Diazepam Metabolism by Human Liver Microsomes Is Mediated by Both S-Mephenytoin Hydroxylase and CYP3A Isoforms

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, temazepam 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-phosphate, 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]; diethyldithiocarbamate (CYP2E1) [22]; and troleandomycin (CYP3A) Following incubation with diazepam, temazepam [23]. Omeprazole was also tested since it is a sub- and N-desmethyldiazepam were the major metabolites strate for S-mephenytoin hydroxylase and CYP3A in all livers studied, while 4-hydroxydiazepam was [24, 25]. The putative inhibitors were studied at one barely detectable under normal incubation conditions. or two concentrations chosen to be selective for the However, with the addition of high concentrations of respective CYP isoforms on the basis of published a-naphthoflavone the size of the chromatographic Ki or Km values. Different potential solvents (DMSO, peak corresponding to 4-hydroxydiazepam increased acetone, propylene glycol, acetonitrile) for the in- substantially. The substrate conversion was low and hibitors were tested in two different livers with within the linear range so secondary metabolism to respect to their influence on diazepam metabolism. oxazepam was not a significant issue. Furthermore, in All solvents were studied at a concentration of 0.5% samples without the NADPH generating system and v/v. DMSO inhibited the formation of both tema- also in samples without substrate minor chromato- zepam and N-desmethyldiazepam by almost 50% graphic peaks with the same retention times as the precluding its use as a solvent for the inhibitors. Ace- two major metabolites, temazepam and N-desmethyl- tonitrile was judged to be the most suitable solvent diazepam, were observed. The origin of these small and inhibited the formation of temazepam by only peaks was not clear, but they seemed to arise from 6.5% and that of N-desmethyldiazepam by less than the microsomal preparation. The peaks were similar 4%. Except for quinidine and omeprazole, which in size independent of substrate concentration and were dissolved in water and water/methanol (< 0.2% were less than 10% of total peak height, except from v/v methanol), respectively, inhibitors were dissolved some samples at the lowest substrate concentration. in acetonitrile resulting in a final concentration in the Since blank samples were included in the experi- incubation of 0.5% v/v. In all cases, inhibited activi- ments the data were corrected for the 'artefactual' ties were compared with activities in control incuba- peaks in all experiments. tions containing 5 gl (0.5% v/v) acetonitrile or 5 gl water as appropriate. Troleandomycin was preincu- Kinetic experiments bated with microsomes and the NADPH generating system for 10 min at 370 C before the reaction was Substrate vs velocity plots for formation of tema- started by addition of the substrate, diazepam, since a zepam and N-desmethyldiazepam by microsomes metabolite formed during troleandomycin incubation from liver H8 in the absence and presence of a-naph- exerts the inhibitory effect on CYP3A enzymes [23]. thoflavone are shown in Figure 1, and the correspond- Otherwise, the incubations were started by adding the ing Eadie-Hofstee plots in Figure 2. The kinetics of NADPH generating system. formation of both metabolites were sigmoidal (Figure Immunoinhibition experiments with an anti-rabbit 1) and this was more marked in the case of tema- CYP2C3 antibody [26] were carried out by preincu- zepam formation. This resulted in atypical Eadie- bating antibody/antisera with microsomes at room Hofstee plots (Figure 2) which did not allow kinetic temperature for 15 min, before starting reactions by parameters to be derived in the usual way. Instead the the addition of diazepam and the NADPH generating data were fitted by a sigmoid Vmax function equiva- system. The total amount of protein (antibody) added lent to the Hill equation. This resulted in an excellent to each incubation was kept constant by the addition fit to the data (Figure 1) and allowed parameters of preimmune antibody. The proportion of antibody describing the data (C50, Vmax and N) to be derived. to microsomal protein content in reaction mixtures These are shown in Table 1. The C50 values for for- was 15:1. mation of the two metabolites were similar but the None of the inhibitors gave rise to any chromato- value of N was significantly greater for temazepam graphic peaks interfering with those of temazepam formation than for N-desmethyldiazepam formation and N-desmethyldiazepam. (P < 0.01). Vmax for temazepam formation was almost All inhibitors, including the antibody, were studied five-fold greater than that for N-desmethyldiazepam in two livers (H8 and H9) and at two different formation (P < 0.001). The sigmoidal nature of the diazepam concentrations (15 and 200 gM). A few kinetics was consistent with a form of substrate acti- inhibitors (including the antibody), judged to be the vation. As a-napthoflavone also activated both re- most relevant ones for the metabolism of diazepam, actions (see below) the kinetic study for one of the were studied in one further liver (H5) and at one livers was repeated in the presence of a concentration 134 T. Andersson et al.

a 4 . .

