【作者】 李春雨；

【导师】 孟宪丽；

【作者基本信息】 成都中医药大学 ， 中药学， 2013， 博士

【摘要】 目的聚乙二醇(PEG)修饰技术是现今国际上蛋白药物二次开发的热点技术。本项目研究重组人胰岛素(Det)的PEG修饰物新型聚乙二醇胰岛素(PEG-Det)的体内代谢过程及安全性,以期阐明PEG修饰技术使蛋白药物发挥长效作用及具有良好安全性的合理性和科学性。方法本研究基于生物技术药物的特点,建立并采用放射免疫(RIA)和酶联免疫(ELISA)的分析方法,研究Det/PEG-Det的Beagle犬药代动力学/药效动力学特征,研究PEG修饰对Det体内过程的影响；研究PEG-Det的Beagle犬毒代动力学/毒效动力学特征、免疫原性、免疫毒性、制剂安全性,综合评价PEG修饰对Det安全性的影响。结果(1)药物体内过程研究结果表明：①与非PEG修饰的Det相比,PEG-Det在Beagle犬体内平均消除半衰期t1/2β延长59倍；清除率CL降低35倍；达峰时间Tmax增加9倍。②Beagle犬单次皮下注射(s.c.) PEG-Det(25、50和100μg/kg)后,峰浓度Cmax和药时曲线下面积AUC、Tmax随剂量增加呈更大比例增大,CL随剂量增大而降低：t1/2β与剂量呈非相关。③Beagle犬多次s.c.PEG-Det (50μg/kg)后，体内无蓄积。④Beagle犬多次s.c. PEG-Det(37.5、75和150μg/kg)在毒性剂量暴露下,血药浓度升高的同时伴随着血糖的降低,随着血药浓度的降低,血糖水平也逐渐升高,药后12h基本恢复到给药前的血糖基础水平,Cmax和AUC随剂量的增加而增大,体内无蓄积。(2)药物安全性研究结果表明：①Beagle犬血清中抗PEG-Det抗体为PEG修饰剂中单甲氧基结构产生,与Det蛋白无关；②PEG-Det对免疫系统补体C3、C4及免疫复合物CIC水平无明显影响,对白细胞总数及分类计数、免疫组织器官未产生明显毒性：③PEG-Det s.c或静脉注射(i.v.)给药时,对注射部位皮肤及肌肉或血管均无刺激反应：④豚鼠全身主动过敏试验和大鼠被动过敏试验结果均呈阴性；⑤体外溶血试验结果呈阴性。结论PEG修饰确实能改善和提高Det的PK/PD特性,延长半衰期,降低清除率,增加暴露量,减小血清峰-谷浓度比率,延缓药物体内作用时间,具有长效作用及良好的安全性。更多还原

【Abstract】 ObjectiveAt present pegylation is the hotspot technology in secondary development of protein drugs. This project study on process in body and safety of pegylated insulin(PEG-Det) to explain the rationality and scientificity of long effective mechanism and safety of pegylation.MethodIn this study, based on characteristic of biotech drugs, two bioanalytical methods, radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) were established and employed to investigate the effect of pegylation on process in body and safety of insulin. The studies performed are listed as follows:①Pharmacokinetics and pharmacodynamics of PEG-Det in Beagle dogs comparison with that of non-pegylated insulin (Det);②Toxicokinetics and dynamicsof PEG-Det;③Immunogenicity and Immunotoxicity of PEG-Det following repeated subcutaneous administration to Beagle dogs;④Safety evaluation of the pegylated Det insulin for injection.Results(1) Process in body results shows:①The pegylated protein exhibited preferred pharmacokinetic properties to Det in Beagle dogs, with a59-fold increase in elimination half life, and a35-fold decrease in serum clearance (CL), as well as a9-fold increase in the time to reach peak serum concentration (Tmax) respectively.②After PEG-Det were injected once subcutaneously to Beagle dogs at a doses of25,50, and100μg/kg, respectively, a greater than proportional increase in Cmax, AUC and Tmax for PEG-Det was observed with the increasing doses, while the rate of clearance decreased with increasing doses. Tmp was found to be dose-independent.③Following repeated subcutaneous administration of PEG-Det at a dose of50μg/kg to Beagle dogs, no accumulation of the drug was found in body.④When the dogs were exposed to PEG-Det at the toxic doses of37.5,75, and150μg/kg respectively, the plasma drug concentrations varied in an oppsite direction to the blood glucose levels, that was to say, an increase in plasma drug concentrations was definitely accompanied by a decrease in blood glucose level, and vice versa. The blood glucose level returned to its baseline value at the twelve hours after the administration of PEG-Det. Both Cmax and AUC increased with the increase of dose and no accumulation in body was observed.(2) Safety results shows:①The anti-PEG-Det antibody was specific to the PEG moiety, which had nothing to do with Det itself.②PEG-Det has no significant effect on the level of C3, C4, CIC, leukocyte counts and differential counts, and has no significant toxicity on immune tissues and organs.③Following subcutaneous and intravenous administration of PEG-Det, no irritation effects was found on the skin, muscle and blood vessel at the injection sites.④The negative results of active systemic allergy test in guinea pigs and of passive allergy test in rats indicated that PEG-Det may not introduce allergic reactions in humans.⑤The results of hemolysis test in vitro was also encouraging.ConclusionThese findings demonstrate that compared to unmodified Det, pegylation of Det possesses more desirable pharmacokinetic and pharmacodynamic properties, including prolonged biological half-life, decreased system clearance, enhanced drug exposure, reduced serum peak-to-trough con-centration ratio,increased safety and increased efficacy.更多还原