Influence of Genetic Polymorphisms on the Effect of High- and Standard-Dose Clopidogrel After Percutaneous Coronary Intervention

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1 Journal of the American College of Cardiology Vol. 59, No. 22, by the American College of Cardiology Foundation ISSN /$36.00 Published by Elsevier Inc. doi: /j.jacc CLINICAL RESEARCH Interventional Cardiology Influence of Genetic Polymorphisms on the Effect of High- and Standard-Dose Clopidogrel After Percutaneous Coronary Intervention The GIFT (Genotype Information and Functional Testing) Study Matthew J. Price, MD,* Sarah S. Murray, PHD, Dominick J. Angiolillo, MD, PHD, Elizabeth Lillie, PHD, Erin N. Smith, PHD, Rebecca L. Tisch, BS, Nicholas J. Schork, PHD, Paul S. Teirstein, MD,* Eric J. Topol, MD,* for the GIFT Investigators La Jolla, California; and Jacksonville, Florida Objectives Background Methods Results Conclusions This study sought to evaluate the influence of single nucleotide polymorphisms (SNPs) on the pharmacodynamic effect of high- or standard-dose clopidogrel after percutaneous coronary intervention (PCI). There is a lack of prospective, multicenter data regarding the effect of different genetic variants on clopidogrel pharmacodynamics over time in patients undergoing PCI. The GRAVITAS (Gauging Responsiveness with A VerifyNow assay Impact on Thrombosis And Safety) trial screened patients with platelet function testing after PCI and randomly assigned those with high on-treatment reactivity (OTR) to either high- or standard-dose clopidogrel; a cohort of patients without high OTR were also followed. DNA samples obtained from 1,028 patients were genotyped for 41 SNPs in 17 genes related to platelet reactivity. After adjusting for clinical characteristics, the associations between the SNPs and OTR using linear regression were evaluated. CYP2C19*2 was significantly associated with OTR at 12 to 24 h (R , p ), 30 days (R , p ), and 6 months after PCI (R , p ), whereas PON1, ABCB C T, and other candidate SNPs were not. Carriers of 1 and 2 reduced-function CYP2C19 alleles were significantly more likely to display persistently high OTR at 30 days and 6 months, irrespective of treatment assignment. The portion of the risk of persistently high OTR at 30 days attributable to reduced-function CYP2C19 allele carriage was 5.2% in the patients randomly assigned to high-dose clopidogrel. CYP2C19, but not PON1 or ABCB1, is a significant determinant of the pharmacodynamic effects of clopidogrel, both early and late after PCI. In patients with high OTR identified by platelet function testing, the CYP2C19 genotype provides limited incremental information regarding the risk of persistently high reactivity with clopidogrel 150-mg maintenance dosing. (Genotype Information and Functional Testing Study [GIFT]; NCT ) (J Am Coll Cardiol 2012;59: ) 2012 by the American College of Cardiology Foundation Clopidogrel, in combination with aspirin, reduces the risk of cardiovascular events in patients with acute coronary syndromes (ACS) and in those undergoing percutaneous coronary intervention (PCI) (1 3). However, the pharmacoki- From the *Division of Cardiovascular Diseases, Scripps Clinic, La Jolla, California; Scripps Genomic Medicine, Scripps Translational Science Institute, La Jolla, California; Division of Cardiology, University of Florida, Jacksonville, Florida; and the Division of Pediatrics and Rady s Children s Hospital, University of California at San Diego School of Medicine, La Jolla, California. This study was funded through an investigator-initiated grant from Bristol-Myers Squibb/sanofi-aventis. The main trial (GRAVITAS) was funded by Accumetrics Inc., and study drug was provided through an investigator-initiated grant from Bristol-Myers Squibb/sanofi-aventis. Dr. Price has received research grant funding from Bristol-Myers Squibb/sanofi-aventis, Accumetrics, and Quest Diagnostics; consulting fees from Daiichi Sankyo/Eli Lilly & Co., AstraZeneca, Bristol-Myers Squibb/sanofi-aventis, Accumetrics, Medicure; lecture fees from Daiichi Sankyo/Eli Lilly & Co., Quest Diagnostics, AstraZeneca, and Nanosphere; and equity interest in Iverson Genetics. Dr. Angiolillo has received consulting fees from Abbott Vascular, Bristol-Myers Squibb/sanofi-aventis, Daiichi Sankyo/Eli Lilly & Co., AstraZeneca, The Medicines Company, Portola, Novartis, Medicure, Accumetrics, Arena Pharmaceuticals, and Merck; and speaking fees from Bristol-Myers Squibb/sanofi-aventis, GlaxoSmithKline, Otsuka, The Medicines Company, Portola, Accumetrics, Schering-Plough, AstraZeneca, Eisai, and Daiichi Sankyo/Eli Lilly & Co. Dr. Teirstein has received consulting fees, research fees, and speaker fees from Accumetrics, Abbott, Medtronic, Boston Scientific, and Daiichi Sankyo/Eli Lilly & Co. Dr. Topol has received consulting fees from Daiichi Sankyo/Eli Lilly & Co., Bristol-Myers Squibb/sanofi-aventis, and Quest Diagnostics. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. A portion of these data was presented as a featured clinical study at the i2/american College of Cardiology Scientific Sessions, New Orleans, Louisiana, April 5, Manuscript received September 14, 2011; revised manuscript received November 17, 2011, accepted November 29, 2011.

