Cytochrome P450 Genetic Polymorphisms and the Response to Prasugrel Relationship to Pharmacokinetic,...

Cytochrome P450 Genetic Polymorphisms and the Responseto Prasugrel

Relationship to Pharmacokinetic, Pharmacodynamic, and Clinical Outcomes

Jessica L. Mega, MD, MPH; Sandra L. Close, PhD; Stephen D. Wiviott, MD; Lei Shen, PhD;Richard D. Hockett, MD; John T. Brandt, MD; Joseph R. Walker, PharmD; Elliott M. Antman, MD;

William L. Macias, MD, PhD; Eugene Braunwald, MD; Marc S. Sabatine, MD, MPH

Background—Both clopidogrel and prasugrel require biotransformation to active metabolites by cytochrome P450 (CYP)enzymes. Among persons treated with clopidogrel, carriers of reduced-function CYP2C19 alleles have significantlylower levels of active metabolite, diminished platelet inhibition, and higher rates of adverse cardiovascular events. Theeffect of CYP polymorphisms on the clinical outcomes in patients treated with prasugrel remains unknown.

Methods and Results—The associations between functional variants in CYP genes, plasma concentrations of active drugmetabolite, and platelet inhibition in response to prasugrel were tested in 238 healthy subjects. We then examined theassociation of these genetic variants with cardiovascular outcomes in a cohort of 1466 patients with acute coronarysyndromes allocated to treatment with prasugrel in the Trial to Assess Improvement in Therapeutic Outcomes byOptimizing Platelet Inhibition With Prasugrel–Thrombolysis in Myocardial Infarction 38 trial. Among the healthysubjects, no significant attenuation of the pharmacokinetic or the pharmacodynamic response to prasugrel was observedin carriers versus noncarriers of at least 1 reduced-function allele for any of the CYP genes tested (CYP2C19, CYP2C9,CYP2B6, CYP3A5, and CYP1A2). Consistent with these findings, in subjects with acute coronary syndromes treatedwith prasugrel, no significant associations were found between any of the tested CYP genotypes and risk ofcardiovascular death, myocardial infarction, or stroke.

Conclusions—Common functional CYP genetic variants do not affect active drug metabolite levels, inhibition of plateletaggregation, or clinical cardiovascular event rates in persons treated with prasugrel. These pharmacogenetic findings arein contrast to observations with clopidogrel, which may explain, in part, the different pharmacological and clinicalresponses to the 2 medications. (Circulation. 2009;119:2553-2560.)

Key Words: cardiovascular diseases � drugs � genetics

Treatment with the thienopyridine clopidogrel reducescardiovascular death and ischemic complications in pa-

tients with acute coronary syndromes (ACS) and thoseundergoing percutaneous coronary intervention (PCI) withstenting and has been widely adopted in clinical practice.1–3

However, the interpatient variability in the pharmacodynamicresponse to clopidogrel is well recognized,4–6 and patientswith coronary artery disease with lesser degrees of plateletinhibition in response to clopidogrel have been shown to be atincreased risk of cardiovascular events.7–9 Prasugrel is a third-generation thienopyridine that achieves greater platelet inhibi-tion than does clopidogrel with less variability.10 The Trial toAssess Improvement in Therapeutic Outcomes by Optimizing

Platelet Inhibition With Prasugrel–Thrombolysis in MyocardialInfarction 38 (TRITON–TIMI 38) trial demonstrated that, com-pared with clopidogrel, treatment with prasugrel resulted in asignificantly lower rate of ischemic events and more bleedingamong patients presenting with ACS with planned PCI.11

Clinical Perspective on p 2560

Both clopidogrel and prasugrel are prodrugs that requirebiotransformation to active metabolites by cytochrome P450(CYP) enzymes. Although the active metabolites of bothdrugs have similar affinity for the P2Y12 receptor in vitro, thein vivo difference in response appears to be mediated pre-dominantly by differences in the metabolic pathways leading

Continuing medical education (CME) credit is available for this article. Go to http://cme.ahajournals.org to take the quiz.Received January 16, 2009; accepted March 12, 2009.From the Thrombolysis in Myocardial Infarction Study Group, Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital and

Harvard Medical School, Boston, Mass (J.L.M., S.D.W., E.M.A., E.B., M.S.S.); Eli Lilly and Company, Indianapolis, Ind (S.L.C., L.S., J.T.B., W.L.M.);Affymetrix, Santa Clara, Calif (R.D.H.); and Daiichi Sankyo, Inc. Parsippany, NJ (J.R.W.).

Guest Editor for this article was Howard A. Rockman, MD.The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.851949/DC1.Correspondence to Jessica L. Mega, MD, MPH, Brigham and Women’s Hospital, TIMI Study Group, 350 Longwood Ave, First Floor, Boston, MA

02115. E-mail [email protected]© 2009 American Heart Association, Inc.

Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.109.851949

2553

Genetics

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to the formation of the active metabolites.12 Esterases shuntthe majority of clopidogrel to a dead-end inactive pathway,with the remaining prodrug requiring 2 separate CYP-dependentoxidative steps.13 In contrast, esterases are part of the activationpathway with prasugrel, and prasugrel is oxidized to its activemetabolite in a single CYP-dependent step, without an apparentdead-end inactive pathway (Figure 1).14

The genes that encode the CYP enzymes are polymorphic,with certain alleles demonstrated to confer reduced enzymaticfunction, thereby interfering with production of the drugmetabolites.15 These reduced-function alleles, particularly inCYP2C19, have been shown to affect the pharmacokineticand pharmacodynamic responses to clopidogrel.16–23 More-over, in the setting of treatment with clopidogrel, patientscarrying reduced-function CYP2C19 alleles compared withnoncarriers have substantially higher rates of major adversecardiovascular events, including stent thrombosis.23–25

The effect of CYP polymorphisms on the clinicaloutcomes in patients treated with prasugrel remains un-known. To address this question, we examined the associ-ation of functional polymorphisms in CYP genes withplasma exposure to prasugrel active metabolite levels andplatelet inhibition in 238 healthy individuals. We thendetermined whether reduced-function CYP alleles wereassociated with adverse cardiovascular outcomes in aseparate cohort of 1466 ACS patients allocated to treat-ment with prasugrel in TRITON–TIMI 38.

