1. CLopidogrel and respOnse Variability Investigation Study “ CLopidogrel and respOnse Variability Investigation Study ” A Randomized Study Comparing the effect of two Clopidogrel LD according to the presence of CYP2C19*2 JP COLLET DISCOSURES: Research Grants : BMS-Sanofi Aventis, Eli Lilly, Medtronic, Cordis,Johnson&Johnson. Consulting Fees : AstraZeneca, BMS, Daiichi Sankyo, Eli Lilly, Sanofi-Aventis. Lecture Fees: AstraZeneca, BMS, Daiichi Sankyo, Eli Lilly, and Sanofi-Aventis TCT, Washington –September 22nd, 2010 Jean-Philippe COLLET* Jean-Sébastien HULOT Ghalia ANZAHA Ana PENA Thomas CHASTRE Johanne SILVAIN Guillaume CAYLA Anne BELLEMAIN-APPAIX Jean-Baptiste VIGNALOU, Farzin BEYGUI Olivier BARTHELEMY Sophie GALIER Vanessa GALOIS Gilles MONTALESCOT *Pitié-Salpêtrière Hospital_URMS_937 75013 Paris-Assistance Publique des Hôpitaux de Paris

2. 2 Odds Ratio, fixed model Bilateral CI, 95% for trials, 95% for MA events / size 01234510 20 Odds Ratio 2C19*2 2C19*1 10/375 8/1014 8/73 4/186 13/247 11/525 10/680 7/1805 41/1375 30/3530 OR 3.45 95% CI: 2.14-5.57, p<0.001, p het =0.78 Total 2C19*2 better 2C19*2 worse STENT THROMBOSIS (n=4905) Mega et al. (0.26) Collet et al. (0.15) Giusti et al. (0.34) Sibbing et al. (0.24) J Am Coll Cardiol 2010; 56(2):134-143

3. Carriers vs Noncarriers Heterozygotes vs Wild Type Homozygotes vs Wild Type Risk Ratio (95% CI) P value 0.51.015.0 2.81 (1.81-4.37) 2.67 (1.69-4.22) 3.97 (1.75-9.02) <.0001 <.001 N = 5772 Risk Higher With CYP2C19 Variant Risk Lower With CYP2C19 Variant Collaborative Meta-analysis: CYP2C19 and Stent Thrombosis in Patients on Clopidogrel Mega JL. American Heart Association; November 2009; Orlando, Florida.

4. FDA boxed warning Clopidogrel has diminished effectiveness in individuals based on their CYP2C19 genotype, specifically in those who harbor two CYP2C19 reduced-function alleles Clinical implications of this genetic-based hazard? – Only early after initiation or during longer-term therapy as well – In the presence of two or at least one CYP2C19 reduced-function allele http://products.sanofi-aventis.us/PLAVIX/PLAVIX.html. Accessed April 20, 2010.

5. Objectives To determine whether high dose of clopidogrel can overcome genetic resistance by – comparing the pharmacokinetic (PK) and pharmacodynamic (PD) responses – to two LDs (LD) of clopidogrel (300mg vs. 900mg) – according to carriage of CYP2C19*2 genetic variant 5

6. 6 Trial organisation ACTION Study Group (Academic Research Organization, Paris) 1-Coordinating Center : 1-Coordinating Center : Institute of Cardiology, Pitié-Salpêtrière Hospital, Paris 2-Sponsor: 2-Sponsor: AP-HP (Assistance Publique-Hôpitaux de Paris) 3-Data center, Statistics: Unité Recherche Clinique, Pitié-Salpêtrière Hospital, Paris 4-Funding: Programme Hospitalier de Recherche Clinique (070117)

7. CLOVIS-2 Study design H0H1H2H4 Aspirine 75 mg/j ±Clopi 75 mg/ j PD PK 900 mg 300 mg H6 PK PD 300 mg H0H4H1 900 mg Phase 1 Phase 2 Wash out 4 weeks H6H2 Young-post MI patients aged <45 years

8. Statistics Sample size: to demonstrate that 300-mg LD would produce a 40% lower RR-RPA in carriers of CYP2C19*2 vs. non carriers Inclusion of 45 carriers was required to yield 90% power with an α-risk error of 0.05 and assuming a 20% SD for the difference between regimens PD was primarily defined as RR-RPA (%) to adjust for pre- treatment status with clopidogrel MD. The absence of carryover effect was checked and comparison between clopidogrel response across genotype groups and the two LDs tested was evaluated by the Kruskall-Wallis test.

