Studies On Cellular Pharmacokinetics Of CP-25 In PBMC And Interaction With P-glycoprotein

Rheumatoid arthritis(RA)is an autoimmune disease.Immune cells play an important role in different stages of RA disease.A complex network of cell-cell interactions was formed to control the occurrence and development of RA,including releasing of inflammatory mediators,induction of cell proliferation and angiogenesis.The study of cellular pharmacokinetics has gradually became a branch of traditional pharmacokinetics and has received increasing attention.The drug enters the cell through passive transport or active transport.At the same time,it would be effluxed by transport proteins.The over-expression of transporters was the main reason for the drug resistance of same drugs.The pharmacokinetic properties of the cells would be changed and the concentration of drug in the cells would be decreased.Multidrug resistance(MDR)was widely regarded as an important cause of treatment failure in patients with tumors and infectious diseases.However,recent studies show that this phenomenon also exists in RA.P-glycoprotein(P-gp),as an important member of the ABC transport superfamily,was closely related to MDR and participates in the transport of multiple disease-modifying anti-rheumatic drugs(DMARDs).Research had shown that paeoniflorin(Pae)is a substrate of P-gp,while Pae can downregulate the expression of P-gp in tumor cells.Paeoniflorin-6?-O-benzene sulfonate(CP-25)is a novel active monomer compound.Immune cells are important target cells for CP-25 to exert anti-inflammatory and immunomodulatory effects.Our current research shows that CP-25 can bind to GRK2 in cytoplasm of immune cells and inhibit the inflammation-related signaling pathways.Therefore,it is important to study the pharmacokinetics of CP-25 in immune cells.At the same time,CP-25 is a structural modification of Pae,is it a substrate of P-gp like Pae and can down-regulate the expression of P-gp.And whether the MDR can be found in the treatment of CP-25.Based on the above background,the UPLC-MS/MS analysis method was established to study absorption and efflux of CP-25 in PBMC and the drug concentration-time curves.Transport protein inhibitors were used to determined whether CP-25 is a substrate for P-gp.Investigating the effect of inhibition and overexpression of P-gp on the concentration of CP-25 in PBMC.Western blot and immunohistochemistry were used to investigate the effect of CP-25 on the expression P-gp in rat PBMC and tissues.It provides a basis for future research on pharmacodynamics and drug combinations.Objective: To study the cellular pharmacokinetics of CP-25 in PBMC and interaction with P-pg.Providing a basis for future research on pharmacodynamics and drug combinations.Method: 1)The UPLC-MS/MS method was established to determination of CP-25 and Pae in rat plasma and PBMC samples.2)The amount of CP-25 and Pae transported by PBMC was measured.The effect of concentration,time and temperature on the transport of CP-25 and Pae was also examined.Detecting the concentration of CP-25 and Pae in plasma and PBMC after single administration.Detecting the concentration of CP-25 and Pae in plasma and PBMC after single and multiple administration,and comparing the differences between plasma and PBMC.Detecting the concentration of CP-25 in plasma and PBMC after single and multiple administration in normal and AA rats.Investigating the effect of AA on the concentration of CP-25 in PBMC.3)The amount of CP-25 effluxed by PBMC was measured.The effects of time,transporter inhibitors and overexpression of P-gp on the efflux of CP-25 were also investigated.Western blot and Rh123 accumulation assay were used to detect the effect of CP-25 on expression and function of P-glycoprotein(P-gp)of PBMC which was stimulated by TNF-?.The relationship between the expression of P-gp and the concentration of CP-25 in PBMC was investigated.4)The expression of P-gp in PBMC was detected by Western blot at different stages after the treatment.The concentration of CP-25 was detected by UPLC-MS/MS at 3 h and 6 h after Administration.After treatment,the rats brain,liver,heart,kidney,spleen,stomach,small intestine,colon and lung were taken.Immunohistochemical staining was used to locate the expression of P-gp in all tissues.Imagepro-plus software was used for semi-quantitative analysis of P-gp.Results:1)The linear range of CP-25 and Pae