Identification of human cytochrome P450 isoforms involved in the metabolism of brotizolam

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 α-OH- and 6-OH-brotizolam by human liver microsomes (n = 3). Vmax for α- 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 μM 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 α- and 6-hydroxylation were 0·05 and 0·07 μM respectively. 4. When incubated with microsomes expressing individual human CYP isoenzymes (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4), brotizolam was metaboli...