3~ . .

E 2. E 0

C: 1 . 0

E 0 -E c U 0 ni - - | - s a - - 0 0 0.005 0.01 0.015 0.02

0.6rb

a 400 0 0.4# Diazepam concentration (pM) a . Figure 1 Substrate vs velocity curves for the formation of temazepam (a) and N-desmethyldiazepam (b) from a 0 diazepam incubated with human liver microsomes from 0.2 U liver H8. - * - without ax-naphthoflavone; - * - in the presence of 25 tM a-naphthoflavone. The lines are the curves of best fit to the sigmoid Vmax model. U S

- .0 on - 0 0.002 0.004 0.006 of a-naphthoflavone that caused maximal activation (Figures 1 and 2). In the case of temazepam for- V/S mation, oc-napthoflavone markedly reduced the sig- Figure 2 Eadie-Hofstee plots for the formation of moidicity of the kinetics (Figure 1) with N being temazepam (a) and N-desmethyldiazepam (b) from reduced from 1.5 in the absence of activator to 1.1 diazepam incubated with human liver microsomes from with oc-napthoflavone present. There was no change liver H8. - * - without x-naphthoflavone; - * - in the in C50 (236 gM with and without a-napthoflavone) presence of 25 gM a-naphthoflavone. and a modest increase (41%) in the Vmax from 4.1 to 5.8 nmol mg- min-' (Table 1). In the presence of x- with or without oc-naphthoflavone) (Figures 1 and 2, naphthoflavone temazepam formation kinetics were Table 1). approximated by Michaelis-Menten kinetics as shown by the Eadie-Hofstee plot (Figure 2a); the N value Inhibition experiments approached 1. With regard to N-desmethyldiazepam formation, addition of ax-naphthoflavone resulted in a Inhibitors selective for CYP2A6 (coumarin 25 glM), decrease in C50 (from 250 to 119 gM) with little CYP2C9/10 (sulphaphenazole 25 giM), CYP2D6 (quini- change in Vmax (0.7 nmol mg-1 min-1 with and 0.8 dine 10 ,UM) or CYP2E1 (diethyldithiocarbamate 50 nmol mg- min-m without a-napthoflavone) or N (1.1 gIM) had little effect (< 15% change) on either path-

Table 1 Computer derived values of C50, Vmax, and N for the formation of temazepam and N-desmethyldiazepam in four livers according to a sigmoid Vmax model (see Methods)

