Xenobiotica the fate of foreign compounds in biological systems

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Diazepam metabolism by rat and human liver in vitro: inhibition by mephenytoin T. V. Beischlag, W. Kalow, W. A. Mahon & T. Inaba To cite this article: T. V. Beischlag, W. Kalow, W. A. Mahon & T. Inaba (1992) Diazepam metabolism by rat and human liver in vitro: inhibition by mephenytoin, Xenobiotica, 22:5, 559-567 To link to this article: http://dx.doi.org/10.3109/00498259209053119

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Date: 27 January 2016, At: 06:49

XENOBIOTICA,

1992,

VOL.

22,

NO.

5, 559-567

Diazepam metabolism by rat and human liver in vitro: inhibition by mephenytoin T. V. BEISCHLAG, W. KALOW, W. A. MAHON and T. INABA

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Department of Pharmacology, University of Toronto, Canada Received 3 July 1991:accepted 3 January 1992

1. Diazepam metabolism and its association with mephenytoin hydroxylase were studied in witro using human and rat livers. 2. Enzyme kinetic parameters were obtained for the formation of p-hydroxydiazepam @-hydroxy-DZP), N-desmethyldiazepam (NDZ), and temazepam (TMZ) from diazepam (DZP) in rat liver fractions. T h e K,,, values for formation in rat of p-hydroxy-DZP, N D Z and T M Z were 14+ 3 ( S E M ) ~ M44+4 , and 63 8, respectively; clearance values calculated from Vmsx/K,,, were 5.7, 3.2 and 4.9 ml/g per min, respectively. 3. Mephenytoin (MP) competitively inhibited, in rat liver, the formation of NDZ, but not the formation of p-hydroxy-DZP or T M Z ; in human liver neither N D Z nor T M Z formation was inhibited by MP. 4. In seven different human livers the formation of p-hydroxy-DZP represented a minor pathway compared to the formation of N D Z and T M Z .

+

Introduction Diazepam (DZP, Valium@)is widely used as an anxiolytic, muscle relaxant and sedative. Large interindividual variability of DZP metabolism was noted in its plasma concentration and pharmacokinetic parameters (Guentert 1984). Studies in vitro have shown that N-demethylation and C3-hydroxylation of D Z P vary over a several-fold range between individuals (Inaba et al. 1988). Nevertheless, while DZP is one of the most commonly prescribed drugs, very little has been done to characterize the individual variability of metabolic pathways involved in its biotransformation. There is evidence to suggest a possible association between diazepam metabolism and the S-mephenytoin hydroxylase polymorphism (Bertilsson et al. 1989). A panel study involving poor and extensive metabolizers (PMs and EMS) of mephenytoin revealed that PMs had significantly lower plasma half-lives than EMS and decreased plasma clearances of DZP and N-desmethyldiazepam (NDZ) after dosing with each. DZP and N D Z have also been shown to competitively inhibit the formation of p-hydroxymephenytoin (the major metabolite of S-mephenytoin) in human liver samples in vitro (Inaba et al. 1985). However, mephenytoin did not inhibit either the N-demethylation or the C3-hydroxylation of DZP in human liver preparations (Tait 1987). If both DZP and N D Z can inhibit mephenytoin hydroxylation and both show altered pharmacokinetics in PMs of mephenytoin, then it would seem reasonable that a metabolic pathway which both DZP and N D Z share may be the pathway affected, namely para-hydroxylation or C3-hydroxylation. It is also worthwhile noting that the para-hydroxylation of mephenytoin chemically resembles that of DZPpara-hydroxylation. Therefore, it is worth investigating whether mephenytoin competes with or specifically inhibits the formation of para-hydroxydiazepam (phydroxy-DZP). 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd.

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To study the possible relationship between the mephenytoin hydroxylation polymorphism and DZP metabolism, in vivo and in vitro studies are required in humans. Much of this work must initially focus on the development of methods by which this phenomenon can be elucidated. At the present stage of development of these methods an animal model has many advantages. T h e rat is an obvious choice; the majority of previous investigations, primarily in vivo studies, have been performed in rat and it has been shown that three different metabolic pathways (figure 1) are involved in the biotransformation of DZP in this species (Schwartz et al. 1967, Reilly et al. 1990). While para-hydroxylation is known to be the predominant metabolic pathway of DZP metabolism in rat, the relative importance of each pathway in humans is not known. The N-demethylation, C3-hydroxylation and para-hydroxylation pathways of DZP metabolism were studied in vitro in rat to characterize each metabolic reaction. Studies in vitro using human liver samples were performed to assess the relative contribution in humans of the para-hydroxylation pathway of DZP metabolism as compared to N-demethylation and C,-hydroxylation. Inhibition by mephenytoin was also examined, to investigate the possible link between diazepam metabolic pathways and mephenytoin hydroxylation polymorphism.

