Department of Pharmacology and PharmacoGenomics Research Center, College of Medicine (M.-J.K., E.S.J, J.-S.P., S.-J.L., J.L.G, J.-G.S., D.-H.K.), and Department of General Surgery, Busan Paik Hospital (C.-S.C.), Inje University, Busan, South Korea.
Drug Metab Dispos. 2014 Jan;42(1):141-52. doi: 10.1124/dmd.113.053017. Epub 2013 Oct 29.
Ticlopidine is a first-generation thienopyridine antiplatelet drug that prevents adenosine 5'-diphosphate (ADP)-induced platelet aggregation. We identified the enzymes responsible for the two-step metabolic bioactivation of ticlopidine in human liver microsomes and plasma. Formation of 2-oxo-ticlopidine, an intermediate metabolite, was NADPH dependent and cytochrome P450 (CYP) 1A2, 2B6, 2C19, and 2D6 were involved in this reaction. Conversion of 2-oxo-ticlopidine to thiol metabolites was observed in both microsomes (M1 and M2) and plasma (M1). These two metabolites were considered as isomers, and mass spectral analysis suggested that M2 was a thiol metabolite bearing an exocyclic double bond, whereas M1 was an isomer in which the double bond was migrated to an endocyclic position in the piperidine ring. The conversion of 2-oxo-ticlopidine to M1 in plasma was significantly increased by the addition of 1 mM CaCl2. In contrast, the activity in microsomes was not changed in the presence of CaCl2. M1 formation in plasma was inhibited by EDTA but not by other esterase inhibitors, whereas this activity in microsomes was substantially inhibited by carboxylesterase (CES) inhibitors such as bis-(p-nitrophenyl)phosphate (BNPP), diisopropylphosphorofluoride (DFP), and clopidogrel. The conversion of 2-oxo-ticlopidine to M1 was further confirmed with recombinant paraoxonase 1 (PON1) and CES1. However, M2 was detected only in NADPH-dependent microsomal incubation, and multiple CYP isoforms were involved in M2 formation with highest contribution of CYP2B6. In vitro platelet aggregation assay demonstrated that M2 was pharmacologically active. These results collectively indicated that the formation of M2 was mediated by CYP isoforms whereas M1, an isomer of M2, was generated either by human PON1 in plasma or by CES1 in the human liver.
噻氯匹定是一种第一代噻吩并吡啶类抗血小板药物，可防止二磷酸腺苷（ADP）诱导的血小板聚集。我们在人肝微粒体和血浆中鉴定了负责噻氯匹定两步代谢生物激活的酶。形成 2-氧代噻氯匹定，一种中间代谢物，是 NADPH 依赖性的，并且细胞色素 P450（CYP）1A2、2B6、2C19 和 2D6 参与了该反应。在微粒体（M1 和 M2）和血浆（M1）中均观察到 2-氧代噻氯匹定转化为硫醇代谢物。这两种代谢物被认为是异构体，质谱分析表明 M2 是一种带有外环双键的硫醇代谢物，而 M1 是一种其中双键迁移到哌啶环中环内位置的异构体。在血浆中，2-氧代噻氯匹定转化为 M1 的速度因加入 1mM CaCl2 而显著增加。相比之下，在 CaCl2 存在下，微粒体中的活性没有变化。M1 在血浆中的形成被 EDTA 抑制，但不是其他酯酶抑制剂，而这种在微粒体中的活性被羧酸酯酶（CES）抑制剂如双（对硝基苯基）磷酸酯（BNPP）、二异丙基氟磷酸（DFP）和氯吡格雷显著抑制。2-氧代噻氯匹定转化为 M1 的反应还通过重组对氧磷酶 1（PON1）和 CES1 得到了进一步证实。然而，仅在 NADPH 依赖性微粒体孵育中检测到 M2，并且多种 CYP 同工酶参与 M2 的形成，其中 CYP2B6 的贡献最大。体外血小板聚集试验表明 M2 具有药理活性。这些结果共同表明 M2 的形成是由 CYP 同工酶介导的，而 M1，M2 的异构体，是在血浆中由人 PON1 产生的，或者是在人肝中由 CES1 产生的。
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