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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.

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  • Authors+Show Affiliations

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    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

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    MeSH

    Antibodies
    Aryl Hydrocarbon Hydroxylases
    Biocatalysis
    Biotransformation
    Cell Line
    Cell Line, Tumor
    Clopidogrel
    Cytochrome P-450 CYP1A2
    Cytochrome P-450 CYP1A2 Inhibitors
    Cytochrome P-450 CYP2B6
    Cytochrome P-450 CYP2C19
    Cytochrome P-450 CYP2C9
    Cytochrome P-450 CYP3A
    Cytochrome P-450 CYP3A Inhibitors
    Cytochrome P-450 Enzyme System
    Enzyme Inhibitors
    Glutathione
    Humans
    Ketoconazole
    Kinetics
    Mephenytoin
    Microsomes
    Microsomes, Liver
    NADP
    Omeprazole
    Oxidation-Reduction
    Oxidoreductases, N-Demethylating
    Platelet Aggregation Inhibitors
    Sulfaphenazole
    Theophylline
    Ticlopidine

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    19812348

    Citation

    Kazui, Miho, et al. "Identification of the Human Cytochrome P450 Enzymes Involved in the Two Oxidative Steps in the Bioactivation of Clopidogrel to Its Pharmacologically Active Metabolite." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 38, no. 1, 2010, pp. 92-9.
    Kazui M, Nishiya Y, Ishizuka T, et al. Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metab Dispos. 2010;38(1):92-9.
    Kazui, M., Nishiya, Y., Ishizuka, T., Hagihara, K., Farid, N. A., Okazaki, O., ... Kurihara, A. (2010). Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 38(1), pp. 92-9. doi:10.1124/dmd.109.029132.
    Kazui M, et al. Identification of the Human Cytochrome P450 Enzymes Involved in the Two Oxidative Steps in the Bioactivation of Clopidogrel to Its Pharmacologically Active Metabolite. Drug Metab Dispos. 2010;38(1):92-9. PubMed PMID: 19812348.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. AU - Kazui,Miho, AU - Nishiya,Yumi, AU - Ishizuka,Tomoko, AU - Hagihara,Katsunobu, AU - Farid,Nagy A, AU - Okazaki,Osamu, AU - Ikeda,Toshihiko, AU - Kurihara,Atsushi, PY - 2009/10/9/entrez PY - 2009/10/9/pubmed PY - 2010/3/20/medline SP - 92 EP - 9 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab. Dispos. VL - 38 IS - 1 N2 - 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. SN - 1521-009X UR - https://www.unboundmedicine.com/medline/citation/19812348/Identification_of_the_human_cytochrome_P450_enzymes_involved_in_the_two_oxidative_steps_in_the_bioactivation_of_clopidogrel_to_its_pharmacologically_active_metabolite_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=19812348 DB - PRIME DP - Unbound Medicine ER -