Metabolism of Sirolimus in the Presence or Absence of Cyclosporine by Genotyped Human Liver Microsomes and Recombinant Cytochromes P450 3A4 and 3A5

Abstract

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 cytochromes P450 (P450s), as well as the impact of the CYP 3A5*3 polymorphism on that of human liver microsomes (HLMs). Two distinct approaches were used; i.e., the measurement of (1) hydroxy-sirolimus and desmethyl-sirolimus production, and (2) sirolimus depletion by the in vitro half-life method. rCYP 3A5 exhibited a lower intrinsic clearance (CL int ) for both hydroxylation (0.11 versus 0.24 l/pmol P450/min) and depletion of sirolimus (0.64 versus 2.36 l/pmol P450/min) than rCYP 3A4. Similar CL int values for hydroxylation, demethylation, and depletion were found when comparing a pool of HLMs carrying at least one CYP 3A5*1 (active) allele with a pool of HLMs not expressing CYP 3A5. 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% versus ؊8% at 0.62 M, 750 g/l cyclosporine), and sirolimus metabolism was slightly less inhibited for HLMs expressing CYP 3A5 than not (؊38% versus ؊56%). In the absence of cyclosporine, the CYP 3A5*3 polymorphism may not influence significantly sirolimus metabolism at the hepatic level. However, strong CYP 3A4 inhibition by cyclosporine could unveil the influence of this polymorphism.

Effect of CYP3A4*22, POR*28, and PPARA rs4253728 on Sirolimus In Vitro Metabolism and Trough Concentrations in Kidney Transplant Recipients

“…We did not find any association between the CYP3A5*1/*3 genotype and SRL blood concentrations. We previously provided in vitro evidence that CYP3A5 genotype had no influence on SRL metabolism by HLM (18 ). We indeed observed similar intrinsic clearance (CL int ) for SRL hydroxylation, demethylation, and depletion between HLM carrying a CYP3A5*1 (active) allele and HLM not expressing CYP3A5.…”

“…SRL (100 g/L) was incubated with individually prepared human liver microsomes (HLM) (n ϭ 31) (donor characteristics are given in Table 1 in the Data Supplement that accompanies the online version of this report at http://www.clinchem.org/content/vol59/ issue12) and pooled HLM as previously described (18 ). Briefly, the incubation mixture consisted of 0.1 mol/L Tris buffer, pH 7.4, 10 mmol/L MgCl 2 , 2 mmol/L NADPH, HLM (0.1 mg protein/mL), and SRL prepared in acetonitrile/water (25/1.25 L) (1.25% acetonitrile in final incubation medium).…”

“…After 10 min, the reaction was stopped using ice-cold acetonitrile (100 L). The remaining concentration of SRL in the incubation mixture was determined by using liquid chromatographytandem mass spectrometry (LC-MS/MS) as described previously (18 ).…”

“…SRL was determined in blood and in vitro samples by using turbulent-flow chromatography-tandem mass spectrometry using a validated, previously described method (18 ).…”

Genes involved in the regulation of vascular homeostasis determine renal survival rate in patients with chronic glomerulonephritis

“…The structure of the primers and PCR conditions for genotyping the DNA polymorphisms are described in detail elsewhere (Agerholm-Larsen et al, 2000;Asai et al, 2001;Jalilian et al, 2008;Lanfear et al, 2005;Picard et al, 2007;Prasad et al, 2006;Yazdanpanah et al, 2007).…”