可解离的人胰高血糖素样肽1与人血清白蛋白融合蛋白的构建表达及其初步药代动力学和药效动力学评价

Construction and expression of releasable glucagon-like peptide-1 and human serum albumin fusion proteins and preliminary evaluation of their pharmacodynamics and pharmacokinetics

目的构建4种在体内以不同速率解离的人胰高血糖素样肽1(GLP-1)与人血清白蛋白融合蛋白,探索各种融合蛋白的疗效与其解离速率之间的关系,获得在药代动力学和药效动力学之间保持平衡的最佳融合蛋白。方法通过重叠延伸PCR扩增出具有不同多肽接头的融合蛋白基因,将其克隆到p PIC9载体,电击转化毕赤酵母GS115,经甲醇诱导,分泌表达各种融合蛋白,将各种融合蛋白分离纯化后进行初步药代学和药效学研究。结果融合蛋白与弗林蛋白酶作用后,不可解离的Gly2-GLP-1-GGGGG-HSA融合蛋白未发生解离,而可解离的融合蛋白Gly2-GLP-1-VTR-HSA为慢解离,Gly2-GLP-1-SARSVRA-HSA为中等解离,Gly2-GLP-1-GRSRVTRSV-HSA为快解离。体外生物学活性表明,各种融合蛋白均具有促进MIN6细胞胰岛素分泌的功能。体内药代学检测结果表明,各种融合蛋白在体内的半衰期大小依次为Gly2-GLP-1-GGGGG-HSA、Gly2-GLP-1-VTR-HSA、Gly2-GLP-1-SARSVRA-HSA、Gly2-GLP-1-GRSRVTRSV-HSA。体内药效学检测结果表明,各种融合蛋白均表现出降糖活性,且其活性高低依次为Gly2-GLP-1-VTR-HSA、Gly2-GLP-1-SARSVRA-HSA、Gly2-GLP-1-GRSRVTRSV-HSA、Gly2-GLP-1-GGGGG-HSA。结论引入蛋白酶切割位点后,GLP-1可在体内从融合蛋白中解离,使其恢复生物学活性。只有控制融合蛋白在体内适当的解离速率才能达到最佳疗效,从而在药代动力学和药效动力学性质之间达到平衡。

Objective To construct four types of glucagon-like peptide-1 （GLP-1） and human serum albumin （HSA） fusion proteins that can be realeased at different rate in vivo by introducing protease cleavage sites between these two moieties. The therapeutic effect and release rate are studied to achieve balanced pharmacokinetics （PK） and pharmacodynamics （PD） of GLP-1 and HSA fusion proteins. Methods The gene with different polypeptide joint of GLP-1 and HSA fusion proteins were synthesized by overlap extension PCR amplification, cloned into expression vector pPIC9 and transformed into Pichia pastoris GS115. Then, fusion proteins were obtained by protein purification after being induced by methanol. The preliminary PK and PD of the fusion proteins were studied after purification. Results The fusion protein Gly2-GLP-1-GGGGG-HSA showed no release while Gly2-GLP-1-VTR-HSA, Gly2-GLP-1-SARSVRA-HSA, and Gly2-GLP- 1-GRSRVTRSV-HSA showed a slow, medium and fast release rate, respectively, after incubation with furin. In vitro biological activity test results dispalyed that each type of fusion protein promoted insulin secretion of MIN6 cells. In vivo PK test indicated the half-life size of fusion proteins was the largest in Gly2-GLP-1-GGGGG-HSA, followed by Gly2-GLP-1- VTR-HSA, Gly2-GLP-1-SARSVRA-HSA, and Gly2-GLP-1-GRSRVTRSV-HSA. In vivo PD test exhibited hypoglycemic activity that was the highest in Gly2-GLP-1-VTR-HSA, followed by Gly2-GLP-1-SARSVRA-HSA, GIy2-GLP-1- GRSRVTRSV-HSA, and Gly2-GLP-1-GGGGG-HSA. Conclusion GLP-1 can be released from fusion proteins with full activity after the introduction of protease cleavage sites. Releasable fusion proteins at an appropriate release rate have the most balanced PK and PD.