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 microM 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 temazepam. 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.

Functional Characterization of Cytochrome P450 2B6 Allelic Variants

“…This P450 isoform is expressed in liver and many extrahepatic tissues including kidney, intestine, and lung (Gonzalez et al, 1992;Gervot et al, 1999). CYP2B6 plays important roles in the metabolism of a number of therapeutic drugs such as the antineoplastic agent cyclophosphamide, antiestrogen tamoxifen, anticonvulsant S-mephenytoin, and benzodiazepine diazepam (Chang et al, 1993;White et al, 1995;Heyn et al, 1996;Ono et al, 1996). In addition, CYP2B6 has been implicated in the metabolism of procarcinogens including aflatoxin B1, 6-aminochrysene, and 7,12-dimethylbenz[a]anthracene (Aoyama et al, 1990;Mimura et al, 1993;Shou et al, 1996).…”

Microsomal Protein Concentration Modifies the Apparent Inhibitory Potency of CYP3A Inhibitors

“…Microsomes were prepared by ultracentrifugation; microsomal pellets were suspended in 0.1 M potassium phosphate buffer containing 20% glycerol and stored at Ϫ80°C until use. Chemical reagents and drug entities were purchased from commercial sources or kindly provided by their pharmaceutical manufacturers (von Moltke et al, 1993(von Moltke et al, , 1994a(von Moltke et al, ,b, 1996aVenkatakrishnan et al, 2000a Venkatakrishnan et al, ,b,c, 2001a.Diazepam 3-hydroxylation to form temazepam was used as the index reaction for CYP3A activity (Andersson et al, 1994;Ono et al, 1996;Jung et al, 1997; Venkatakrishnan et al, 2000). Methanolic solutions of diazepam (100 M) were added to 2-ml microcentrifuge tubes, and the solvent was evaporated to dryness under conditions of mild vacuum at 40°C.…”

Metabolic Activation of Benzodiazepines by CYP3A4

“…The major metabolic pathways of diazepam are 3-hydroxylayion by CYP3A4 and N-desmethylation by CYP2C9 (Schwartz et al, 1965;Ono et al, 1996). Thus, desmethyldiazepam, temazepam, and oxazepam are metabolites of diazepam (Fig.…”