Received for publication June 27, 2003.
Revised July 16, 2003.
Accepted for publication September 30, 2003.

POTENT MECHANISM-BASED INHIBITION OF HUMAN CYP2B6 BY CLOPIDOGREL AND TICLOPIDINE

Abstract

The thienopyridine derivates, ticlopidine and clopidogrel, are inhibitors of ADP-induced platelet aggregation. Pharmacological activity of these prodrugs depends on cytochrome P450 (CYP)-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 CYP 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 concentration- dependent, and as shown by dialysis experiments, it was irreversible and dependent on NADPH, suggesting a mechanism-based mode of action. Inactivation was of non- pseudo-first-order type with maximal rates of inactivation (K_inact) for clopidogrel and ticlopidine in microsomes (recombinant CYP2B6) of 0.35 min^-1 (1.5 min^-1) and 0.5 min^{- 1} (0.8 min^-1), respectively, and half- maximal inactivator concentrations (K_I) were 0.5 µM (1.1 µM) for clopidogrel and 0.2 µM (0.8 µM) 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.

Key words: clopidogrel, cytochrome P450, drug interaction, homology modelling, mechanism-based inhibition, suicide inhibitor

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