Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases.
N-Deacetyl ketoconazole (DAK) is the major metabolite of orally administered ketoconazole. This major metabolite has been demonstrated to be further metabolized predominately by the flavin-containing monooxygenases (FMOs) to the secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK) by adult and postnatal rat hepatic microsomes. Our current investigation evaluated the FMO isoform specificity of DAK in a pyrophosphate buffer (pH 8.8) containing the glucose 6-phosphate NADPH-generating system. cDNA-expressed human FMOs (FMO1, FMO3, and FMO5) and cDNA-expressed rabbit FMOs (FMO1, FMO2, FMO3, and FMO5) were used to assess the metabolism of DAK to its subsequent FMO-mediated metabolites by HPLC analysis. Human and rabbit cDNA-expressed FMO3 resulted in extensive metabolism of DAK in 1 h (71.2 and 64.5%, respectively) to N-hydroxy-DAK (48.2 and 47.7%, respectively) and two other metabolites, metabolite 1 (11.7 and 7.8%, respectively) and metabolite 3 (10.5 and 10.0%, respectively). Previous studies suggest that metabolite 1 is the nitrone formed after successive FMO-mediated metabolism of N-hydroxy-DAK. Moreover, these studies display similar metabolic profiles seen with adult and postnatal rat hepatic microsomes. The human and rabbit FMO1 metabolized DAK predominately to the N-hydroxy-DAK in 1 h (36.2 and 25.3%, respectively) with minimal metabolism to the other metabolites (</=5%). Rabbit FMO2 metabolized DAK to N-hydroxy-DAK (15.9%) and metabolite 1 (6.6%). Last, DAK did not appear to be a substrate for human or rabbit FMO5. Heat inactivation of cDNA-expressed FMOs abolished DAK metabolite formation. These results suggest that DAK is a substrate for human and rabbit FMO1 and FMO3, rabbit FMO2, but not human or rabbit FMO5.
Rodriguez
R J
RJ
Department of Pharmaceutical Sciences, Oregon State University, Corvallis 97331-3507, USA. Rosita.Rodriguez@orst.edu
Miranda
C L
CL
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007654:Ketoconazole / Q000031:analogs & derivatives* / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity; D013997:Time Factors
D007527:Isoenzymes; C035996:deacylketoconazole; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D007654:Ketoconazole