MethodsPharmacokinetics and PharmacodynamicsHealthy subjects in 6 studies (n�238) involving prasugrel wereincluded in the pharmacokinetic and pharmacodynamic analyses(Table I in the online-only Data Supplement). Plasma concentrationsof prasugrel active metabolites were measured by liquid chromatog-raphy with mass spectrometry.26 The area under the plasmaconcentration-time curve from time of dose to last measurableconcentration (AUC0-t) of prasugrel active metabolite was computedby noncompartmental methods of analysis with the log-linear trap-ezoidal method. The pharmacodynamic response, assessed with theuse of light transmission aggregometry in response to 20 �mol/L

ADP, was expressed as absolute reduction in maximal plateletaggregation from baseline (�MPA).

Clinical OutcomesIn the TRITON–TIMI 38 trial, patients with ACS with planned PCIwho were randomly allocated to treatment with prasugrel received a60-mg loading dose followed by 10 mg daily for up to 15 months.11

The primary efficacy end point was a composite of cardiovasculardeath, myocardial infarction, or stroke. A key prespecified secondaryend point was definite or probable stent thrombosis as defined by theAcademic Research Consortium.27 Safety end points included non–coronary artery bypass graft–related TIMI major or minor bleeding.All outcomes were adjudicated by a blinded clinical eventscommittee.

The prasugrel pharmacogenetic analysis included 1466 patientswho provided a DNA sample (Table II in the online-only DataSupplement). All studies were approved by institutional reviewboards, and written informed consent was obtained from subjects.

Genotyping MethodologyGenotyping was performed with the use of the Affymetrix TargetedHuman DMET (drug-metabolizing enzymes and transporters) 1.0Assay (Affymetrix, Santa Clara, Calif; 98% of genotypes)28 andbidirectional sequencing or exon-specific polymerase chain reactionamplification followed by restriction fragment length polymorphismgel electrophoresis in the case of CYP2C19*17 or a no-call on theDMET chip (2% of genotypes). A total of 54 alleles, comprising theknown major functional variants, were determined with the use ofclinically validated assays for CYP2C19, CYP2C9, CYP2B6,CYP3A5, CYP3A4, and CYP1A2 (Table III in the online-only DataSupplement). Of note, the tested alleles in CYP3A4 were notpolymorphic, leaving 5 genes for analysis. Genotypes were pre-sumed to be in Hardy-Weinberg equilibrium if the P value was�0.001 (0.05/50 alleles�0.001).

CYP Genotype ClassificationsEach allele of the CYP genes was classified a priori by its knowneffect on enzymatic function according to the literature and with theuse of the established common-consensus star allele nomencla-ture.15,30 For each CYP gene, subjects were dichotomized a prioriinto 2 groups on the basis of whether or not they possessed at least1 significantly reduced-function allele. Table IV in the online-onlyData Supplement describes the measured alleles, observed geno-types, and their classification.

CYP3ACYP2B6CYP2C9 CYP2C19

R-95913(inactive)

Esterases

Prasugrel

H3 CS

N

O

FO

OO

SN

O

FHOOC N

O

FSHR-138727(active)

SN

Cl

OCH3O

Cl

N

OCH3O

SO

SN

Cl

OHO

NCl

OCH3O

HS

HOOC

-oxO-2lergodipolCclopidogrel

SR26334 (inactive)

CYP1A2CYP2C19CYP2B6

CYP3ACYP2C9

CYP2C19 CYP2B6

R-130964(active)85%

Esterases

Figure 1. Schematic representation of themetabolism of clopidogrel and prasugrel.

2554 Circulation May 19, 2009

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Statistical AnalysisThe associations between genetic variation and pharmacokinetic andpharmacodynamic parameters were tested with the use of likelihoodratio tests based on linear mixed-effects models, with the primaryoutcomes being exposure to active drug metabolite [log(AUC0-t)] andplatelet inhibition (�MPA). The models contained subject as arandom effect, reduced-function allele carrier status as the predictorof main interest, and other fixed effects including study, dose, andethnicity and, for pharmacodynamics, time, dose-by-time interac-tion, and baseline MPA. To account for other potential baselinedifferences, additional demographic variables including age, sex,weight, and smoking were included as determined by forwardselection for each model. Two-sided P values were calculated, and asignificance threshold of P�0.01 was used to correct for multiplehypotheses testing for the 5 CYP genes.

