Effect Of Mycophenolic Acid On The Expression Of Surface Antigens Of Host T Cells After Transplantation And Its Pharmacokinetic-pharmacodynamic Relationship

Mycophenolic acid?MPA?exerts its effect through selectively inhibiting the proliferation of T cell and B cell.The pharmacokinetic?PK?process of MPA has been researched widly.However,the studies on the relationship between the PK and pharmacodynamics?PD?of MPA is relatively scarce.The purpose of the present study was to research the impact of MPA on the T cell surface antigen expression of host after transplantation and the PK-PD correlation.Firstly,we established a liquid chromatography-tandem mass spectrometry?LC-MS/MS?method for the simultaneous determination of the concentration of various immunosuppressive agents,including MPA,cyclosporine A?CsA?,tacrolimus?TAC?and sirolimus?SRL?in plasma or whole blood.Plasma and whole blood were treated by protein precipitation or liquid-liquid extraction assay,respectively.The resultant was eluted through a gradient elution assay,ESI cation method with multi-reaction monitoring assay was used to quantify the various immunosuppressive agents.The linear ranges were as follows:4-1000 ng/mL?r=0.9997?for CsA,0.2-50 ng/mL?r=0.9995?for TAC,0.2-50 ng/mL?r=0.9998?for SRL,0.204-51?g/mL?r=0.9976?for MPA,the relative standard deviation?RSD?of QCs in both plasma and whole blood were less than 15%.The extract efficacy and matrix in plasma was 72.38%-98.52%and 65.61%-99.19%;in total blood was85.34%-99.76%and 75.94%-97.67%,respectively.The results of CsA or TAC in whole blood were consistent determined by Enzyme multiplied immunoassay technique?EMIT?or the present LC-MS/MS assay;At the same time,the correlation between the results in whole blood and plasma determined by LC-MS/MS were also good.The method established in this study is valuable for monitoring multiple immunosuppressive agents simultaneously for patients after organ transplantation.The skin transplantation model was established by using BALB/c inbred line and C57BL/6 mice.After MPA and CsA therapy,the cell markers including T cell surface antigen and the expression of donor specific antibody?DSA?of BALB/c mice was determined by flow cytometry technique,including the expression of CD4⁺CD25⁺,CD69⁺and DSA?MHC Class 1 H2Db?.The MPA PKs of the mice were also determined.The results showed that 14 days after skin transplantation,CD4⁺CD25⁺of mice elevated significantly.After treatment of CsA or MPA,CD4⁺CD25⁺decreased significantly.On the other hand,as an early activated antigen,CD69⁺elvated significantly in the early stage after transplantation.The expression of DSA?MHC Class 1 H2 Db?is relatively stable,with minor change of the expression at different time points.After medication,there is a certain delay of PK and PD,and there is correlation between PD and MPA exposure.Blood samples 0-12h after administration of MMF or EC-MPS were collected from 61 cases of renal transplantation patients.Plasma concentration were determined using the EMIT or LC-MS/MS method.The expression of different antigens was determined.The C₀,C_max,AUC and PD index i.e.AEC_IMPDH,or expression of T cell antigen?CD4⁺CD25⁺and CD3⁺CD69⁺?were collected.The results showed that the expression of CD4⁺CD25⁺in regulatory T cells increased with the MPA exposure,indicating that MPA contributed to the generation of immune tolerance.Previous studies showed that MPA was negatively correlated with IMPDH inhibition.In the present study,AEC_IMPDH showed the opposite trend with CD4⁺CD25⁺,which was consistent with previous studies.The correlation of CD3+CD69+expression with MPA exposure was not significant.The present study established a LC-MS/MS assay for the mornitoring various immunosuppressive agents simultaneously;establishing an animal transplantation model and collecting data from renal transplant patients for the study the relationshipe between MPA PK and PD.We found there is correlation between MPA exposure and PD,which provide evidence for the rational therapy with MPA.