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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 observe the toxic reaction of insulin glargine injection and to determine the dose-free level (NOAEL) of Beagle dogs by long-term toxicity test and its accompanying immunogenicity test, so as to provide reference for clinical adverse reaction monitoring and prevention. Methods Forty healthy Beagle dogs were randomly divided into three groups: low, medium and high doses (0.5,1.0,2.0 IU / kg) of insulin glargine injection, vehicle control group and control group (2.0 IU / kg) Eight animals in each group, half male and half female. Continuous sc for 30 days, withdrawal stopped 16 days. During the experiment, the general signs of observation, food intake, body weight, rectal temperature, blood glucose and ECG examination; determination of hematology and coagulation indicators, serum biochemistry, urine routine and other indicators; bone marrow cell morphology examination, Check and histopathological examination. Indirect ELISA was used to detect the anti-drug-binding antibody of serum in different periods. The activity of neutralizing antibody-producing positive serum was detected by bioactivity HPLC in vitro. Results On the 8th day and the 10th day of the administration period, one females in the control group and the high dose group each had symptoms of seizures and salivation caused by hypoglycemia 5 to 6 hours after the administration, in which high dose The abnormal animals in the group died on the following day. The electrocardiogram examination on the 11th day of the administration showed that the T wave inversion ratio in the high-dose group was slightly higher than that in the vehicle control group (5/7 vs 1/8) The remaining surviving animals showed no abnormal changes in body weight, rectal temperature, food intake, urine routine, hematology, serum biochemistry and histopathology. Immunogenicity results showed that only one high-dose group of animals in the dosing period on the 12th produce drug-resistant antibodies, antibody titers 1:16, the positive rate was 14.3%, produced antibody-binding serum samples were detected as Non-neutralizing active antibodies. Conclusions Under the test conditions, the NOAEL of insulin glargine injection in Beagle dogs was 1.0 IU / kg, which is equivalent to twice the clinical dose. The drug may be less immunogenic for individual Beagle dogs at higher doses.