Identification of rat cytochrome P450 forms involved in the metabolism of clausenamide enantiomers.

Abstract

To identify which cytochrome P450 (CYP) isoform(s) are responsible for the metabolism of clausenamide (CLA) enantiomers in rats, effects of various CYP isoform inducers and inhibitors on the formation of CLA metabolites were investigated in liver microsomes. In incubations with rat liver microsomes, CLA enantiomers were mainly converted to 4-hydroxy, 5-hydroxy, and 7-hydroxy-metabolites. 4-OH-CLA was the major metabolite of (+)-3R, 4S, 5S, 6R-CLA [(+)-CLA], while 7-OH-CLA was the major one of (-)-3S, 4R, 5R, 6S-CLA [(-)-CLA]. In induction studies, enzymatic parameters were used to assess the role of different CYP forms in CLA hydroxylation reactions. A marked increase in the rate of metabolism of CLA enantiomers was observed in microsomes of dexamethasone treated rats, V(max)/K(m) values for 4-OH-(+)-CLA, 7-OH-, 5-OH-, and 4-OH-(-)-CLA were 5.3, 6.5, 3.0, and 5.9 times higher than those in control microsomes, respectively. Rifampicin treatment caused corresponding 1.7-, 2.6-, 3.1-, and 2.8-fold increases. Dex and Rif also increased in the amount of (+)-5- and (+)-7-OH-CLA that were not detectable in the control group. These results suggested that inducible CYP3A1 was involved in the hydroxylation of CLA enantiomers. In inhibition studies, ketoconazone (6.25 microM) completely inhibited the production of main metabolites of (-)-CLA (100%) and (+)-CLA (97%). Triacetyloleandomycin (12.5 microM) strongly inhibited the corresponding metabolites by 34-85%. These findings also indicated that institutive CYP3A2 shared a major role in the hydroxylation of CLA enantiomers with CYP3A1 in untreated rats. Together, the data suggested that CYP3A was the predominant isoform responsible for the metabolism of CLA enantiomers.