目的 通過(guò)小鼠單次給藥毒性試驗(yàn)、Beagle犬重復(fù)毒性及免疫原性試驗(yàn)和豚鼠全身主動(dòng)過(guò)敏試驗(yàn)，考察精蛋白重組人胰島素注射液（Insulin NPH）的毒副反應(yīng)、毒性靶器官或靶組織，為開(kāi)展臨床試驗(yàn)提供依據(jù)。方法 ①小鼠單次給藥毒性試驗(yàn)：采用最大給藥量法分別sc生理鹽水、溶媒和Insulin NPH（2092~2488 IU/kg），監(jiān)測(cè)給藥后小鼠一般狀態(tài)、體質(zhì)量、臟器異常。②Beagle犬重復(fù)毒性試驗(yàn)：sc溶媒、原研對(duì)照藥（Humulin NPH，1.5 IU/kg），低、中和高劑量（0.5、1.0和1.5IU/kg）的Insulin NPH，每天1次，連續(xù)30 d，停藥恢復(fù)14 d；在給藥期和恢復(fù)期內(nèi)觀察動(dòng)物的一般體征和注射部位的局部刺激性，進(jìn)行體質(zhì)量、肛溫、血糖及心電圖檢查，測(cè)定血液學(xué)、血清生化、尿液常規(guī)等指標(biāo)，并進(jìn)行臟器質(zhì)量及組織病理學(xué)檢查；免疫原性試驗(yàn)采用間接ELISA法檢測(cè)不同給藥期Beagle犬血清中抗藥結(jié)合抗體。③豚鼠主動(dòng)全身過(guò)敏試驗(yàn)：分別sc低和高劑量（4和12 IU/kg）的Insulin NPH、生理鹽水和溶媒，另設(shè)卵清白蛋白為陽(yáng)性對(duì)照，使用以上劑量進(jìn)行5次致敏試驗(yàn)后，iv 3倍致敏劑量進(jìn)行激發(fā)試驗(yàn)，觀察豚鼠過(guò)敏癥狀。結(jié)果 小鼠sc 165倍臨床常用劑量的Insulin NPH后，未見(jiàn)明顯毒性反應(yīng)；Beagle犬重復(fù)毒性試驗(yàn)中1.0 IU/kg是Insulin NPH的無(wú)毒反應(yīng)劑量（NOAEL），該劑量相當(dāng)于臨床擬用劑量的2倍，免疫原性試驗(yàn)各劑量組均未發(fā)現(xiàn)抗藥結(jié)合抗體；豚鼠主動(dòng)全身過(guò)敏試驗(yàn)中未見(jiàn)明顯過(guò)敏癥狀。結(jié)論 在本試驗(yàn)條件下未觀察到Insulin NPH明顯毒性反應(yīng)。

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.

天津創(chuàng)新藥物安全評(píng)價(jià)技術(shù)平臺(tái)（2013ZX09302301）