in plasma samples was 10-800 ng/m L.The linear range of CP-25 in PBMC samples was 0.4~5 ng/m L,the linear range of Pae in PBMC samples was 5~20 ng/m L.The accuracy and accuracy,recovery rate,matrix effect and stability of the two methods were all in line with the provisions and requirements of detection and analysis of biological samples.Two methods can meet the requirements in our experiment.2)The transport of CP-25 and Pae in PBMC was gradually decreasing with the increase of incubation time,and reaching dynamic equilibrium after 1 h.Temperature has no effect on transport of CP-25 and Pae.After single administration(100 mg/kg),the concentration of CP-25 in plasma was 1.33 times that of Pae,and the concentration of CP-25 in PBMC was 1.94 times as much as Pae.After multiple administration(50mg/kg),the concentration of CP-25 in PBMC was similar to that of CP-25 in plasma.After single administration(50 mg/kg),the average PBMC/plasma ratio of CP-25 concentration was 71.05 %(range from 45.42 % to 89.99 %).The concentration in PBMC has some correlation with in plasma(r=0.814,P<0.001).After single administration(50 mg/kg),the AUC(0-?)and MRT(0-t)of AA rats was significantly lower than that of normal rats(AUC(0-?): 154.663±20.775 vs 199.752±36.955 ug/L*h,P<0.05;MRT(0-t): 3.129±0.064 vs 3.362±0.092 h,P<0.01).After multiple administration(50mg/kg),the AUC(0-t)and MRT was significantly lower than that of normal rats(AUC(0-t):113.517±19.856 vs 136.866±21.391 ug/L*h,P<0.01;MRT(0-t): 2.047±0.179 vs3.077±0.053 h,P<0.01;MRT(0-?): 4.124±0.796 vs 7.576±2.480 h,P<0.05).3)The efflux of CP-25 by PBMC was gradually increasing with the increase of time before 30 min,which follows a linear trend and gradually decreasing after 30 min.Verapamil could significantly increase the concentration of CP-25 in PBMC.Three kinds of P-gp inhibitors could significantly increase the concentration of CP-25 in PBMC after CP-25 was removed from medium,and concentration dependence was observed within certain range.The efflux of CP-25 was dramatically increased by PBMC which was stimulated by TNF-?.The concentration of CP-25 in PBMC which was stimulated by TNF-? was less than half of the normal PBMC after 30 min,and as same as concentration of CP-25 in normal PBMC after 60 min.In addition,the initial concentration of CP-25 in PBMC which was stimulated by TNF-? was significantly lower than that of normal PBMC.CP-25 could inhibit expression and function of P-gp on PBMC which was stimulated by TNF-?,and time and concentration dependence were observed.With the increasing of incubation time,the concentration of CP-25 in PBMC tends to increase.The concentration of CP-25 in PBMC at 24 h was significantly higher than at 2 h,but was as same as at 36 h.4)From fourth days after the treatment to the end of the treatment,CP-25 could gradually reduce the expression of P-gp in AA rats PBMC,and dose dependence were observed.From twelfth days after treatment(50 mg/kg),the concentration of CP-25 in AA rat PBMC was significantly increased at 6 h after administration compared with 6 h after the first day of administration.No significant changes were observed at 3 h after administration.However,from eighth days after treatment,the mean drug concentration ratio of 6 h to 3 h was higher than that prior treatment,and there was a markedly difference from the twelfth days.The expression of P-gp was found in all examined tissues.Both the AA and CP-25 have no effect on the expression of P-gp in the capillaries of brain,spleen,stomach,colon,heart and lung.However,AA could increase the expression of P-gp in brain vessels,hepatocytes and bile canaliculus and small intestinal epithelial cells,and decrease P-gp expression in proximal and distal tubules of kidney.CP-25 could regulate the abnormal expression of P-gp in the tissues,and the effect of 100 and 50 mg/kg groups was the most obvious.Conclusions:1.Passive transport was the main pattern of CP-25 and Pae which were absorbed by PBMC.The absorption of CP-25 by PBMC was better than that of Pae;2.The concentration of CP-25 in PBMC was closely related to the concentration in plasma.The AUC and MRT of CP-25 in PBMC were reduced by AA;3.P-gp was involved in the efflux of CP-25 in PBMC.CP-25 could reduce the expression and function of P-gp and increase the concentration of CP-25 in PBMC;4.CP-25 could regulate the abnormal expression of P-gp in PBMC and tissues of AA rats.