ROLE OF CDNA-EXPRESSED HUMAN CYTOCHROMES P450 IN THE METABOLISM OF DIAZEPAM

The metabolic conversion of diazepam (DZ) to temazepam (TMZ, a C3-hydr oxylation product of DZ) and N-desmethyldiazepam (NDZ, an N1-demethyla tion product of DZ) was studied using cDNA-expressed human cytochrome P450 (CYP) isozymes 1A2, 2B6, 2C8, 2C9, 2C9(R144C), 2E1, 3A4, and 3A5 and human liver microsomes from five organ donors. Of the CYPs examine d, 3A5, 3A4, and 2B6 exhibited the highest enzymatic activities with t urnovers ranging from 7.5 to 12.5 nmol of product formed/min/nmol for the total metabolism of DZ, while 2C8, 2C9, and 2C9(R144C) showed less er and moderate activities. 1A2 and 2E1 produced insignificant amounts of metabolites of DZ. The regioselectivity of CYPs was determined, an d 2B6 was found to catalyze exclusively and 2C8, 2C9, and 2C9(R144C) p referentially the N1-demethylation of DZ to form NDZ. 3A4 and 3A5 cata lyzed primarily the C3-hydroxylation of DZ, which was more extensive t han the N1-demethylation. The ratios of TMZ to NDZ farmed in the metab olism of DZ by 3A4 and 3A5 were approximately 4:1. Enzyme kinetic stud ies indicated that 2B6- and 2C9-catalyzed DZ metabolism followed Micha elis-Menten kinetics, whereas 3A4 and 3A5 displayed atypical and non-l inear curves in Lineweaver-Burk plots. Human liver microsomes converte d DZ to both TMZ and NDZ at a ratio of 2:1. Our results suggest that h epatic CYP3A, 2C, and 2B6 enzymes have an important role In the metabo lism of DZ by human liver. (C) 1998 Elsevier Science Inc.