Identification of enzymes responsible for rifalazil metabolism in human liver microsomes.
1. The major metabolites of rifalazil in human are 25-deacetyl-rifalazil and 32-hydroxy-rifalazil. Biotransformation to these metabolites in pooled human liver microsomes, cytosol and supernatant 9000g (S9) fractions was studied, and the enzymes responsible for rifalazil metabolism were identified using inhibitors of esterases and cytochromes P450 (CYP). 2. The 25-deacetylation and 32-hydroxylation of rifalazil occurred in incubations with microsomes or S9 but not with cytosol, indicating that both the enzymes responsible for rifalazil metabolism were microsomal. Km and Vmax of the rifalazil-25-deacetylation in microsomes were 6.5 microM and 11.9 pmol/min/mg with NADPH, and 2.6 microM and 6.0 pmol/min/mg without NADPH, indicating that, although rifalazil-25-deacetylation did not require NADPH, NADPH activated it. Rifalazil-32-hydroxylation was NADPH dependent, and its Km and Vmax were 3.3 microM and 11.0 pmol/min/mg respectively. 3. Rifalazil-25-deacetylation in microsomes was completely inhibited by diisopropyl fluorophosphate, diethyl p-nitrophenyl phosphate and eserine, but not by p-chloromercuribenzoate or 5,5'-dithio-bis(2-nitrobenzoic acid), indicating that the enzyme responsible for the rifalazil-25-deacetylation is a B-esterase. 4. Rifalazil-32-hydroxylation in microsomes was completely inhibited by CYP3A4-specific inhibitors (fluconazole, ketoconazole, miconazole, troleandomycin) and drugs metabolized by CYP3A4 such as cyclosporin A and clarithromycin, indicating that the enzyme responsible for the rifalazil-32-hydroxylation is CYP3A4.
Takasago Research Laboratories, Kaneka Corporation, Hyogo, Japan.
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Mae
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Konishi
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D000107:Acetylation / Q000187:drug effects; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000494:pharmacology; D000995:Antitubercular Agents / Q000494:pharmacology; D001711:Biotransformation / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D016572:Cyclosporine / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004359:Drug Therapy, Combination; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004950:Esterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006634:Histamine H1 Antagonists / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation / Q000187:drug effects; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015394:Molecular Structure; D009249:NADP / Q000378:metabolism; D012294:Rifamycins / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics
D000900:Anti-Bacterial Agents; D019380:Anti-HIV Agents; D000995:Antitubercular Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006634:Histamine H1 Antagonists; D012294:Rifamycins; C068980:KRM 1648; D007531:Isoflurophate; D009249:NADP; D016572:Cyclosporine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D004950:Esterases