2 JACC Vol. 59, No. 22, 2012 May 29, 2012: Price et al. The GIFT Study 1929 netics and pharmacodynamics of clopidogrel vary widely among individuals (4 7). Single-center studies using a candidate gene approach have identified associations between reduced-function variants of the cytochrome 450 (CYP) 2C19 isozyme and on-treatment reactivity (OTR) while receiving clopidogrel (8 13), and a genome-wide association study identified the reduced-function CYP2C19*2 allele as the major determinant of adenosine diphosphate induced platelet aggregation in healthy Amish subjects treated with clopidogrel (14). These findings are consistent with a 2-step oxidative process required for transformation of clopidogrel to its labile active metabolite, in which CYP2C19 contributes substantially to each step (15). Candidate gene studies have also detected associations between other single nucleotide polymorphisms (SNPs) and clopidogrel pharmacodynamics and outcomes after PCI, in particular the 3435 C T polymorphism of the ABCB1 gene that encodes the intestinal efflux transporter P-glycoprotein (16 18), and the Q192R polymorphism of the PON1 gene that encodes the esterase paraoxonase-1 (19). The latter observation has led to a suggested alternative pathway of clopidogrel active metabolite formation that does not significantly involve CYP2C19, challenging the mechanistic basis underlying the association between reduced-function CYP2C19 allele carriage and clinical outcomes in ACS and PCI patients treated with clopidogrel (18,20 24). However, the clinical impact of the ABCB1 and PON1 polymorphisms on clopidogrel efficacy has been inconsistent (25,26), and therefore their effect on clopidogrel pharmacodynamics in patients undergoing PCI requires further validation. A higher clopidogrel maintenance dose (MD) could potentially provide a more intense antiplatelet effect in patients with a genetic predisposition to a diminished response to standarddose therapy by providing greater substrate for biotransformation into the active metabolite. However, the relationships between common genetic polymorphisms and clopidogrel pharmacodynamics using different dosing strategies have not been examined in the setting of a large clinical trial of patients undergoing PCI, where this antiplatelet drug is mostly used. The objective of the current study was to assess the genetic determinants of clopidogrel response variability during the acute and maintenance phases of therapy in a multicenter, randomized, double-blind trial of high-dose compared with standard-dose clopidogrel after PCI. Methods Study population. Patients were enrolled as part of a pre-specified genetic substudy of the GRAVITAS (Gauging Responsiveness with A VerifyNow assay Impact on Thrombosis And Safety) trial. The study design, entry criteria, baseline characteristics, and primary results of this trial were described previously (27,28). The GRAVITAS trial was a prospective, multinational, multicenter, doubleblind, randomized, active-controlled trial. Patients with stable coronary artery disease or ACS who had undergone PCI with at least 1 drug-eluting stent and on a specified periprocedural clopidogrel dosing regimen underwent platelet function assessment with the VerifyNow P2Y12 test 12 to 24 h after PCI. Patients with high on-treatment platelet reactivity, defined as 230 P2Y12 reaction units (PRU), were randomly assigned by an interactive voice response system to treatment with high-dose clopidogrel (600 mg loading dose followed by a 150-mg MD) or standard-dose clopidogrel (no additional loading dose, 75-mg Abbreviations and Acronyms ACS acute coronary syndrome(s) CI confidence interval CYP cytochrome 450 MD maintenance dose OTR on-treatment reactivity PCI percutaneous coronary artery PRU P2Y12 reaction unit SNP single nucleotide polymorphism MD). A randomly selected cohort of patients with OTR 230 PRU were selected by the interactive voice response system to be followed in a blinded fashion and treated with a 75-mg MD of clopidogrel, and the remainder exited the study after platelet function testing per protocol. In those treated with study drug, platelet function was reassessed in a blinded fashion 30 7 days and days after enrollment. The genetic substudy was conducted at 48 participating sites, was approved by all institutional review boards, and written informed consent was obtained from all participants. All patients who were entered into the GRAVITAS trial interactive voice response system were eligible to be enrolled in the genetic substudy. A