The Development of a Cocktail CYP 2 B 6 , CYP 2 C 8 , and CYP 3 A 5 Inhibition Assay and a Preliminary Assessment of Utility in a Drug Discovery Setting

Tools for studying the roles of CYP2B6, CYP2C8, and CYP3A5 in drug metabolism have recently become available. The level of interest in these enzymes has been elevated because investigations have revealed substrate promiscuity and/or polymorphic expression. In this study, we aimed to develop a single cocktail inhibition assay for the three enzymes and assess its utility in drug discovery. Bupropion hydroxylation, amodiaquine N-deethylation, and midazolam 1 -hydroxylation were chosen as probe reactions for CYP2B6, CYP2C8, and CYP3A5 and were analyzed using liquid chromatography-tandem mass spectrometry. Kinetic analyses were performed to establish suitable conditions for inhibition assays, which were subsequently automated. CYP2B6, CYP2C8, and CYP3A5 IC50 values were determined for marketed drugs and almost 200 AstraZeneca discovery compounds from 16 separate discovery projects. For the marketed drugs, results obtained were comparable with literature values. Data were also compared with IC50 values determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. In this dataset, the majority of compounds were more potent inhibitors of CYP2C9, CYP2C19, CYP2D6, and CYP3A4 than of CYP2B6, CYP2C8, or CYP3A5. The potential impact of these findings on a cytochrome P450 inhibition strategy is discussed. Drug discovery programs traditionally study CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 when the metabolism and enzyme inhibition potential of new chemical entities (NCEs) are being investigated. The tools for studying other P450s have more recently become available and the roles of CYP2B6, CYP2C8, and CYP3A5 in drug metabolism have come to the fore because they have been implicated in the metabolism of a number of marketed drugs and physiologically important endogenous molecules. In addition, increased variability in the pharmacokinetics of drugs metabolized by CYP2B6, CYP2C8, and CYP3A5, as a consequence of polymorphic expression, makes consideration of these enzymes important. CYP2B6 is estimated to make up at least 5% of the total hepatic P450 content (Code et al., 1997; Ekins et al., 1998; Stresser and Kupfer, 1999). It metabolizes a range of drugs including efavirenz (Ward et al., 2003) and cyclophosphamide (Huang et al., 2000) as well as drugs of abuse such as cocaine and ecstasy (Aoki et al., 2000; Kreth et al., 2000). In addition, large interindividual variations in hepatic CYP2B6 have been demonstrated because of its highly inducible nature and the existence of polymorphisms (Ariyoshi et al., 2001; Lang et al., 2001). CYP2C8 is a major human hepatic P450, constituting 7% of the total microsomal content in the liver (Shimada et al., 1994; Rendic and Di Carlo, 1997). It has been shown to be involved in the metabolism of a variety of drugs including amiodarone, amodiaquine, diclofenac, and troglitazone (Yamazaki et al., 1999; Ohyama et al., 2000; Tang, 2003; Walsky and Obach, 2004), as well as physiologically important endogenous molecules such as arachidonic acid (Rifkind et al., 1995). CYP2C8 is polymorphic; the most common variant alleles are CYP2C8*2 and CYP2C8*3, both of which result in a functional change of the enzyme (Dai et al., 2001). The CYP2C8*2 variant occurs in African-American populations with an allele frequency of 18%, but it is relatively uncommon in Caucasian populations, whereas CYP2C8*3 is more common in Caucasian individuals (23%) and quite rare in African-American subjects (Totah and Rettie,