| Liver transplantation is the only effective means to solve end stage liver disease.Recipients need to take conventional tacrolimus and other immunosuppressive agents to prevent rejection.Tacrolimus as a new calcineurin inhibitor,it is the first-line medication by recommended guidelines because of strong immunosuppressive effect and fewer adverse reactions.Tacrolimus has a narrow therapeutic window with wide inter-individual variability in pharmacokinetics.So it needs therapeutic drug monitoring.Hugan Tablets consists of radix bupleuri,herba artemisiae,radix isatidis,fructus schisandrae,pig bile powder,mung bean,which is good for liver and spleen.It can also lower transaminase.In clinical,organ transplant recipients,especially liver transplant recipients who often take tacrolimus at the same time take Hugan Tablets.We found that Hugan Tablets may increase tacrolimus plasma concentration in the follow-up of recipients.So,is there any drug interaction between Hugan Tablets and tacrolimus? As the liver transplant recipients taking more types of drugs,there is interference in the study of the interaction between the two drugs.Therefore,the animal experiment will be designed to study the effect of Hugan Tablets on the pharmacokinetics of tacrolimus and to provide the basis and reference for the clinical study.At the same time,we will establish a method for the determination of various components in Hugan Tablets,and provide experimental data for further study on the pharmacological effects of tacrolimus on Hugan tablets.Part one Effect of Hugan Tablets on pharmacokinetics of tacrolimus in beaglesObjective: To study the effects of Hugan Tablets on the pharmacokinetics of tacrolimus in beagles,and to observe drug interactions of the two drugs.Methods: The control group(n = 6): tacrolimus alone,test group(n = 6): tacrolimus combined with Hugan Tablets.Self-control method was used.Six beagles were orally administered with a single dose of tacrolimus 1 mg.After the wash-out period,six beagles were orally administered with an additional single dose of tacrolimus 1 mg combined with Hugan Tablets(2 tablets each).Fasting for 12 h before administration,freely drinking water during the period.1m L blood samples were collected from small saphenous vein of the beagles at 0,0.167,0.33,0.5,0.67,0.833,1,1.5,2,3,4,6,8,12,and 24 h after each cycle of the administration and then were put in anticoagulant tube.The concentration of tacrolimus in the beagles was determined by ARCHITECT i1000 SR.The main pharmacokinetic parameters of tacrolimus were calculated by DAS(version 2.1.1)software,including: area under concentration-time curve(AUC0-t,AUC0-?),elimination half time,time of maximum concentration,maximum concentration,apparent volume of distribution,clearance.The data of the plasma concentration at different time and pharmacokinetic parameters of tacrolimus in two groups were compared with SPSS 21.0 statistical software.The measurement data were expressed as mean ± standard deviation,and the paired t test was used for comparison between groups.P <0.05 was considered statistically significant.Results: The pharmacokinetic parameters of tacrolimus before and after co-administration with Hugan Tablets were as follows: AUC0-24h(54.23±33.95)VS.(74.54±32.85);t1/2(13.84±9.05)VS.(8.71±4.49)h;T max(0.72±0.40)VS.(0.86±0.37)h;C max(17.17±7.80)VS.(22.93±8.10)?g·L-1;CL/F(2.22±1.67)VS.(1.42±0.67)L·h-1;V/F(37.54±24.77)VS.(17.50±11.82)L.Compared to tacrolimus alone,the AUC0-24 h and C max of tacrolimus after co-administration of Hugan Tablets had an average increment of 37.5% and 34%,respectively,an average decrement of 36% and 53% for CL/F and V/F,respectively.The difference was statistically significant(P<0.05).T max delayed and t1/2 slowed down.The difference was not statistically significant(P>0.05).Conclusions: Hugan Tablets can improve the concentration and bioavailability of tacrolimus in the whole blood of Beagle dogs,and reduce the drug clearance rate and apparent distribution volume.Part Two Simultaneous determination of Chlorogenic Acid,Schisandrol A, Schisandrin B,Schisandrin B in Hugan Tablets by HPLC with wavelength switching detection methodObjective: To establish a HPLC method of wavelength switching for simultaneous determination the contents of Chlorogenic Acid,Schisandrol A,Schisandrin A,Schisandrin B in Hugan Tablets,and to provide a basis for further study of tacrolimus on the pharmacological effects of Hugan Tablets.Methods: ZORBAX SB-C18(4.6 mm×150 mm,5 ?m)column was used for seperation.The mobile phase was 0.2% phosphoric acid solution(A)-acetonitrile(B)with linear gradient elution [ 0~5 min,B(13%);5~15 min,B(13%~55%);15~23 min,B(55%~62%);23~25 min,B(62%~70%);25~29 min,B(70%~75%);29~35 min,B(75%);35~35.10 min,B(75%~13%);35.10~45 min,B(13%)] at the flow rate of 1.0 m L·min-1.The column temperature was controlled at 35?,the detection wavelength was 327 nm(0-6 min)for Chlorogenic Acid,250 nm(16-45 min)for Schisandrol A,Schisandrin A,Schisandrin B.The samples size was 10 ?L.Results: A HPLC method of wavelength switching was established for simultaneous determination of the contents of Chlorogenic Acid,Schisandrol A,Schisandrin A,Schisandrin B in Hugan Tablets.Chlorogenic Acid,Schisandrol A,Schisandrin A,Schisandrin B were well separated,less impurity interference,R>1.5,the retention time of Chlorogenic Acid,Schisandrol A,Schisandrin A,Schisandrin B were 4.02 min(4.35 min),18.29 min,29.32 min and 31.29 min,respectively.The standard curve equations of Chlorogenic Acid was Y=28.26330X+24.9888,r=0.99939(n=5),the linear ranges was 4~100 ?g·m L-1;the standard curve equation of Schisandrol A was Y=21.21935X-8.43444,r=0.99994(n=5),the linear ranges was 4~100 ?g·m L-1;the standard curve equation of Schisandrin A was Y=20.07794X-2.83327,r=0.99993(n=5),the linear ranges was 1~25 ?g·m L-1;the standard curve equation of Schisandrin B was Y=17.76046X-2.51254,r=0.99992(n=5),the linear ranges was 2~50 ?g·m L-1.RSD(n=6)of precision tests were 0.32%,0.58%,0.14%,0.12%,respectively;RSD(n=6)of repeatability tests were 1.03%,0.82%,0.91%,0.53%,respectively;RSD(n=6)of stability tests were 0.94%,0.31%,0.77%,0.21%,respectively;The average recovery of the four components were 103.24%,101.21%,101.01%,98.77%,RSD(n=6)were 2.09%,1.66%,1.77%,1.48%.Conclusions: The method is simple,accurate and reproducible.It can be used to determine Chlorogenic Acid,Schisandrol A,Schisandrin A,Schisandrin B in Hugan Tablets with the same chromatogram condition,and to provide the experimental basis for further study of tacrolimus on the pharmacological effects of Hugan Tablets. |
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