For clinical outcomes, rates of the end points were expressed asKaplan–Meier estimates at 15 months and were compared betweencarriers and noncarriers of at least 1 reduced-function CYP allele.Consistent with the primary trial analyses, the Gehan-Wilcoxon testwas used for the primary efficacy end point, and the log-rank testwas used for other end points.11 Hazard ratios (HRs) and 95%confidence intervals (CIs) were calculated on the basis of Coxproportional hazards regression models with clinical syndrome(non–ST-elevation ACS versus ST-elevation myocardial infarction)as a stratification factor. If a statistically significant relationship wasfound between genotype and prasugrel pharmacokinetics and phar-macodynamics, then this genotype was to be carried forward as theprimary hypothesis with a P value threshold of �0.05. Because nonewas found, the associations between CYP genes and clinical out-comes were conducted in an exploratory manner, with 2-sided Pvalues calculated and a significance threshold of P�0.01 used tocorrect for multiple hypotheses testing for the 5 genes.

To assess whether statistically significant heterogeneity exists inthe effect of CYP2C19 genetic variation on pharmacokinetic andpharmacodynamic parameters in prasugrel-treated subjects versusour previous observations in these same study populations forclopidogrel-treated subjects,23 formal interaction terms were testedin linear mixed-effects models containing data from the healthysubject studies involving treatment with both clopidogrel and pra-sugrel (N�346). In an analogous fashion, a formal interaction termwas tested in a Cox proportional hazards model in the entireTRITON–TIMI 38 genetic data set (N�2943).

In keeping with the informed consent and privacy policies, allgenetic data reside with the sponsor in a deidentified database behinda firewall and were analyzed by statisticians distinct from those withaccess to the clinical database. The genetic studies were designedand performed as a collaboration between the TIMI Study Group andthe sponsors, Eli Lilly and Daiichi Sankyo. Academic authorsdirected and had access to all the analyses as well as the full clinicaldatabase, wrote all drafts of the manuscript, decided to publish theresults, and vouch for the accuracy and completeness of the data.

ResultsPharmacokinetics and PharmacodynamicsDNA samples were available for 238 healthy subjects for thepharmacokinetic and pharmacodynamic analyses. Their av-

erage age was 33.9�14.6 years, and 26.9% were women.After 4 hours, treatment with a 60-mg dose of prasugrelresulted in an absolute reduction of MPA of 70.6�11.1percentage points.

The association between being a carrier of a reduced-function CYP allele and plasma exposure to the prasugrelactive metabolite and platelet inhibition is presented in Figure2. Carrier status for a reduced-function allele was not asso-ciated with an attenuation of either the pharmacokinetic orpharmacodynamic response to prasugrel for any of theCYP genes evaluated. Moreover, even when an extendedCYP2C19 genotypic classification was used (ultrarapid, ex-tensive, intermediate, and poor metabolizer genotypes; TableIV in the online-only Data Supplement), no association wasfound between CYP2C19 genotype and pharmacokinetic orpharmacodynamic response (Figure in the online-only DataSupplement). In an analysis of the healthy subjects studiesinvolving treatment with both clopidogrel and prasugrel, theinteraction between CYP2C19 and thienopyridine treatmentgroup was significant for both the pharmacokinetic effect(P�0.0001) and the pharmacodynamic effect (P�0.015),demonstrating heterogeneity in the pharmacological impactof CYP2C19 reduced-function alleles between subjectstreated with clopidogrel and those treated with prasugrel.

Clinical OutcomesDNA samples were available in 1466 subjects allocated totreatment with prasugrel in the TRITON–TIMI 38 trial. Theiraverage age was 60.3�10.7 years, 27.3% were female, 69.8%presented with non–ST-segment elevation ACS, and 30.2%presented with ST-segment elevation myocardial infarction.

Consistent with the pharmacokinetic and pharmacody-namic results, no significant associations between any of thetested CYP genotypes and the TRITON–TIMI 38 primaryefficacy end point (cardiovascular death, myocardial infarc-tion, or stroke) were observed (Table 1). Notably, prasugrel-treated patients exhibited no association between CYP2C19genotype and the primary efficacy end point (8.5% forcarriers versus 9.8% for noncarriers; HR, 0.89; 95% CI, 0.60to 1.31; P�0.27; Figure 3). In an analysis of the entireTRITON–TIMI 38 genetic data set, the interaction betweenCYP2C19 and thienopyridine treatment group was significant(P�0.046), demonstrating heterogeneity in the clinical im-pact of CYP2C19 reduced-function alleles between subjectstreated with clopidogrel and those treated with prasugrel.

When we examined each component of the primary effi-cacy end point, no difference in hazard was observed among

P value

-6.1 0.06

-5.3 0.27

-0.4 0.90

-0.8 0.82

-3.5 0.47

Gene

CYP2C19

CYP2C9

CYP2B6

CYP3A5

CYP1A2

-15

scimanydocamrahPscitenikocamrahP

-10 -5 0 5 10 15-40 -20 200-50 -30 -10 3010

P value

-1.3 0.63

-1.7 0.42

-0.6 0.65

1.3 0.38

-1.6 0.37

% Differencein AUC0-t

AbsoluteDifferencein ∆MPA

Relative Percent Difference in AUC0-t (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function Allele

Absolute Difference in ∆MPA (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function Allele

Figure 2. Model-based estimates and95% CIs for genetic effects on the phar-macokinetic (percent difference inAUC0-t) and pharmacodynamic (�MPA)parameters of prasugrel in 238 healthysubjects (doses combined). The P valuethreshold for significance was �0.01.

