Characterization of quinidine 3-hydroxylation as a probe for the CYP 3A enzyme using a novel capillary electrophoresis technique.
Capillary electrophoresis (CE) with a direct injection technique was used to characterize the formation of (3S)-3-hydroxyquinidine (3-OHQ) as a probe for the CYP 3A isoenzymes in rat liver microsomes. Detection was performed either in the absorbance mode or by employing laser-induced fluorescence (LIF) detection. Michaelis-Menten parameters (mean values+/-S.D.) K(m) and V(max) for the formation of 3-OHQ from the probe drug quinidine sulfate (QS) in rat liver microsomes were 37+/-4.6 micro g/ml (47.1+/-5.9 micro M) and 321+/-4 ng/mg/h (942+/-11.7 pmol/mg/h), respectively. Inhibition studies were performed to evaluate the specificity of 3-OHQ as a probe for the CYP 3A enzyme. Ketoconazole and fluconazole were found to be inhibitors of 3-OHQ formation and exhibited K(i) values of 0.19 and 20.1 micro M, respectively. Inhibition with the weak inhibitor, erythromycin could only be estimated using LIF detection due to lack of sensitivity in the absorbance mode. The formation of 3-OHQ in rat liver microsomes can be used as a model for the screening of the CYP 3A enzyme. Direct injection, ensures faster analysis time due to the lack of sample preparation and the low volume capabilities of the technique makes it attractive for the screening of a large number of compounds.
Department of Pharmaceutics, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980533, Richmond, VA 23298-0533, USA.
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Bhoopathy
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Sarkar
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Karnes
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis; D051544:Cytochrome P-450 CYP3A; D019075:Electrophoresis, Capillary / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004917:Erythromycin / Q000494:pharmacology; D015725:Fluconazole / Q000494:pharmacology; D006900:Hydroxylation; D007654:Ketoconazole / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011802:Quinidine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D015415:Biomarkers; D004791:Enzyme Inhibitors; C013569:3-hydroxyquinidine; D004917:Erythromycin; D015725:Fluconazole; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D011802:Quinidine; D007654:Ketoconazole