OXIDATION OF TIENILIC ACID BY HUMAN YEAST-EXPRESSED CYTOCHROMES P-4502C8, 2C9, 2C18 AND 2C19 - EVIDENCE THAT THIS DRUG IS A MECHANISM-BASED INHIBITOR SPECIFIC FOR CYTOCHROME-P-450 2C9

Oxidation of tienilic acid by human cytochromes P-450 (CYP) 2C9, 2C18, 2C8 and 2C19 was studied using recombinant enzymes expressed in yeast . CYP 2C9 was the best catalyst for 5-hydroxylation of tienilic acid ( K-m = 5 +/- 1 mu M, k(cat) = 1.7 +/- 0.2 min(-1)), 30-fold more potent in terms of k(cat)/K-m than CYP 2C18 (K-m = 150 +/- 15 mu M, k(cat) = 1.8 +/- 0.2 min(-1)) and 300-fold more potent than CYP 2C8 (K-m = 145 +/- 15 mu M, k(cat) = 0.2 +/- 0.1 min(-1)). CYP 2C19 was unable to ca talyze this hydroxylation under our experimental conditions. During th is study, a marked effect of the ionic strength on the activities (hyd roxylations of tienilic acid and tolbutamide) of these cytochromes P-4 50 expressed in the yeast strain 334 was observed. The effect was part icula!ly great in the case of CYP 2C18, with a tenfold decrease of act ivity upon increasing ionic strength from 0.02 to 0.1. Specific-covale nt binding of tienilic acid metabolites to cytochrome P-450 (incubatio ns in the presence of 5 mM glutathione) was markedly higher upon tieni lic acid oxidation by CYP 2C9 than by CYP 2C18 and CYP 2C8. Mechanism- based inactivation of cytochrome P-450 during tienilic acid oxidation was observed in the case of CYP 2C9 but was not detectable with CYP 2C 18 and CYP 2C8. Tienilic acid thus appears to be a mechanism-based inh ibitor specific for CYP 2C9 in human liver. Experiments performed with human liver microsomes confirmed that tienilic acid 5-hydroxylase und erwent a time-dependent inactivation (apparent t(1/2) = 10 +/- 5 min) during 5-hydroxylation of tienilic acid.