Genotyping: Which One Should we Perform? How to Interpret the Data? Is it Ready for Clinical Practice? Paul A. Gurbel, M.D. Sinai Center for Thrombosis Research Baltimore, Maryland, U.S.A. 1 1

Paul A. Gurbel, MD DISCLOSURES Consulting Fees AstraZeneca, Bayer AG, Medtronic CardioVascular, Inc. Grants/Contracted Research AstraZeneca, Bayer AG, Boston Scientific Corporation , Haemoscope, Medtronic CardioVascular, Inc., POZEN Inc. Honoraria Bayer AG, Schering and Millennium

Clopidogrel Response Variability: A PK Problem Intestinal Absorption Esterases 90% P-glycoprotein (ABCB1 gene polymorphism) ? Limited absorption Two Step Conversion Hepatic P450 Cytochromes 10% 2C19, 1A2, 2B6 *2,*17 Genetic polymorphisms 2C19, 2C9, 3A4, 2B6 Variable Active Metabolite Generation Wide Pharmacodynamic Response, Nonresponsiveness Worse Clinical Outcome Adapted from Kauzi M et al. Drug Metab Dispos. 2009; Gurbel et al. J Inv Cardiol. 2009 3

4 4 Linkage Disequilibrium and CYP2C19 Haplotypes and Diplotypes 4 Due to linkage disequilibrium between CYP2C19 *2 loss-of-function (slow metabolizer) and *17 gain-of-function (fast metabolizer) variants, Only 3 (of 4 possible) haplotypes exist: *1----*17 (fast) *1----non-*17 (normal) *2----non-*17 (slow) These 3 haplotypes result in 6 diplotypes which define 3 main phenotypes: Diplotypes Phenotypes fast/fast, fast/normal Extensive metabolizers (EM) normal/normal, fast/slow Normal metabolizers (NM) normal/slow, slow/slow Poor metabolizers (PM) Gurbel et al. ACC 2009 4 4 4

Polymorphisms, Platelet Reactivity, Outcomes on Clopidogrel Study Patients (n) Gene Variants High Platelet Reactivity SNP/ Studied Outcomes 1. Taubert et al (2006) PCI (60) ABCB1 3435T  CLP absorption/active metabolite 2. Suh et al (2006) Healthy (16) CYP3A5 Nonexpressor more drug-drug interactions PCI (348) Nonexpressor- more CV events 3. Giusti et al (2007) PCI (1419) P2Y12, CYP3A4, CYP2C19*2 CYP2C19*2 7. Geisler et al (2008) PCI (235) CYP2C19, CYP3A4, CYP3A5 CYP2C19*2 9. Trenk et al. (2008) PCI (797) CYP2C19*2 CYP2C19*2 10. Gladding et al (2008) PCI (60) CYP2C19, CYP2C9, CYP3A4, CYP2C19*2/3, CYP2C9*11 CYP3A5, CYP2B6, P2Y12, ABCB1 CYP2C19*2,*4,*17 11. Frere et al (2008) NSTE-ACS CYP2C19, CYP3A4, CYP3A5 CYP2C19*2 12. Simon et al (2008) Acute MI (2009) CYP2C19, CYP3A5, ABCB1, ABCB1, CYP2C19*2,*3, *4,*5 -HPR P2Y12 13. Mega et al (2008) Healthy (162) CYP2C19, CYP3A4, CYP2B6, CYP2C19*2 ACS (1477) CYP3A5, CYP1A2 CYP2C19-  1.53x- MACE, 3x- ST 14. Collet et al (2009) MI (259) CYP2C19  CV events including ST 15. Shuldiner et al (2009) Amish (429) GWAS CYP2C19*2 PCI (227) 2C19*2  1 year CV events 16. Sibbing et al (2009) PCI (2485) 2C19*1,*2  30 d ST 17. Giusti et al (2009) PCI (772) 2C19*2  6 month ST and ST + cardiac mortality 18. Bhatt et al (2009) Stable(4862) CYP2C19*2, *3, and *17 No relation between 2C19* hetero and outcomes, ? effect of homo 5 5

