In vitro metabolism of quinidine: The (3S)-3-hydroxylation of quinidine isa specific marker reaction for cytochrome P-4503A4 activity in human livermicrosomes

The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-ox idation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity i n human liver microsome preparations. An HPLC method was developed to assay the metabolites (3S)-3-hydroxyquinidine (3-OH-Q) and quinidine N-oxide (Q- N-OX) formed during incubation with microsomes from human liver and from Sa ccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Q formation c omplied with Michaelis-Menten kinetics (mean values of V-max and K-m: 74.4 nmol/ mg/h and 74.2 mu M, respectively). Q-N-OX formation followed two-site kinetics with mean values of V-max, K-m and V-max/K-m for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 mu M and 0.03 ml/mg/h, respectively. 3-OH- Q and Q-N-OX formations were potently inhibited by ketoconazole, itraconazo le, and triacetyloleandomycin. Isozyme specific inhibitors of CYP1A2, -2C9, -2C19, -2D6, and -2E1 did not inhibit 3-OH-Q or Q-N-OX formation, with Ki values comparable with previously reported values. Statistically significan t correlations were observed between CYP3A4 content and formations of 3-OH- Q and Q-N-OX in 12 human liver microsome preparations. Studies with yeast-e xpressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-h ydroxylation. CYP3A4 was the most active enzyme in Q-N-OX formation, but CY P2C9 and 2E1 also catalyzed minor proportions of the N-oxidation. In conclu sion, our studies demonstrate that only CYP3A4 is actively involved in the formation of 3-OH-Q. Hence, the (3S)-3-hydroxylation of quinidine is a spec ific probe for CYP3A4 activity in human liver microsome preparations, where as the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demo nstrated by different approaches.