total of 1,170 patients consented and provided blood samples for genotyping either at the time of screening platelet function assessment 12 to 24 h after PCI or during follow-up (Fig. 1). Patients were excluded from the analysis if they were not of Caucasian ancestry (self-identified) to avoid potential effects of population stratification. Genotype analysis. Forty-three SNPs of 17 genes related to platelet reactivity and/or previously implicated for having an effect on clopidogrel responsiveness were genotyped using the MassARRAY platform (Sequenom, San Diego, California). Polymerase chain reaction assays and extension primers for these SNPs were designed using the Mass ARRAY design software, version 3.1. SNPs were genotyped using the iplex Gold assay (Sequenom, Inc., San Diego, California), based on multiplex polymerase chain reaction, followed by a single base primer extension reaction. The masses of the primer extension products correlating with genotype were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Final genotypes were called using the MassARRAY Typer, version 4.0. Seven samples were removed due to low call rates (sample call cutoff 90%). The mean call rate across the 43 SNPs was 99.6% (range, 93.3% to 100%). Hardy-Weinberg equilibrium was tested in a cohort of 611 patients who consented and had their genetic sample drawn at screening 12 to 24 h after PCI (i.e., patients who were and were not

3 1930 Price et al. JACC Vol. 59, No. 22, 2012 The GIFT Study May 29, 2012: excluded 94 non-caucasian ethnicity 9 ethnicity unknown 7 with low call rate 26 unable to process specimen 1 major protocol deviation 1,170 patients with samples for genetic analysis 1,028 patients with platelet function assessment at randomization hrs after PCI Analysis population hrs after PCI 578 with High On-Treatment (PRU 230) Randomized to: 450 without High On-Treatment (PRU <230) Allocated to: 285 high-dose clopidogrel 293 standard-dose clopidogrel 163 standard-dose clopidogrel 287 exited per study protocol 272 with platelet function assessment at 30-days 275 with platelet function assessment at 30-days 155 with platelet-function assessment at 30-days Analysis population at 30-days 260 with platelet function assessment at 6-months 264 with platelet function assessment at 6-months 149 with platelet function assessment at 6-months Analysis population at 6-months Figure 1 Patient Flow Patient flow in the study from enrollment through follow-up. PCI percutaneous coronary intervention; PRU P2Y12 reaction unit. selected to be followed on study drug) because the distribution of OTR in this cohort should better reflect the general PCI population. Equilibrium was observed for 41 of the 43 SNPs, and therefore these were included in subsequent analyses (Table 1). Endpoints. The primary pharmacodynamic endpoint was OTR. Pre-specified analyses included the relationship between SNPs and OTR at screening 12 to 24 h after PCI and at 30-day and 6-month follow-up; the change in OTR from screening to 30 days; and the rate of high OTR, defined as 230 PRU. The primary efficacy endpoint was a composite of death from cardiovascular causes, nonfatal myocardial infarction, or stent thrombosis. A clinical events committee blinded to treatment assignment and genotype and independent of the sponsor adjudicated all suspected primary efficacy end points, as previously described (28). Statistical analyses. SNPs were tested for their association with OTR by linear regression using a codominant model after adjusting for clinical characteristics associated with OTR at an 0.05 level. These covariates were determined separately for the analyses of OTR at the time of randomization (12 to 24 h after PCI) and at 30 days; 6-month analyses used the same covariates as at 30 days. Bonferroni-adjusted p value cutoffs were used to determine statistical significance. Associations between the 41 genotyped SNPs and on-treatment platelet reactivity were thus evaluated at the level of significance (i.e., ). Multivariate generalized linear regression models were built for significant polymorphisms that included relevant clinical characteristics as fixed covariates. The specific portion of variance explained by genotype and clinical characteristics were determined by calculating the partial 2 from this model. The population-attributable risk of persistently high reactivity was determined by subtracting the incidence of high reactivity at 30 days in noncarriers from the incidence in carriers and noncarriers combined. Categorical variables are reported as counts (percentages) and continuous variables as mean SD where appropriate. Because this was a

