| Baicalin(BG)is one of the active and quality control ingredients in Scutellaria baicalensis Georgi(SR).BG has been shown to exert anti-inflammatory,antiviral,antioxidant pharmacological effects,widely used in the treatment of acute and chronic hepatitis and hepatitis of chronic defer sex.Baicalin and baicalensis compound preparation have been usually used in clinical trials,the interaction with other drugs are also frequently reported.The drug interaction is important reason for drug adverse reactions,and the interactions between baicalin and other drugs study has a guiding significance about the evaluation of rational drug use.Liver transplantation is the choice of patients with various acute or chronic liver diseases that cannot be controlled or cured with other treatments.By November 2009,China Liver Transplant Registration(CLTR)registered a total of 11,457 liver transplants performed between 1993 and 2007.About 75% of the recipients were hepatitis B patients.Cyclosporine(Cs A)as a potent immunosuppressant with narrow therapeutic ranges and it has great individual difference,which effectively inhibits organ rejection in patients after transplantation.The 5-year survival rate of liver transplantation can reach 70% to 75%.Cs A is mainly metabolized by CYP3 A in the liver and small intestine,and it is also a substrate for P-glycoprotein(P-gp).For liver transpiant patients need long-term use of cyclosporine,and at the same time need antiviral treatment,which increases the opportunities for clinical use of cyclosporine and baicalin.Therefore,studies of the effect and mechanism of baicalin on cyclosporine pharmacokinetics in rats to provide a theoretical reference for the clinical application of drugs.Methods 1.Effects of BG on the pharmacokinetics of Cs A 1.1 Determination of Cs A concentration in rat blood by LC-MS/MS The method for analysis of Cs A in whole blood was established and its specificity,accuracy,precision,recovery and stability were investigated.1.2 Administration and blood collection 1.2.1 Effect of intravenous administration BG on pharmacokinetics of Cs A in rats In the i.v.group,single-dose of Cs A(3 mg·kg-1)was given to rats via tail vein with a 3-day washout period between treatments.BG(80 mg·kg-1)were injected via the tail vein for 7 days and Cs A(3 mg·kg-1)was given to rats immediately after the multiple dose of BG.Blood samples(0.2 m L)were collected in vacutainer tubes containing EDTA at 0,0.0167,0.083,0.33,0.67,1,1.5,2,3,4,6,8,12,24,36 and 48 h after intravenous cyclosporine.The whole blood was obtained and frozen at-80 °C prior to analysis.1.2.2 Effect of oral administration BG on pharmacokinetics of Cs A in rats In the oral group,twelve rats were randomly separated into control and BG treatment groups(n=6).Oral administration was given via gastric gavage.Saline was used as a vehicle control and was injected into the rats at the time of BG(80 mg·kg-1)administration for 7 days.Cs A(3mg kg-1)was immediately given to rats after the last administration of BG.Blood samples(0.2 m L)were collected in vacutainer tubes containing EDTA at 0,0.33,0.67,1,1.5,2,3,4,6,8,12,24,36 and 48 h after oral cyclosporine.The specimens were stored at-80°C until analyzed by LC–MS/MS.2.The effect of BG on the expression of CYP3 A and P-gp in liver and intestine2.1 Preparation of microsomes and tissues After the last blood collection,the rats were fasted for 12 hours and blood was taken from the abdominal aorta.The liver and intestine were removed immediately microsomes were prepared by differential centrifugation method.Take liver and intestine tissue respectively,and add RIPA lysis at a ratio of 1:10 to fully homogenize in 4°C condition.The supernatant is the toal protein after centrifugation and protein concentration was determined using the Bradfosrd method,then stored at-80 °C.2.2 Measurement the protein expression of CYP 3A and P-gp The effect of multiple doses of BG(80 mg·kg-1,7 days,i.g.)on the expression of CYP3 A and P-gp in the liver and intestine was detected by Western Blot.3.Effect of BG on P-gp activity and protein expression in MDCK-P-gp cells 3.1 Determination of R123 concentration by LC-MS/MS The method for analysis of R123 in cell lysate are examined,which included recovery,precision,stability,sensibility and linear range and so on.3.2 BG on P-gp activity After 48 h of BG(1,2,10 and 20 μM)administration of MDCK-Pgp cells,the