Oxidation of tienilic acid by human cytochromes P-450 (CUP) 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, = 5 2 1 pM, k,,, = 1.7 +-0.2 min-I), 30-fold more potent in terms of k,,,/K,,, than CYP 2C18 (K, = 150?15 pM, k,, = 1.850.2min-I) and 300-fold more potent than CYP 2C8 ( K , = 145 +-15 pM, k,,, = 0.2 +-0.1 min-I). CYP 2C19 was unable to catalyze this hydroxylation under our experimental conditions. During this study, a marked effect of the ionic strength on the activities (hydroxylations of tienilic acid and tolbutamide) of these cytochromes P-450 expressed in the yeast strain 334 was observed. The effect was particularly great in the case of CYP 2C18, with a tenfold decrease of activity upon increasing ionic strength from 0.02 to 0.1. Specific-covalent binding of tienilic acid metabolites to cytochrome P-450 (incubations in the presence of 5 mM glutathione) was markedly higher upon tienilic 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 2C18 and CYP 2C8. Tienilic acid thus appears to be a mechanism-based inhibitor specific for CYP 2C9 in human liver. Experiments performed with human liver microsomes confirmed that tienilic acid 5-hydroxylase underwent a time-dependent inactivation (apparent tllZ = 10 +-5 min) during 5-hydroxylation of tienilic acid.
