Human liver microsomal diazepam metabolism using cDNA-expressed cytochrome P450s: Role of CYP2B6, 2C19 and the 3A subfamily

Abstract

1. We have examined the metabolism of diazepam by ten human cytochrome P450 forms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) expressed in HepG₂ cells using a recombinant vaccinia virus system.

2. Among the P450 forms tested, diazepam was significantly demethylated by CYP2B6, 2C9, 2C19, 3A4 and 3A5, with 2C19 exhibiting the highest rate at concentrations < 0.1 mM, and hydroxylated only by the latter three enzymes, with 3A5 being the most active. The N-demethylation activity of diazepam by 2C19 at a concentration of 20 μM was six times of that by 3A4. However, that by 2C9 was detected at only a trace level.

3. CYP2C19, 3A4 and 3A5 of the ten human P450s catalysed the 3-hydroxylation of nordiazepam, and 2B6, the 2C subfamily and the 3A subfamily catalysed the N-demethylation of temazspam. CYP3A4 exhibited the highest activity of nordiazepam 3-hydroxylation and temazepam N-demethylation.

4. Diazepam N-demethylation by human liver microsomes correlated with diazepam 3-hydroxylation, but not S-mephenytoin 4′-hydroxylation.

5. Our results suggest that in the human liver, the metabolism of diazepam to nordiazepam is mediated by CYP3A4, which has been reported as the most abundant P450 form in human liver as well as 2C19, which has been reported as a polymorphic enzyme.