Mega et al CYP Genetic Polymorphisms and Prasugrel 2555



prasugrel-treated patients carrying a CYP2C19 reduced-function allele compared with noncarriers for cardiovasculardeath (0.99% versus 1.58%; HR, 0.80; 95% CI, 0.26 to 2.47;P�0.70), nonfatal myocardial infarction (6.6% versus 8.1%;HR, 0.82; 95% CI, 0.53 to 1.27; P�0.37), or nonfatal stroke(1.0% versus 0.82%; HR, 1.30; 95% CI, 0.39 to 4.31;P�0.67). Likewise, no significant relationship betweenCYP2C19 genotype and stent thrombosis was observed (0.5%for carriers versus 1.0% for noncarriers; HR, 0.58; 95% CI,0.13 to 2.69; P�0.48; Figure 4) among patients allocated totreatment with prasugrel.

Rates of non–coronary artery bypass graft–related TIMImajor or minor bleeding did not differ statistically by CYPgenotype among prasugrel-treated subjects (Table 2).

DiscussionTreatment with the thienopyridine clopidogrel is frequentlyused after an ACS or PCI with stenting to reduce ischemiccomplications. However, there has been a growing apprecia-tion of the variability in the pharmacological as well as theclinical response to clopidogrel, with genetic polymorphismsin CYP genes explaining some of this variation. In contrast,prasugrel has been found to display less pharmacologicalvariability than clopidogrel. We now show that commonfunctional CYP genetic variants do not affect active metab-olite levels, platelet inhibition, or clinical cardiovascularevents among individuals treated with prasugrel.

Clopidogrel and prasugrel are both prodrugs and requireoxidation by similar CYP enzymes (Figure 1). However,CYP genetic variants may have different influences on thepharmacological and clinical responses to clopidogrel andprasugrel because of the differences in the metabolic path-ways of the 2 thienopyridines. Approximately 85% of clopi-dogrel is shunted into an inactive metabolite by esterases. Assuch, genetic variants that slow the first CYP-oxidative stepinvolved in clopidogrel metabolism may divert the prodrugpreferentially into a dead-end pathway. In contrast, esterasesare part of the activation pathway for prasugrel, and even inthe setting of delayed CYP oxidation, no inactivation path-way is involved in prasugrel metabolism. Additionally, clo-pidogrel requires 2 CYP-oxidative steps, whereas prasugrelrequires only 1.

Genetic variants in CYP2C19 have been shown to alter thepharmacokinetics and pharmacodynamics of clopidogrel. Themost influential of these appears to be the CYP2C19*2 allele,a loss-of-function polymorphism that encodes a cryptic splicevariant that leads to loss of enzymatic activity.31 In severalplatelet aggregation studies, platelet inhibition in response toclopidogrel was significantly less in carriers compared with

Table 1. Efficacy End Point by Genotype Status at 15 Monthsin Patients Treated With Prasugrel

Gene

Event Rates for CV Death, MI, orStroke

HR (95% CI) P

Carriers ofReduced-

Function Allele Noncarriers

Rate(%) n/N

Rate(%) n/N

CYP2C19 8.5 34/407 9.8 99/1048 0.89 (0.60–1.31) 0.27

CYP2C9 11.2 25/234 9.2 108/1213 1.20 (0.78–1.85) 0.58

CYP2B6 11.2 36/329 8.1 62/798 1.45 (0.96–2.18) 0.15

CYP3A5 9.3 99/1095 9.1 14/157 1.03 (0.59–1.80) 0.81

CYP1A2 12.1 9/75 9.0 94/1093 1.42 (0.72–2.81) 0.11

CV indicates cardiovascular; MI, myocardial infarction. Rates are expressedas Kaplan–Meier cumulative incidence estimates over 15 months, and so arenot equal to the numeric percentages. Not all alleles for all genes weresuccessfully genotyped, and not all patients could be classified as a carrier ornoncarrier.

Hazard Ratio 0.89(95% CI 0.60-1.31)

P=0.27

9.8

8.5

1048 991 982 951 849 750 541407 383 376 364 320 276 188

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

0 30 90 180 270 360 450

Number at Risk:Days After Randomization

Non-CarrierCarrier

CV

Dea

th, M

I, or

Str

oke

(%) Non-carriers

Carriers

Non-carriers of a CYP2C19reduced function allele

Carriers Figure 3. Cumulative incidence curves for thecomposite of cardiovascular death, myocardialinfarction, or stroke in carriers of a reduced-function CYP2C19 allele versus noncarriers in1455 subjects treated with prasugrel, who couldbe classified as CYP2C19 carriers or noncarriers inTRITON–TIMI 38.




noncarriers of this variant.16,17,20–22 We have recently shownnot only that genetic variants in CYP2C19, particularlyCYP2C19*2, significantly diminish both the pharmacokineticand pharmacodynamic responses to clopidogrel by approxi-mately one quarter to one third but also that the same variantsare associated with a �50% higher rate of cardiovasculardeath, myocardial infarction, or stroke and a 3-fold higherrate of stent thrombosis in patients treated with clopidogrel inthe TRITON–TIMI 38 trial.23 In another study, only homozy-gotes for a CYP2C19 reduced-function allele variant werefound to be at risk of increased cardiovascular events.24

However, 3 additional studies demonstrate that individualswho carry at least 1 copy of CYP2C19*2 have worse clinicaloutcomes than noncarriers in the setting of treatment withclopidogrel.25,32,32a Thus, the totality of the pharmacokinetic,

pharmacodynamic, and clinical outcomes data suggests thatboth heterozygotes and homozygotes for the CYP2C19*2allele are at increased risk of adverse events in the setting oftreatment with clopidogrel.