1) First genome-wide association study (~400,000 SNP’s): Event-Free Survival Over 1 Year of Follow-up in Sinai Hospital of Baltimore Patients Treated With Clopidogrel Following Percutaneous Coronary Intervention, Stratified by CYP2C19*2 Genotype 1) First genome-wide association study (~400,000 SNP’s): - Pharmacogenomics of Antiplatelet Intervention (PAPI) study - Identify genes associated with clopidogrel response variability - Homogeneous founder population - Healthy Older Order Amish (n=429) - Minimized confounding factors-medications, lifestyle, etc. - Clopidogrel 300 mg load then 75 mg qd x 6 days - Association analyses between SNP’s and ADP-induced aggregation by variance component model (additive effect of genotype) Genotyping was performed with the Affymetrix GeneChip Human Mapping 500K or 1 M (version 6.0) arrays (Affymetrix Inc, Santa Clara, CA) 2) Replication and clinical outcome study in PCI patients (n=227) TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA)

1) Genome-Wide Association Study of Adenosine Diphosphate-Stimulated Platelet Aggregation in Response to Clopidogrel 13 SNP’s 1) 13 SNP’s cluster (within and flanking CYP2C18-2C19-2C9-2C8 cluster) (1.5 megabases on 10q24) strongly associated with clopidogrel response (p <10-7). 2) SNP’s in strong linkage disequilibrium. 3) None of the SNP’s associated with pre-clopidogrel platelet function or response to ASA. 4) No other region with signals meeting genome-wide significance (p <10-7). 5) No relation of ABCB1 variant or CYP2C19*17 with clopidogrel response. 6) CYP2C19*2 accounted for most or all of the 10q24 association signal. (~12% of the variable response) 7) Majority of clopidogrel response variability remains unexplained. .

Patients With Ischemic Events (%) 20 µM ADP-Induced Aggregation (%) CYP 2C19*2 Frequency, Platelet Function and Ischemic Events Patients (n) Patients With Ischemic Events (%) 160 64 3 40 80 120 -/- -/*2 *2/*2 (Frequency = 30%) CYP2C19*2 Frequency in Total Population Relation of Ischemic Events Frequency to Genotype 5 10 15 20 25 21% 10% + CYP2C19*2 (n=67) - CYP2C19*2 (n=160) p=0.02 (n = 14) (n = 16) 15 30 45 60 75 -/- -/*2 *2/*2 p=0.02 Without Events With Events p=0.004 20 µM ADP-Induced Aggregation (%) Platelet Function Measurement 8 8 8

High Platelet Reactivity (HPR) and CYP 2C19*2 Frequency and Their Relation to Post-PCI Ischemic Event Occurrence Total Patients No Events With Events Neither 56% HPR 13% Both 20% CYP*2 11% Neither 61% HPR 11% Both 10% CYP*2 18% 25% 29% 17% Specificity CYP*2 = 72% HPR = 79% Sensitivity CYP*2 = 46% HPR = 46% Gurbel PA et al Presented at i2 Summit Symposium, Orlando 2009 9

Cox Model HRs by Treatment Arm CHARISMA Genomics Study Cox Model HRs by Treatment Arm Effect p value HR 95% CI for HR Effect of Genotype in Placebo Group *2/WT vs WT/WT 0.54 0.852 (0.51, 1.42) *2/*2 vs WT/WT 0.19 1.852 (0.74, 4.65) *2/*17 vs WT/WT 0.47 1.285 (0.65, 2.54) *17/WT vs WT/WT 0.052 1.486 (1.00, 2.21) *17/*17 vs WT/WT 0.71 0.841 (0.33, 2.11) Effect of Genotype in Clopidogrel Group *2/WT vs WT/WT 0.63 1.112 (0.72, 1.71) *2/*2 vs WT/WT 0.022 2.383 (1.14, 5.00) *2/*17 vs WT/WT 0.36 1.322 (0.72, 2.42) *17/WT vs WT/WT 0.72 0.927 (0.61, 1.40) *17/*17 vs WT/WT 0.44 0.696 (0.28, 1.74) *2/*2 has increased risk in clopidogrel arm, but much of this risk is also present in placebo arm Presented at TCT by Bhatt et al.

