目的 通過(guò)Beagle犬長(zhǎng)期毒性試驗(yàn)及其伴隨的免疫原性試驗(yàn)，觀察甘精胰島素注射液的毒性反應(yīng)，確定未觀察到臨床不良反應(yīng)的劑量水平（NOAEL），為臨床不良反應(yīng)監(jiān)測(cè)及防治提供參考。方法 健康Beagle犬40條，隨機(jī)分為甘精胰島素注射液低、中和高（0.5、1.0、2.0 IU/kg）劑量組、溶媒對(duì)照組及原研對(duì)照組（來(lái)得時(shí)，2.0 IU/kg），每組8只動(dòng)物，雌雄各半。連續(xù)sc給藥30天，停藥恢復(fù)16天。試驗(yàn)期間進(jìn)行一般體征觀察、進(jìn)食量、體質(zhì)量、肛溫、全血血糖及心電圖檢查；測(cè)定血液學(xué)常規(guī)與凝血指標(biāo)、血清生化、尿液常規(guī)等指標(biāo)；進(jìn)行骨髓細(xì)胞形態(tài)學(xué)檢查、大體剖檢檢查以及組織病理學(xué)檢查。采用間接ELISA法檢測(cè)不同時(shí)期動(dòng)物血清抗藥的結(jié)合抗體；采用體外生物活性HPLC法檢測(cè)產(chǎn)生抗藥抗體的陽(yáng)性血清的中和活性。結(jié)果 在給藥期的第8和10天，原研對(duì)照組和高劑量組各有1只雌性動(dòng)物在給藥后5～6 h出現(xiàn)抽搐和流涎等由低血糖所致的異常癥狀，其中高劑量組的該異常動(dòng)物于次日死亡；給藥期第11天的心電圖檢查發(fā)現(xiàn)高劑量組的T波倒置比例略高于溶媒對(duì)照組（5/7 vs 1/8），停藥后恢復(fù)正常；其余存活動(dòng)物的體質(zhì)量、肛溫、進(jìn)食量、尿常規(guī)、血液學(xué)、血清生化和組織病理學(xué)等均未見(jiàn)毒理學(xué)意義的異常改變。免疫原性結(jié)果顯示，僅有高劑量組1只雄性動(dòng)物于給藥期第12天產(chǎn)生抗藥抗體，抗體滴度為1:16，陽(yáng)性率為14.3%，產(chǎn)生結(jié)合抗體的血清樣品經(jīng)檢測(cè)為非中和活性抗體。結(jié)論 在本試驗(yàn)條件下，Beagle犬sc重復(fù)給予甘精胰島素注射液的NOAEL為1.0 IU/kg，該劑量相當(dāng)于臨床擬用劑量的2倍。該藥在高劑量下可能對(duì)個(gè)別Beagle犬具有較弱的免疫原性。

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.

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