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 α-OH- and 6-OH-brotizolam by human liver microsomes (n = 3). V_max for α- and 6-hydroxylation of brotizolam were 1470 ± 259 and 8983 ± 7740 pmol/min/mg protein respectively, and the corresponding K_m 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. K_i 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 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 α- and 6-hydroxylation of brotizolam in human liver microsomes.