Materials and methods Drugs and chemicals Diazepam and N-desmethyldiazepam were obtained from Hoffman-LaRoche Inc. (Nutley, NJ, USA); temazepam (3-hydroxydiazepam) from Sandoz Canada Inc. (Dorval, Quebec, Canada), parahydroxydiazepam from Dr R. H. Waring (Dept of Biochemistry, University of Birmingham, UK). Glucose 6-phosphate dehydrogenase, NADP’ and glucose 6-phosphate were obtained from Sigma Chemical Co (St Louis, MO, USA). All other chemicals were of analytical reagent grade and were obtained from either Fisher Scientific Co. (Fair Lawn, NJ, USA), BDH Chemicals Ltd. (Toronto, Ontario, Canada) or Caledon Laboratories Ltd. (Georgetown, Ontario, Canada). Rat liver experiments Three male Long-Evans hooded rats (350g, Charles River Canada Inc., Quebec, Canada) were killed by decapitation. Their livers were immediately removed and placed in ice-cold 1.15% KCI solution. The

NDZ I ___* U

p-OliDZI’

0

0

I

Figure 1. Postulated pathways of diazepam oxidation in humans and rats. I =N-demethylation; I1 = C,-hydroxylation; 111 =para-hydroxylation. DZP= diazepam, NDZ = N-desmethyldiazepam, TMZ = temazepam = C,-hydroxydiazepam, p-OHDZP = p hydroxydiazepam.

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supernatant fraction (S9) from a 9000g centrifugation was prepared according to the method of Inaba et al. (1988). Kinetic studies were performed using the S9 fraction. Incubations were carried out as previously described (Inaba et al. 1988). Briefly, each incubation mixture contained: 0.2 ml liver S9, 0.6 ml of phosphate buffer ( 0 2 M, pH 7.4) and 0 2 ml of a NADPHgenerating system ( 0 . 4 m ~NADPH', 4mM glucose 6-phosphate, 0.4 units of glucose 6-phosphate dehydrogenase, 2 mM MgCl in phosphate buffer). The mixture in 1 ml total volume contained approx. 3 mg of protein. The concentrations of DZP ranged from 4 to 800 p ~DZP ; was dissolved in 10 p1 acetone. Samples were incubated for 15 min at 37°C. The reaction was stopped by removing each tube from the bath, immersing in an ice bath and adding 2 ml diethyl ether-isopropanol(7 : 3 v/v). At this point 0 5 pg of the internal standard flurazepam (in 2 0 4 methanol) was added to each tube. Each tube was vortexed and centrifuged before the organic layer was transferred to a 3 ml tube and then dried under nitrogen. Before g.1.c. analysis, the residue was dissolved in acetone (50-1OOpl) and a 1-2pl aliquot was analysed. Gas chromatographic analysis Analysis was carried out with a Shimadzu GC-9A gas chromatograph equipped with an electron capture detector (63Ni,ECD). A DB-17 30m x 0.54mm Megabore column (J.&W.Scientific) was used. Oven temp. was 245°C and the injector port and detector temp. were maintained at 300°C. Carrier gas (nitrogen) flow was kept at 20 ml/min (inlet pressure was 1.80 kg/cm*).The development of this method is is discussed elsewhere (Beischlag and Inaba 1992). Standard curves were constructed for each experiment using 0.5-5.0pg/ml incubation mixture of pure reference standards, NDZ, T M Z and p-hydroxy-DZP. Enzyme kinetics in rat liver tissue The timeactivity curves for the generation of NDZ, T M Z and p-hydroxy-DZP by the rat liver S9 fraction were determined at 40 p~ and 4 0 0 p DZP. ~ At the lower substrate concentration the dependence of metabolite formation with time is linear up to 20 min for all three metabolites monitored. At the higher substrate concentration this relationship is linear past 30min. The formation of NDZ, T M Z and p-hydroxy-DZP as a function of protein concentration was measured with DZP concentrations of 40 and 4 0 0 p ~ incubating , samples for 15 min. At 4 0 p ~rates , of formation for all three metabolites are linear with respect to protein concentration up to 5 mg proteinlml incubation mixture and at 4 0 0 p ~up to 7.5 mg proteinlml incubation mixture (Beischlag 1990). Rat liver S 9 preparations with an NADPH-generating system were preincubated for 10min with varying concentrations of the cytochrome P-450 inhibitor SKF-525A (&SO0 p~ final concentration, dissolved in 1.15%KCI). The substrate (DZP 4 0 ~ was ~ added, ) and this mixture was incubated for a further 15 min. Extraction and analysis were performed as described above. SKF-525A caused a concentration-dependent decrease in the enzyme activity responsible for the production of p-hydroxyDZP with the IC,, value of 20pM. The ability of mephenytoin to inhibit the formation of NDZ, T M Z and p-hydroxy-DZP was determined by measuring the velocity of production of these metabolites from DZP ( 8 N 0 0 p ~in) rat liver S9 fraction in the presence of either 0,80,200 or 4 0 0 p mephenytoin ~ (dissolved in 20 pl acetone). All other incubation conditions were the same as described above. Human liver experiments All seven human livers were from kidney donors; details of donors (K18, K21, K22, K23, K24, K25 and K28) have been previously described (Campbell et al. 1987). The livers, stored at -70°C in cubes, were thawed to prepare the S9 fraction as described for rat liver. Incubation conditions and extraction of metabolites were the same as described above for the rat liver with slight modifications. The mephenytoin inhibition study was carried out as for rat liver, but no inhibition of the formation of NDZ and T M Z was observed and the inhibitory effect on@-hydroxy-DZP could not be reliably determined in human tissue. The formation by human cytochrome P-450 of NDZ, T M Z and p-hydroxy-DZP from DZP was monitored at two different substrate concentrations (80 and 200 p ~ in) seven different human livers. This was done in order to assess the relative importance of the three different metabolic pathways of DZP in humans. Treatment of data Estimation of Michaelis-Menten parameters was achieved with the aid of the graphical technique of Hofstee (1952). By using the method of Dixon and Webb (1964), in which 1 / Vwas plotted against [ I ] for two or more concentrations of substrate, Ki values were estimated. Because this method cannot distinguish competitive from mixed-type inhibitions, the method of Cornish-Bowden (1974) was also used to evaluate the type of inhibition.

