; bradycardia can be simulated by pacing at low rate.

37. The Second in vitro Model: Purkinje/Papillary EXAMPLE: • 1 Hz

38. Action Potential: Source of Variability a: nœud sinusal b: tissu atrial (humain) c: fibre de Purkinje (chien) d: tissu épicardique (chien) e: tissu endocardique (chien) f: tissu myocardique (rat)

39. IKr Control Control Control IKs IK1 INa-Ca IKto1 IKto2 ICa Action Potential: Effect of Various Channels INa But also:

40. ACTION POTENTIAL EXPERIMENTS • Not be used as a stand-alone experiment to appreciate the risk of TdP linked to hERG inhibition; • Global view on the AP, taking • into account all effects on the various channels; • Considered as follow-up studies (in addition to hERG and in vivo tests) and can be used to perform the Integrated Risk Assessment before First-entry-Into-Man and then at every time new information is coming either from clinical trials or fromlong-term toxicological studies.

41. DDQTc Time DDQTc Time of Cmax ANALYSIS Central Tendency: Method 1 = Maximum Mean DDQTc Method 2 = Mean DDQTc at Cmax

42. Drug QTc Baseline Time DDQTc Placebo Time DQTc QTc Baseline Time Time CALCULATING MAX MEAN CHANGE Maximum Mean DDQTc Mean DDQTc Baseline Subtracted: DQTc = Response adjusted (D) for baseline effect Placebo Subtracted: DDQTc = Response adjusted (DD) for baseline & placebo effect

43. Drug QTc Baseline Baseline Subtracted: DQTc = Response adjusted (D) for baseline effect Placebo Subtracted: DDQTc = Response adjusted (DD) for baseline & placebo effect Time Placebo DQTc QTc DDQTc Baseline Time Time Time CALCULATING MEAN CHANGE AT CMAX Mean DDQTc at Cmax Mean DDQTc Time of Cmax