The Study Of Immunosuppressive Effects And Pharmacokinetics Of Demethylzeylasteral

Tripterygium wilfordii Hook f.(Celastraceae) is commonly known in China as Lei-Gong-Teng (Thunder God vine), and is widely distributed in Eastern and Southern China, Korea and Japan. Over the last30years, it has been used for the treatment of rheumatoid arthritis, chronic nephritis, ankylosing spondylitis and various skin diseases. The herb is able to inhibit the immune reaction at various levels, including inhibiting interleukin-2(IL-2), activating suppressor T cells, and inhibiting the activation of cytotoxic T lymphocytes and macrophages, natural killer cells and the generation of antibodies.Several diterpenoids and triterpenoids, as well as other compounds, have so far been isolated from T. wilfordii, and the immunosuppressive and antifertility activities of some fractions prepared from metabolites of cultured cells of the plant have been reported. We have isolated an immunosuppressive monomer, demethylzeylasteral (T-96), from the root xylem of T. wilfordii.In this study, we aimed to investigate the immunosuppressive activity of demethylzeylasteral (T-96), isolated from the traditional Chinese herbal medicine, Tripteiygium wilfordii Hook f. Its immunosuppressive effect was investigated using mouse splenocytes in vitro, and in an in vivo beagle dog kidney transplant model. These results demonstrate the strong immunosuppressive activity of T-96and suggest a possible clinical use for T-96as an immunosuppressive agent in the fields of organ transplantation and autoimmune disorders.Part I:The immunosuppressive effect of T-96in vitro and in vivoObjectives:To study the immunosuppressive effect of T-96on proliferation of immune activating cells and on the production and release of cytokine in vitro and to explore the possible mechanism of the immunosuppressive effect of T-96in vivo.Material and methods:Spleen cell suspension of BALB/c mouse was prepared and cultured in the10%FBS with RPMI-1640medium. Various concentrations of T-96, triptolide and CsA were added into it. The cell proliferation was measured by3H-TdR uptake assay and splenocyte survival was detected with MTT assay. Cell factors were measured by ELISA. In addition, the dinitrochlorobenzene (DNCB)-induced delayed type hyper sensitivity (DTH) model was employed.Results:It revealed that T-96below the concentration of1μg/ml did not have significant cytotoxicity on lymphocytes, while T-96could inhibit the ConA-induced T lymphocytes proliferation effect even at the concentration of0.0001μg/ml. The mechanism of the immunosuppressive effect of T-96was inhibiting the T lymphocytes proliferation almost the same mechanism as CsA. In the dinitrochlorobenzene (DNCB)-induced delayed type hypersensitivity (DTH) model.0.1mg/kg of T-96inhibited the DNCB-induced DTH. In acute toxic study, the LD50of T-96was above30mg/kg.Conclusions:There was different mechanism between the immunosuppressive effect of T-96and the cell toxic effect of triptolide. The inhibiting effect of T-96on immune reaction was stronger than that of triptolide, and may reach the level of CsA, when certain concentration was achieved, suggesting that the inhibiting effect of T-96on the host-to-graft rejection may act through several levels.Part Ⅱ:Establish kidney transplantation model in beagle dogsObjectives:To establish a simple and reliable kidney transplantation model in beagle dogs, and postoperative serum creatinine (Cr) and blood urea nitrogen (BUN) of transplanted kidney were observed.Material and methods:The beagle dogs were randomly divided into2treatment groups as follows:1, the transplant group (n=6) and2, the shamed group (n=6). Heterotopic renal transplantation with native nephrectomy was performed in the transplant group, while the left nephrectomy was performed in the shamed group. Postoperative serum creatinine (Cr) and blood urea nitrogen (BUN) of transplanted kidney were observed each day.Results:The result showed that serum creatinine and blood urea nitrogen of transplanted kidney were raised rapidly3days after operation. The serum creatinine was varied from71.17 ±7.52μmol/L to1026.4±110.06μ mol/L, and BUN was varied from4.38+0.75mmol/L to42.08±5.58mmol/L, but the serum creatinine and BUN of the shamed group were remained steadily.Conclusions:We established a simple and reliable kidney transplantation model in beagle dogs. It can be used as a model to study transplantation immunity and evaluate immunosuppressant.Part Ⅲ:The immunosuppressive effect of T-96in a beagle dog kidney transplantation modelObjectives:To study and evaluate the immunosuppressive effect of T-96using a beagle dog kidney transplantation model.Material and methods:In this study, renal allografts were undertaken between beagle dogs. Recipient and donor male beagle dogs were obtained from different breeders to ensure MHC mismatching. Dogs were randomly divided into6groups following kidney transplantation, and different doses of T-96or CsA were administered to each group:1, control group treated with normal saline (n=6);2, CsA-treated group (5mg/kg/day)(n=6);3, T-96-treated group (5mg/kg/day)(n=6);4, T-96-treated group (10mg/kg/day)(n=6);5, T-96-treated group (20mg/kg/day)(n=6); and6, T-96+CsA-treated group (10mg/kg/day,5mg/kg/day, respectively)(n=6).Results:The results showed that T-96alone at a dosage of10mg/kg/day/dog prolonged graft survival up to10.83±1.47days. Moreover, a combination of T-96and CsA also significantly prolonged survival:10mg/kg/day T-96with5mg/kg/day CsA increased the survival time to13.33±1.75days.Conclusions:These results demonstrate the strong immunosuppressive activity of T-96and suggest a possible clinical use for T-96as an immunosuppressive agent in the fields of organ transplantation and autoimmune disorders.Part IV:Establish and evaluate the LC-MS/MS method of T-96in beagle dogs Objectives:To develop the LC-MS/MS method for determining T-96in beagle dog plasma. To evaluate whether accuracy, precision, sensitivity, specificity and linearity range of the method meets the requirement of biological sample analysis from T-96pharmacokinetic study in beagle dogs.Material and methods:By establishing a calibration curve of T-96in beagle dogplasma, to evaluate specificity, precision, recovery, absolute recovery, matrix effect, stability and dilution test of the method.Results:T-96determination in beagle dog is not interfered by endogenous substances, relative recovery is>85%, inter-and intra-day RSD is<5%, lower limit of quantitation is2.02ng/ml and linearity range is2.02-505ng/mL.Conclusions:The LC-MS/MS method for determining T-96in beagle dog plasma is accurate, precise, specific and sensitive. The method can be used to study the pharmacokinetics of T-96in beagle dog plasma.Part V:The pharmacokinetic study of T-96in beagle dogs using LC-MS/MS methodObjectives:To determine T-96in beagle dog plasma and to evaluate the pharmacokinetic characteristics of T-96in6beagle dogs using the developed LC-MS/MS method in part Ⅱ.Material and methods:To study pharmacokinetics of T-96in6beagle dogs after an oral administration of20,40and80mg/kg and after a foreleg intravenous injection of5mg/kg. to obtain pharmacokinetic parameters including elimination half-life (ti/2), mean residence time (MRT), plasma peak concentration (Cmax), time to peak concentration (tmax), AUC0-24, AUC0-∞and absolute bioavailability using a non-compartment model.Results:After an oral administration of20(40and80) mg/kg, T-96t1\2, Cmax, tmax, AUC0-24, AUC0-∞were10.75±1.14h (11.70±1.21h and11.20±1.43h), 418.42±73.61ng/ml (842.49±99.43ng/ml and1383.61±143.05ng/ml),8.7±1.0h (11.70±1.1h and10.7±1.0h),7691.96±2361.70ng·h/ml (16225.59±3081.34ng·h/ml and26027.86±5150.72ng-h/ml),7788.33±2365.32ng·h/ml (16553.42±3289.75ng-h/ml and26517.20±5461.39ng-h/ml), respectively. After an intravenous injection of5mg/kg, T-96t1/2, MRT, Co. CIT, VD, AUC0-12, AUC0-∞were9.85±0.76h,6.94±1.65h,47701.35±2559.10ng/ml,0.10±0.03L/h/Kg,0.74±0.39L/Kg,54615.76±19147.38ng·h/ml,54786.26±19161.47ng-h/ml, respectively.Conclusions:Compare AUC of an oral administration of40mg/kg to that of an intravenous injection of5mg/kg in6healthy beagle dogs after dose correction, T-96had an absolute bioavailability of4.2%±1.9%(2.3%-6.1%).