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Heterologously expressed human drug-metabolizing enzyme systems in a variety of hosts (mammalian cell lines, baculovirus/insect cells, yeast and Escherichia coli) have their distinct advantages for particular studies of biotransformation or mechanistic processes. In contrast to classical in vitro systems such as tissue slices, hepatocytes or subcellular fractions, expressed enzymes allow the study of single enzyme reactions in isolation. Furthermore, metabolic reactions of enzymes expressed only in minor amounts in human tissue can be assessed. Here we present an overview of how recombinant enzymes are being used in biotransformation studies and we will present several examples of applications of recombinant cytochrome P450 preparations during drug development. It is beyond the scope of this overview to describe all experimental procedures in detail since they are based on published techniques unless otherwise indicated. Examples of applications of recombinant cytochrome P450 preparations include the involvement of human metabolizing enzymes in a metabolic pathway and their metabolic products, mechanistic studies to determine specific drug-drug interactions at the metabolic level and detection of mechanism-based inactivation of drug-metabolizing enzymes. Heterologous expression systems offer a constant and reproducible source of human drug-metabolizing enzymes that are easily available by standard laboratory techniques. Considering the constraints on the availability and use of human tissue it is likely that these systems will be widely used in the future. But, because of the unequal distribution of individual drug-metabolizing enzymes in man, extrapolation needs to be done carefully.

To determine the metabolic profile of minimally absorbed orlistat in obese/overweight patients, an open-label, single-dose study was performed in eight obese/overweight volunteers between 23 and 68 years of age. Each subject received a single oral dose of 360 mg orlistat containing approximately 400 muCi of 14C-labeled orlistat. Serial blood samples were collected at specified times over 10 hours after administration of orlistat for determination of total radioactivity, unchanged orlistat, and major metabolites in the plasma. Urine samples were collected over 24 hours and analyzed to evaluate the urinary recovery of total radioactivity and the profile of orlistat metabolites in the urine. In addition, all fecal samples were collected and analyzed for total radioactivity. Urinary and fecal recovery of the administered dose of total radioactivity were 1.13 +/- 0.50% (24-hour data only) and 96.4 +/- 18.1% (n = 7), respectively. Maximum observed concentration (Cmax) and time to Cmax (tmax) values of plasma total radioactivity were 150 +/- 51 ng.eq/mL and 6.8 +/- 1.5 hrs, respectively. All these parameters obtained in obese/ overweight subjects were similar to those reported previously in healthy subjects. On the basis of the area under the concentration-time curve from 0 to 10 hours (AUC0-10), two major metabolites comprise a total of approximately 42% of the total radioactivity in plasma. The primary metabolite (M1) has a short half-life (approximately 2 hours), whereas the secondary metabolite (M3) disappeared at a slower rate. No strikingly apparent difference in the urinary metabolic profile was observed between two gender groups. It is concluded that the disposition of orlistat appears to be similar between normal and obese/overweight subjects. Of the minimal fraction of the dose that was absorbed systemically, the presence of two major metabolites accounts for approximately 42%.

The arotinoid mofarotene is a novel potent anticancer compound. The metabolic profiles obtained from rat, dog, and human plasma showed a good correlation with the corresponding in vitro profiles observed with liver microsomes and hepatocytes. Interspecies differences in its metabolism were investigated using microsomes prepared from the livers of the mouse, rat, dog, cynomolgus monkey, and humans. These in vitro experiments showed that, both qualitatively and quantitatively, the metabolic profiles obtained with cynomolgus monkey liver samples were similar to those observed with human liver material. However, rat and dog were also confirmed to be suitable species for assessing the safety of mofarotene, and were used in toxicology. The involvement of cytochrome P450 (CYP) in the metabolism of mofarotene was examined with human liver microsomes. CYP3A4 plays a major role in the metabolism, and CYP1A2 might be responsible for a minor pathway. Finally, the potential induction by mofarotene of four major CYP isoenzymes was investigated in rats. These experiments showed that CYP1A1 was clearly induced, whereas a slight induction of CYP3A and CYP2B was observed. Repeated administration of mofarotene had no effect on CYP2E1. These studies with liver microsomes and hepatocytes aided the selection of appropriate species for toxicology, and have provided information that will help to predict potential drug-drug interactions in clinical trials.