contents of R123 and Cs A specific substrates of P-gp in cells were determined by LC-MS.Protein concentration was determined by the BCA method and the activity was determined by the amount of R123 and Cs A per mg of protein in the cells.3.3 BG on P-gp a protein expression After being treated with BG(1,2,10 and 20 μM)for 48 h,MDCK-Pgp cells were extracted with total protein.The effect of BG on the expression of Pgp protein was detected by Western Blot.4.Statistical analysisWinolin software was used to calculate the pharmacokinetic parameters of cyclosporine in rats,and SPSS17.0 software for statistical analysis of the data.One-way ANOVA was used to test the effect of BG on the pharmacokinetics of cyclosporine in rats.If experimental data does not meet the normal distribution,then apply the rank sum test in the non-parametric test for analysis.A value of P <0.05 considered statistically significant.Result 1.Effects of BG on the pharmacokinetics of Cs A 1.1 Analytical method Endogenous substances in whole blood did not interfere with the determination of the concentration of cyclosporine,the baseline noise was small,and the linear relationship of cyclosporine was well within the concentration range of 10 ng·m L-1 to 3000 ng·m L-1(r=0.9985).Precision,accuracy,recovery and stability all meet the requirements of biological sample determination.The method established in this article is sensitive and reliable,and can be used to determine the concentration of cyclosporine in the whole blood of rats.1.2 Effect of intravenous administration BG on pharmacokinetics of Cs A in rats The main pharmacokinetic parameters of Cs A in rats administrated with single dose Cs A(3 mg·kg-1,i.v.)and combined with BG(80 mg·kg-1,7 days,i.v.)were as follows: the Cmax were 2.91±0.51 and 2.56±0.28 μg·ml-1,AUC0~∞ were 10.57±0.35 and 11.47±0.99 h·μg·ml-1,CL/F were 0.29±0.01 and 0.28±0.03 L·h-1,respectively.The results indicated that intravenous administration BG did not affect Cs A pharmacokinetic parameters 1.3 Effect of oral administration BG on pharmacokinetics of Cs A in rats After treatment with saline or BG(80 mg·kg-1,7 days,i.g.),the keypharmacokinetics of Cs A in control group and BG group were as follows: Cmax were 0.53±0.04 and 0.33±0.02 μg·ml-1,AUC0~∞ were 4.16±0.29 and 3.12±0.19 h·μg·ml-1,CL/F were 0.74±0.05 and 0.98±0.06 L·h-1,respectively.The results suggest that oral administration of BG significantly reduced the absorption of Cs A in rats.2.Effects of BG on the expression of CYP3 A and P-gp in rat liver and intestine Compared with the control group,multiple dose BG(80 mg·kg-1,7 days,ig)had no effect on the expression of CYP3 A protein in the liver and intestine,and also did not change the protein expression of P-gp in the liver,but it was significantly upregulated the expression of P-gp protein in the intestine is about 71%.It is suggested that BG can significantly change the expression of P-gp in the intestine of rats after multiple administrations.3.Effects of BG on P-gp activity and protein expression in MDCK-P-gp cells 3.1 Analytical method The method was high specificity and reproducibility,high sensitivity,extraction recovery and precision conforming to the requirements of sample determination,and can provide convenient and rapid experimental means for evaluating the activity and function of P-gp in cells.3.2 Effect of BG on P-gp activity BG at(1,2,10 and 20 μM)significantly decreased the intracellular accumulation of R123 by 41%,41%,35% and 42 %,respectively(P<0.01).Similarly,BG significantly decreased the intracellular accumulation of Cs A by 23,30,33 and 22 %,respectively.The results suggest that BG can be increased P-gp activity and the amount of substrate accumulation in the cells can be reduced.3.3 Effect of BG on P-gp protein expressionBG at 2,10 and 20μM treatment led to a significant increase in the protein expression levels of P-gp by 136,165 and 173%,respectively(P<0.05).It can be seen that BG can increase the expression of P-gp protein.Conclusion 1.The clinically equivalent dose of BG intravenous administration had no effect on the pharmacokinetics of Cs A in rats.2.The clinically equivalent dose of BG oral administration can significantly reduce the Cmax and AUC0~∞ of Cs A in rats,and the mechanism may be related to BG induced intestinal P-gp expression.3.BG can increase the P-gp activity and protein expression of MDCK-Pgp cells. |
