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...

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.

ABSTRACT:The risk of idiosyncratic drug toxicity (IDT) is of great concern to the pharmaceutical industry. Current hypotheses based on retrospective studies suggest that the occurrence of IDT is related to covalent binding and daily dose. We determined the covalent binding of 42 radiolabeled drugs in three test systems (human liver microsomes and hepatocytes in vitro and rat liver in vivo) to assess the risk of IDT. On the basis of safety profiles given in official documentation, tested drugs were classified into the safety categories of safe, warning, black box warning, and withdrawn. The covalent binding in each of the three test systems did not distinguish the safety categories clearly. However, when the log-normalized covalent binding was plotted against the log-normalized daily dose, the distribution of the plot in the safety categories became clear. An ordinal logistic regression analysis indicated that both covalent binding and daily dose were significantly correlated with safety category and that covalent binding in hepatocytes was the best predictor among the three systems. When two separation lines were drawn on the correlation graph between covalent binding in human hepatocytes and daily dose by a regression analysis to create three zones, 30 of 37 tested drugs were located in zones corresponding to their respective classified safety categories. In conclusion, we established a zone classification system using covalent binding in human hepatocytes and daily dose for the risk assessment of IDTs.

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

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

The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrelÕs active metabolite. J Thromb Haemost 2007; 5: 1545-51.Summary. Background and methods: Prasugrel is a novel orally active thienopyridine prodrug with potent and longlasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y 12 receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated. Results: Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5¢-diphosphate (ADP) (10 lM) with an IC 50 value of 1.8 lM. Clopidogrel AM similarly inhibited platelet aggregation with an IC 50 value of 2.4 lM. Similar results were also observed for ADP-induced (10 lM) decreases in prostaglandin E 1 -stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities. Conclusions: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.

ABSTRACT:Acyl glucuronides (AGs) formed from carboxylic acid-containing drugs have been considered to be a cause of idiosyncratic drug toxicity (IDT). Chemical stability of AGs is supposed to relate to their reactivity. In this study, the half-lives of 21 AGs of carboxylic drugs in potassium phosphate buffer (KPB), human serum albumin (HSA) solution, and human fresh plasma were analyzed in relation to the IDT risk derived from these drugs. The carboxylic drugs were classified into three safety categories of "safe," "warning," and "withdrawn" in terms of their IDT risk. As for the results, the half-lives of AGs in KPB correlated with the IDT risk better than those in HSA solution or in human fresh plasma with regard to the separation of the safe drugs from the warning drugs or the withdrawn drugs. In KPB, whereas the half-lives in the safe category were 7.2 h or longer, those in the withdrawn category were 1.7 h or shorter. The classification value of the half-life in KPB, which separated the safe drugs from the withdrawn drugs was calculated to be 3.6 h by regression analysis. In conclusion, this is the first report that clearly shows the relationship between the IDT risk and chemical stability of AGs in several in vitro systems. The KPB system was considered to be the best for evaluating the stability of AGs, and the classification value of the half-life in KPB serves as a useful key predictor for the IDT risk.

No abstract