| Abstract
| - Experiments using recombinant yeast-expressed human liver cytochromes P450 confirmedprevious literature data indicating that ticlopidine is an inhibitor of CYP 2C19. The present studiesdemonstrated that ticlopidine is selective for CYP 2C19 within the CYP 2C subfamily. UV−visible studieson the interaction of a series of ticlopidine derivatives with CYP 2C19 showed that ticlopidine binds tothe CYP 2C19 active site with a Ks value of 2.8 ± 1 μM. Derivatives that do not involve either theo-chlorophenyl substituent, the free tertiary amine function, or the thiophene ring of ticlopidine did notlead to such spectral interactions and failed to inhibit CYP 2C19. Ticlopidine is oxidized by CYP 2C19with formation of two major metabolites, the keto tautomer of 2-hydroxyticlopidine (1) and the dimers ofticlopidine S-oxide (TSOD) (Vmax = 13 ± 2 and 0.4 ± 0.1 min-1). During this oxidation, CYP 2C19 wasinactivated; the rate of its inactivation was time and ticlopidine concentration dependent. This processmeets the chemical and kinetic criteria generally accepted for mechanism-based enzyme inactivation. Itoccurs in parralel with CYP 2C19-catalyzed oxidation of ticlopidine, is inhibited by an alternative well-known substrate of CYP 2C19, omeprazole, and correlates with the covalent binding of ticlopidinemetabolite(s) to proteins. Moreover, CYP 2C19 inactivation is not inhibited by the presence of 5 mMglutathione, suggesting that it is due to an alkylation occurring inside the CYP 2C19 active site. Theeffects of ticlopidine on CYP 2C19 are very analogous with those previously described for the inactivationof CYP 2C9 by tienilic acid. This suggests that a similar electrophilic intermediate, possibly a thiopheneS-oxide, is involved in the inactivation of CYP 2C19 and CYP 2C9 by ticlopidine and tienilic acid,respectively. The kinetic parameters calculated for ticlopidine-dependent inactivation of CYP 2C19, i.e.,t1/2max = 3.4 min, kinact = 3.2 10-3 s-1, KI = 87 μM, kinact/KI = 37 L·mol-1·s-1, and r (partition ratio) =26 (in relation with formation of 1 + TSOD), classify ticlopidine as an efficient mechanism-based inhibitoralthough somewhat less efficient than tienilic acid for CYP 2C9. Importantly, ticlopidine is the first selectivemechanism-based inhibitor of human liver CYP 2C19 and should be a new interesting tool for studyingthe topology of the active site of CYP 2C19.
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