Br J clin Pharmac 1994; 38: 13 1-137 Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms TOMMY ANDERSSON" 2, JOHN 0. MINERS', MAURICE E. VERONESE' & DONALD J. BIRKETT' 'Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia and 2Clinical Pharmacology, Astra Hassle AB, S-431 83 M6lndal, Sweden 1 The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temaze- pam and N-desmethyldiazepam. 2 The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3 a-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 gIM a-naphthoflavone the kinetics for both pathways were approximated by Michaelis- Menten kinetics. 4 Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase. Keywords diazepam human microsomes kinetics CYP isoforms Introduction Diazepam is widely used as a muscle relaxant, seda- forms, and the isoform responsible for the formation tive, anxiolytic or anticonvulsant. It has a low hepatic of N-desmethyldiazepam seems to be the same as clearance and the metabolites formed are pharmaco- that hydroxylating S-mephenytoin [9-11]. However, logically active. Its clearance varies substantially mephenytoin did not inhibit diazepam metabolism in between individuals, however, and is dependent on vitro [12, 13]. age, gender, concomitant medication and on genetic The in vitro kinetics of diazepam have not been variability [1-6]. There is a correlation between the investigated systematically and the specific CYP polymorphic hydroxylation of S-mephenytoin and isoforms responsible for the different metabolic trans- the metabolism of diazepam since slow metabolisers formations of the drug have not been identified of S-mephenytoin are also slow metabolisers of definitively. diazepam [4, 6]. Diazepam has also been seen to be a This report presents data on the formation kinetics potent inhibitor of S-mephenytoin hydroxylation in of the two major diazepam metabolites, temazepam vitro [7, 8]. Other in vitro studies report that the two and N-desmethyldiazepam, together with the effects major pathways for diazepam metabolism-the for- of chemical inhibitors and antibodies selective for mation of temazepam and N-desmethyldiazepam-are various CYP isoforms on these reactions. All experi- catalysed by different cytochrome P450 (CYP) iso- ments were performed in human liver microsomes. Correspondence: Dr Tommy Andersson, Clinical Pharmacology, Astra Hassle AB, S-431 83 Molndal, Sweden 131 132 T. Andersson et al. Methods started by addition of the NADPH generating system. After 10 min (formation of both metabolites was Chemicals shown to be linear up to 10 min), reactions were ter- minated by the addition of 4 ml dichloromethane Diazepam, temazepam, N-desmethyldiazepam, 4- and by cooling the samples on ice. Thereafter, 1 ml hydroxydiazepam and clonazepam (used as internal of carbonate buffer (1 M) was added to the mixture standard) were obtained from Roche Australia. followed by 150 ,ul of the internal standard solution Coumarin, diethyldithiocarbamate and troleando- (clonazepam, 2 mg 1-' methanol). Extraction was per- mycin were purchased from Sigma Chemical Co. formed on a vortex mixer for 1 min immediately (St Louis, MO). Other drugs were obtained from the followed by centrifugation for 5 min (1000 g). A 3.5 following sources: a-naphthoflavone from Aldrich ml aliquot of the organic phase was evaporated to Chemical Co. (Milwaukee, WI), sulphaphenazole dryness under nitrogen. Residues were reconstituted from Ciba-Geigy, Aust. (Sydney, Australia), R,S- in 150 ,ul of the mobile phase, and a 50 gl aliquot mephenytoin from Sandoz Ltd (Basel, Switzerland), was injected onto the h.p.l.c. column. Retention times quinidine from Burroughs Wellcome Aust. (Sydney, of clonazepam, temazepam, N-desmethyldiazepam Australia) and omeprazole from Astra Hassle AB and diazepam were 6 min, 9 min, 12 min and 14 (Molndal, Sweden). NADP+, glucose 6-phosphate and min, respectively. The retention time of 4-hydroxy- glucose 6-phosphate dehydrogenase were purchased diazepam was 7.5 min. Extraction efficiency was from Sigma Chemical Co. (St Louis, MO). All other essentially quantitative for temazepam (92%), N-des- reagents and solvents were of analytical grade. methyldiazepam (92%) and clonazepam (112%), with a coefficient of variation (CV) less than 5% in each Liver samples case. Within-day assay