Correlation Between Endogenous Cortisol Metabolism And The Ratio Of CsA Blood Concentrations To Dose

Objective The present study was undertaken to demonstrate if a correlation can be seen between cortisol (F) and cortisone (E) 6β-hydroxylation metabolism and the ratio of cyclosporine (CsA) blood concentrations to dose.Method Simultaneous determination of F and E in human urine was performed by high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV). The separation was achieved on a Diamonsil C18 column (200×4.6mm,5μm), monitored by UV absorption at 254nm and operated at 1.0 mL·min^-1 using the gradient elution of methanol and water containing 0.1% formic acid. The extraction solvent was dichlormethane. Simultaneous determination of 6β-hydroxycorisol (6β-OHF) and 6β-hydroxycortisone (6β-OHE) in human urine was also performed by HPLC-UV. The separation was achieved on a Diamonsil C18 column (200×4.6mm,5μm), monitored by UV absorption at 244nm and operated at 1.0 mL·min^-1 using the gradient elution of acetonitrile and water containing 0.1% formic acid. The procedure consisted of steps as followed: elution of the urine sample with ethyl acetate-diethyl ether on an Oasis HLB extraction cartridge and subsequent wash of the organic extracts with alkaline and acidic solutions saturated with sodium sulfate. In the study on correlation between F and E 6β-hydroxylation metabolism and the C/D ratio of CsA in renal transplant recipients, 105 renal transplant recipients (80 males and 25 females) participate in this study. Blood and urine samples were collected before CsA administration on the study day. 6β-OHF,6β-OHE,F and E in urine were determined by HPLC-UV. Trough concentrations of CsA in whole blood were assayed by FPIA. Correlations between 6β-OHF+6β-OHE/F+E and the C/D ratio of CsA, 6β-OHF+6β-OHE/F+E and weight-normalized C/D ratio of CsA were examined by Spearman's rank correlation analysis. Parameter values obtained under different conditions were compared using Wilcoxon's rank sum test.Result A good separation of F,E and internal standard was obtained. The lower limits of quantitation were 5.0 ng/ml for F and E in urine. Accuracy determined at three concentrations ranged between 89.74% and 107.64%. The intra-day and inter-day relative standard deviations were less than 12.21%. Good linear correlations in urine were found between 5.0 and 320.0 ng-ml^-1 for F, 5.0 and 400.0 ng-ml^-1 for E, respectively. A good separation of 6β-OHF,6β-OHE and internal standard was also obtained. The lower limits of quantitation were 5.0 ng/ml for F and E. Accuracy determined at three concentrations ranged between 92.16% and 109.77%. The intra-day and inter-day relative standard deviations were less than 7.4%. Good linear correlations were found between 5.0 and 500.0 ng-ml^-1 for 6β-OHF, 5.0 and 400.0 ng-ml^-1 for 6β-OHE, respectively. In the study on correlation between F and E 6β-hydroxylation metabolism and the C/D ratio of CsA in renal transplant recipients, there were inverse correlations between 6β-OHF+6β-OHE/F+E and the C/D ratio of CsA, 6β-OHF+6β-OHE/F+E and weight-normalized C/D ratio of CsA in all, male and female subjects. Correlations were also found during different postoperative periods. An inverse correlation between them was weaker with calcium channel blockers co-administration. Significant differences were seen in the C/D ratio of CsA and weight-normalized C/D ratio of CsA during different postoperative periods, but not in 6β-OHF+6β-OHE/F+E. 6β-OHF+6β-OHE/F+E and the C/D ratio of CsA showed sexual dimorphism while weight-normalized C/D ratio of CsA didn't. A decrease was also seen in 6β-OHF+6β-OHE/F+E with calcium channel blockers co-administration. But no significant differences were found in the C/D ratio of CsA and weight-normalized C/D ratio of CsA.Conclusion The present study provides two simple, sensitive and accurate techniques for simultaneous determination of 6β-OHF and 6β-OHE or F and E in human urine. An inverse correlation has been found between 6β-OHF+6β-OHE/F+E and the C/D ratio of CsA. It may be used as a predictor of pharmacokinetics of CYP3A and P-gp substrates belonged to the biopharmaceutical classification system (BCS)Ⅱ, but the more precise indexes will be studied in our further researches.