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 59-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 CaCl 2 . In contrast, the activity in microsomes was not changed in the presence of CaCl 2 . 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 NADPHdependent 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.

Carboxylesterase 1-Mediated Drug–Drug Interactions between Clopidogrel and Simvastatin

“…Consistent with previous report, 19) clopidogrel was found to be an inhibitor of CES1 with an IC 50 value of 24.7 µM on PNPA hydrolysis (Fig. 4).…”

Association of Liver Injury From Specific Drugs, or Groups of Drugs, With Polymorphisms in HLA and Other Genes in a Genome-Wide Association Study

“…35, 36 Ticlopidine is subject to extensive metabolism by several cytochrome P450 isoforms and carboxyesterase. 37 A study in rats suggests that adducts are formed following metabolism by cytochrome P450 with evidence for toxicity after biliary excretion of glutathione-conjugated metabolites via MRP2-facilitated transport. 38 In previous studies, 22 Japanese patients with ticlopidine DILI showed an association with an HLA haplotype including A*33:03 (odds ratio 13).…”

Glutathione trapping of reactive drug metabolites produced by biomimetic metalloporphyrin catalysts

“…The fragmentation pattern and increased retention time compared to ticlopidine suggested that metabolite M5 was an N‐oxide. Based on the fragmentation pattern and literature, M6 was most likely 2‐oxoticlopidine . Metabolites M4–M6 were abundant in both S9 fraction incubations and in the BMO incubations.…”

“…Based on the fragmentation pattern and literature, M6 was most likely 2-oxoticlopidine. [18] Metabolites M4-M6 were abundant in both S9 fraction incubations and in the BMO incubations. M5 was produced with very high abundance in the BMO incubations.…”