Temazepam N-desmethyldiazepam Liver C50 Vmax C50 Vrnax number (pM) (nmol mg-I min-1) N (pM) (nmol mg-I min-1) N H5 188 14.4 1.4 190 3.0 1.2 H8 236 4.1 1.5 250 0.8 1.1 H9 164 3.6 1.6 144 0.6 1.2 H10 168 5.1 1.5 151 1.1 1.2 Mean 188 6.8* 1.5** 184 1.4* 1.2** s.d. 33 5.1 0.1 49 1.1 0.1 *Comparison of Vmax values; P < 0.001. **Comparison of N values, P < 0.01. Diazepam metabolism in human liver microsomes 135 way of diazepam metabolism. This was the case at tially by the anti-CYP2C3 antibody, and to a minor low (15 gM) and high (200 gM) diazepam concentra- extent by mephenytoin (S-mephenytoin hydroxylase) tions. ax-Naphthoflavone at a concentration of 2 gM, (Figure 3b). Consistent with this, omeprazole (100 gM) which inhibits CYP1A2, caused activation of the which is a substrate for both CYP3A and S-mepheny- formation of both temazepam and N-desmethyl- toin hydroxylase [25] markedly inhibited N-des- diazepam. This activation was more marked at higher methyldiazepam formation (Figure 3b). a-naphthoflavone concentrations and depended also on the substrate (diazepam) concentration. The formation of temazepam in the presence of 10 gM cx- naphthoflavone was activated by 201%, 105% and Discussion 14% at diazepam concentrations of 4, 15 and 200 gM, respectively. The corresponding extents of activation A metabolic scheme for diazepam is proposed in of N-desmethyldiazepam formation were 96%, 55% Figure 4. The Vmax for formation of temazepam was and 25%, respectively. almost 5-fold greater than that for N-desmethyl- The effects of other inhibitors and the inhibitory diazepam. However, the activity ratio decreased with anti-CYP2C antibody on the formation rates of the decreasing diazepam concentration because of the two metabolites are shown in Figure 3. Because of more marked sigmoidicity of the formation kinetics the atypical kinetics of diazepam, inhibitors were of temazepam. At the lowest diazepam concentration tested at three substrate concentrations (4 gM, 15 gM used (4 gM), the rates of formation of the two and 200 gM diazepam). At all diazepam concentra- metabolites were approximately the same (data not tions, temazepam formation was almost totally inhib- shown). This is consistent with in vivo studies in- ited by the CYP3A inhibitor troleandomycin (Figure dicating that formation of N-desmethyldiazepam 3a). The other inhibitors, including the CYP2C3 accounts for 50 to 60% of total diazepam clearance antibody, had only minor inhibitory effects, and [4, 27-29]. Clearly in vitro studies with high dia- mephenytoin caused some activation at the lowest zepam concentrations cannot be used to predict rela- diazepam concentration. tive in vivo partial clearances because of the complex N-desmethyldiazepam formation was inhibited par- in vitro kinetics of both metabolic pathways. A simi- tially by the CYP3A inhibitor (troleandomycin), par- lar conclusion was reached by Bertilsson et al. [30]. The other potential metabolite, 4-hydroxydiazepam, a Temazepam formation (n=3; mean and s.d.) was not detected under normal incubation conditions. However, in the presence of higher concentrations of Omeprazole 25 pM ax-naphthoflavone a peak corresponding to 4-hydroxydiazepam was apparent. This is consistent with for- Omeprazole IIl 100 PM Mephenytoin 1 000 PM CH3 O CYP2C3-AntibodyI 15:1 ClAN ci N Troleandomycin 10 PM . -110(0 -75 -50 -25 0 25 50 75 100 Diazepam N-desmethyidiazepam formation /PA (CYP3A?) S-CYP3A b ((n=3; mean and s.d.) CYP3A/ . CpA -MPH

Omeprazole , , CH3. O H 25 pM Omeprazole f 100 PM ci N cis d Mephenytoin 1000 PM I=- I-H Temazepam OH N-desmethyldiazepam CYP2C3-Antibody 15:1 p-hydroxydiazepam Troleandomycin 10 PM -100 -75 -50 -25 0 25 50 75 1100 % Inhibition % Activation Figure 3 Effects of omeprazole, mephenytoin, troleandomycin and anti-CYP2C3 antibody on formation of temazepam (a) and N-desmethyldiazepam (b) from diazepam incubated with human liver microsomes. The Oxazepam data are the mean and standard deviation of results with Figure 4 Proposed scheme for diazepam metabolism microsomes from livers H5, H8 and H9. Inhibitory effects in humans showing the CYP isoforms involved in are shown at three diazepam concentrations (4 O, 15 Rxi, and N-demethylation to N-desmethyldiazepam and C3- 200 gM *). hydroxylation to temazepam. 136 T. Andersson et al.