Although reduced-function polymorphisms in CYP2C19have been linked to lesser pharmacological responses andworse clinical outcomes among individuals treated withclopidogrel, no other clinical studies have been able toexamine a group of subjects that could be considered acontrol group: those with the same CYP genetic variantstreated with an alternative thienopyridine. It is possible thatvariants in CYP2C19 would alter the pharmacological re-sponse to all thienopyridines. Moreover, CYP2C19 variantsthemselves could be associated with an increase in adversecardiovascular events in patients after an ACS, regardless oftreatment with clopidogrel.

However, our data show that a significant impact ofreduced-function CYP2C19 alleles appears to be confined topatients taking clopidogrel, as CYP2C19 variants do notsignificantly affect pharmacological or clinical outcomes inpatients treated with prasugrel (Figure 5). Specifically,among persons treated with prasugrel, carriers of a CYP2C19reduced-function allele did not have lower prasugrel activemetabolite levels or lesser degrees of platelet inhibitioncompared with noncarriers. Furthermore, carriers and noncar-riers of such alleles treated with prasugrel experienced similarrates of cardiovascular death, myocardial infarction, orstroke. Notably, the 95% CI in this study for the risk ofcardiovascular death, myocardial infarction, or stroke inCYP2C19 carriers versus noncarriers treated with prasugrelexcludes a HR down to 1.31, well below what was seen in the3 aforementioned associations evaluating CYP2C19 geneticvariants and clinical outcomes among patients treated withclopidogrel.23–25,32,32a Additionally, significant interactions

Hazard Ratio, 0.58(95% CI 0.13-2.69)

P=0.48

1.0

0.5

1000 992 990 969 870 764 550379 374 371 363 323 276 189

0

1

2

3

4

0 30 90 180 270 360 450

Def

inite

or P

roba

ble

Sten

tThr

ombo

sis

(%)

Number at Risk:Days After Randomization

Non-CarrierCarrier

Non-carriers

Carriers

Non-carriers of a CYP2C19reduced function allele

Carriers

Figure 4. Cumulative incidence curves for Aca-demic Research Consortium–defined definite orprobable stent thrombosis in carriers of areduced-function CYP2C19 allele versus noncarri-ers in 1379 subjects treated with prasugrel whounderwent PCI with stenting and could be classi-fied as CYP2C19 carriers or noncarriers inTRITON–TIMI 38.

Table 2. Safety End Point by Genotype Status at 15 Monthsin Patients Treated With Prasugrel

Gene

Event Rates for Major or MinorBleeding

HR (95% CI) P

Carriers ofReduced-

Function Allele Noncarriers

% n/N % n/N

CYP2C19 4.5 17/405 3.8 38/1047 1.17 (0.66–2.07) 0.60

CYP2C9 5.5 12/233 3.7 42/1211 1.48 (0.78–2.82) 0.23

CYP2B6 2.3 7/329 4.2 31/795 0.55 (0.24–1.25) 0.15

CYP3A5 3.7 39/1092 5.5 8/157 0.71 (0.33–1.52) 0.38

CYP1A2 2.7 2/75 3.8 39/1090 0.77 (0.19–3.19) 0.72

Rates are expressed as Kaplan–Meier cumulative incidence estimates over15 months. Not all alleles for all genes were successfully genotyped, and notall patients could be classified as a carrier or noncarrier.




were found between CYP2C19 and the thienopyridine treat-ment group, demonstrating that the effect of CYP2C19reduced-function polymorphisms on pharmacological andclinical outcomes was different in clopidogrel-treated indi-viduals versus in prasugrel-treated individuals. Becausereduced-function variants of CYP2C19 are common, occur-ring in 30% of whites, 40% of blacks, and �55% of EastAsians,33–33b the effective use of clopidogrel might involveconsideration of CYP2C19 genotyping or pharmacodynamicmonitoring. Alternatively, use of prasugrel would avoid thisissue, albeit with an increased risk of bleeding.

Although no consistent effect of genotype was foundacross pharmacokinetic, pharmacodynamic, and clinical out-comes with any of the tested CYP genes for prasugrel, weobserved that carriers of a reduced-function CYP2B6 orCYP1A2 allele exhibited nonsignificant trends toward morecardiovascular events and, in the case of CYP2B6, fewerbleeding events. These findings may warrant further study;however, they should be considered exploratory given thelack of biological consistency with no effect on the pharma-cokinetic or pharmacodynamic response to prasugrel, lack ofstatistically significant clinical findings, and the observationthat CYP1A2 does not contribute to prasugrel metabolism.

This study has several potential limitations. Although wegenotyped multiple known functional variants in the relevantCYP genes, CYP genetic variants exist that were not part of

the DMET 1.0 Assay and therefore were not tested, such as*27 and *5 in CYP2B6, and other non-CYP genes that may bepertinent to the pharmacological response prasugrel anddeserve exploration.34 Second, this analysis tested the asso-ciation between CYP genetic variants and a 60-mg loadingdose and 10-mg maintenance dose of prasugrel; other doseswere not evaluated. Additionally, the pharmacokinetic andpharmacodynamic sampling was conducted in 6 healthycohorts of subjects. Because of the need for precise samplehandling, these assessments could not be uniformly con-ducted in the several hundred sites around the world enrollingpatients in TRITON–TIMI 38. Finally, the absence of anotherlarge prasugrel clinical trial precludes replication of theclinical findings; however, in an independent cohort ofhealthy subjects, we did not observe any impact of the CYPvariants on active metabolite levels and platelet inhibition inresponse to prasugrel. Given the established associationbetween the degree of platelet inhibition with thienopyridinesand clinical outcomes,7–9 the totality of our data supports thenotion that these variants do not have a clinical impact.

