This title is available in both hardbound and paperback editions. The paperback edition is sold subject to the condition that it shall not, by way of trade or otherwise, be lent, resold, hired out, or otherwise circulated without the publisher's prior consent in any form of binding or cover other than that in which it is published and without a similar condition including this condition being imposed on the subsequent purchaser.All rights reserved. No part of this book may be reprinted, or reproduced or utilized in any form or by an electronic, mechanical or other means, now PrefaceAlthough the scientific literature on drug metabolism is extensive, it suffers from the disadvantage that the material is diffuse and consists largely of specialist monographs dealing with particular aspects of the subject. In addition, although there are a few excellent texts on drug metabolism in print, these tend to be earlier publications and hence do not take into account the many recent advances in this area. Our motivations for writing this book therefore arose from the clear need for a recent and cohesive introductory text on this subject, specifically designed to cater for the needs of undergraduate and postgraduate students. Much of the subject matter in this text is derived from various courses on drug metabolism given at the University of Surrey and the University of Glasgow to basic science students in pharmacology, biochemistry, nutrition and nursing studies, to pre-clinical medical students and to undergraduate and post-graduate students in toxicology. Therefore, it is our intention that this text will serve as a primer in drug metabolism to a variety of students in the life sciences taking courses in this subject.The term 'drug metabolism' in its broadest sense may be considered as the absorption, distribution, biotransformation and excretion of drugs. To cover all these facets of drug metabolism in a single text is a voluminous task and therefore we have focused primarily on the biotransformation aspects of the subject. Having said this, the text is not solely a list of drug metabolism pathways, but rather it uses biotransformation reactions to rationalize many ix some of their published material. Finally, we express our thanks to the staff of Chapman and Hall for their excellent co-operation and for giving us the opportunity to bring this project to fruition.

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Hypolipidaemic drugs induce peroxisomal proliferation in the liver and many induce the formation of the hepatic endoplasmic reticulum in general and the formation of cytochrome P-450 in particular. We have induced the formation of rat liver microsomal cytochrome P-450 by the administration of the hypolipidaemic drug clofibrate, isolated the endoplasmic reticulum, solubilized the cytochrome P-450 from these membranes and subdivided the cytochrome P-450 into four fractions by the use of hydrophobic, anionic, cationic and adsorption chromatography. One of these fractions (cytochrome P-450 fraction 1) was highly purified to a specific content of 17nmol of cytochrome P-450/mg of protein and the protein was active in a reconstituted enzyme system towards the 12- and 11-hydroxylation of the fatty acid, dodecanoic (lauric) acid, with preferential activity towards the 12-hydroxy metabolite. This reconstituted activity was absolutely dependent on NADPH, NADPH-cytochrome P-450 reductase and cytochrome P-450, indicating the role of the mixed-function oxidase system in the metabolism of lauric acid. Another fraction of the haemoprotein (cytochrome P-450 fraction 2) preferentially formed 11-hydroxylauric acid, whereas a third fraction (cytochrome P-450 fraction 3) exhibited only trace laurate oxidase activity and was similar to the phenobarbitone form of the haemoprotein in that these last two cytochromes rapidly turned-over the drug benzphetamine. The molecular weights and spectral properties of these cytochrome P-450 fractions are reported, along with the phenobarbitone-induced form of the enzyme and the nature of the cytochrome(s) induced by clofibrate pretreatment are discussed in the terms of possible haemoprotein heterogeneity.

The effect of insulin-dependent diabetes on the hepatic microsomal activity of cytochrome P450III and P450IV family proteins was investigated in rats pretreated with streptozotocin. In order to discern between the effects of the diabetogen per se and those of the ensuing diabetes, streptozotocin-treated rats received in addition either nicotinamide to prevent the onset of diabetes or daily treatment with insulin to antagonize the effects of diabetes. Streptozotocin-treated rats displayed higher ethylmorphine and erythromycin N-demethylase activities and lauric acid hydroxylase activity. Increases were also detected immunologically by using monospecific polyclonal antibodies against the P450III and P450IV families. All effects were prevented by nicotinamide and effectively antagonized by insulin. In order to evaluate the role of the ketone bodies in the diabetes-induced increases in the above activities, rats were rendered hyperketonaemic by dietary administration of medium-chain triacylglycerols. These hyperketonaemic animals displayed high laurate hydroxylase activity and P450IV apoprotein levels, similar to those seen in the diabetic animals. Hyperketonaemia induced by dietary means caused a modest increase in the demethylation of erythromycin and had no significant effect on the N-demethylation of ethylmorphine. Furthermore, no marked increases were evident in the P450III apoprotein levels in the hyperketonaemic animals. It is concluded that insulin-dependent diabetes induces proteins of the P450III and P450IV families, and that the hyperketonaemia that accompanies diabetes is largely responsible for the changes in the latter family.

