ABSTRACT:The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.Clopidogrel is a thienopyridine antiplatelet agent that has been widely used in the management of cardiovascular diseases, including atherothrombosis, unstable angina, and myocardial infarction (Savi and Herbert, 2005). Clopidogrel is an inactive prodrug that needs to be converted to the pharmacologically active metabolite in vivo through the hepatic metabolism to exhibit the antiplatelet effect (Savi et al., 1992). Clopidogrel is first converted by the action of cytochrome P450 (P450) to 2-oxo-clopidogrel (a thiolactone) then in a second step converted to the pharmacologically active, thiol-containing metabolite as shown in Fig. 1 (Savi et al., 2000). The P450 isoforms involved in the bioactivation of clopidogrel have been suggested to be CYP1A2 in rats (Savi et al., 1994) and CYP3A in humans (Clarke and Waskell, 2003), although the contribution of these P450s to produce the active metabolite was still unclear. In addition, several recent clinical studies demonstrated that CYP3A4, CYP3A5, and CYP2C19 have a significant role in the formation of the active metabolite from clopidogrel (Hulot et al., 2006;Suh et al., 2006;Brandt et al., 2007;Farid et al., 2007Farid et al., , 2008. Furthermore, Brandt et al. (2007) reported that loss of function of CYP2C19 due to polymorphisms resulted in decreased exposure to...

Summary. Background: Thienopyridines are metabolized to active metabolites that irreversibly inhibit the platelet P2Y 12 adenosine diphosphate receptor. The pharmacodynamic response to clopidogrel is more variable than the response to prasugrel, but the reasons for variation in response to clopidogrel are not well characterized. Objective: To determine the relationship between genetic variation in cytochrome P450 (CYP) isoenzymes and the pharmacokinetic/pharmacodynamic response to prasugrel and clopidogrel. Methods: Genotyping was performed for CYP1A2, CYP2B6, CYP2C19, CYP2C9, CYP3A4 and CYP3A5 on samples from healthy subjects participating in studies evaluating pharmacokinetic and pharmacodynamic responses to prasugrel (60 mg, n = 71) or clopidogrel (300 mg, n = 74). Results: In subjects receiving clopidogrel, the presence of the CYP2C19*2 loss of function variant was significantly associated with lower exposure to clopidogrel active metabolite, as measured by the area under the concentration curve (AUC 0-24 ; P = 0.004) and maximal plasma concentration (C max ; P = 0.020), lower inhibition of platelet aggregation at 4 h (P = 0.003) and poor-responder status (P = 0.030). Similarly, CYP2C9 loss of function variants were significantly associated with lower AUC 0-24 (P = 0.043), lower C max (P = 0.006), lower IPA (P = 0.046) and poor-responder status (P = 0.024). For prasugrel, there was no relationship observed between CYP2C19 or CYP2C9 loss of function genotypes and exposure to the active metabolite of prasugrel or pharmacodynamic response. Conclusions: The common loss of function polymorphisms of CYP2C19 and CYP2C9 are associated with decreased exposure to the active metabolite of clopidogrel but not prasugrel. Decreased exposure to its active metabolite is associated with a diminished pharmacodynamic response to clopidogrel.

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Ticlopidine, clopidogrel, and prasugrel are thienopyridine prodrugs that inhibit adenosine-5'-diphosphate (ADP)-mediated platelet aggregation in vivo. These compounds are converted to thiol-containing active metabolites through a corresponding thiolactone. The 3 compounds differ in their metabolic pathways to their active metabolites in humans. Whereas ticlopidine and clopidogrel are metabolized to their thiolactones in the liver by cytochromes P450, prasugrel proceeds to its thiolactone following hydrolysis by carboxylesterase 2 during absorption, and a portion of prasugrel's active metabolite is also formed by intestinal CYP3A. Both ticlopidine and clopidogrel are subject to major competing metabolic pathways to inactive metabolites. Thus, varying efficiencies in the formation of active metabolites affect observed effects on the onset of action and extent of inhibition of platelet aggregation (IPA). Knowledge of the CYP-dependent formation of ticlopidine and clopidogrel thiolactones helps explain some of the observed drug-drug interactions with these molecules and, more important, the role of CYP2C19 genetic polymorphism on the pharmacokinetics of and pharmacodynamic response to clopidogrel. The lack of drug interaction potential and the absence of CYP2C19 genetic effect result in a predictable response to thienopyridine antiplatelet therapy with prasugrel. Current literature shows that greater ADP-mediated IPA is associated with significantly better clinical outcomes for patients with acute coronary syndrome.

