Experimental Approaches

Biotransformation studies have benefited from advances in both molecular biology and biochemistry. Molecular biology provided new reagents for mechanistic studies and screening experiments in the form of expressed drug-metabolizing enzymes [4]. Some of these expression systems include cytochrome P450, glucuronide transferases, and sulfotransferases [5,6]. More recently, drug-transport proteins, such as ^-glycoprotein, have been cloned and expressed. Transport proteins are now recognized as a major factor that contributes to the ADME of drugs [7]. Drug interactions traditionally thought to occur most frequently with P450 metabolizing enzymes are beginning to be recognized with transport systems as well [7]. Improved cell/tissue culture and handling techniques allow for the harvest and cryopreservation of primary hepatocytes that are an important tool for the approximation of in vivo biotransformation [8]. Additionally, the development of human intestinal epithelial cancer cell lines (CaCO2 cells) allowed the generation of in vitro absorption models such that the role of transport and biotransformation in absorption may be studied now on a routine basis [7].

Biotransformation studies and metabolite identification are now feasible at the screening stages of drug discovery. Related screening paradigms have been established to characterize compounds with respect to important metabolic properties, such as metabolic stability, P450 inhibition, P450 induction, P450phenotyping, and membrane permeability [9-13]. Increasingly, liquid-handling robots are used to conduct these in vitro assays and to perform sample preparation for subsequent rapid liquid chromatography-mass spectrometry (LC-MS) analyses. The implementation of automation and parallel processing [14] have increased the throughput of metabolic screening and compound characterization to the point where drug-metabolism scientists can start to analyze a significant fraction of the compounds that are synthesized by combinatorial chemistry and related parallel synthetic approaches.

Taken as a whole, the characterization of metabolic pathways in early drug discovery has become both an important and realistic objective. Recognition of the value of these experiments has been only one half of the equation; the availability of greatly improved hardware and software for the characterization of biotransformations has made it possible to ask more questions.

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