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Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases
1Department of Pharmaceutical Sciences, Oregon State University, Corvallis 97331-3507, USA. Rosita.Rodriguez@orst.edu
Abstract:
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 (
Insights
N-Deacetyl ketoconazole (DAK) is metabolized by flavin-containing monooxygenases (FMOs). Human and rabbit FMO3 and FMO1 are key enzymes in DAK metabolism, while FMO5 is not involved.
Area of Science:
- Pharmacology
- Drug Metabolism
- Enzymology
Background:
- N-Deacetyl ketoconazole (DAK) is the primary metabolite of oral ketoconazole.
- Flavin-containing monooxygenases (FMOs) are crucial in metabolizing DAK to N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK).
- Understanding FMO isoform specificity is vital for predicting drug interactions and metabolic pathways.
Purpose of the Study:
- To investigate the specific FMO isoforms responsible for metabolizing N-Deacetyl ketoconazole (DAK).
- To compare the metabolic activity of human and rabbit FMO isoforms on DAK.
- To identify the major metabolites of DAK produced by different FMOs.
Main Methods:
- Utilized cDNA-expressed human (FMO1, FMO3, FMO5) and rabbit (FMO1, FMO2, FMO3, FMO5) FMOs.
- Incubated DAK with FMOs in a pyrophosphate buffer with a NADPH-generating system.
- Analyzed DAK metabolism and metabolite formation using High-Performance Liquid Chromatography (HPLC).
Main Results:
- Human and rabbit FMO3 extensively metabolized DAK (71.2% and 64.5%), producing N-hydroxy-DAK, metabolite 1, and metabolite 3.
- Human and rabbit FMO1 also metabolized DAK (36.2% and 25.3%) primarily to N-hydroxy-DAK.
- Rabbit FMO2 showed moderate DAK metabolism, while FMO5 isoforms did not metabolize DAK. Heat inactivation abolished metabolite formation.
Conclusions:
- N-Deacetyl ketoconazole (DAK) is a substrate for human and rabbit FMO1 and FMO3, and rabbit FMO2.
- Human and rabbit FMO5 isoforms do not appear to metabolize DAK.
- These findings clarify the role of specific FMO isoforms in the metabolic fate of DAK.