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A fully automated assay to determine the enzymology of drug oxidation by …


Biology Articles » Biochemistry » Enzymology » Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s » Introduction

Introduction
- Automated Definition of the Enzymology of Drug Oxidation by the Major Human Drug Metabolizing Cytochrome P450s

Much interest is currently focused on the early identification of the drug-metabolizing enzymes responsible for the biotransformations commonly encountered in drug development (Becquemont et al., 1998). Such information may help identify the key organs for clearance and explain or even predict the observed variability in pharmacokinetics with some substrates and prioritize drug-drug interaction studies. Because most (~60%) marketed compounds are cleared metabolically by cytochrome P450 (CYP)2 enzymes, the major activity in this area has focused on this family of enzymes (Bertz and Granneman, 1997).

Traditionally, human liver microsomes (HLM) have been the in vitro tool for these studies and have provided both qualitative, e.g., identifying which CYP isoform(s) metabolize the compound of interest (Pichard et al., 1990; Andersson et al., 1993; Otton et al., 1990; Jacqz-Aigrain et al., 1993; Doecke et al., 1991; Wester et al., 2000; Yasumori et al., 1993; Kroemer et al., 1993) and quantitative information, e.g., predicted CLint (Houston, 1994; Rodrigues, 1994; Carlile et al., 1999). Identifying the enzymology of metabolism by human CYPs has proved somewhat labor- and time-intensive, requiring comparative kinetics across a bank of characterized HLM, chemical, and/or antibody inhibition followed by the use of recombinant CYP isoforms (Rodrigues, 1999). The routine access to recombinant CYPs has facilitated direct identification of the isoform(s) responsible for the oxidative metabolism of the drug of interest, although their use in vitro has generally been to support HLM data (Aoyama et al., 1990; Tassaneeyakul et al., 1992; Kroemer et al., 1993; Rodrigues et al., 1994; Yamazaki et al., 1997; Von Moltke et al., 1998; Rodrigues, 1999).

With the advent of combinatorial chemistry and parallel synthesis techniques, there is an expectation to achieve both higher throughput and faster turnaround times in many biological assays. There is an increasing emphasis within drug metabolism in the pharmaceutical industry to develop enhanced throughput frontline in vitro models, including those to determine both the extent and route of the metabolism of new chemical entities (NCEs) and to screen for inducers and inhibitors of drug-metabolizing enzymes (Ayrton et al., 1998; Moody et al., 1999).

The ability to predict directly the human enzymology using enhanced throughput methods would represent a major breakthrough in this technology (Becquemont et al., 1998; Roy et al., 1999) in a similar manner to that adopted for CYP inhibition assays (Crespi et al., 1998; Moody et al., 1999). This laboratory has demonstrated that the five major human hepatic CYPs expressed in Escherichia coli (CYP1A2, -2C9, -2C19, -2D6, and -3A4) are faithful surrogates for their human liver counterparts with respect to their kinetic profiles and inhibition properties (McGinnity et al., 1999; Moody et al., 1999). In this study, the application of recombinant enzymes as a first line approach for identifying the CYP(s) responsible for metabolizing NCEs has been proposed. A fully automated assay has been developed using the major drug-metabolizing human hepatic cytochrome P450s (CYP1A2, -2C9, -2C19, -2D6, and -3A4) coexpressed functionally in E. coli with human NADPH-P450 reductase, to predict the CYP isoform(s) involved in the oxidative metabolism of NCEs.



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