CYCLOSPORINE METABOLISM BY RAT-LIVER MICROSOMES - EVIDENCE FOR INVOLVEMENT OF ENZYME(S) OTHER THAN CYTOCHROMES P-450 3A

Cyclosporine (CyA) metabolism was investigated in liver microsomes obt ained from untreated male and female Sprague-Dawley rats, and rats pre treated with ethinyl estradiol (EE), dexamethasone (DX), and phenobarb ital (PB). Total hepatic microsomal cytochrome P-450 content of DX- an d PB-treated male and female rats was significantly higher than that o f their respective control or EE-treated rats. However, CyA metabolism was significantly increased, by all drug pretreatments, both in male and female rats. EE increased (2-5 fold) the formation of AM9 (a hydro xylated metabolite) and AM1c (a cyclized-hydroxylated product) over th e CyA concentration range tested (0.2-42 muM). DX and PB significantly increased (2- to 20-fold) all detected metabolites (AMI, another hydr oxylated metabolite; AM9; AM4N, an N-demethylated product; and AM1c), especially at high substrate concentrations (above 1.25 muM). Immunobl ot analyses revealed that the microsomal P-450 3A2 content was decreas ed in EE-treated male rats, but markedly induced in those treated with either DX or PB. P-450 3A1 was undetectable in untreated and EE-treat ed female rats, but greatly induced in DX-treated male and female rats . Examination of P-450 3A activity, using 6beta-hydroxytestosterone fo rmation as a probe, confirmed the immunoblot results. These studies su ggest that enzyme(s), other than P-450s 3A1 and 3A2 also play a signif icant role in CyA metabolism. Further analyses using immunoblots and v arious specific P-450 functional markers revealed considerably decreas ed or undetected hepatic microsomal content and/or activities of P-450 s 2B1/2, 2C11, 2E1, and 2A2 from EE-treated male and female rats, indi cating that these P-450s were not responsible for the enhanced CyA met abolism in these animals. Although hepatic microsomal P-450 2C6 conten t, as revealed by Western immunoblotting and the corresponding progest erone 21-hydroxylase were markedly induced in both EE-treated male and female rats, no detectable CyA metabolism was observed in functionall y reconstituted, purified rat hepatic P-450 2C6 or 2C7 systems. The is oenzyme(s) responsible for CyA metabolism in untreated and EE-treated male and female rats remains to be identified.