precision, tested on the same batch of microsomes (n = 6), was between 6 and 7% Human liver samples were obtained from renal trans- for both temazepam and N-desmethyldiazepam. Stan- plant donors and relevant details of the donors of the dard curves for the metabolites were constructed in livers used in the present study have been published the concentration range of interest, and linearity (r = elsewhere [14]. Liver samples were stored at -80° C 1.0) was obtained for both metabolites. Unknown until used. Microsomes were prepared by differential concentrations were determined by comparison of centrifugation as described previously [15], and micro- metabolite-to-internal standard peak-height ratio with somal protein concentration was measured according those of the calibration curve. to Lowry et al. [16] using crystalline bovine serum albumin as standard. Ethical approval was obtained to Kinetics of diazepam metabolism use the livers for drug metabolism studies. Nine different concentrations of diazepam, ranging Measurement of diazepam metabolites in human liver from 4 to 500 gM were used in the kinetic experi- microsomes ments; four livers (H5, H8, H9 and H1O) were studied. Since atypical Eadie-Hofstee plots were The assay of Hooper et al. [12] was used to measure obtained for both metabolites, the data could not be for temazepam and N-desmethyldiazepam with minor fitted to the Michaelis-Menten expression. Instead, a modifications. A Model LC 1100 solvent-delivery sigmoid Vmax model equivalent to the Hill equation system, a Model LC 1200 variable wavelength UV- was fitted to the data according to: VIS detector (ICI instruments, Melbourne, Australia), CN a Model 7125 Rheodyne injector and a Model SE 120 max BBC Goetz Metrawatt recorder were used. Absorbance V = was monitored at 236 nm. The column was Spherisorb C5N + CN C18 5 ,um (25 cm x 4.6 mm), ICI Instruments Pty Ltd (Victoria, Australia), operated at ambient tempera- where V is the velocity of the reaction at substrate ture. The mobile phase, delivered at a flow rate of 1.2 concentration C, Vmax is the maximal velocity, C50 is ml min-, comprised methanol (62.5%), 1 M H3PO4 the substrate concentration at half maximal velocity, (0.7%), and triethylamine (0.1%) in water. Reaction and N is a parameter describing the sigmoidicity of mixtures contained 0.5 mg human liver microsomal the curve. When N is 1, the expression reduces to protein (temazepam and N-desmethyldiazepam for- the usual Michaelis-Menten expression. The data mation was linear up to 1 mg of protein), diazepam were fitted by weighted least squares regression with (4-500 gM) and 0.25 ml NADPH generating system a weighting of l/y. containing 1 mM NADPH+, 10 mm glucose 6-phos- phate, 2 units glucose 6-phosphate dehydrogenase, Activation by ac-naphthoflavone and 5 mM MgCl2 in a final volume of 1.0 ml of 0.1 M KH2PO4 buffer (pH 7.4). Diazepam, temazepam, Optimal activation of the formation of temazepam N-desmethyldiazepam, 4-hydroxydiazepam and clon- and N-desmethyldiazepam was obtained at an cx- azepam were dissolved in methanol. The final con- naphthoflavone concentration of 25 gM (concentra- centration of methanol in the incubation mixture was tions of 1-100 gM were tested). In one of the livers 1% v/v; this concentration inhibited temazepam for- studied the kinetic experiment was, therefore, re- mation by 8% and N-desmethyldiazepam formation peated with 25 gM a-naphthoflavone present at each by 28%. Reactions were carried out at 37°C and were concentration of diazepam. Diazepam metabolism in human liver microsomes 133 Inhibition experiments further diazepam concentration (4 gM). Duplicate samples were used in all inhibition experiments. The effects of inhibitors or substrates (potential com- petitive inhibitors) selective for various CYP iso- Analysis of results forms on diazepam metabolic pathways were studied. The isoform selective inhibitors or alternative sub- All results are presented as individual data and/or strates used were ox-naphthoflavone (CYPIA in- mean ± s.d. hibition at low concentrations and normally CYP3A activation at higher concentrations) [14]; coumarin (CYP2A6) [17, 18]; sulphaphenazole (CYP2C9/10) [19]; R,S-mephenytoin (S-mephenytoin hydroxylase) Results [20]; quinidine (CYP2D6) [7, 21]; diethyldithiocarba-