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, IC50 values for CYP3A (0.03–0.24 μM) were at least 100-fold lower than all other non-CYP3A enzymes except CYP2J2 (∼50-fold lower). The IC50 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 (IC50 values <10 μM) 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 μM azamulin in the presence of NADPH for 10 min inhibited ∼95% of testosterone 6β-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 μM, 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 μM. Azamulin represents an important new chemical tool for use in characterizing the contribution of CYP3A to the metabolism of xenobiotics.
Footnotes
-
↵1 Unless otherwise specified, the term “CYP3A” refers to CYP3A4 and CYP3A5; other members of this subfamily include CYP3A7 and CYP3A43. It is generally accepted that only CYP3A4 and CYP3A5 can be found in liver at detectable levels and that, of the two, CYP3A4 is predominant in terms of abundance and importance in drug metabolism (Wrighton and Thummel, 2000). The relative contribution of CYP3A5 to the metabolism of drugs is considered to be much less but is an area of active research.
-
↵2 Abbreviations used are: HLM, human liver microsome; P450, cytochrome P450; DBF, dibenzylfluorescein; AMMC, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin; BFC, 7-benzyloxy-4-trifluoromethylcoumarin; CEC, 3-cyano-7-ethoxycoumarin; MFC, 7-methoxy-4-trifluoromethylcoumarin; AAA, 94% acetonitrile/6% glacial acetic acid; HPLC, high performance liquid chromatography; TAO, triacetyloleandomycin; DMSO, dimethyl sulfoxide; LTB4, leukotriene B4; LTB4 20-OH, 20-hydroxyleukotriene B4; Ks APP, apparent spectral dissociation constant; MIC, metabolite intermediate complex; KTZ, ketoconazole; AZA, azamulin.
-
Portions of this work were presented at the International Society for the Study of Xenobiotics meeting, Orlando, Florida, October 27–31, 2002; Abstract No. 355.
- Received February 25, 2003.
- Accepted September 22, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|