Study On The Combination Safety Of Yiqi Fumai Lyophilized Injection(YQFM) And The Immobilization Of Thermolysin

The safety study of Yiqi Fumai Lyophilized Injection combined with 9 common injectionsObjective:To evaluate the pharmacological safety of Yiqi Fumai Lyophilized Injection in combination with 9 common injections,the mouse pseudo-allergic test,rabbit infusion high temperature test,and hemolysis or coagulation test has been taken.Methods:(1)The mouse pseudo-allergic reaction model was developed by injecting test substance in 0.4% Evans blue(EB)solutions via tail vein.The positive control group was given Compound 48/80-0.4% EB saline solution,the negative control group was given 0.4% EB saline solution,and the drug compatibility group was given "drug-0.4% EB" saline solution.According to the standards of mice ear blue staining test,the scores of ear blue staining was evaluated,and the EB exudation of mice ear was measured.(2)In the concentration test of histamine in mice,except that 0.4% EB saline solution was replaced by saline solution,the administration method was all the same as that of mice ear blue staining test.Blood was collected after 30 min,and the histamine content in blood of mice was determined by chemical method.(3)Febrile reaction test in rabbit: the normal saline solutions of the test substances were instilled into the ear.The body temperature of rabbits was measured,and the range of body temperature changes in rabbits was evaluated according to the criteria for pyrogen reaction.(4)Hemolysis or coagulation test,test tube method: The test substances solutions of the 5 times,10 times,15 times of the rabbit equivalent dose were added into 2% red blood cell suspensions.The positive control group was given purified water;the negative control group was given normal saline.Incubation of the solutions at 37 °C in a constant temperature water bath for 3 h and judgement of the hemolysis or coagulation reaction was taken according to the hemolysis or coagulation test standards.The test tube after water bathing was centrifuged,and the supernatant was moved out.The absorbance of hemoglobin released from the rupture of red blood cells in the supernatant was measured at 545 nm by a spectrophotometer,and the hemolysis rate was calculated.Result:(1)Vitamin B6 and naloxone hydrochloride for injection administered alone or in combination with YQFM showed positive results in mice ear blue staining test and the other results were negative.(2)There was no significant change in histamine contents in the blood of mice before and after compatibility.(3)More than one rabbit temperature after administration of single and mixed solution of aminophylline injection increased more than 0.6 °C,indicating that the rabbit had a febrile reaction.(4)The hemolysis or coagulation reaction measured by test tube method were all negative after adding of single and mixed administration group.(5)There were some differences between the spectrophotometric method and the test tube method.In the single administration group,the hemolysis rate of three doses of ozagrel and cyclophosphamide for injection were less than 5%.In the mixed administration group except aminophylline,vitamin B6 injection and ozagrel injection,the hemolysis rate was less than 5%.Conclusion:(1)Comparing the mixed administration group with the single administration group respectively,there was no increase in the risk of pseudo-allergic reaction in mice through mice ear blue staining test and histamine detection.(2)There was no significant difference in febrile reaction test in rabbit between the mixed administration group and the single administration group.The febrile reaction was not related to the mixed administration.(3)The test tube method and the spectrophotometry method are inconsistent in the results of hemolysis or coagulation experiments.Spectrophotometry can quantify the degree of hemolysis and the results may be more accurate.Among the results of the two methods,the mixed administration group did not increase the hemolysis coagulation reaction compared with the single administration group.The Study on Preparation of Low Molecular Protamine by Biomimetic Immobilization of ThermolysinObjectives: To obtain low molecular weight protamine(LMWP)thermolysin(TLN)has been immobilized by biomimetic silicification.Methods:(1)Biomimetic TLN immobilization method: tetramethyl orthosilicate(TMOS)as a precursor of biomimetic silicification,providing framework materials for immobilized enzymes;Poly-L-Lysine(PLL)as a biological template.The immobilized TLN can be prepared by adding the acidified TMOS to the TLN and PLL mixture solution and reacting for 5 min.(2)Isolation and purification of LMWP: the immobilized TLN was added into the protamine solution to obtain a mixture of protamine hydrolysate containing LMWP.The target product LMWP was separated and purified by the Fast protein liquid chromatography system(FPLC).(4)Optimization of the LMWP isolation and purification method: Before the elution gradient of mobile phase B concentration of 55%,a low concentration gradient was inserted,so that the mixed impurity could be eluted before LMWP.(2)Optimization of the biomimetic immobilization method: DOE experimental design scheme have been used to design experiment to optimize the bioimmobilization condition by changing the proportion between TLN,TMOS,PLL,and reaction time,and then Minitab system is used to analyze the experiment result.Results:(1)Protein purification process: A: Tris-hydrochloric acid buffer and B: Tris-hydrochloric acid and sodium chloride buffer as mobile phase.According to previous LMWP elution gradient(mobile phase B concentration of 55%),a mixture peak was obtained,and the target product LMWP could not be completely separated.A gradient of 40% of the mobile phase B concentration was inserted before the LMWP elution gradien,and the elution time was appropriately extended to elute the mixed impurities absolutely.The optimized elution conditions are: B salt concentration: 0-20 min: 33%;20-50 min: 40%;50-70 min: 55%;70-80 min: 100%.(2)Using hydrolysis efficiency of protamine as an indicator,the best hydrolyzing conditions had been determined by changing the ratio between TLN,TMOS and PLL.Finally,TMOS concentration 7.5%,PLL 0.3 mg,TLN 0.3 mg were selected as the optimal experimental condition.