sp2 May 2008 28 Facilitating the identification of toxic metabolites early in the drug development process and enabling the diversification of lead compounds through the generation of a broad range of hydroxylated derivatives may lead to the discovery of safer, more potent drugs with a reduced risk of failure in clinical trials. In humans and animals most xenobiotic compounds including drugs are metabolised and eliminated via a reaction sequence carried out predominantly in the liver.1 Commonly in drug metabolism the first step is catalysed by cytochrome P450 monooxygenases, forming primary metabolites that are hydroxylated, de-alkylated, or oxidised in other ways.2,3 During preclinical and clinical testing, drugs frequently fail not due to problems with the drug candidate itself but due to problems arising from the metabolites.4 For this reason, the FDA has published guidance that strongly recommends that all significant metabolites be characterised early in the drug development process.5 It is highly desirable that such characterisation be done prior to the initiation of human testing. Often, once the site of hydroxylation or other modification is identified, a drug can be redesigned to improve its activity and/or half-life. A common strategy is to replace a reactive hydrogen atom with fluorine, blocking P450-catalysed hydroxylation at that site and thereby eliminating the formation of a toxic or problematic metabolite and/or extending the serum half-life for a drug. Metabolites are not always the source of negative data for a drug. In a number of cases metabolites have been shown to display biological activities superior to or different from that of the drug from which they are derived. The identification of these so-called ‘active metabolites’ can lead to safer, more active drug substances. A wellknown example is the antihistamine fexofenadine (AllegraTM), which is a metabolite of the older drug terfenadine (SeldaneTM). Fexofenadine was found to have all the biological activity of terfenadine but cause fewer adverse reactions in patients, leading to its approval in 1996 by the FDA. As a result, the metabolite fexofenadine is now marketed as an improved drug in place of the original drug substance terfenadine.6 Producing human metabolites in vitro in amounts sufficient for structural identification and preliminary toxicity and activity testing poses significant challenges. Chemical synthesis has been traditionally employed to produce metabolites, but often the synthetic route requires many steps and months of time to develop and carry out, resulting in a low yield and a high cost in both time and materials. An alternative to chemical synthesis is to use cytochrome P450 enzymes to generate the desired metabolites. The most common source is pooled human liver microsomes (HLM), but these microsomal preparations contain a mixture of many different enzymes, and their cost, batch-to-batch variability in activity, and restriction on availability limit the usefulness of human liver microsomes for preparative synthetic work. Liver microsomes from alternative animal sources have been used as in in vitro alternative to HLM.7,8 Although lower in cost than the human analogues, animal liver microsomes suffer from most of the same caveats as HLM and, in addition, have the disadvantage of sometimes producing different metabolites compared to their human counterparts. For the laboratory preparation of drug metabolites, better systems for production were needed.