mation of this minor metabolite being mediated by diazepam) of the other. Our data were well fitted by CYP3A, which is activated by cx-naphthoflavone. the sigmoid Vmax expression, a version of the Hill The inhibitor results presented here afforded strong equation in which. C50 is used instead of a substrate evidence that formation of temazepam is a CYP3A binding constant.. The observation that activation by mediated reaction. This is supported by the almost ax-naphthoflavone was much greater at low diazepam complete inhibition of formation of this metabolite by concentrations, suggested that activation by x-naph- troleandomycin, particularly at low diazepam concen- thoflavone and diazepam were not additive. This was trations, and by the marked activation seen with oc- supported by the kinetic study of temazepam for- naphthoflavone. The anti-CYP2C inhibitory antibody mation in the presence of a maximally activating had only a minor effect as did inhibitors selective for concentration of a-naphthoflavone. Under these cir- CYP 1 A2 (ax-naphthoflavone), CYP2A6 (coumarin), cumstances the kinetics approximated to the classical CYP2C9/10 (sulphaphenazole), CYP2D6 (quinidine), Michaelis-Menten function, indicating that substrate CYP2E1 (diethyldithiocarbamate) and S-mephenytoin activation by diazepam did not occur in the presence hydroxylase (mephenytoin). of a-naphthoflavone. Schwab et al. [32] showed simi- N-desmethyldiazepam formation was inhibited par- larly that the kinetics of progesterone 6f8-hydroxyla- tially by troleandomycin to the extent of 60% at the tion were 'linearised' by addition of a-napthoflavone lowest diazepam concentration used (4 rM). At this with both human and rabbit liver microsomes. The diazepam concentration, the CYP2C3 antibody also data are consistent with a-naphthoflavone and dia- caused substantial (about 40%) inhibition and zepam binding to an allosteric site distinct from the mephenytoin also caused some degree of inhibition. active site and causing a conformational change The marked inhibitory effect of omeprazole is consis- which results in activation of the reaction. tent with this, as omeprazole is a substrate for both The effects of putative inhibitors of N-desmethyl- CYP3A and S-mephenytoin hydroxylase isoforms [25]. diazepam formation suggested that at least two CYP Activation by a-naphthoflavone was weaker than for isoforms (CYP3A and S-mephenytoin hydroxylase) temazepam formation, which is consistent with N- are involved in this reaction, making the kinetics desmethyldiazepam formation being only partly complex and difficult to interpret. The atypical kinet- CYP3A mediated. Selective inhibitors of CYP1A2, ics observed for this reaction were not as marked as CYP2A6, CYP2C9/10, CYP2D6 and CYP2E1 had for temazepam formation, which is consistent with little or no effect. This is in agreement with in vivo CYP3A having only a partial involvement. As with studies indicating that the clearance of diazepam temazepam formation, the atypical kinetics for N- is impaired in poor metabolisers of S-mephenytoin desmethyldiazepam formation were 'linearised' sub- [4-6]. Bertilsson et al. [43] further showed that clear- stantially by addition of a-naphthoflavone, indicating ance to N-desmethyldiazepam is lower in poor meta- a similar activation mechanism for this reaction. bolisers of S-mephenytoin, but there was substantial In summary, our data indicate that temazepam for- (43%) residual clearance by this route in the poor mation from diazepam is mediated predominantly by metabolisers. This is consistent with the present in CYP3A whereas N-desmethyldiazepam formation is vitro inhibition data which indicate that about half of mediated partly by this isoform(s) and partly by the the diazepam N-demethylation activity in human liver S-mephenytoin hydroxylase. The atypical kinetics of microsomes is mediated by S-mephenytoin hydroxy- the CYP3A mediated formation of temazepam have lase and half is CYP3A mediated. been fitted by an expression (the Hill equation) for The atypical formation kinetics for temazepam reactions exhibiting positive cooperativity, in this have been reported previously [12, 30]. Similar atypi- case substrate activation. The non-additive effects of cal kinetics have been reported for other CYP3A the substrate (diazepam) and a-naphthoflavone sug- mediated reactions including caffeine 8-oxidation gest that both compounds act by a similar mechanism [31] and progesterone 6f-hydroxylation [32]. The involving binding at a site distinct from the cyto- sigmoid nature of the velocity vs substrate curves chrome P450 active site. suggested that a form of substrate activation was occurring. This is also consistent with the report by This work was supported in part by funding from the Hooper et al. [12] that temazepam and N-desmethyl- National Health and Medical Research Council of diazepam could each activate the formation (from Australia.

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