The Effect Of CYP3A5and MDR1Gene Polymorphism On The Metabolic Rate Of Cortisol And Cortisone Evaluating Cclosporine Metabolism In Vivo

Aim:The aim of the present study was to develop an (HPLC)-UV that can simultaneous determine urinary cortisol(F), cortisone(E),6β-hydroxycortisol(6β-OHF) and6β-hydroxycortisone(6β-OHE); and explore the effect of CYP3A5and MDR1gene polymorphism on HOM evaluating cyclosporine metabolism in vivo. HOM is the urinary ratio of6β-OHF and6β-OHE to cortisol and cortisone (6β-OHF+6β-OHE)/(F+E) in Chinese renal transplant recipients.Methods:①Establish an HPLC-UV to simultaneous determine urinary F, E,6β-OHF and6β-OHE. Separation was performed on a reversed-phase C18column (200×4.6mm,5μm, Agilent) monitored by UV absorbance at245nm and operated at1.0mL.min-1using the gradient elution of methanol,acetonitrile and pure water. The internal standard was dexamethasone. The column temperature was maintained at30℃. The method involved a solid phase extraction of the compounds from urine. The sample size was30ul.②Explore the effect of CYP3A5and MDR1gene polymorphism on HOM evaluating cyclosporine metabolism in vivo. On the morning of the study day, blood samples were collected before administration for genotyping and determining cyclosporine steady state concertration. Urine samples were collected at08:00-10:00for determining HOM (6β-OHF+6β-OHE)/(F+E). The demographic parameters, dosing regimens, co-medications and laboratory examinations were recorded. Analysis the effect of CYP3A5*3and MDR1C3435T gene polymorphism on cyclosporine C/D and urinary HOM respectively;Analysis the effect of calcium antagonist on cyclosporine C/D and HOM respectively; Analysis the gender difference of cyclosporine C/D and HOM respectively. Analysis the correlation between cyclosporine C/D and HOM, cyclosporine C/D and age,weight respectively by Spearman test.Results:①The analytes,6β-OHF,6β-OHE, E, F and internal standard DM, were well separated under the described chromatographic conditions. The linear relationship of F in5～320μg·L-1concentration range and E、6β-OHF、6β-OHE in5～400μg·L-1concentration range are good, and the LLOQ of four analytes were5μg·L-1. Precision and accuracy of the assay and the extraction recoveries at low, medium and high quality control samples of the four analytes can meet the standard of biological samples analysis.②There were no significant effect of CYP3A5*3and MDR1C3435T gene polymorphism on cyclosporine C/D and HOM respectively (p>0.05); Also cyclosporine C/D and HOM had no significant gender difference respectively (p>0.05). Co-administration of calcium antagonists also had no significant effect on C/D and HOM (p>0.05).③There was no significant correlation between cyclosporine C/D and age or weight et al.There was significant negative correlation between cyclosporine C/D and HOMγ=-0.739(p<0.01), and the correlation of (CYP3A5*3*3+MDR1CT,TT) group was better than that of (CYP3A5*1*1,*1*3+MDR1CC) group, but the difference was small.Conclusions:①we developed a simple and accurate reversed-phase HPLC assay to quantify simultaneousry E,F,6β-OHF and6β-OHE in human urine, which can optimize the detection and shorten the analysis time. The method offered an effective analysis approach for further study the total6β-hydroxylation clearance of F and E.②There was good negative correlation between HOM and cyclosporine C/D, but CYP3A5*3and MDR1C3435T gene polymorphism had no effect on HOM evaluating cyclosporine metabolism in vivo. The research provided certain theoretical basis on cyclosporine clinical individualized therapy. This study contained12Figures,12tables and84references.