Pharmacokinetic And Pharmacodynamic Studies Of Recombinant Human Thrombopoietin Injection In Rhesus Monkeys

Objective:The rhTPO injection studied in this experiment is a recombinant factor expressed by Chinese hamster ovary cells（CHO cells）produced by Shenyang Sansheng Pharmaceutical Co.,Ltd.The main purposes of this experiment:To increase the indication for rhTPO-cute radiation sickness.This thesis studies the following aspects:（1） the relative bioavailability and pharmacokinetic characteristics of the subcutaneous and intramuscular injections is compared in normal rhesus monkeys;（2）the similarities and differences of the pharmacokinetic characteristics between normal and irradiated rhesus monkeys is studied;（3）the pharmacodynamic testing is performed at the same time as pharmacokinetics,and the correlation of PD and PK is analyzed.Method:In this paper,the concentration of rhTPO in rhesus monkeys was determined by double-antibody sandwich enzyme-linked immunosorbent assay（ELISA）,and methodologies such as specificity,accuracy,precision,dilution linearity,parallelism,and stability were verified.The method was applied to the pharmacokinetic study of normal and irradiated rhesus monkeys and the influence of rhTPO on peripheral blood was measured with a blood cell analyzer while pharmacokinetics were studied.Result:（1）It has been verified that the specificity,accuracy,precision,dilution linearity,parallelism,and stability of the method meet the requirements of the Pharmacopoeia.（2）Results of pharmacokinetic and pharmacodynamic studies in normal rhesus monkeys:After subcutaneous injection and intramuscular injection of rhTPO in rhesus monkeys,there are no statistically significant difference in elimination half-life(t_1/2),peak time（T_m）,and average retention time(MRT_last)in vivo,t_1/2 are 15.00±2.40 h and 16.01±3.29 h、T_m are 5.33±2.07 h and 4.00±1.07 h、MRT_last are 14.71±2.34 h and 13.63±1.83 h,respectively.But the relative bioavailability:im/sc=394.34%.Peripheral blood test found that rhTPO can only promote the increase of platelet counts when acting on normal animals,and there is no obvious trend of changes in other blood cell counts.（3）The experimental results of pharmacokinetics and pharmacodynamics in irradiated rhesus monkeys are as follows:After intramuscular injection of different doses of rhTPO(5μg·kg^-1and 10μg·kg^-1)in irradiated macaques,and the results show that the elimination half-life(t_1/2)of these two doses are 23.56±5.71 h and 19.96±2.30 h,the peak time of serum concentration(T_max)are 3.5±1 h and 3.2±1.1 h,the apparent volume of distribution（V_z）are1229.22±752.48 m L·h^-1·kg^-1 and 835.34±195.43 m L·h^-1·kg^-1,the serum clearance（CL）rae 33.75±14.74 m L·h^-1·kg^-1 and 29.05±6.82 m L·h^-1·kg^-1,the average residence time(MRT_last)are 25.63±5.62h and 20.11±3.13h,respectively,and the difference is not statistically significant.The maximum serum concentration(C_max)are8450.44±4659.9 pg·m L^-1 and 21136.3±8005.77 pg·m L^-1,respectively,which are significantly different（p<0.05）.The area under the average blood concentration-time curve(AUC_last)are 170678.89±87206.45 h·pg·m L^-1 and 356149.59±90119.22h·pg·m L^-1,respectively,the difference is statistically significant（p<0.01）with a ratio of 1:2.09 and a dose ratio of 1:2.Peripheral blood test find that rhTPO can promote the recovery of multi-line hematopoietic system when acting on radiation model animals,not only increase the platelet counts,but also increase the counts of white blood cells,neutrophils,monocytes,and lymphocytes in the peripheral blood,thus reducing the mortality.Conclusion:（1）The results of methodology validation show that:Double antibody sandwich enzyme-linked immunosorbent assay（ELISA）is suitable for quantitative analysis of macromolecular protein drugs rhTPO,and can be used for the preclinical pharmacokinetics study of rhTPO injection.（2）In normal rhesus monkeys,intramuscular injection and subcutaneous injection have similar metabolic processes,the elimination is slower,the terminal half-life is longer,and the relative bioavailability of the two is quite different,indicating that rhTPO injection has better absorption and higher bioavailability by intramuscular injection.In order to study the effect of rhTPO on the recovery of peripheral blood cells,it is found that in normal macaques,rhTPO can only promote the increase of platelets,and has no obvious promotion effect on other blood cells,showing monophyletic specificity.PD-PK correlation analysis show:The peak concentration of serum rhTPO and the duration of the increase in concentration alone are not directly related to the increase in PLT.However,the systemic exposure of rhTPO is related to the increase of PLT.（3）In irradiated rhesus monkeys,different doses of rhTPO are injected intramuscularly,and the area ratio under the drug time curve of different doses is approximately equal to the dose ratio,indicating that the linear kinetic characteristics of intramuscular injection of rhTPO(5-10μg·kg^-1)is observed in irradiated rhesus monkeys.The recovery of radiation-induced pancytopenia is observed.It is found that rhTPO could promote the recovery of multi lineage hematopoiesis,not only increase the platelet counts,but also promote the production of white blood cells,red blood cells and neutrophils.（4）For the new indication of rhTPO injection-acute radiation sickness,compared with subcutaneous injection,intramuscular injection has better absorption in rhesus monkeys,higer bioavailability,and convenient administration,which is a better method of administration.