Research Articles
Polymorphism in Diazepam Metabolism in Wistar Rats

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Abstract

We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats of Wistar strain we examined showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals at the substrate concentrations less than 5 μM. We classified them as extensive metabolizer (EM) and poor metabolizer (PM) of diazepam. No sex difference was observed in the frequency of appearance of EM. Activities of the primary metabolic pathways of diazepam were examined to elucidate the cause of this polymorphism in male Wistar rats. No significant differences were observed in activities of neither diazepam 3-hydroxylation or N-desmethylation between EM and PM rats, while activity of diazepam p-hydroxylation was markedly (more than 200 times) higher in EM rats, indicating that this reaction is responsible for the polymorphism of diazepam metabolism in Wistar rats. We examined the expression levels of CYP2D1, which was reported to catalyze diazepam p-hydroxylation in Wistar rats to find no differences in the expression levels of CYP2D1 between EM and PM rats. The kinetic study on diazepam metabolism in male Wistar rats revealed that EM rats had markedly higher Vmax and smaller Km in diazepam p-hydroxylation than those of PM rats, indicating the presence of high affinity high capacity p-hydroxylase enzyme in EM rats. As a consequence, at low concentrations of diazepam, major pathways of diazepam metabolism were p-hydroxylation and 3-hydroxylation in male EM rats, while in male PM rats, 3-hydroxylation followed by N-desmethylation. Due to this kinetic nature of p-hydroxylase activity, EM rats had markedly higher total CLint of diazepam than that of PM rats. Polymorphism in diazepam metabolism in humans is well documented, but this is the first report revealing the presence of the polymorphism in diazepam metabolism in rats. The current results infer polymorphic expression of new diazepam p-hydroxylating enzyme with lower Km than CYP2D1 in EM Wistar rats. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1271–1278, 2004

Section snippets

INTRODUCTION

Polymorphic expressions of drug metabolizing enzymes are of clinical importance. Metabolic activities of a number of drugs have been reported to show marked interindividual or interracial differences in a human population due to their genetic polymorphism of drug metabolizing enzymes. Those who have the high drug metabolizing activities are usually classified to be extensive metabolizers (EM) and the low drug metabolizing activities, poor metabolizers (PM). Administration of a drug at the

Materials

Diazepam and the internal standard, nitrazepam, were purchased from Wako Pure Chemicals Co. (Osaka, Japan). The three primary diazepam metabolites, p-hydroxy-diazepam, 3-hydroxy-diazepam, and N-desmethyl-diazepam, and the two secondary diazepam metabolites, 3-hydroxy-N-desmethyl-diazepam and p-hydroxy-N-desmethyl-diazepam were gifted from Japan Hoffman La Rosch Pharmaceutical Co. (Tokyo, Japan). Glucose-6-phosphate (G-6-P), Glucose-6-phosphate dehydrogenase (G-6-PDH), and β-nicotinamid-adenine

RESULTS

We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats (3 of 18) showed two times higher activities of diazepam metabolism in their liver microsomes than the rest of animals at the substrate concentrations less than 5 μM. We classified them as an EM of diazepam, and the remaining rats with low diazepam metabolism activity, the PM. We found the same frequency of EM in

DISCUSSION

We observed variations in the metabolism of diazepam in Wistar rats. We classified them as EM and PM of diazepam. About 17% of male rats of Wistar strain we examined (EM) showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals (PM) at the substrate concentrations less than 5 μM. Neville et al. reported that p-hydroxylation, 3-hydroxylation, and N-desmethylation of diazepam proceeded in adult male Wistar rat liver microsomes, and that they were

Acknowledgements

This study was supported by Grants-in-Aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, Culture and Technology, to S.F. (Grants 11358009 and 11306021) and A.K. (Grant 09306021 and 14657091). This study was also supported by Grant-in-Aid for the Development of Innovative Technology and Grant-in-Aid for Scientific Research on Priority Areas (A) (Grant 13027201) of the Japanese Ministry of Education, Science, Sports, Culture and Technology.

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    Copyright © 2004 Wiley-Liss, Inc. Published by Elsevier Inc. All rights reserved.