4 JACC Vol. 59, No. 22, 2012 May 29, 2012: Price et al. The GIFT Study 1931 Candidate at 12 to 24SNPs, h, Candidate 30Their Days, Frequencies, SNPs, and 6Their Months Frequencies, andafter Their PCI* Associations and Their Associations With On-Treatment With On-Treatment Table 1 at 12 to 24 h, 30 Days, and 6 Months After PCI* On-Treatment After PCI At 30 Days At 6 Months SNP Allele Allelic Frequency R 2 p Value R 2 p Value R 2 p Value rs CYP2C19* rs CYP2B6*1B rs CYP2B6*1C rs CYP2B6*1C rs CYP2C19* rs PEAR rs5918 ITGB rs VAV3/NTNG rs VAV rs ITPR rs CYP2B6* rs CYP2B6* rs ABCB1 3435C T rs PEAR rs PEAR rs RAF rs662 PON1 Q192R rs PEAR rs MAP2K rs PDGFC rs PEAR rs ITGA1/ITGA rs P2RY rs CYP2C19* rs ABCB1 1236C T rs ITGA2/ITGA rs F2R (PAR-1) rs P2RY rs RAF rs RAF rs CYP3A5* rs ITPR rs CYP3A5*2A rs CYP2C19* rs CYP2C19* NA NA NA NA rs MAPK rs IRS rs CYP2C19* rs CYP2C19*5 0 NA NA NA NA NA NA rs CYP2C19*7 0 NA NA NA NA NA NA rs CYP3A5*3F 0 NA NA NA NA NA NA *R 2 values are adjusted for clinical covariates and at 30 days and 6 months for treatment assignment. Allelic frequencies were obtained from the cohort of 611 patients in whom genetic samples were obtained at the time of platelet function assessment after PCI and before study drug assignment or study exit to avoid bias due to the study design. See Methods for more details. A cutoff of p was determined a priori as the level at which to determine statistical significance to correct for multiple comparisons. No carriers were present in the population enrolled at screening 12 to 24 h after PCI. NA not available because no carriers of the given allele were identified; PCI percutaneous coronary intervention; SNP single nucleotide polymorphism. substudy, a target sample size was not pre-specified. The sample size that was achieved provided 90% power to detect an effect size (f 2 ) of and 80% power to detect an effect size of with Analyses were performed with SAS version 9.2 (SAS Institute Inc., Cary, North Carolina). The sponsors had no role in the collection, management, analysis, or interpretation of the data or in the preparation, review, or approval of this paper. Results A total 1,170 patients provided DNA samples over the course of the study, of which 1,028 were included in the analysis of OTR 12 to 24 h after PCI (Fig. 1). Their mean age was years, 301 patients (29.3%) were women, 286 patients (27.8%) had diabetes mellitus, 118 patients (11.5%) presented with ACS, and 450 (44%)

5 1932 Price et al. JACC Vol. 59, No. 22, 2012 The GIFT Study May 29, 2012: displayed normal OTR. A total of 741 patients were assigned to and followed on the study drug: 285 patients displayed high OTR and were randomly assigned to highdose clopidogrel, 293 patients displayed high OTR and were randomly assigned to standard-dose clopdiogrel, and 157 patients displayed normal OTR and were treated with standard-dose clopidogrel. Repeat platelet function assessment on the study drug was available at 30 days in 702 patients (95%) and at 6 months in 672 patients (91%). Genetic determinants of OTR after PCI. Table 1 shows the relationships between the candidate SNPs and OTR 12 to 24 h after PCI adjusted for clinical covariates significantly associated with OTR according to univariate analysis (age, sex, body mass index, current smoking, creatinine clearance 60 ml/min, diabetes mellitus, history of congestive heart failure, hypertension, or hyperlipidemia). The CYP2C19*2 reduced-function allele was significantly associated with OTR (R , p ), whereas the PON1 Q192R, ABCB C T, and CYP2C19*17 alleles were not (R , p 0.42; R , p 0.97; and R , p 0.08, respectively). There was a strong trend for an association between the CYP2B6*1B allele and OTR (R , p ). No interaction was observed between the CYP2C19 and CYP2B6 genotypes (p 0.11). Carriers of reduced-function CYP2C19 alleles present in the study population (i.e., *2, *3, *4, *6, and *8) were pooled for further analyses. Compared with noncarriers, the risk of high OTR 12 to 24 h after PCI was 2.5-fold greater for carriers of 1 reduced-function CYP2C19 allele and approximately 4.5-fold greater for carriers of 2 reduced-function CYP2C19 alleles (Fig. 2). Genetic determinants of OTR during follow-up. The CYP2C19*2 allele was significantly associated with OTR at 30 days (R , p ) and at 6 months (R , p ). Carriage of PON1 Q192R and ABCB C T were not associated with OTR at 30 days or 6 months; an association between