title: Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite.
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.
authors.affiliation: Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-Ku, Tokyo, 140-8710, Japan. kazui.miho.yk@daiichisankyo.co.jp
authors.forename: Miho
authors.initials: M
authors.lastname: Kazui
authors.forename: Yumi
authors.initials: Y
authors.lastname: Nishiya
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ishizuka
authors.forename: Katsunobu
authors.initials: K
authors.lastname: Hagihara
authors.forename: Nagy A
authors.initials: NA
authors.lastname: Farid
authors.forename: Osamu
authors.initials: O
authors.lastname: Okazaki
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Ikeda
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kurihara
mesh_terms: D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D055162:Biocatalysis; D001711:Biotransformation / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D000077144:Clopidogrel; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065702:Cytochrome P-450 CYP2B6; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008617:Mephenytoin / Q000031:analogs & derivatives / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D009853:Omeprazole / Q000494:pharmacology; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics; D013426:Sulfaphenazole / Q000494:pharmacology; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: D000906:Antibodies; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; C454675:N-3-benzylnirvanol; D010975:Platelet Aggregation Inhibitors; C540904:2-oxo-clopidogrel; D013426:Sulfaphenazole; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D000077144:Clopidogrel; D013806:Theophylline; C050131:furafylline; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C585599:CYP2B6 protein, human; C045793:CYP2C19 protein, human; D019388:Cytochrome P-450 CYP1A2; D065702:Cytochrome P-450 CYP2B6; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione; D009853:Omeprazole; D013988:Ticlopidine; D008617:Mephenytoin; D007654:Ketoconazole