All GUSTO Bleeding Events: Logistic Regression ORs by Treatment Arm Effect p value OR 95% CI for OR Treatment vs Placebo <0.0001 2.4110 (1.978, 2.943) Effect of Genotype in Placebo Group *2/WT vs WT/WT 0.6392 1.0640 (0.821, 1.374) *2/*2 vs WT/WT 0.3528 0.7250 (0.340, 1.400) *2/*17 vs WT/WT 0.5803 1.1180 (0.748, 1.641) *17/WT vs WT/WT 0.8618 0.9790 (0.769, 1.243) *17/*17 vs WT/WT 0.3477 0.7910 (0.469, 1.278) Effect of Genotype in Clopidogrel Group 0.1712 0.8550 (0.683, 1.070) 4 x 10-4 0.3290 (0.160, 0.619) 0.0652 0.7270 (0.513, 1.020) 0.9222 1.0100 (0.824, 1.238) 0.2236 1.2770 (0.860, 1.891) Significantly reduced risk of bleeding events for the *2/*2 genotype vs wt/wt on clopidogrel Significantly more bleeding events in wt/wt subjects on clopidogrel compared with placebo Bleeding risk in *2/*2 is not significantly different between treatments Presented at TCT by Bhatt et al.

2C19*17, Platelet Aggregation and Bleeding 1524 Post PCI Patients 6.4 M ADP-Induced Aggregation 30- Day Incidence of TIMI Bleedings Caveats: - *2 not analyzed - relation of platelet function to bleeding not reported - *2 and *17 variants are 19,959 base pairs apart and in linkage disequilibrium. - Therefore, pts. with one or two *17 alleles are less likely to carry the *2 allele. - Pts with zero *17 alleles (“wild type” homozygotes at the *17 locus) are more likely to carry the *2 allele. - Greater clopidogrel response in *17 group can be partly or completely attributed to a decreased frequency of the *2 allele. Sibbing D et al. Circulation 2010;121:512-518 12

Clinical Outcomes in High Risk Patients (PCI/ACS) Conclusions Pharmacogenetic Measurement (CYP2C19*2) Pharmacokinetic Measurement (reduced clopidogrel active metabolite) Pharmacodynamic Measurement (high on-treatment platelet reactivity) ? Independent Effect CHARISMA Placebo Group Clinical Outcomes in High Risk Patients (PCI/ACS) Definitive association between: pharmacogenetic, pharmacokinetic and pharmacodynamic measurements and clinical outcomes has not yet been confirmed in a single adequately powered prospective study

Future of Personalized Antiplatelet Therapy Larger prospective studies are needed to confirm the Association of : - CYP2C19*2 - High on-treatment platelet reactivity - Increased ischemic risk - CYP2C19*17- Low on-treatment platelet reactivity - Increased bleeding risk - Establish the role of genotyping vs. platelet function measurements for personalized antiplatelet therapy (alternative or complementary?). VerifyNow Multiplate Analyzer Blood Volume ~ 3 mL Time ~ 30 minutes POC Candidates for Measurement of Phenotype 14

POC Candidates for Measurement of Genotype Blood Volume = 1 mL Single Cell Cartidge Time ~ 3 hours Blood Volume = 1 mL 24-Well Plate Format Time ~ 3 hours 15

Genotyping: Which One Should we Perform? None at this time Most of data are on 2C19*2 How to Interpret the Data? Genotype is not a surrogate for phenotype *2 – ischemic risk *17- Independent bleeding risk? Diplotype analysis may enhance risk assessment Is it Ready for Clinical Practice? Not yet- No prospective studies available 16 16