In the present studies, a novel form of highly purified cytochrome P-450 (cytochrome P-452) isolated from the hepatic microsomes of clofibrate-pretreated rats has been compared to the major isozymes isolated from the hepatic microsomes of rats pretreated with phenobarbital (cytochrome P-450) and 2-naphthoflavone (cytochrome P-447) using a number of biochemical criteria.The results show that these three isozymes exhibit marked structural differences from each other as judged by a complete lack of immunochemical cross-reactivity between the isozymes and the heterologous rabbit serum antibodies using Ouchterlony double diffusion, and non-identity between the limited proteolytic digestion maps of the three isozymes obtained in the presence of chymotrypsin, papain and Staphylococcus aureus V, proteases. Furthermore, the three isozymes exhibited clear differences in their monomeric molecular weights determined on calibrated sodium dodecyl sulphate/polyacrylamide gel electrophoresis in gels of varying acrylamide concentration. Substantial differences were also observed in the substrate specificities of the isozymes, which were reflected in differences in the turnover rates and positional selectivities of the hemoproteins for some model substrates. In addition, the isozymes differed in their substrate binding affinities and their ability to interact with purified hepatic microsomal cytochrome b,, as judged using difference spectrophotometry. Finally, subtle differences were detected in the ultraviolet visible absorbance spectra of the hemoproteins in the ferric, ferrous, and carbonmonoxyferrous states.Taken collectively, the above data provides compelling evidence that fundamental differences exist between these cytochrome P-450 isozymes, further establishing the uniqueness of the major form of cytochrome P-450 induced by clofibrate pretreatment.Cytochrome P-450 is the terminal hemoprotein component of the hepatic microsomal electron-transfer chain which functions in the oxidation of a structurally diverse number of xenobiotics and endogenous compounds [I]. Considerable evidence has been presented in support of the existence of Much confusion exists regarding the terminology of cytochrome P-450 isozymes primarily because there is no uniform classification system which has been adopted to make unequivocal assignments of the similarity between isozymes isolated in different laboratories. Accordingly, the above three isozymes described in this paper have been classified according to the wavelength maxima of the ferrouscarbonmonoxy adducts. In the current paper, limited use is made of the term cytochrome P-450 in a general sense to collectively indicate all the cytochrome P-450 isozymes. Where appropriate, we make extended use of the term cytochrome P-450 to indicate that major isozyme induced by phenobarbital pretreatment in rat liver. This is regarded as necessary because to make a distinction for this latter isozyme and rename it would add further confusion to the literature. Similarly, throughout this paper, the terms c...

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The anaerobic reduction kinetics of purified rat liver ferric cytochrome P-450 from phenobarbital-treated rat liver microsomes, reconstituted with saturating NADPH-cytochrome P-450 reductase, have been investigated and were shown not to be monophasic. From experiments correlating changes in the rate of fast-phase reduction with the spin state of the heme iron existing at preequilibrium, data were obtained consistent with a model for spin-state control of cytochrome P-450 reduction wherein the high-spin form of the hemoprotein is more rapidly reduced than the low-spin form. In addition, the temperature dependence of the reduction process in the presence of the substrate benzphetamine was studied. From the results obtained it is suggested that the endothermic nature of the low- to high-spin transition largely accounts for the apparent activation energy observed for the reduction of high-spin cytochrome P-450 being relatively temperature insensitive when compared to the rate constant for reduction of the membrane-bound form of the hemoprotein.