title: Identification of human cytochrome P450 isoforms involved in the metabolism of brotizolam.
abstract: 1. To identify the cytochrome P450 (CYP) isoenzyme(s) responsible for the major metabolic pathways of brotizolam in man, we examined the metabolism of brotizolam using human liver microsomes and microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4). 2. Brotizolam was metabolized to alpha-OH- and 6-OH-brotizolam by human liver microsomes (n = 3). Vmax for alpha- and 6-hydroxylation of brotizolam were 1470 +/- 259 and 8983 +/- 7740 pmol/min/mg protein respectively, and the corresponding Km were 49 +/- 9.3 and 595 +/- 580 microM respectively. 3. Among CYP inhibitors examined (furafylline, sulphaphenazole, quinidine, ketoconazole and cimetidine), ketoconazole showed the most potent inhibitory effect on brotizolam metabolism by human liver microsomes. Ki of ketoconazole for alpha- and 6-hydroxylation were 0.05 and 0.07 microM respectively. 4. When incubated with microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4), brotizolam was metabolized only by CYP3A4. 5. Brotizolam metabolism in human liver microsomes was almost completely inhibited by anti-CYP3A4 antiserum. 6. These results suggest that CYP3A4 is predominantly responsible for both alpha- and 6-hydroxylation of brotizolam in human liver microsomes.
authors.affiliation: Department of Biochemistry, Kawanishi Pharma Research Institute, Nippon Boehringer Ingelheim Co., Hyogo, Japan.
authors.forename: C
authors.initials: C
authors.lastname: Senda
authors.forename: W
authors.initials: W
authors.lastname: Kishimoto
authors.forename: K
authors.initials: K
authors.lastname: Sakai
authors.forename: A
authors.initials: A
authors.lastname: Nagakura
authors.forename: T
authors.initials: T
authors.lastname: Igarashi
mesh_terms: D001381:Azepines / Q000378:metabolism*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D006993:Hypnotics and Sedatives / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D012680:Sensitivity and Specificity
chemical_list: D001381:Azepines; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D006993:Hypnotics and Sedatives; D007527:Isoenzymes; C020960:brotizolam; D003577:Cytochrome P-450 Enzyme System