目的 通过Beagle犬长期毒性试验及其伴随的免疫原性试验，观察甘精胰岛素注射液的毒性反应，确定未观察到临床不良反应的剂量水平（NOAEL），为临床不良反应监测及防治提供参考。方法 健康Beagle犬40条，随机分为甘精胰岛素注射液低、中和高（0.5、1.0、2.0 IU/kg）剂量组、溶媒对照组及原研对照组（来得时，2.0 IU/kg），每组8只动物，雌雄各半。连续sc给药30天，停药恢复16天。试验期间进行一般体征观察、进食量、体质量、肛温、全血血糖及心电图检查；测定血液学常规与凝血指标、血清生化、尿液常规等指标；进行骨髓细胞形态学检查、大体剖检检查以及组织病理学检查。采用间接ELISA法检测不同时期动物血清抗药的结合抗体；采用体外生物活性HPLC法检测产生抗药抗体的阳性血清的中和活性。结果 在给药期的第8和10天，原研对照组和高剂量组各有1只雌性动物在给药后5～6 h出现抽搐和流涎等由低血糖所致的异常症状，其中高剂量组的该异常动物于次日死亡；给药期第11天的心电图检查发现高剂量组的T波倒置比例略高于溶媒对照组（5/7 vs 1/8），停药后恢复正常；其余存活动物的体质量、肛温、进食量、尿常规、血液学、血清生化和组织病理学等均未见毒理学意义的异常改变。免疫原性结果显示，仅有高剂量组1只雄性动物于给药期第12天产生抗药抗体，抗体滴度为1:16，阳性率为14.3%，产生结合抗体的血清样品经检测为非中和活性抗体。结论 在本试验条件下，Beagle犬sc重复给予甘精胰岛素注射液的NOAEL为1.0 IU/kg，该剂量相当于临床拟用剂量的2倍。该药在高剂量下可能对个别Beagle犬具有较弱的免疫原性。

Objective To determine the no-observed-adverse-effect level (NOAEL) of insulin glargine injection (IGI) by observation of toxicity reactions in long-term toxicity study in Beagle's dogs and its accompanying immunogenicity test, and provide reference for clinical adverse reaction monitoring and prevention. Methods 40 healthy Beagle dogs were randomly divided into five groups, including low-, mid-, and high-dose (0.5, 1.0, and 2.0 IU/kg) groups of IGI, vehicle control group, and original research group (Lantus, 2.0 U/kg). Each group included eight Beagle's dogs, half male and half female. All animals were sc administered for 30 d continuously, and then recovered for 16 d with no dosing. General observations, food intake, body weight, rectal temperature, blood glucose, and electrocardiographic examination were conducted. Routine hematology and coagulation, serum biochemistry, routine urianlysis, and other indicators were also determined. Meanwhile, bone marrow cell morphology, gross necropsy examination and histopathological examination were carried out during the study. Drug-resistant binding antibody at different periods in dog serum was determined by an indirect ELISA method. The neutralizing activity of resistant antibody in positive serum was measured by using biological activity HPLC assay in vitro. Results During the dosing period of d8 and d10, one female animal showed the abnormal symptoms of salivation and convulsions caused by hypoglycemia after dosing 5-6 h in original research group and high-dose group respectively, and the abnormal animal in high-dose group died the next day. The proportion of T wave inversion in high-dose group respectively, and the abnormal animal in high-dose group died the next day. The proportion of T wave inversion in high-dose group was found slightly higher than that in vehicle control group (5/7 vs 1/8) by electrocardiographic examination, which was back to normal after stopping dosing. There were no significant toxicological changes in body weight, rectal temperature, food intake, routine urianlysis, routine hematology, serum biochemistry, and histopathology of the remaining surviving animals. Immunogenicity test indicated that only one male animal in high-dose group produced drug-resistant binding antibody during the dosing period of d12. The antibody titer was 1:16 and the positive rate was 14.3%. The binding antibody in serum sample was tested to be non-neutralizing activity antibody. Conclusion Under the experimental conditions, the NOAEL of IGI in Beagle's dogs with repeat-dose of 1.0 IU/kg, which was equivalent to two times of clinical therapeutic dose. This medicine at high dose may produce weak immunogenicity in particular Beagle's dog.

天津创新药物安全评价技术平台（2013ZX09302301）