目的 通过小鼠单次给药毒性试验、Beagle犬重复毒性及免疫原性试验和豚鼠全身主动过敏试验，考察精蛋白重组人胰岛素注射液（Insulin NPH）的毒副反应、毒性靶器官或靶组织，为开展临床试验提供依据。方法 ①小鼠单次给药毒性试验：采用最大给药量法分别sc生理盐水、溶媒和Insulin NPH（2092~2488 IU/kg），监测给药后小鼠一般状态、体质量、脏器异常。②Beagle犬重复毒性试验：sc溶媒、原研对照药（Humulin NPH，1.5 IU/kg），低、中和高剂量（0.5、1.0和1.5IU/kg）的Insulin NPH，每天1次，连续30 d，停药恢复14 d；在给药期和恢复期内观察动物的一般体征和注射部位的局部刺激性，进行体质量、肛温、血糖及心电图检查，测定血液学、血清生化、尿液常规等指标，并进行脏器质量及组织病理学检查；免疫原性试验采用间接ELISA法检测不同给药期Beagle犬血清中抗药结合抗体。③豚鼠主动全身过敏试验：分别sc低和高剂量（4和12 IU/kg）的Insulin NPH、生理盐水和溶媒，另设卵清白蛋白为阳性对照，使用以上剂量进行5次致敏试验后，iv 3倍致敏剂量进行激发试验，观察豚鼠过敏症状。结果 小鼠sc 165倍临床常用剂量的Insulin NPH后，未见明显毒性反应；Beagle犬重复毒性试验中1.0 IU/kg是Insulin NPH的无毒反应剂量（NOAEL），该剂量相当于临床拟用剂量的2倍，免疫原性试验各剂量组均未发现抗药结合抗体；豚鼠主动全身过敏试验中未见明显过敏症状。结论 在本试验条件下未观察到Insulin NPH明显毒性反应。

Objective To investigate the toxic reaction, toxic organs or target tissues of protamine recombinant human insulin (Insulin NPH), and provide basis for clinical trials by single dose toxicity test in mice, repeated toxicity and immunogenicity of Beagle's dogs, and systemic active allergy in guinea pig. Methods ① Using maximum dose method, mice in single dose toxicity test were sc injected with normal saline (NS), vehicle, and Insulin NPH (2092—2488 IU/kg), the toxic reactions after injection were monitored. ② In repeated toxicity study, Beagle's dogs were sc administrated with vehicle, the original (Humulin NPH, 1.5 IU/kg) and different doses of Insulin NPH (0.5, 1.0 and 1.5 IU/kg) for 30 d continuously, followed by a 14-d recovery. During the administration and recovery period, general observation, local irritation, body weight, anus temperature, blood glucose, and electrocardiogram (ECG) were checked, moreover, hematology, serum biochemistry and urine were detected. Also, organic weights and histopathological examination were conducted. Binding antibodies in dog serum were measured by indirect ELISA method in immunogenicity test. ③ In systemic active allergy study, cavies were sc injected with low- and high-dose (4 and 12 IU/kg) Insulin NPH, normal saline and vehicle. Besides, ova as positive control was also included. After five times of sensitization test with above doses, the excitation reactions of iv injection with tripled sensitizing doses were observed. Results No obvious toxicity was observed in mice after injected with 165 times of usual clinical dose of Insulin NPH. Repeated toxicity study of Beagle's dogs revealed that 1.0 IU/kg was the no-toxic-effect dose (NOAEL) for Insulin NPH, which was equivalent to 2 times of clinical dose. No bindingantibodies were found in immunogenicity test. There was no obvious allergic symptom in the active systemic allergy study of guinea pig. Conclusion Under the experimental conditions, no serious toxicity of Insulin NPH is found.

天津创新药物安全评价技术平台（2013ZX09302301）