CYP2C19*17 carriage and reduced levels of OTR became apparent at 30 days (R , p ), although the association did not reach the threshold of significance at 6 months (R , p 0.01) (Table 1). Body mass index, diabetes mellitus, age, and current smoking also contributed significantly to the variance in OTR at both time points in the multivariate model incorporating the CYP2C19 genotype (Table 2). Clopidogrel MD strategy, CYP2C19 genotype, and OTR. Figure 3 shows the change in OTR from randomization to 30 days according to treatment arm stratified by the CYP2C19 genotype. After adjustment for treatment assignment, characteristics significantly associated with the change in reactivity from randomization to 30 days on multivariate analysis were CYP2C19 genotype ( , p ), age ( , p 0.001), and body mass index ( , p 0.032). CYP2C19 reduced-function allele carriage was associated with a substantially greater risk of high OTR at 30 days and A At Randomization B 200/ / ( ) 1or 223/ / ( ) 23/29 346/ ( ) No.ofPatientsatRisk C At 30 Days 128/ / ( ) 1 or 145/ / ( ) 17/21 158/ ( ) At 6 Months 111/ / ( ) 1or 128/ / ( ) 16/21 130/ ( ) Figure Adjusted s for High On-Treatment by CYP2C19 Genotype At randomization (A), at 30 days (B), and at 6 months (C). Odd ratios are versus noncarriers, adjusted for clinical covariates at randomization and for clinical covariates and treatment arm at 30 days and 6 months. The number of patients with high on-treatment reactivity and the total number of patients at risk of high on-treatment reactivity are presented for each comparison. The p values for trend are for the CYP2C19 genotype and the risk of high on-treatment reactivity for all time points. Patient numbers may vary due to the lack of complete data for multivariate analysis in some patients. CI confidence interval. 6 months, particularly for carriers of 2 reduced-function alleles, irrespective of maintenance dosing strategy (Fig. 4). Among the patients with high OTR after PCI who were randomly assigned to clopidogrel 150-mg MD, the portion of the risk of persistently high reactivity attributable to reduced-function allele carriage (population-attributable risk) was 5.2%. The population-attributable risk was 7.3%

6 JACC Vol. 59, No. 22, 2012 May 29, 2012: Price et al. The GIFT Study 1933 Full at 30Multivariate Days and Full6Multivariate Generalized Months After Linear Generalized PCI Models Linear of On-Treatment Models of On-Treatment Table 2 at 30 Days and 6 Months After PCI 30 Days 6 Months Characteristic Partial 2 * p Value Partial 2 * p Value Study arm CYP2C19 genotype Body mass index Diabetes mellitus History of congestive heart failure Age Creatinine clearance 60 ml/min Current smoking Calcium channel blocker use History of hyperlipidemia Bivalirudin use during index PCI Proton pump inhibitor use ACS presentation Sex UFH use during index PCI * 2 is the specific portion of variance explained by the characteristic. CYP2C19 genotype was classified according to 0, 1, or 2 loss-of-function alleles. ACS acute coronary syndrome(s); PCI percutaneous coronary intervention; UFH unfractionated heparin. using a cut point of PRU 208 for persistently high reactivity. The CYP2C19 genotype was also significantly associated with a greater risk of persistently high reactivity during follow-up among the patients with high OTR randomly assigned to standard-dose clopidogrel (p ) (Fig. 4). Clinical outcomes. The primary clinical endpoint of death due to cardiovascular causes, nonfatal myocardial infarction, or stent thrombosis occurred in 14 patients (1.9%) during follow-up. Carriers of 2 reduced-function alleles were at significantly higher risk of the primary endpoint compared with noncarriers (1 event in 24 patients at-risk compared with 11 events in 466 patients at-risk; hazard ratio: 1.58; 95% confidence interval [CI]: 1.04 to 2.41; p 0.03). There was no association between carriage of 1 reducedfunction CYP2C19 allele and the primary endpoint (2 events in 244 patients at risk; hazard ratio: 1.07; 95% CI: 0.91 to 1.25; p 0.42). There was no association between PON1 Q192R carriage and clinical outcomes (carriers of 1 allele compared with noncarriers: odds ratio: 0.97; 95% CI: 0.83 to 1.14; p 0.73; carriers of 2 alleles compared with noncarriers: odds ratio: 0.90; 95% CI: 0.69 to 1.17; p 0.44). Discussion In this prospective substudy of a large, randomized, multicenter clinical trial, we tested the association between OTR and several gene variants that plausibly could be related to interindividual variability in adenosine