The Preliminary Study On Releasable Glucagon-like Peptide-1 And Human Serum Albumin Fusion Proteins

Glucagon-like peptide-1 is secreted by intestinal L-cells and made of 30 residue peptide. GLP-1 plays an important role in stimulating insulin secretion in a glucose dependent manner, slowing the emptying of the stomach, regulation of feeding, inhibiting glucagon secretion, increasing B cell proliferation, inhibiting B cell apoptosis and without low blood glucose dangerous. After binding with GLP-1 receptors which are on intestinal beta-cells, GLP-1 can control insulin secretion in the form of glucose dependent. Type Ⅱ diabetes is often characterized by GLP-1 damaged secretion. But islet beta cells are normal to react to GLP-1. Therefore GLP-1 has a good application prospect in the treatment of patients with type 2 diabetes. The active GLP-1 produced by the body itself was easily degraded by dipeptide peptidase and became inactivation. At the same time it was easily cleaned up by the kidneys. So its biological half-life is only 2-6 min. Thus its clinical application and curative effect to a great extent is limited.By combining with human serum albumin (HSA) or soluble inert polymer polyethylene glycol (PEG) through chemical coupling method, the short peptide or small molecule protein drugs with a short half-life could obviously improve the pharmacokinetic characteristics in vivo. However, due to the space steric effect of human serum albumin, the traditional human serum albumin fusion technology often improves pharmacokinetic at the expense of pharmacodynamics. By inserting peptide sequence cleaved by furin between the GLP-1 and human serum albumin, this study made GLP-1 being released from the fusion proteins in a controlled and targeted way in the body and recovered of biological activities. At the same time Ala replaced with Gly on the second sequence of GLP-1 to stop DPP-Ⅳ degradation. Thus pharmacokinetics achieved balanced with pharmacodynamics and GLP-1 can show maximum efficacy.This study aimed to construct GLP-1 and HSA fusion proteins that could be realeased at different rate in vivo by introducing protease cleavage sites between these two moieties. Therapeutic effect and release rate was researched to achieve balanced pharmacokinetics and pharmacodynamics of GLP-1 and HSA fusion protein. The major methods and conclusions are below:1、The gene with different polypeptide joint of GLP-1 and HSA fusion protein was synthesized by multistep PCR amplification, cloned into expression vector pPIC9 and transformed into Pichia Pastoris GS115. Then fusion proteins were obtaind by protein purification after being induced by methanol.2、The different kinds of fusion proteins were incubated with furin, And the enzyme-digested products were tested by Western-blot. The results showed that nonreleasable fusion protein was not released under the action of furin. And releasable fusion proteins Gly2-GLP-1-VTR-HSA was with a slow rate; Gly2-GLP-1-SARSVRA-HSA was with a medium rate; Gly2-GLP-1-GRSRVTRSV-HSA was with a fast rate.3、Through the test of insulin secretion assay, the different kinds of fusion proteins in vitro biological activities were assigned. Fusion proteins stimulated MIN6 cells with 2 hours with a final concentration of 40μg/ml and then the cell culture supernatant was detected by insulin mouse elisa kit. The experimental results showed that in glucose concentration of 25mM and 0.5mM, insulin release quantity were higher than the blank control group and they had the extremely significant difference. But there was no significant difference among the fusion proteins.4、By subcutaneous injection 100μg of the different kinds of fusion proteins, tail vein blood was tested at different time points. Sandwich elisa was used to detect the serum concentration of fusion proteins and determine pharmacokinetic properties of different kinds of fusion proteins. The results showed that the half-life of various kinds of fusion proteins size in the order was:Gly2-GLP-1-GGGGG-HSA、Gly2-GLP-1-VTR-HSA、Gly2-GLP-1-SARSVRA-HSA、Gly2-GLP-1-GRSRVTRSV-HSA。5、The experimental mice were subcutaneous injection of saline and different fusion proteins with a dose of 40 nmol/kg immediately after the celiac injection of glucose alone. Blood was collected at different time points and blood glucose levels were determined to evaluate in vivo pharmacodynamics properties of all kinds of fusion protein. Results showed that all kinds of fusion proteins were exhibited hypoglycemic activity, and the active high or low in the order was Gly2-GLP-1-VTR-HSA、Gly2-GLP-1-SARSVRA-HSA. Gly2-GLP-1-GRSRVTRSV-HSA、Gly2-GLP-1-GGGGG-HSA.GLP-1 can release from the fusion proteins with full activity after the introduction of protease cleavage sites. Releasable fusion proteins with appropriate release rate had the most balanced PK and PD. Releasable human serum albumin fusion technology used in this study would provide an effective solution for long-term protein in drug development which was faced with the contradiction between the pharmacodynamics and pharmacokinetics. At the same time, GLP-1 and human serum albumin fusion proteins constructed in this study will also provide innovative drug research and development of solid experimental basis.