In conclusion, among healthy individuals treated withprasugrel, no CYP genetic variants were found that affectedactive drug metabolite levels and platelet inhibition. In ACSpatients treated with prasugrel who underwent PCI, no CYPgenetic variants affected cardiovascular outcomes. Thesegenetic observations explain some of the differences in the

-32.4 0.00006

CLOPIDOGREL

-15 -10 -5 0 5 10

-40 -20 200-50 -30 -10 3010

-9.0 0.00054

Relative Percent Difference in AUC0-t (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function CYP2C19 Allele

Absolute Difference in ∆MPA (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function CYP2C19 Allele

Less platelet inhibition More platelet inhibition

Less active metabolite More active metabolite

2.5 2.0 1.5 1.0 0.5

More ischemic events Fewer ischemic events

2.5 2.0 1.5 1.0 0.5

Hazard Ratio for CV Death, MI, or Stroke (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function CYP2C19 Allele

1.53 0.01

-6.1 0.061

-40 -20 200-50 -30 -10 3010

-1.3 0.63

Relative Percent Difference in AUC0-t (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function CYP2C19 Allele

Absolute Difference in ∆MPA (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function CYP2C19 Allele

-15 -10 -5 0 5 10

Hazard Ratio for CV Death, MI, or Stroke (95% CI)in Carriers vs. Non-Carriers of a Reduced-Function CYP2C19 Allele

0.89 0.27

PRASUGRELPh

arm

acok

inet

ics

Phar

mac

odyn

amic

sC

linic

al O

utco

mes

Less platelet inhibition More platelet inhibition

Less active metabolite More active metabolite

More ischemic events Fewer ischemic events

InteractionP value

<0.0001

0.046

0.015

Figure 5. For individuals treated with clopidogrel and for those treated with prasugrel, model-based estimates and 95% CIs forCYP2C19 genetic effects on the pharmacokinetic (percent difference in AUC0-t) and pharmacodynamic (�MPA) parameters in healthysubjects and clinical outcomes in subjects in TRITON–TIMI 38. Numbers to the right of each graph are the point estimate and corre-sponding P value. P values for the interaction terms between carriage of a reduced-function CYP2C19 allele and thienopyridine treat-ment group are presented on the far right. Clopidogrel data adapted from Mega JL, Close SL, Wiviott SD, Shen L, Hockett RD, BrandtJT, Walker JR, Antman EM, Macias W, Braunwald E, Sabatine MS. Cytochrome P-450 polymorphisms and response to clopidogrel.N Engl J Med. 2009;360:354–362.23




pharmacological and clinical response to treatment withprasugrel compared with clopidogrel and potentially could beused to help tailor pharmacotherapy in the future.

Sources of FundingThis study was supported by research grants from Daiichi SankyoCompany, Limited and Eli Lilly and Company.

DisclosuresThe TIMI Study Group receives research grant support from DaiichiSankyo, Eli Lilly, Sanofi-Aventis, and Schering-Plough. In addition,Dr Mega reports receiving research grant support from Johnson &Johnson and Schering-Plough; Dr Wiviott, research grant supportfrom Daiichi Sankyo, Eli Lilly, and Sanofi-Aventis, consulting feesfrom Astra-Zeneca, Portola, and Sanofi-Aventis, and lecture feesfrom Daiichi-Sankyo, Eli Lilly, and Astra-Zeneca; Dr Antman,research grant support from Daiichi Sankyo and Eli Lilly, consultingfees from Sanofi-Aventis, and consulting fees from Daiichi Sankyoand Eli Lilly for a Food and Drug Administration Advisory Panel; DrBraunwald, research grant support from Daiichi Sankyo, Eli Lilly,Merck, Sanofi-Aventis, and Schering-Plough, consulting fees fromDaiichi Sankyo, Eli Lilly, Merck, and Schering-Plough, and lecturefees from Eli Lilly and Schering-Plough; Dr Sabatine, research grantsupport from Sanofi-Aventis and Schering-Plough, consulting feesfrom Astra-Zeneca, Bristol-Myers Squibb, and Sanofi-Aventis, andlecture fees from Bristol-Myers Squibb, Daiichi Sankyo, Eli Lilly,and Sanofi-Aventis. Dr Walker reports being an employee of DaiichiSankyo and holding equity ownership or stock options therein. DrsClose, Shen, Hockett, Brandt, and Macias report being employees ofEli Lilly and holding equity ownership or stock options therein.Additionally, Dr Hockett reports receiving consulting fees fromAffymetrix.

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CLINICAL PERSPECTIVEClopidogrel has been shown to reduce cardiovascular events in acute coronary syndrome patients. However, interpatientvariability in the response to clopidogrel is well recognized, and individuals with lesser degrees of platelet inhibition areat increased risk of death and ischemic complications. Prasugrel is a newer, third-generation thienopyridine that achievesgreater platelet inhibition than clopidogrel with less variability. Clopidogrel and prasugrel are prodrugs and requirebiotransformation to an active metabolite by cytochrome P450 (CYP) enzymes, although the exact activation pathwaysdiffer between the 2 drugs. Among persons treated with clopidogrel, carriers of reduced-function CYP2C19 genetic variants(�30% of the population) have been shown to have significantly lower levels of active drug metabolite, diminished plateletinhibition, and higher rates of adverse cardiovascular events. In this study, we examined the associations betweenfunctional variants in CYP genes (CYP2C19, CYP2C9, CYP2B6, CYP3A5, and CYP1A2) and the pharmacological responseto prasugrel in 238 healthy subjects and the association of these genetic variants with cardiovascular outcomes(cardiovascular death, myocardial infarction, or stroke) in a cohort of 1466 patients with acute coronary syndrome allocatedto treatment with prasugrel in the Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet InhibitionWith Prasugrel–Thrombolysis in Myocardial Infarction 38 trial. No difference was found in the active drug metabolitelevels, platelet inhibition, and rates of adverse cardiovascular events in carriers versus noncarriers of at least 1reduced-function allele for any of the CYP genes tested. These pharmacogenetic findings with prasugrel are in contrast toobservations with clopidogrel, which may explain, in part, the different pharmacological and clinical responses to the 2medications.