Results Figure 2 shows typical gas-chromatograms of DZP, NDZ, T M Z and ~ in rat liver S9 fraction and after p-hydroxy-DZP after incubation of 1 O p DZP

T. V. Beischlag et al.

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TIME (min)

Figure 2 .

4 ! o

TIMI ( m i 4

Characteristic gas chromatograms of extracted diazepam (DZP) and its metabolites after incubation in rat and human liver S9 fractions.

Incubations were for 15min, using 1 O p ~ DZP in rat S9 preparation, and 200pM in human s9 preparation.

2 0 0 p ~DZP in human liver S9 fraction. The conversion for all three metabolites never exceeded 16% of total substrate at the lowest concentration ( 4 p in ~ rat). The kinetic studies of the formation of NDZ, T M Z and p-hydroxy-DZP were performed in the S9 fraction from pooled rat livers. Figure 3 shows representative Eadie-Hofstee plots; linearity for each metabolite indicates a single binding site. values for DZP metabolism Table 1 lists the K,, V,,, and clearance (Vmax/Km) ) to indicating that para-hydroxylation has the highest affinity ( K , = 14 p ~compared and 63 PM, respectively, yet the N D Z and T M Z formation, with K , values of 44 lowest maximum velocity of formation. The clearance value for p-hydroxy-DZP, however, is the highest among the three metabolic pathways. In human liver, the DZP conversion to N D Z and T M Z was not inhibited by mephenytoin, confirming our previous data (Tait 1987). However, the formation of N D Z in rat liver was inhibited by mephenytoin (figure 4 C) with a K ivalue of 400 pM. T h e parallelism in the Cornish-Bowden plot (figure 4 d) indicates competitive

563

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A

.04

.02

0

.06

I.

8

VJS Figure 3.

Eadie-Hofstee plots for the formation of N-desmethyldiazepam (NDZ), temazepam (TMZ) and p-hydroxydiazepam @-hydroxy-DZP) in rat liver.

Protein concentration was 3.5 mg/ml, incubation was for 15 min and substrate concentration of diazepam (DZP) ranged from 4 to 800pM. N D Z (m), T M Z ( 0 )and p-hydroxy-DZP (A).