9. Clovis-2 Flow Chart 9 224 never treated with clopidogrel and 50 without genotype 106 patients available for final analysis 566 patients <45 years enrolled (1996 up to 2009) 51 carriers agreed to participate 292 patients <45 years eligible with clinical follow-up 182 declined to participate 8 Homozygous carriers (*2/*2) were matched with 16 non carriers (wt/wt) 8 Homozygous carriers (*2/*2) were matched with 16 non carriers (wt/wt) 43 Heterozygous carriers (wt/*2) were matched with 43 non carriers (wt/wt) 43 Heterozygous carriers (wt/*2) were matched with 43 non carriers (wt/wt)

10. Study Endpoints – Relative reduction in residual platelet aggregation (% RPA 6 hours )–(% RPA baseline )/(% RPA baseline )x100 (LTA 20µ Mol ADP) – Area under the plasma concentration (AUC 0-6 )–time curve of H4 active metabolite (in ng.h/mL) from baseline to six-hours post loading (electrospray liquid chromatography tandem mass spectrometry ) 10

11. Patients characteristics 11 CharacteristicsOverallCYP2C19*2 genotype N=106 wt/wt N=58 wt/*2 N=41 *2/*2 N=7 Age- yr Mean±SD40.1±4.840.1±4.640.2±4.739.9±4.7 BMI (kg/m²) Mean±SD26.1±3.826.2±3.526.1±4.326.0±3.6 Race or Ethnic Background (%) White european North african Black Asian 81.1 12.3 0.9 5.6 84.5 12.1 1,7 1.7 78.1 9.8 0 12.1 71.4 28.6 0 Risk factors-no (%) Familial history of CAD38.041.435.028.5 Active cigarette smoking56.960.343.914.3 Dyslipidemia68.9 68.371.4 Arterial hypertension28.334.521.914.3 Diabetes mellitus15.119.012.20

12. Patient characteristics 12 CharacteristicsOverallCYP2C19*2 genotype N=106 wt/wt N=58 wt/*2 N=41 *2/*2 N=7 Number of vessel (%) Single Double Triple 60.0 22.9 16.2 61.4 24.6 14.0 63.4 19.5 14.6 28.6 28.5 42.8 Revascularization (%) Bypass only PCI only Both 5.6 88.8 5.6 3.4 93.1 3.5 4.9 95.1 0 28.5 71.5 0 Multiple vascular disease* (%)7.58.64.914.3 Drug therapy during follow up (%) Clopidogrel at inclusion Aspirin Statins Beta-blockers Proton pump inhibitors 82.1 92.4 95.0 76.4 44.3 89.6 93.1 100 81.0 48.3 78.1 92.7 100 65.8 43.9 42.8 85.7 97.6 100 14.3

13. 13 wt/wtwt/*2*2/*2 0 25 50 75 100 p<0.02 for all CYP2C19*2 genotype p=0.16p<0.04 p=0.03 ADP 20 µmol/L-induced Residual Platelet Aggregation (%) Baseline Residual Platelet Aggregation

14. Relative Reduction in RPA wt/wtwt/*2*2/*2 0 25 50 75 100 300mg-LD <0.003 for all* 0.030.04 p=0.0045 wt/wtwt/*2*2/*2 900mg-LD <0.0005 for all* 0.20<0.001 p<0.001 Relative change of ADP 20  mol/L-induced residual platelet aggregation (%)

15. Active Metabolites Formation 0 10 20 30 40 50 wt/wtwt/*2*2/*2 p<0.01 for all 300mg-LD 0.038 0.153 p=0.02 wt/wtwt/*2*2/*2 900mg-LD p<0.001 for all 0.059 0.10 p=0.012 AUC 0-6 H4 Active metabolite (ng.h/ml)

16. High-On Treatment Platelet Reactivity 0 20 40 60 80 ADL 20 µmol/L-induced MPA 300mg-LD <0.0017 for all* 0.024<0.0004 p<0.0001 wt/wtwt/*2 *2/*2 900mg-LD <0.0005 for all* 0.1<0.0001 p<0.0001 wt/wtwt/*2 *2/*2

17. PK/PD correlations 17 R 2 =0.20 for 300mg-LD and 0.23 for 900mg-LD (p<0.0001 for both curves). significant between AUC 0-6 and RR-RPA for both LDs (right-shift of the 900 mg-LD curve) <10 ng.h/mL  great variability of PD, not observed with the 900 mg-LD. >20 ng.h/ml  meaningful inhibitory effect of clopidogrel loading

18. Conclusions Co-dominant effect of the 2C19*2 loss-of function allele High clopidogrel LD overcomes poor response in wt/*2 but not in *2/*2 carriers *2/*2 carriers deserve alternate therapy Define a clinical strategy to exploit this pharmacogenetic information to optimize outcomes with clopidogrel