Prasugrel and clopidogrel inhibit platelet aggregation through active metabolite formation. Prasugrel's active metabolite (R-138727) is formed primarily by cytochrome P450 (CYP) 3A and CYP2B6, with roles for CYP2C9 and CYP2C19. Clopidogrel's activation involves two sequential steps by CYP3A, CYP1A2, CYP2C9, CYP2C19, and/or CYP2B6. In a randomized crossover study, healthy subjects received a loading dose (LD) of prasugrel (60 mg) or clopidogrel (300 mg), followed by five daily maintenance doses (MDs) (15 and 75 mg, respectively) with or without the potent CYP3A inhibitor ketoconazole (400 mg/day). Subjects had a 2-week washout between periods. Ketoconazole decreased R-138727 and clopidogrel active metabolite Cmax (maximum plasma concentration) 34-61% after prasugrel and clopidogrel dosing. Ketoconazole did not affect R-138727 exposure or prasugrel's inhibition of platelet aggregation (IPA). Ketoconazole decreased clopidogrel's active metabolite AUC0-24 (area under the concentration-time curve to 24 h postdose) 22% (LD) to 29% (MD) and reduced IPA 28% (LD) to 33% (MD). We conclude that CYP3A4 and CYP3A5 inhibition by ketoconazole affects formation of clopidogrel's but not prasugrel's active metabolite. The decreased formation of clopidogrel's active metabolite is associated with reduced IPA.

ABSTRACT:The biotransformation of prasugrel to R-138727 (2- [

Prasugrel and clopidogrel, thienopyridine prodrugs, are each metabolized to an active metabolite that inhibits the platelet P2Y(12) ADP receptor. In this open-label, 4-period crossover study, the effects of the proton pump inhibitor lansoprazole on the pharmacokinetics and pharmacodynamics of prasugrel and clopidogrel were assessed in healthy subjects given single doses of prasugrel 60 mg and clopidogrel 300 mg with and without concurrent lansoprazole 30 mg qd. C(max) and AUC(0-tlast) of prasugrel's active metabolite, R-138727, and clopidogrel's inactive carboxylic acid metabolite, SR26334, were assessed. Inhibition of platelet aggregation (IPA) was measured by turbidimetric aggregometry 4 to 24 hours after each treatment. Lansoprazole (1) decreased R-138727 AUC(0-tlast) and C(max) by 13% and 29%, respectively, but did not affect IPA after the prasugrel dose, and (2) did not affect SR62334 exposure but tended to lower IPA after a clopidogrel dose. A retrospective tertile analysis showed in subjects with high IPA after a clopidogrel dose alone that lansoprazole decreased IPA, whereas IPA was unaffected in these same subjects after a prasugrel dose. The overall data suggest that a prasugrel dose adjustment is not likely warranted in an individual taking prasugrel with a proton pump inhibitor such as lansoprazole.

ABSTRACT:Prasugrel, a prodrug, is a novel and potent inhibitor of platelet aggregation in vivo. The metabolism of prasugrel and the elimination and pharmacokinetics of its active metabolite, 2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid (R-138727), three inactive metabolites, and radioactivity were determined in five healthy male subjects after a single 15-mg (100 Ci) Clopidogrel and ticlopidine are thienopyridine prodrugs that are activated in vivo to pharmacologically active metabolites that bind irreversibly to the platelets' P2Y 12 receptor, thus inhibiting platelet aggregation. Studies with [14 C]clopidogrel showed that the major metabolic pathway in humans is hydrolysis of clopidogrel to an inactive acid analog; no further metabolites were reported (Lins et al., 1999). Prasugrel (Fig. 1) is a novel and potent thienopyridine prodrug that also inhibits platelet aggregation in vivo. The structure of the thiol-containing active metabolite of prasugrel, R-138727, was previously reported (Sugidachi et al., 2000(Sugidachi et al., , 2001. Similarly, the structure of the thiol-containing active metabolite of clopidogrel was determined by in vitro studies (Pereillo et al., 2002). The platelet-inhibitory activity of prasugrel and initial pharmacokinetic data were recently reported . In vivo, prasugrel is rapidly hydrolyzed to a pharmacologically inactive thiolactone (R-95913), followed by cytochrome P450-dependent ring opening to form the active metabolite R-138727 (Rehmel et al., 2006). The active metabolite of prasugrel possesses two chiral centers, and its four isomers were shown to possess varying degrees of activity toward inhibition of platelet aggregation (Hasegawa et al., 2005).The prasugrel metabolites measured in human plasma in initial studies (Asai et al., 2006) indicated that after formation of R-95913, two thiol-containing compounds are formed, R-138727 and M4, which are further metabolized by S-methylation to R-106583 and R-100932, or conjugation with cysteine to R-119251 and R-118443 (Fig. 1). Compounds R-95913, R-106583, and R-100932 were measured in plasma as indicators for absorption and exposure to prasugrel and its active metabolite. In this report, the physiologic disposition of prasugrel in healthy subjects following a 15-mg (100 Ci) p.o. dose of [14 C]prasugrel is presented. Materials and MethodsRadiolabeled Drug and Chemicals. Prasugrel hydrochloride was provided by Sankyo Co., Ltd. (Tokyo, Japan). Article, publication date, and citation information can be found at