Potent Mechanism-Based Inhibition of Human CYP2B6 by Clopidogrel and Ticlopidine

Abstract

The thienopyridine derivatives ticlopidine and clopidogrel are inhibitors of ADP-induced platelet aggregation. Pharmacological activity of these prodrugs depends on cytochrome P450 (P450)-dependent oxidation to the active antithrombotic agent. In this study, we investigated the interaction potential of clopidogrel and ticlopidine by using human liver microsomes and recombinantly expressed P450 isoforms. Both clopidogrel and ticlopidine inhibited CYP2B6 with highest potency and CYP2C19 with lower potency. Clopidogrel also inhibited CYP2C9, and ticlopidine also inhibited CYP1A2, with lower potency. Inhibition of CYP2B6 was time-and concentrationdependent, and as shown by dialysis experiments, it was irreversible and dependent on NADPH, suggesting a mechanismbased mode of action. Inactivation was of nonpseudo-firstorder type with maximal rates of inactivation (K inact ) for clopidogrel and ticlopidine in microsomes (recombinant CYP2B6) of 0.35 (1.5 min Ϫ1 ) and 0.5 min Ϫ1 (0.8 min Ϫ1 ), respectively, and half-maximal inactivator concentrations (K I ) were 0.5 M (1.1 M) for clopidogrel and 0.2 M (0.8 M) for ticlopidine. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. A chemical mechanism is discussed based on the known metabolic activation of clopidogrel and on the finding that hemoprotein integrity of recombinant CYP2B6 was not affected by irreversible inhibition. These results suggest the possibility of drug interactions between thienopyridine derivates and drug substrates of CYP2B6 and CYP2C19.

Metabolism of Endosulfan-Alpha by Human Liver Microsomes and its Utility as a Simultaneous In Vitro Probe for CYP2B6 and CYP3A4

“…Ticlopidine, a potent mechanism-based chemical inhibitor to CYP2B6 (Richter et al, 2004), and ketoconazole, a selective chemical inhibitor to CYP3A4 (Baldwin et al, 1995) were purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions of ticlopidine were prepared in distilled water and stored at room temperature.…”

“…Protocols for CYP2136 and CYP3A4 inhibition by ticlopidine and ketoconazole utilized methods previously established by Richter et al (2004) and Nomeir et al (2001), respectively. In the case of ticlopidine, a mechanism-based inhibitor of CYP2B6, a 3 min pre-incubation at 37VC of ticlopidine (5 jiM) with HLMs (100 jig) or rCYPs (5 pmol) in 50 mM potassium phosphate buffer (with 3.3 mM MgC1 2 and lmM EDTA) in combination with an NADPH regenerating system (final concentration of 0.5 mM NADP+, 5 mM glucose-6-phosphate, and 4 U/mL glucose-6-phosphate dehydrogenase), occurred prior to the addition of endosulfan-a (20 jiM).…”

Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

“…The results demonstrate a surprisingly broad and potent induction of P450 enzyme activities by most statins. (Richter et al, 2004). S-Mephenytoin was a kind gift from Professor Urs Meyer (Biozentrum, Basel, Switzerland).…”

Ticlopidine-induced hepatotoxicity is associated with specific human leukocyte antigen genomic subtypes in Japanese patients: a preliminary case–control study

“…9 Immune-mediated mechanisms via such covalent binding are believed to be associated with idiosyncratic drug-induced hepatotoxicity. 10,11 We therefore hypothesized that ticlopidine-induced hepatotoxicity might be mediated by a similar mechanism, and searched for genes encoding CYPs that catalyze the metabolic activation of ticlopidine, [12][13][14][15][16][17][18] ticlopidine-reactive metabolite(s)-detoxifying enzymes such as glutathione S-transferase, 19 and associated proteins focusing on polymorphisms that might affect enzyme activity and amino-acid substitution as well as those for which variants have been confirmed in Japanese (as published in the literature and online searchable databases). Furthermore, since there is evidence supporting a relationship between idiosyncratic drug-induced hepatotoxicity and human leukocyte antigen (HLA) genotype, [20][21][22][23][24] three HLA class I loci (HLA-A, B and C) and 3 HLA class II loci (HLA-DRB1, DQB1 and DPB1) were also investigated.…”