Vol. 25, Issue 12, 1383-1388, 1997

In Vitro Biotransformation and Identification of Human Cytochrome P450 Isozyme-Dependent Metabolism of Tazofelone

Sekhar S. Surapaneni, Michael P. Clay, Larry A. Spangle, Jonathan W. Paschal, and Terry D. Lindstrom

Department of Drug Disposition (S.S.S., M.P.C., T.D.L.), Department of Research Technology and Proteins (L.A.S., J.W.P.), Lilly Research Laboratories, Eli Lilly and Company

Tazofelone is a new inflammatory bowel disease agent. The biotransformation of tazofelone in human livers and the cytochrome P450 responsible for the biotransformation has been studied. Two metabolites of tazofelone were formed in vitro. A sulfoxide metabolite was identified by cochromatography with authentic standards, and a quinol metabolite of tazofelone was identified by mass spectrometry and proton NMR. Sulfoxidation was catalyzed by a single enzyme system while formation of the quinol metabolite was catalyzed by a two enzyme system. The K_m and V_max values for sulfoxidation were 12.4 µM and 0.27 nmol/min/mg protein, respectively. The high affinity K_m and V_max values for the formation of the quinol metabolite were 7.5 µM and 0.17 nmol/min/mg protein, respectively. Tazofelone was incubated at 20 µM concentration with human microsomes to determine which of the cytochrome P450 isozyme(s) is involved in the oxidation of tazofelone. A strong correlation was found between the immunoquantified concentrations of CYP3A and the rates of formation of the sulfoxide and quinol metabolites of tazofelone. Similarly, significant correlations were observed between the formation of midazolam 1-hydroxylation and the rates of formation of both metabolites of tazofelone. Inhibition studies have indicated that triacetyloleandomycin, a CYP3A specific inhibitor, almost completely inhibited the formation of both of these tazofelone metabolites. Incubations with specific cDNA expressed microsomes indicated that the formation of both the sulfoxide and quinol metabolites was highest with CYP3A4 containing microsomes. The correlation data was confirmed by inhibition studies and cDNA expressed cytochrome P450 systems demonstrating that the biotransformation of tazofelone to its metabolites is primarily mediated by CYP3A.