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ABSTRACT:Azamulin [14-O-(5-(2-amino-1,3,4-triazolyl)thioacetyl)-dihydromutilin] is an azole derivative of the pleuromutilin class of antiinfectives. We tested the inhibition potency of azamulin toward 18 cytochromes P450 using human liver microsomes or microsomes from insect cells expressing single isoforms. In a competitive inhibition model, IC 50 values for CYP3A (0.03-0.24 M) were at least 100-fold lower than all other non-CYP3A enzymes except CYP2J2 (ϳ50-fold lower). The IC 50 value with heterologously expressed CYP3A4 was 15-fold and 13-fold less than those of CYP3A5 and CYP3A7, respectively. The reference inhibitor ketoconazole was less selective and exhibited potent inhibition (IC 50 values <10 M) for CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP4F2, and CYP4F12. Inhibition of CYP3A by azamulin appeared sigmoidal and well behaved with the substrates 7-benzyloxy-4-trifluoromethylcoumarin, testosterone, and midazolam. Preincubation of 4.8 M azamulin in the presence of NADPH for 10 min inhibited ϳ95% of testosterone 6-hydroxylase activity compared with preincubation in the absence of NADPH. Catalytic activities of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 were unaffected by similar experiments. Incubation of azamulin with heterologously expressed CYP3A4 yielded a type I binding spectrum with a spectral dissociation constant of 3.5 M, whereas no interaction was found with CYP2D6. Azamulin exhibited good chemical stability when stored in acetonitrile for up to 12 days. Aqueous solubility was found to be >300 M. Azamulin represents an important new chemical tool for use in characterizing the contribution of CYP3A to the metabolism of xenobiotics.Along with other experimental approaches, enzyme-selective chemical inhibitors are commonly used in reaction phenotyping studies to determine cytochrome P450 isoform contribution to a metabolic reaction (Clarke, 1998). Chemical inhibitors provide a simpler and more cost-effective alternative to immunoinhibitory antibodies and can be used in cells. However, proper use of chemicals may require foreknowledge of the reaction kinetics under investigation. For example, competitive inhibitors should be used with a substrate concentration near or below the apparent K M . In addition, specificity is often lost when the inhibitor concentration is too high. The effect of microsomal protein concentration and incubation time may also need to be considered, if the inhibitor is also a substrate.Chemicals used as selective inhibitors of CYP3A include triacetyloleandomycin, gestodene, and ketoconazole. Ketoconazole is most widely used, probably because of advantages in potency, selectivity, commercial availability, and ease of use (e.g., preincubation steps are not required) (Maurice et al., 1992;Baldwin et al., 1995;Newton et al., 1995;Bourrie et al., 1996;Sai et al., 2000;Zhang et al., 2002). However, selectivity of ketoconazole for CYP3A is often less than ideal. For example, CYP1B1, CYP2B6, and CYP2C8...
