title: Multiple cytochrome P450 isoforms are involved in the generation of a pharmacologically active thiol metabolite, whereas paraoxonase 1 and carboxylesterase 1 catalyze the formation of a thiol metabolite isomer from ticlopidine.
abstract: 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.
authors.affiliation: 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.
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Kim
authors.forename: Eun Sook
authors.initials: ES
authors.lastname: Jeong
authors.forename: Jung-Soon
authors.initials: JS
authors.lastname: Park
authors.forename: Su-Jun
authors.initials: SJ
authors.lastname: Lee
authors.forename: Jong Lyul
authors.initials: JL
authors.lastname: Ghim
authors.forename: Chang-Soo
authors.initials: CS
authors.lastname: Choi
authors.forename: Jae-Gook
authors.initials: JG
authors.lastname: Shin
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
mesh_terms: D000328:Adult; D043303:Aryldialkylphosphatase / Q000378:metabolism*; D043182:Carboxylesterase / Q000037:antagonists & inhibitors / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D000077144:Clopidogrel; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004950:Esterases / Q000378:metabolism; D006801:Humans; D008297:Male; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism* / Q000494:pharmacology; D013988:Ticlopidine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D055815:Young Adult
chemical_list: D004791:Enzyme Inhibitors; D010975:Platelet Aggregation Inhibitors; D020033:Protein Isoforms; D013438:Sulfhydryl Compounds; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D000077144:Clopidogrel; D004950:Esterases; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human; D043182:Carboxylesterase; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D013988:Ticlopidine