diphosphate induced platelet reactivity in patients treated with clopidogrel, including those encoding the CYP450 enzyme family, cell surface receptors, kinases, and other signaling molecules (29). This study provides strong evidence that: 1) CYP2C19 is a key genetic determinant of OTR while receiving clopidogrel early and late after PCI; 2) identification of the CYP2C19 genotype in patients with high OTR according to platelet function testing provides only limited information regarding the risk of persistently high reactivity with clopidogrel 150-mg MD; and 3) it is unlikely that PON1 or ABCB1 contributes significantly to clopidogrel response variability and, by extension, to clopidogrel metabolism. Despite the clinical risk that appears to be imparted by the CYP2C19 genotype in PCI patients treated with clopidogrel (21,22), the pharmacodynamic effect of CYP2C19 among this patient population has not been previously examined in a prospective, multicenter setting. Our finding of a very robust association between the CYP2C19 genotype and OTR is strengthened by several factors: the moderateto-large effect size (R 2 between and 0.10 during follow-up, adjusted odds ratios 2.5 for high OTR in carriers of at least 1 reduced-function allele); the magnitude of the p value ( ); statistical correction for multiple comparisons; and the low likelihood of population stratification given the homogeneous ethnicity of the analysis cohort. We thereby avoided common pitfalls of candidate gene studies that can lead to false-positive reporting. We believe that these results provide definitive evidence supporting a pharmacodynamic basis for the reported association between CYP2C19 and cardiovascular outcomes. Our findings that, after adjustment for clinical and procedural characteristics, reduced-function CYP2C19 allele carriage explains approximately 10% of clopidogrel response variability in the long-term phase of therapy at 30 days and 7% at 6 months extend previous observations of a similar contribution in the acute setting (14,30), and supports the continued influence of CYP2C19 on clopidogrel pharmacodynamics over time.

7 1934 Price et al. JACC Vol. 59, No. 22, 2012 The GIFT Study May 29, 2012: A Unadjusted On-Treatment (P2Y 12 Reaction Unit) B Adjusted On-Treatment (P2Y 12 Reaction Unit) Figure No. of patients No. of patients P=.001 P=.008 High Dose, High On-Treatment P<.001 P=.076 Standard Dose, High On-Treatment Treatment Group P= P<.001 P=.004 High Dose, High On-Treatment P<.001 No LOF Alleles 1LOFAllele 2LOFAlleles Standard Dose, Not High On-Treatment In the GRAVITAS trial, high-dose clopidogrel compared with standard-dose clopidogrel did not reduce the incidence of cardiovascular events among patients with high OTR after PCI (28). An insufficient pharmacodynamic response may have contributed in part to the lack of clinical effectiveness observed with high-dose therapy (31). The current study demonstrates that among patients with high OTR according to platelet function testing after PCI, CYP2C19 is a key determinant of a persistently heightened level of OTR with a standard clopidogrel MD and a diminished antiplatelet effect with a high clopidogrel MD. However, in patients randomly assigned to the 150-mg MD of clopidogrel, the portion of the risk of persistently high OTR at 30 days attributable to reduced-function allele carriage was only 5% using the pre-specified definition of high reactivity ( 230 PRU) and only 7% using a post hoc definition ( 208 PRU) that has been associated with subsequent cardiovascular events (31). P=.11 Standard Dose, High On-Treatment Treatment Group P=.97 Standard Dose, Not High On-Treatment Change in On-Treatment From Randomization to 30 Days by CYP2C19 Genotype and Treatment Group Unadjusted (A) and adjusted (B) for clinical covariates. In the group of patients without high on-treatment reactivity followed on standard-dose clopidogrel, only 1 patient was a carrier of 2 reduced-function CYP2C19 alleles and is not included. Central markers denote the mean, and error bars the SDs. p Values are corrected for multiple comparisons using the Bonferroni method. LOF loss of function. The association that we observed between the CYP2C19*17 allele and an increased antiplatelet effect of clopidogrel was inconsistent, with a significant relationship noted at 30 days but not reaching the threshold for significance after PCI or at 6-month follow-up. Although some observational studies have shown increased platelet inhibition in carriers of the so-called gain-of-function