Received for publication July 24, 2006.
Revised December 4, 2006.
Accepted for publication December 5, 2006.

METABOLISM OF SIROLIMUS IN THE PRESENCE OR ABSENCE OF CYCLOSPORIN BY GENOTYPED HUMAN LIVER MICROSOMES AND RECOMBINANT CYTOCHROMES P450 3A4 AND 3A5

Abstract

Sirolimus is an immunosuppressive drug currently used alone or in combination with cyclosporine. Both drugs undergo extensive metabolism by the CYP 3A enzymes. This study aimed at comparing the activity of recombinant CYP (rCYP) 3A4 and 3A5 toward sirolimus; investigating the effect of cyclosporine on the metabolic rate of these two CYPs, as well as the impact of the CYP 3A5*3 polymorphism on that of human liver microsomes (HLM). Two distinct approaches were used, i.e. the measurement of: (i) hydroxy-sirolimus and desmethyl-sirolimus production; and (ii) sirolimus depletion by the in vitro half-life method. rCYP 3A5 exhibited a lower intrinsic clearance (CL_int) for both hydroxylation (0.11 vs 0.24 µl/pmol CYP/min) and depletion of sirolimus (0.64 vs 2.36 µl/pmol CYP/min) than rCYP 3A4. Similar CL_int of hydroxylation, demethylation and depletion were found when comparing a pool of HLM carrying at least one CYP 3A5*1 (active) allele with a pool of HLM not expressing CYP3A5. This was further confirmed for sirolimus depletion using individual microsome preparations (p=0.42). A deeper inhibitory effect of cyclosporine on the CL_int of sirolimus depletion was found for rCYP 3A4 than for rCYP 3A5 (i.e. -44 % vs -8 % at 0.62 µM, i.e.750 µg/l cyclosporine) and sirolimus metabolism was slightly less inhibited for HLM expressing CYP 3A5 than not (-38 % vs -56%). In the absence of cyclosporine, the CYP 3A5*3 polymorphism may not influence significantly sirolimus metabolism at the hepatic level. However, CYP3A4 strong inhibition by cyclosporine could unveil the influence of this polymorphism.

Key words: cytochrome P450, drug-drug interactions, mass spectrometry, microsomes, pharmacogenetics