Table 1. Michaelis-Menten parameters for the paro-hydroxylation, N-demethylation and C,hydroxylation of diazepam (DZP) in rat liver S9 fraction. ~~

~

Metabolite p-hydroxy-DZP NDZ TMZ

~

~~

~

~

(PM)

(pmol/mg protein per 15 min)

VmaX/K,,, (clearance) (ml/g per min)

14k2.7 44+43 63 If: 8.0

1.19f0.11 2.1 1f0.41 4.61 k087

5.7 3.2 4.9

K m

Vmax

Values are derived from the means of seven experiments, fSEM.

inhibition. Figures 4a and 4 b show Dixon plots for the formation of p-hydroxyDZP and T M Z , respectively and there was no indication of inhibition by mephenytoin. Due to the relatively slow production of p-hydroxy-DZP in human livers compared to the other pathways, the kinetic constants could not be reliably obtained. After incubating with 200pM DZP, the average velocities of formation of NDZ, T M Z andp-hydroxy-DZP in seven different liver preparations were 11.0 5.4 (SD), 2 3 * 4 + 18.5 and 1.3 f1.2 pmol/mg protein per min, respectively. Figure 5 shows individual velocities of formation of these three metabolites in different livers. I n six of the seven livers tested, T M Z was the major metabolite formed, while in one liver (K2.5) N D Z accounted for the major metabolite. For 8 0 p DZP ~ similar variation was seen in the metabolic capacities to form NDZ, T M Z , and p-hydroxy-DZP. Liver K18 was the only tissue tested in which para-hydroxylation of DZP accounted for a significant portion of its elimination (about 15%). In the remaining six livers, para-hydroxylation accounted for less than 7% of the metabolites formed.

Discussion This study describes the measurement, by g.l.c., of the three different metabolites of DZP formed in rat and human liver S9 preparations. Also presented are

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Figure 4. (a-c) Dixon plots for the effect of mephenytoin on p-hydroxydiazepam (p-hydroxy-DZP), ternazepam (TMZ) and N-desrnethyldiazeparn(NDZ) formation in rat liver S9 preparation; ( d ) Cornish-Bowden plot of mephenytoin inhibition of NDZ formation. Velocity is expressed as pmol/mg protein per 15 min. DZP concentrations used were 40, 80 and 200 PM. Mephenytoin concentrationswere 0,40,80 and 200 PM. Each point represents the mean of four experiments.

experiments to characterize the kinetic parameters of each reaction involved, and the effect of mephenytoin on the formation of NDZ, T M Z and p-hydroxy-DZP. Ackermann and Richter (1977) reported that the formation of N D Z and T M Z from DZP was catalysed by the cytochrome P-450 in rat liver microsomes. The formation of these metabolites has been observed in male Wistar rat liver preparations (Nau and Liddiard 1980). T h e inhibitory effect of SKF-525A on phydroxy-DZP formation in our rat liver studies indicates that this reaction is also catalysed by cytochrome P-450. T h e K , data (table 1) are in accord with in wiwo studies performed by other investigators to determine the metabolic fate of diazepam in rat (Andrews and Griffiths 1984, Trennery and Waring 1985, Chenery et al. 1987). It has also been proposed that more than one enzyme is responsible for the formation of T M Z (Bertilsson et al. 1989, 1990). Several investigators have suggested, after analysis of urine samples, that p-hydroxy-DZP was the major product of DZP biotransformation in rat while N D Z and T M Z were somewhat less important (Andrews and Griffiths 1984, Trennery and Waring 1985, Chenery et al. 1987). Earlier, Schwartz et al. (1967) reported a

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Diazepam metabolism in rat and man

Kl8

KZ1

KZZ

KZ3

KZ4

K25

K28

Figure 5.

Comparison of velocities of formation of N-desmethyldiazepam (NDZ), temazepam (TMZ) and p-hydroxydiazepam @-hydroxy-DZP) in seven different human livers. Substrate concentration of diazepam (DZP) was 2 0 0 ~N~D.Z = 0 ; TMZ= I; p-hydroxy-DZP = Q.

