Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P4502C19

Experiments using recombinant yeast-expressed human liver cytochromes P450 confirmed previous literature data indicating that ticlopidine is an inhibi tor of CYP 2C19. The present studies demonstrated that ticlopidine is selec tive for CYP 2C19 within the CYP 2C subfamily. UV-visible studies on the in teraction of a series of ticlopidine derivatives with CYP 2C19 showed that ticlopidine binds to the CYP 2C19 active site with a K-s value of 2.8 +/- 1 muM. Derivatives that do not involve either the o-chlorophenyl substituent , the free tertiary amine function, or the thiophene ring of ticlopidine di d not lead to such spectral interactions and failed to inhibit CYP 2C19. Ti clopidine is oxidized by CYP 2C19 with formation of two major metabolites, the keto tautomer of 2-hydroxyticlopidine (1) and the dimers of ticlopidine S-oxide (TSOD) (V-max = 13 +/- 2 and 0.4 +/- 0.1 min(-1)). During this oxi dation, CYP 2C19 was inactivated; the rate of its inactivation was time and ticlopidine concentration dependent. This process meets the chemical and k inetic criteria generally accepted for mechanism-based enzyme inactivation. It occurs 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 ticlopidine metabolite(s) to pr oteins. Moreover, CYP 2C19 inactivation is not inhibited by the presence of 5 mM glutathione, suggesting that it is due to an alkylation occurring ins ide the CYP 2C19 active site. The effects of ticlopidine on CYP 2C19 are ve ry analogous with those previously described for the inactivation of CYP 2C 9 by tienilic acid. This suggests that a similar electrophilic intermediate , possibly a thiophene S-oxide, is involved in the inactivation of CYP 2C19 and CYP 2C9 by ticlopidine and tienilic acid, respectively. The kinetic pa rameters calculated for ticlopidine-dependent inactivation of CYP 2C19, i.e ., t(1/2max) = 3.4 min, k(inact) = 3.2 10(-3) s(-1), K-1 = 87 muM, k(inact) /K-1 = 37 L . mol(-1).s(-1), and r (partition ratio) = 26 (in relation with formation of 1 + TSOD), classify ticlopidine as an efficient mechanism-bas ed inhibitor although somewhat less efficient than tienilic acid for CYP 2C 9. Importantly, ticlopidine is the first selective mechanism-based inhibito r of human liver CYP 2C19 and should be a new interesting tool for studying the topology of the active site of CYP 2C19.