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1

SUPPLEMENTAL MATERIAL

2

Supplemental Table 1. Healthy Subject Pharmacokinetic and Pharmacodynamic Studies Study Prasugrel Doses # of Subjects PK PD

Study 1 10 or 60 mg LD 41 40 40

Study 2 60 mg LD, 10 mg MD 28 28 27

Study 3 60 mg LD, 10 mg MD 28 27 27

Study 4 60 mg LD, 10 mg MD 22 22 22

Study 5 60 mg LD, 10 mg MD 89 88 89

Study 6 10 mg MD 30 30 0

TOTAL 238 235 205 Time points in the pharmacodynamic analyses included before first dose and 4 and 24 hours postdose for loading and maintenance dosing. LD denotes loading dose, MD maintenance dose, PD pharmacodynamics, PK pharmacokinetics. 1. Li YG, Ni L, Small D, et al. Inhibition of platelet aggregation with prasugrel and clopidogrel. Am J Cardiol

2008;102:Suppl:76i (Abstract).

2. Farid NA, Small DS, Payne CD, et al. Effects of atorvastatin on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel in healthy subjects. Pharmacotherapy. 2008:28:1483-94.

3. Payne CD, Li YG, Small DS, et al. Increased active metabolite formation explains the greater platelet inhibition with prasugrel compared to high-dose clopidogrel. J Cardiovasc Pharmacol. 2007;50:555-562.

4. Small DS, Farid NA, Li YG, Ernest CS II, Payne CD, Salazar DE, Winters KJ. Effect of ranitidine on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel. Curr Med Res Opin. 2008;24:2251-2257.

5. Small DS, Kothare PA, Yuen ES, et al. The pharmacokinetics and pharmacodynamics of prasugrel in healthy Chinese, Japanese, and Korean subjects compared with healthy Caucasian subjects. Clin Pharmacol Ther. 2008;83 (Suppl 1):S56-57 (Abstract).

6. Small DS, Wrishko RE, Ernest CS II, et al. The pharmacokinetics and pharmacodynamics of prasugrel metabolites in elderly healthy patients. Am J Geriatr Cardiol. 2008;17:137-143 (Abstract).

3

Supplemental Table 2. Baseline Characteristics of Subjects Allocated to Treatment with Prasugrel in the TRITON-TIMI 38 Genetic Study and the Overall Trial

Genetic Substudy Overall Trial

Total (N) 1,466 6,813

Age (y, mean ± SD) 60.3±10.7 60.9±11.2

Female (N, [%]) 400 (27.3) 1,705 (25.0)

Body Weight (kg, mean ± SD) 82.3±15.6 83.6±16.8

Ethnicity (N, [%])

Caucasian 1,429 (97.5) 6,263 (91.9)

Hispanic 19 (1.3) 269 (4.0)

African 9 (0.6) 205 (3.0)

Asian 4 (0.3) 60 (0.9)

Other 5 (0.3) 16 (0.2)

Region (N, [%])

North America 113 (7.7) 2,164 (31.8)

South America 25 (1.7) 270 (4.0)

Western Europe 294 (20.1) 1,779 (26.1)

Eastern Europe 957 (65.3) 1,657 (24.3)

Other 77 (5.3) 943 (13.8)

Hypertension (N, [%]) 939 (64.1) 4,370 (64.1)

Hypercholesterolemia (N, [%]) 702 (47.9) 3,790 (55.6)

Diabetes mellitus (N, [%]) 299 (20.4) 1,576 (23.1)

Tobacco Use (N, [%]) 595 (40.6) 2,612 (38.3)

Prior MI 254 (17.3) 1,226 (18.0)

Prior TIA or Stroke (N, [%]) 47 (3.2) 181 (2.7)

Creatinine Clearance

<60 ml/min

125 (8.5) 717 (10.7)

Index diagnosis (N, [%])

UA/NSTEMI 1,023 (69.8) 5,044 (74.0)

STEMI 443 (30.2) 1,769 (26.0) Ethnicity was self-reported.

4

Supplemental Table 3. Cytochrome P450 Genes and Measured Alleles

Gene Star Alleles

CYP2C19 *1A, *2A, *3, *4, *5A, *6, *7, *8, *9, *10, *12, *13, *14, *17a

CYP2C9 *1A, *2A, *3A, *4, *5, *6, *8, *9, *10, *11A, *12

CYP2B6 *1A, *1C, *6, *8, *9, *11, *12, *13, *14, *15

CYP3A5 *1A, *3A, *3B, *3D, *3F, *6, *8, *9, *10

CYP3A4 *1A, *17, *18

CYP1A2 *1A, *1C, *1D, *1E, *1K, *1L, *7

a CYP2C19*17 allele measured by conventional polymerase chain reaction (PCR) followed by restriction

fragment length polymorphism analysis. All remaining alleles genotyped by the Affymetrix Targeted Human Drug Metabolizing Enzymes and Transporters (DMET) 1.0 Assay (Affymetrix, Santa Clara, CA, USA). In the case of missing genotype data from the DMET chip, additional genotyping was conducted using bi-directional sequencing or exon-specific polymerase chain reaction amplification followed by restriction fragment length polymorphism gel electrophoresis.

