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Vol. 289, Issue 1, 31-37, April 1999

In Vitro Metabolism of Quinidine: The (3S)-3-Hydroxylation of Quinidine Is a Specific Marker Reaction for Cytochrome P-4503A4 Activity in Human Liver Microsomes

Torben Leo Nielsen, Birgitte Buur Rasmussen, Jean-Pierre Flinois, Philippe Beaune and Kim Brøsen

Department of Clinical Pharmacology, Institute of Medical Biology, Odense University, Denmark (T.L.N., B.B.R., K.B.); and Institut National de la Santé et de la Recherche Médicale U490, Toxicologie Moleculaire, Centre Universitaire des Saints-Pères, Paris, France (J-P.F., P.B.)

The aim of this study was to evaluate the (3S)-3-hydroxylation and the N-oxidation of quinidine as biomarkers for cytochrome P-450 (CYP)3A4 activity in 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 Saccharomyces cerevisiae strains expressing 10 human CYPs. 3-OH-Q formation complied with Michaelis-Menten kinetics (mean values of Vmax and Km: 74.4 nmol/mg/h and 74.2 µM, respectively). Q-N-OX formation followed two-site kinetics with mean values of Vmax, Km and Vmax/Km for the low affinity isozyme of 15.9 nmol/mg/h, 76.1 µM and 0.03 ml/mg/h, respectively. 3-OH-Q and Q-N-OX formations were potently inhibited by ketoconazole, itraconazole, 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 significant 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-expressed isozymes revealed that only CYP3A4 actively catalyzed the (3S)-3-hydroxylation. CYP3A4 was the most active enzyme in Q-N-OX formation, but CYP2C9 and 2E1 also catalyzed minor proportions of the N-oxidation. In conclusion, 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 specific probe for CYP3A4 activity in human liver microsome preparations, whereas the N-oxidation of quinidine is a somewhat less specific marker reaction for CYP3A4 activity, because the presence of a low affinity enzyme is demonstrated by different approaches.

0022-3565/99/2891-0031$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics


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