similar profile on examining the intestinal contents of rat after D Z P administration, and Inaba et al. (1974) also supported this after analysis of bile samples. While thephydroxy-DZP pathway has the lowest V,,, of the three reactions studied, it also has the highest affinity. At the concentrations of D Z P in vivo, which are much lower than the K , values, the production of p-hydroxy-DZP is favoured over N D Z and T M Z . p-Hydroxydiazepam and T M Z form glucuronide conjugates, and the conjugates are readily excreted in the urine while non-polar N D Z persists in plasma. Mutual inhibition tests were conducted. If mephenytoin strongly and competitively inhibits the formation of one or more of the DZP metabolites measured, it may be the substrate for the cytochrome P-450 whose activity it affects. As previously mentioned, since both D Z P and N D Z competitively inhibited human mephenytoin metabolism (Inaba et al. 1988), they might be potential substrates of mephenytoin hydroxylase, and the metabolic pathway affected would probably be common to the biotransformation of both D Z P and NDZ. In rat, the only major pathways both compounds share are para-hydroxylation and C,-hydroxylation. However these two pathways were found not to be inhibited by mephenytoin (figures 4 a and 4 b). T h e lack of inhibition of mephenytoin on the formation of p-hydroxy-DZP indicates that this pathway is probably not catalysed by mephenytoin hydroxylase, at least in rat. Recently, a member of the P450IIIA family has been proposed as the enzyme responsible for C3-hydroxylation in rat (Reilly et al. 1990), making it unlikely that this pathway is linked to the S-mephenytoin P-450 polymorphism (P450I I C family). Unexpectedly, the N-demethylation pathway showed competitive inhibition by mephenytoin in rat liver experiments (Ki= 400 /AM, figures 4 c and 4 d). This is distinctly different from previous findings using human liver tissue, in which

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6 0 - 4 8 0 p ~mephenytoin failed to inhibit the in oitro formation of N D Z from DZP (Tait 1987). Shimada et al. (1986) also reported that human mephenytoin phydroxylase and diazepam N-demethylase are different cytochrome P-450 enzymes. The relative velocities of formation of NDZ, T M Z and p-hydroxy-DZP from diazepam indicate that NDZ and T M Z are major metabolites while p-hydroxy-DZP is relatively minor (usually less than 7%) in humans, (figure 5). Even if the parahydroxylation reaction is mediated by the same polymorphic enzyme that controls S-mephenytoin hydroxylation, then it would be unlikely to be responsible for the considerable variation of DZP elimination seen between different subjects, since it represents such a small proportion of DZP metabolism. Although these pathways were studied at only two different substrate concentrations (80 and ~ O O ~ M they ), were well above the K , observed in rat for the formation of p-hydroxy-DZP and within the range used for the characterization of the N-demethylation and C,-hydroxylation pathways previously investigated in humans (Inaba et al. 1988). One cannot make inferences as to the mean K , of this enzyme in human liver, or the range over which it varies, but it does seem to have a very low capacity compared with the enzymes responsible for the formation of N D Z and T M Z . Previous studies on the metabolites of DZP in human samples indicated that para-hydroxylation may have been overlooked as a metabolic route (Sisenwine et al. 1972, Mahon et al. 1976). However, this study confirms the recent work of a British group that demonstrated para-hydroxylation of D Z P was minor in humans (Seddon et al. 1989); any para-hydroxylated metabolites of D Z P were below the detection limit of their assay methods. T h e present study demonstrates that parahydroxylation is probably not the link with the mephenytoin polymorphism, or if it is, it is of little clinical consequence.

Acknowledgements This work was supported by the Medical Research Council of Canada. Send reprint requests to: Dr T. Inaba, Department of Pharmacology, Faculty of Medicine, University of Toronto, Toronto M5S 1A8, Canada. We wish to thank Agnes Bleiwas for discussion, and for critically reading the paper. References ACKERMANN, E., and RICHTER, K., 1977, Diazepam metabolism in human foetal and adult liver. European Journal of Clinical Pharmacology, 11, 4 3 4 9 . ANDREWS, S. M., and GRIFFITHS, L. A., 1984, The metabolism and disposition of [2-'4C]diazepam in the streptozotocin-diabetic rat. Xenobiotica, 14, 751-760. BEISCHLAC, T. V. P. C., 1990, The effect of mephenytoin on diazepam metabolism in rat and man. MSc thesis, University of Toronto. BEISCHLAG, T . V., and INABA,T . , 1992, Determination of nonderivatized para-hydroxylated metabolites of diazepam in biological fluids using a GC Megabore column system. Journal of Analytical Toxicology (In press). BERTILSSON, L., HENTHORN, T. K., SANZ,E., TYBRING, G., SAWE, J., and VILLEN, T., 1989, Importance of genetic factors in the regulation of diazepam metabolism: relationship to S-mephenytoin, but not debrisoquine hydroxylation phenotype. Clinical Pharmacology and Therapeutics,45,348-355. BERTILSSON, L., BAILLIE, T. A., and REVIRIEGO, J., 1990, Factors influencing the metabolism of diazepam. Pharmacology and Therapeutics, 45, 85-91. CAMPBELL, M. E., GRANT, D. G., INABA,T., and KALOW, W., 1987, Biotransformation of caffeine, paraxanthine, theophylline and theobromine by polycyclic aromatic hydrocarbon inducible cytochrome P-450 in human liver microsornes. Drug Metabolism and Disposition, 15, 237-249. CHENERY, R. J., AYRTON, A., OLDHAM, H. G., STANDRING, P., NORMAN, S. J., SEDDON, T.,and KIRBY, R., 1987, Diazepam metabolism in cultured hepatocytes from rat, rabbit, dog, guinea pig, and man. Drug Metabolism and Disposition, 15, 312-316.

