Effects Of CP-25 On The Pharmacokinetics,Tissue Distribution And Excretion Of Leflunomide In Rats

Paeoniflorin-6′-O-benzene sulfonate（CP-25）is a novel ester derivative of paeoniflorin（Pae）.CP-25 has significantly higher lipophilicity,bioavailability and biological activity than Pae.Following oral administration in rats,CP-25 has the advantage of exhibiting good absorption,good distribution,a low clearance rate,long average retention time and moderate bioavailability.CP-25 has significant therapeutic effect on animal models of collagen-induced arthritis（CIA）and Adjuvant arthritis（AA）.It can also inhibit the abnormal proliferation and migration of rat fibroblast-like synoviocytes（FLS）.Therefore,CP-25 is a potential anti-inflammatory and immunomodulatory agent.Leflunomide（LEF）is an disease modifying antirheumatic drug（DMARD）that can be used to treat autoimmune diseases,such as rheumatoid arthritis,psoriasis,systemic lupus erythematosus and sarcoidosis.The use of LEF may involve serious adverse reactions in the blood,liver,immune system and skin system,as well as side effects such as diarrhea.LEF is a prodrug,and when administered orally,it is rapidly and almost completely converted into its active metabolite,teriflunomide（TER）.TER,as LEF,has been approved for the treatment of multiple sclerosis and inhibits the synthesis of dihydroorate dehydrogenase and pyrimidine.The pharmacokinetics of TER are not affected by food,age or sex.Previous studies have shown that CP-25 combined with LEF can increase efficacy and reduce hepatotoxicity in AA rats,but the mechanism has not been clarified.In this study,we attempted to explore the synergistic mechanism of CP-25 and LEF from the perspective of pharmacokinetics.Objective:1.To study the effects of CP-25 on the pharmacokinetics,tissue distribution and excretion of LEF in rats.2.To investigate the effect of CP-25 on TER absorption in rat peripheral blood mononuclear cells（PBMCs）.Methods:We developed an UPLC-MS/MS-based method for the determination of levels of TER（an active metabolite of LEF）,and to verify its selectivity,lower limit of quantitation,linearity,precision and accuracy,recovery,matrix effect and stability.This method was used to determine TER concentrations in the plasma,urine,feces,and bile,heart,liver,spleen,lung,kidney,intestinal,brain,and synovial tissues,and peripheral blood mononuclear cells（PBMCs）of rats in the control[LEF（10 mg/kg）]and combined[CP-25（50 mg/kg×7d）plus LEF（10 mg/kg）]groups.Results:1.Endogenous substances in the biological samples did not interfere with the determination of the samples,and good baseline separation from TER and IS was achieved.The retention time of TER in plasma,urine,feces,bile and tissue analysis of rats was about 0.5 min,and that of IS was about 0.40 min.There was no interference of impurity peaks near the drug peak and IS peak,and endogenous substances in biological samples did not interfere with the content determination in vivo.The concentration of the lower limit of quantification was determined by a signal to noise ratio of 5:1.The RSD and RE of LLOQ samples from 5 different sources（plasma,urine,feces,bile,heart,liver,spleen,lung,kidney,brain,small intestine,synovium and PBMC）were investigated,and the RSD and RE were all within 20%,meeting the requirements of biological sample detection conditions.The standard curve equation was obtained by linear regression with sample concentration as abscissa and the ratio of TER peak area to IS peak area as ordinate,and its R2 were all greater than 0.999,which met the requirements of biological sample detection.The intra-day and intra-day precision and accuracy of QC samples were investigated.Precision is expressed by RSD and accuracy is expressed by RE between the measured concentration and the concentration added.The RSD and RE of QC samples were within±15%,which met the requirements of biological samples.The recovery and matrix effect of TER in different biological samples of rats ranged from 85%to 115%,and the RSD of QC samples was within±15%,which met the requirements of sample analysis method.RSD and RE of stability of QC samples of TER under different conditions were within±15%,which met the requirements of test conditions of sample analysis method.2.The mean plasma concentration-time distribution curve of TER in control group and combined group showed that TER elimination was accelerated in rats after combined treatment with LEF and CP-25.Compared with the control group,the AUC,T_max,MRT,T_1/2α,T_1/2βof TER in the combined group decreased（P<0.05）,CL increased（P<0.01）,suggesting that CP-25 can promote the excretion or metabolism of LEF in rats.3.Compared with the control group,TER excretion in urine of rats in the combined group was significantly increased at 0-6h,6-12h,12-18h,18-24h,24-36h and 36-48h.Within 48h,the cumulative excretion of TER in urine of rats in the combined group was about 2.4 times that in the control group.Compared with the control group,the excretion of TER in feces of rats in the combined group was significantly increased during 0-6h,6-12h and 12-24h.Within 48h,the cumulative excretion of TER in feces of rats in the combined group was about 2.5 times that in the control group.Compared with the control group,the excretion of TER in bile of rats in the combined group was significantly increased at 0-4h and 12-18h.Within 24h,the cumulative excretion of TER in bile of rats in the combined group was about 1.7 times that in the control group.4.CP-25 combined with LEF could reduce TER distribution in heart,spleen,lung and intestine at 2h and 9h,but had no effect at 24h.CP-25 could decrease TER distribution at 2h,9h and 24h in liver.CP-25 could reduce the distribution of TER in kidney and brain at 2h and 9h,but the distribution at 2h was not statistically significant,which was due to the large individual difference.CP-25 increased TER distribution at 9h and 24h in synovium.5.The combined administration of CP-25 and LEF significantly improved the ability of PBMCs to uptake TER and increased TER content in PBMCs both in vivo or in vitro.Conclusions:Oral administration of CP-25 combined with LEF can promote the excretion of TER,and reduce its contents in most tissues and organs,especially its exposure to the liver,this is one of the reasons why CP-25 can reduce liver damage caused by LEF.CP-25also increased TER exposure in the synovium and its absorption by PBMCs,and this could explain the synergistic effects of CP-25 and LEF.