5

Supplemental Table 4. Genotyping Results in Prasugrel Allocated Subjects by Gene and Predicted Metabolic Phenotype

Number of Subjects (%) Gene

Dichotomous classification

Predicted Phenotype Observed Genotypesa PK/PD TRITON-TIMI 38

UM *17/*17, *1A/*17 37 (16) Non-carrier

EM *1A/*1A 93 (41) 1048 (72) b

IM *1A/*2A, *1A/*3, *1A/*4, *1A/*8 78 (35) 372 (26) Carrier

PM *2A/*2A, *2A/*3, *2A/*4

*2A/*5A, *2A/*8 18 (8) 35 (2)

CYP2C19

n/a Unknownc *1A/*9, *1A/*10, *2A/*17, *6/*17 NIc NIc

Non-carrier EM *1A/*1A, *1A/*2A, *1A/*11A, *1A/*12 213 (90) 1213 (84)

IM *1A/*3A, *2A/*2A, *2A/*3A, *2A/*11A, *2A/*12, *3A/*11A, *3A/*12, *6/*11A

23 (10) 226 (16) Carrier

PM *3A/*3A 0 (0) 8 (1) CYP2C9

n/a Unknownc *1A/*5, *1A/*8, *1A/*9, *9/*9 NIc NIc

Non-carrier EM *1A/*1A, *1A/*1C, *1C/*1C 155 (67) 798 (71)

IM *1A/*6, *1A/*9, *1C/*6 *1C/*9, *1C/*13, *6/*9

75 (33) 271 (24) CYP2B6 Carrier

PM *9/*9 0 (0) 58 (5)

EM *1A/*1A 11 (5) 6 (0) Non-carrier

IMd *1A/*3A, *1A/*6, *2A/*3A 53 (23) 151 (12) CYP3A5

Carrier PM *3A/*3A, *3A/*3F, *3A/*6 170 (73) 1095 (87)

Non-carrier EM *1A/*1A, *1A/*18 238 (100) 1377 (100)

IM None CYP3A4 Carriere

PM None

Non-carrier EM *1A/*1A, *1A/*1D, *1A/*1E, *1D/*1D, *1D/*1E, *1D/*1L, *1E/*1L, *1L/*1L

199 (87) 1093 (94)

IM *1A/*1C, *1C/*1D, *1C/*1E 30 (13) 74 (6) Carrier

PM *1C/*1C 0 (0) 1 (0)

CYP1A2

n/a Unknownc *1A/*7 NIc NIc a The frequencies of CYP genotypes were similar to previous reports and within Hardy-Weinberg equilibrium. b CYP2C19*17 was not measured in TRITON-TIMI 38. Thus, ultra-rapid and extensive metabolizer status could not be distinguished and were combined as non-carriers of a reduced-function allele. In addition, *2A/*17, *6/*17 genotypes would be classified as heterozygous carriers of a reduced-function allele. c The predicted metabolic phenotypes of these observed genotype combinations are unknown. They were therefore excluded from analyses. d For CYP3A5, intermediate metabolizer genotypes confer near-normal activity and were therefore a priori combined with the extensive metabolizer genotypes. e No associations with CYP3A4 alleles were tested because no subjects carried the assayed alleles. CYP denotes cytochrome P450, EM extensive metabolizer, IM intermediate metabolizer, NI not included in analyses, PK/PD pharmacokinetics/pharmacodynamics, PM poor metabolizer, UM ultra-rapid metabolizer.

6

Supplemental Figure. Relationship between CYP2C19 Genetic Classification and Pharmacologic Responses to Prasugrel A. Pharmacokinetic Response B. Pharmacodynamic Response

-1.5

-1.0

-0.5

0.0

0.5

UM EM IM PMn=35 n=73 n=66 n=14

log[

AU

C(0

-tla

st)

(uM

*hou

r)]

Prasugrel 60 mg

-1.5

-1.0

-0.5

0.0


log[

AU

C(0

-tla

st)

(uM

*hou

r)]

Prasugrel 10 mg

020

4060

8010

0


Red

uctio

n in

MP

A (

%)

at 2

4-ho

ur

Prasugrel 60 mg

020

4060

8010

0


Red

uctio

n in

MP

A (

%)

at 2

4-ho

ur

Prasugrel 10 mg

Relationship between CYP2C19 genetic classification and pharmacokinetic (left) and pharmacodynamic (right) parameters following loading and maintenance doses of clopidogrel in 238 healthy subjects. EM denotes extensive metabolizer, IM intermediate metabolizer, MPA maximal platelet aggregation, PM poor metabolizer, and UM ultra-rapid metabolizer.

S. SabatineBrandt, Joseph R. Walker, Elliott M. Antman, William L. Macias, Eugene Braunwald and Marc Jessica L. Mega, Sandra L. Close, Stephen D. Wiviott, Lei Shen, Richard D. Hockett, John T.

Pharmacokinetic, Pharmacodynamic, and Clinical OutcomesCytochrome P450 Genetic Polymorphisms and the Response to Prasugrel: Relationship to

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