CYP2C19*17 allele (32,33), other studies have shown no effect (14,21,34 36). The findings of clinical outcome studies are similarly inconsistent (18,25,32,37,38). The comparative strength of the association of CYP2C19*2 in our cohort further supports the supposition that reducedfunction alleles play the dominant genetic role in determining on-clopidogrel platelet reactivity (36), and, by extension, in influencing clinical outcomes. We investigated the influence of polymorphisms of the ABCB1 gene, which encodes the P-glycoprotein efflux transporter believed to mediate the intestinal absorption of clopidogrel (16). Data regarding the effect of the ABCB C T polymorphism on clopidogrel pharmacodynamics is limited and inconsistent (14,17,35,36). The impact of the ABCB1 genotype on clinical outcomes after PCI has also been discordant between studies (17,18,25). In the present study, none of the common polymorphisms of ABCB1, including 3435 C T, influenced OTR after PCI, at 30 days, or at 6 months, thereby challenging the biological plausibility of an effect of the ABCB1 genotype on clinical outcomes in clopidogrel-treated patients in the acute or maintenance phases. We assessed the influence of PON1, which encodes the paroxonase-1 enzyme that has been proposed to mediate the rate-limiting step in clopidogrel metabolism (19). A case-cohort and replication study observed that the PON1 Q192R polymorphism was associated with lower paraoxonase-1 plasma activity, lower active metabolite levels, lower platelet inhibition, and a 10-fold greater risk of stent thrombosis in PCI patients receiving clopidogrel (19). We did not detect any influence of PON1 Q192R on the antiplatelet effect of clopidogrel, either in the acute or chronic phase of therapy after PCI. Therefore, according to our findings, there does not appear to be a pharmacodynamic basis for a clinical effect of PON1 on cardiovascular outcomes in clopidogrel-treated patients. A retrospective case-cohort study also did not confirm an effect of PON1 Q192R in patients with stent thrombosis (26), and a retrospective, post hoc single-center study of elective PCI patients also did not observe a relationship between the PON1 genotype and the effect of clopidogrel on residual platelet aggregation by light transmittance aggregometry (39). Clinical implications. On-treatment reactivity while receiving clopidogrel has been associated with clinical outcomes in a patient-level meta-analysis and in a pharmacodynamic analysis of the GRAVITAS trial (31,40). The findings of the current study have several implications regarding optimal antiplatelet therapy after PCI. The

8 JACC Vol. 59, No. 22, 2012 May 29, 2012: Price et al. The GIFT Study 1935 A High-dose clopidogrel, 30 days B High-dose clopidogrel, 6 Months 42/96 55/ ( ) 34/89 44/ ( ) 1or 49/105 55/ ( ) 1or 40/98 44/ ( ) 7/9 55/ ( ) 6/9 44/ ( ) C Standard-dose clopidogrel, 30 days D Standard-dose clopidogrel, 6 Months 72/98 90/ ( ) 63/91 76/ ( ) 1or 73/102 76/ ( ) 1 or 82/109 90/ ( ) 10/11 90/ ( ) 10/11 76/ ( ) Figure 4 Adjusted s for High On-Treatment at 30 Days and 6 Months by CYP2C19 Genotype According to Maintenance Dose Assignment Odds ratios are versus noncarriers and are adjusted for clinical covariates. (A) High-dose clopidogrel MD at 30 days; p trend for the CYP2C19 genotype. (B) High-dose clopidogrel at 6 months; p trend (C) Standard-dose clopidogrel at 30 days; p trend (D) Standard-dose clopdiogrel at 6 months; p trend Patient numbers may vary due to the lack of complete data for multivariate analysis in some patients. CI confidence interval; MD maintenance dose. CYP2C19 genotype is predictive of high OTR in both the early and late phases after PCI. Carriers of 2 reducedfunction CYP2C19 alleles are highly likely to display levels of OTR associated with adverse outcomes, and a 150-mg MD of clopidogrel had a particularly marginal pharmacodynamic effect in these patients. Therefore, in patients who are identified to be poor metabolizers by genotyping, the use of alternative P2Y 12 antagonists that are not influenced by CYP2C19 should be considered if an adequate pharmacodynamic effect is desired. In patients who have undergone platelet function testing and are thereby determined to be at higher risk of ischemic events, additional CYP2C19 genotyping provides little help in identifying who will adequately respond to a 150-mg MD of clopidogrel. Whether even larger doses of clopidogrel may provide an improved response in reduced-function CYP2C19 allele carriers is being examined in the