低温乙醇法和硫酸铵盐析法制备抗人T细胞猪免疫球蛋白的非临床安全性评价

Nonclinical safety evaluation of anti-human T lymphocyte porcine immunoglobulin prepared by Cohn fractionation and ammonium sulphate precipitation

目的采用低温乙醇法制备抗人T细胞猪免疫球蛋白(anti-human T lymphocyte porcine immunoglobulin,P-ATG),并对其进行多项非临床研究,同时与原硫酸铵盐析工艺制品(市售制品)进行非临床对比研究,评价2种制品在毒理特性方面的一致性。方法给予新西兰兔的血管重复刺激性试验,评价2种制品对注射部位的局部反应;新西兰兔红细胞的体外溶血试验,评价2种制品的溶血性;单次给药静脉注射SD大鼠进行毒性试验,评价2种制品的急性毒性;4周重复给药静脉注射SD大鼠进行毒性并伴随局部刺激性、免疫原性和毒代动力学试验,评价2种制品的长期毒性。结果2种工艺制品连续5 d静脉注射新西兰兔,未见局部刺激性;2种工艺制品体外对新西兰兔红细胞无溶血作用,不引起新西兰兔红细胞凝聚;供试品组(低温乙醇法)在累计剂量1560 mg/kg和市售对照组(硫酸铵盐析法)在累计剂量1640 mg/kg的情况下,单次静脉注射SD大鼠产生的急性毒性反应相同,未显示出明显毒性,P-ATG(低温乙醇法)最大耐受量(maximum tolerance dose,MTD)>1560 mg/kg,为等效剂量的10.4倍,临床拟用剂量的62.4倍;供试品组(低温乙醇法)低、中、高剂量组分别在每日剂量195、310、780 mg/kg的情况下,重复静脉注射SD大鼠未出现明显毒性;供试品组(低温乙醇法)中剂量组在每日剂量310 mg/kg和市售对照组(硫酸铵盐析法)在每日剂量410 mg/kg的情况下,重复静脉注射SD大鼠产生的毒性特征基本一致,毒性结果变化趋势也较为接近,P-ATG(低温乙醇法)的未见明显毒性作用剂量(no observed adverse effect level,NOAEL)为780 mg/kg,为等效剂量的5.2倍,临床拟用剂量的31.2倍。结论低温乙醇法制品和硫酸铵盐析法制品的毒理特性具有良好的一致性。

Objective Anti-human T lymphocyte porcine immunoglobulin(P-ATG)was prepared by Cohn fractionation,and several non-clinical studies were conducted on it,and also non-clinical comparative studies were conducted with the original ammonium sulphate precipitation product(commercial product),which was to evaluate the consistency of the toxicological properties of the two products.Methods The vascular irritation test was repeatedly given to New Zealand rabbits to evaluate the local reaction of the two products to the injection site;the in vitro hemolysis test was given to New Zealand rabbit erythrocytes to evaluate the hemolysis of the two products;the single dose toxicity test was given to SD rats by intravenous injection to evaluate the acute toxicity of the two products;the 4-weeks repeated dose toxicity test accompanied by local irritation,immunogenicity and toxicokinetic tests were given to SD rats by intravenous injection to evaluate the long-term toxicity.Results No local irritation was observed in rabbits.No hemolysis or agglutination of rabbit erythrocytes was observed in vitro.Under the cumulative dose of 1560 mg/kg in experimental group and 1640 mg/kg in control group,the acute toxicity was comparable,no local irritation was observed when the two products were given intravenously to New Zealand rabbits for 5 consecutive days;the two products had no hemolytic effect on rabbit erythrocytes in vitro and did not cause rabbit erythrocytes coagulation;the acute toxicity reactions produced by a single intravenous injection to SD rats at a cumulative dose of 1560 mg/kg in the test group(Cohn fractionation)and 1640 mg/kg in the control group(ammonium sulphate precipitation)were comparable,no obvious toxicity was shown.The maximum tolerated dose(MTD)for Cohn fractionation procedure group was not less than 1560 mg/kg,which was 10.4 times of the equivalent dose,and 62.4 times of the clinical dose.The test group(Cohn fractionation)with high,middle and low dose at a daily dose of 195,310,780 mg/kg,inoculated intravenously to SD rats,the results shows no obvious toxicity;characteristic of toxicity in SD rat was basically the same repeatedly inoculated with P-ATG intra-veneously at a daily dose of 410 mg/kg in control group and 310 mg/kg in test group,and the trends of toxicity results were also relatively similar.The no observed adverse effect level(NOAEL)of P-ATG(cryoethanol method)was 780 mg/kg,which was 5.2 times of the equivalent dose,and 31.2 times of the clinical dose.Conclusion The toxicological properties of Cohn fractionation and ammonium sulphate precipitation products are in good agreement.