IN-VITRO METABOLISM OF CYCLOSPORINE-A WITH RABBIT RENAL OR HEPATIC MICROSOMES - ANALYSIS BY HPLC-FPIA AND HPLC-MS

Cyclosporin A (CsA) is in vivo mainly metabolized by hepatic cytochrom e P450 IIIA to more than 21 metabolites, the major ones known as: M1, M17 and M21. The aim of this work is to explore the in vitro metabolis m of CsA after incubation, in the presence of NADPH, with renal or hep atic microsomes obtained from rabbits pretreated with rifampycin (enzy me inducer) or erythromycin (enzyme inhibitor). The presumed metabolit es were separated by semi-preparative high-performance liquid chromato graphy (HPLC) and identified in each collected fraction by fluorescenc e polarization immunoassay (FPIA) (HPLC-FPIA) using a non-specific pol yclonal antibody. They were also analyzed by HPLC-mass spectrometry (M S) using fast atom bombardment (HPLC-MS-FAB). Five collected fractions gave positive results with FPIA. The major metabolites found were M1, M17 and M21 after identification by HPLC-MS-FAB and comparison with t hree corresponding standard metabolites. The CsA biotransformation rat es were calculated by the amount of unmetabolized CsA and were linear with time. These mean rates (V-m) for 12-min incubation by renal micro somes of rabbits treated with rifampicin or erythromycin or untreated (control) were 0.11, 0.02 and 0.04 nmol/min x mg microsomal protein, r espectively. These rates were 15-, 37-, and 30-fold lower than those o btained with hepatic microsomes of rabbits treated identically. As CsA metabolites are less cytotoxic than the parent drug, this weak renal biotransformation of CsA after in vitro incubation should be one of th e mechanisms of its in vivo nephrotoxicity.