ELEVATE TIMI 56 (Escalating Clopidogrel by Involving a Genetic Strategy Thrombolysis In Myocardial Infarction 56) trial (NCT ). Study limitations. A potential limitation of the present study is that the GRAVITAS trial, by design, preferentially enrolled patients with higher levels of OTR. This selection bias potentially reduced the power of our association tests. Procedural characteristics and concomitant medications, including proton pump inhibitor use, were not available for the entire cohort at randomization, but were available and included in the adjusted analyses at follow-up. We did not examine the pharmacodynamic effect of CYP2C19 on different clopidogrel loading doses, although our findings are consistent with those reported by a single-center casecontrol study (41). The study was not powered to detect the influence of genotype on clinical events, and the significant association between carriage of 2 reduced-function CYP2C19 alleles and outcomes must be considered exploratory and hypothesis generating, although it is consistent with previous reports (18,21,22). Conclusions The findings of this pharmacogenetic analysis of a multicenter, randomized clinical trial demonstrate a strong and consistent association between the CYP2C19 genotype and OTR while receiving clopidogrel after PCI, present during both the early and long-term phases after the procedure and irrespective of MD. In patients with high OTR identified by platelet function testing, the CYP2C19 genotype provides only limited incremental information regarding the risk of persistently high reactivity with a treatment strategy of clopidogrel 150 mg daily. Our findings do not support a pharmacodynamic basis for the

9 1936 Price et al. JACC Vol. 59, No. 22, 2012 The GIFT Study May 29, 2012: influence of PON1 or ABCB1 on clinical outcomes after PCI in clopidogrel-treated patients. Acknowledgment The authors acknowledge Judy Sheard of the Scripps Translational Science Institute for her assistance in the management of this study. Reprint requests and correspondence: Dr. Matthew J. Price, Scripps Clinic, North Torrey Pines Road, Maildrop S1056, La Jolla, California scrippshealth.org. REFERENCES 1. Yusuf S, Zhao F, Mehta SR, Chrolavicius S, Tognoni G, Fox KK. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 2001;345: Steinhubl SR, Berger PB, Mann JT 3rd, et al. Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial. JAMA 2002;288: Sabatine MS, Cannon CP, Gibson CM, et al. 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10 JACC Vol. 59, No. 22, 2012 May 29, 2012: Price et al. The GIFT Study 1937 PRINC (Plavix Response in Coronary Intervention) trial. J Am Coll Cardiol Intv 2008;1: Tantry US, Bliden KP, Wei C, et al. First analysis of the relation between CYP2C19 genotype and pharmacodynamics in patients treated with ticagrelor versus clopidogrel: the ONSET/OFFSET and RESPOND genotype studies. Circ Cardiovasc Genet 2010;3: Pare G, Mehta SR, Yusuf S, et al. Effects of CYP2C19 genotype on outcomes of clopidogrel treatment. N Engl J Med 2010;363: Tiroch KA, Sibbing D, Koch W, et al. Protective effect of the CYP2C19 *17 polymorphism with increased activation of clopidogrel on cardiovascular events. Am Heart J 2010;160: Trenk D, Hochholzer W, Fromm MF, et al. Paraoxonase-1 Q192R polymorphism and antiplatelet effects of clopidogrel in patients undergoing elective coronary stent placement. Circ Cardiovasc Genet 2011;4: Brar SS, ten Berg J, Marcucci R, et al. Impact of platelet reactivity on clinical outcomes after percutaneous coronary intervention: a collaborative meta-analysis of individual participant data. J Am Coll Cardiol 2011;58: Collet JP, Hulot JS, Anzaha G, et al. High doses of clopidogrel to overcome genetic resistance: the randomized crossover CLOVIS-2 (Clopidogrel and Response Variability Investigation Study 2). J Am Coll Cardiol Intv 2011;4: Key Words: clopidogrel y CYP2C19 y genotype y platelet function testing y thienipyridine. APPENDIX: GIFT INVESTIGATORS A. Abbas, M. Amine, D. Angiolillo, R. Applegate, J. Aragon, H. Aronow, W. Batchelor, P. Berger, M. Buchbinder, L. Cannon, N. Chronos, D. Cohen, E. Fry, M. Fugit, R. Gammon, P Gordon, K. Garratt, A. Jacobs, M.Z. Jafar, M. Lucca, M. Lurie, E. Mahmud, A. Malik, T. Mann, S. Manoukian, S. Marshalko, T. McGarry, B. McLaurin, J.C. Merrit, R. Minutello, V. Misra, E.D. Nukta, C. O Shaughnessy, S. Plante, D. Purdy, S. Puri, D. Rizik, M. Robbins, J. Saucedo, M. Schweiger, D. Spriggs, R. Stoler, P. Teirstein, M. Turco, R. Waksman, S. Ward, M. Warshofsky, G. Wong.