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CORNISH-BOWDEN, A., 1974, A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors. Biochemical Journal, 137, 143-144. DIXON,M., and WEBB,E. C., 1964, Enzymes, 2nd edn (London: Longmans Green), pp. 315-331. GUENTERT, T. W., 1984, Pharmacokinetics of benzodiazepines and of their metabolites, in Progress in Drug Metabolism, vol. 8, edited by J. W. Bridges and L. F. Chasseaud (London: Taylor & Francis), pp. 241-386. HOFSTEE, B. H. J., 1952, On the evaluation of the cosntants V,,,and K,,, in enzyme reactions. Science, 116, 329-33 1. INABA, T . , TSUTSUMI, E., MAHON, W. A,, and KALOW, W., 1974, Biliary excretion of diazepam in the rat. Drug Metabolism and Disposition, 2, 429-432. INABA, T . , JURIMA, M., MAHON, W. A., and KALOW, W., 1985, I n vitro inhibition studies of two isozymes of human liver cytochrome P-450: mephenytoin p-hydroxylase and sparteine monooxygenase. Drug Metabolism and Disposition, 13, 443448. A,, NAKANO, M., MAHON, W. A,, and KALOW, W., 1988, Metabolism of diazepam in INABA, T., TAIT, vitro by human liver: Independent variability of N-demethylation and C,-hydroxylation. Drug Metabolism and Disposition, 16, 605-608. T., UMEDA,T . , TSUTSUMI, E., and STONE,R., 1976, Biliary elimination of MAHON,W. A,, INABA, diazepam in man. Clinical Pharmacology and Therapeutics, 19, 443450. C., 1980, Postnatal development of sex-dependent differences in the metabolism NAU,H., and LIDDIARD, of diazepam by rat liver. Biochemical Pharmacology, 29, 447449. P. E. B., THOMPSON, D. A,, MASON,S. R., and HOOPER, W. D., 1990, Cytochrome P450IIIA REILLY, enzymes in rat liver microsomes: involvement in C,-hydroxylation of diazepam and nordiazepam but not N-dealkylation of diazepam and temazepam. Molecular Pharmacology, 37, 767-774. P., and VANE,F. M., 1967, Diazepam metabolites in the rat: Characterization SCHWARTZ, M. A,, BOMMER, by high-resolution mass spectrometry and nuclear magnetic resonance. Archives of Biochemistry and Biophysics, 121, 508-516. SEDDON, T . , MICHELLE, I., and CHENERY, R. J., 1989, Comparative drug metabolism of diazepam in hepatocytes isolated from man, rat, monkey and dog. Biochemical Pharmacology, 38, 1657-1665. F. P., 1986, Human liver microsomal P450mephenytoin SHIMADA, T . , MISONO,K. S., and GUENGERICH, 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Journal of Biological Chemistry, 261, 909-921. S. F., Tro, C. O., SHRADER, S. R., and RUELIUS, H. W., 1972, The biotransformation of SISENWINE, oxazepam (7-chloro-1,3-dihydro-3-hydroxy-5-phenyl-2H-l,4-benzodiazepin-2-one) in man, miniature swine and rat. Arzneimittel Forschung. (Drug Research), 22, 682-687. TAIT,A., 1987, I n vitro metabolism of diazepam in man, MSc thesis, University of Toronto. TRENNERY, P. N., and WARING, R. H., 1985, T h e influence of an experimental liver cirrhosis upon the metabolism of diazepam and imipramine HCl in the rat. Xenobiotica, 15, 813-823.

Diazepam metabolism by rat and human liver in vitro: inhibition by mephenytoin.

1. Diazepam metabolism and its association with mephenytoin hydroxylase were studied in vitro using human and rat livers. 2. Enzyme kinetic parameters...
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