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backs to hepatocytes and are poor for rate measurements; (d) whole isolated organs, which are good for metab-olite production and physiological scaling but human tissues cannot be used; and (e) heterologous expressed enzyme systems using yeast, bacteria, or mammalian cell lines, which provide an idea of the specific enzyme involvement but not the total interrelationship of a whole tissue activity. One of the more important aspects of in vitro metabolism is the potential for the early prediction of human kinetic difficulties. |
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IV. Metabolic Isoforms and Kinetic Variability |
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There are myriad factors that control the drug levels in individual patients [24], including environment, patient compliance, and genetic differences. Although we do not know the specific contribution of each, increasingly we are finding that genetic variation is a major contributor to the clinical variability in steadystate drug levels. Thus, in addition to providing early information on the rates, routes, and metabolic profiles and kinetic scaling, in vitro metabolic studies may give an insight into future kinetic problems that may occur by providing information on which specific enzymes the drug is metabolized and whether this enzyme is under polymorphic control. Genetic polymorphism occurs when the gene of an individual contains different codons that express a particular isozyme compared to the normal wild form. This can occur for a number of isozymes, including the cytochromes 2D6, 2C9, but also the nonmicrosomal enzymes, N-acetyl transferase, Glutathione-S-transferase, choline esterase, and others [25]. This detection of multiple hidden alleles in a number of enzymes provides an understanding of why there can be a large variability of metabolic rates, not just at the extremes, but within the normal distribution curve. It also shows that simple classifications into fast or slow metabolizers is a gross oversimplification of the real situation. |
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Thus for CYP 2D6, at least 19 different allele forms exist, producing extremes from ultrafast to very slow metabolizers and many variants in between [26]. A survey of UK companies [23] indicates that most have started to routinely screen their drugs for polymorphism using a variety of techniques (Fig. 3). These include in vitro methods such as (a) inhibition by a specific enzyme inhibitor; (b) metabolism by an isolated heterologous expressed enzyme; and (c) cross correlation of a genotyped bank of human microsomes and in vivo methods (a) kinetic measurements in geno- or phenotyped individuals using single or cocktails of compounds after single or repeated dose population screening, (b) polymodality of metabolic ratios, or (c) multiple interaction studies. |
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Up until recently, it was thought that animal in vitro metabolic studies could provide useful information, not only on the types of metabolic pathways, but also on the specific isozymes responsible for the metabolism in humans. Al- |
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