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Glucagon-like peptide-1 mutant polypeptide, its preparation method and application thereof

A technique for glucagon and mutants, applied in the field of novel glucagon-like peptide-1 mutant polypeptides

Active Publication Date: 2012-11-07
TIANJIN INSTITUTE OF PHARMA RESEARCH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the half-life of GLP-1 receptor agonists such as Liraglutide on the market has been extended, patients still need to inject the drug every day, and there is still room for improvement in the comfort of drug use

Method used

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  • Glucagon-like peptide-1 mutant polypeptide, its preparation method and application thereof
  • Glucagon-like peptide-1 mutant polypeptide, its preparation method and application thereof
  • Glucagon-like peptide-1 mutant polypeptide, its preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0101] Example 1 Synthesis of GLP-1 mutant tandem polypeptides

[0102] 1.1: Determination, preparation and sequencing verification of the GLP-1 polypeptide sequence. Using the amino acid protected by Fmoc at the amino terminal as the raw material, it was synthesized by the solid-phase synthesis method on the Liberty1 single-channel fully automatic microwave peptide synthesis system (purchased from the Beijing Office of Paian Technology Co., Ltd., USA). The synthesis method refers to the manufacturer's instrument manual. conduct. After deprotection, coupling and final cleavage reaction of Fmoc amino acid (purchased from Shanghai Jier Biochemical Co., Ltd.), the target polypeptide is finally formed. The obtained polypeptide was confirmed by sequencing by Shanghai Sangon Biotechnology Company.

[0103] 1.2: Determination, preparation and sequencing verification of Exendin-4(31-39). Using the amino acid protected by Fmoc at the amino terminal as the raw material, it was synt...

Embodiment 2

[0107] Example 2 In vitro GLP-1 receptor binding experiment of GLP-1 mutant tandem polypeptides

[0108] The receptor binding experiments were performed using the GLP-1 receptor high-expressing cell line INS-1 (rat β-insulinoma cells, ATCC, Manassas, VA). First, INS-1 cells were seeded on a 12-well plate, and the cells were washed with PBS buffer 2 hours before the experiment, and then the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After cell counting, 10 5 Each cell / well was co-incubated with GLP-1 mutant tandem polypeptide (SEQ ID NO6-14) and natural GLP-1 for 2 hours respectively. After washing the unbound polypeptides with PBS, the GLP-1 ELISA kit was used for detection. It can be seen from Table 1 that the GLP-1 mutant tandem polypeptide greatly improves its binding ability to the GLP-1 receptor, compared with GLP-1.

[0109] Table 1, GLP-1 mutant tandem polypeptides and GLP-1 polypeptides

[0110] Binding Constants for GLP-1 Recep...

Embodiment 3

[0112] Example 3 Experiment of hypoglycemic function of GLP-1 mutant tandem polypeptide in mice

[0113] Intravenously inject mice with 9 different GLP-1 mutant tandem polypeptides according to the present invention (injection dose is 100 μg / kg), with Exendin-4 as a positive control, respectively at 0.5, 4, 6, and 8 hours after administration. , 12, 24 and 48 hours inject glucose (2g / kg), measure the blood glucose level half an hour after the injection, from figure 1 It can be seen that Exendin-4 completely loses its hypoglycemic function after 4 hours, but the nine GLP-1 mutant tandem peptides still have the hypoglycemic function after 48 hours of administration.

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Abstract

The invention provides a glucagon-like peptide-1 mutant polypeptide, its preparation method and an application thereof. The mutant series polypeptide is formed by adding Cys series Exendin-4 (31-39) to the C terminal of the glucagon-like peptide-1 mutant. And the mutant series polypeptide folds itself to form a disulfide bond. The glucagon-like peptide-1 mutant polypeptide has an amino acid sequence as shown in SEQ ID NO 1, which is generated by point mutation to Cys occurring at the 9th Asp, 16th Gly or 27th Val of amino acid residues and the addition of Cys to the C terminal. The Exendin-4(31-39) has an amino acid sequence as shown in SEQ ID NO 5. The glucagon-like peptide-1 mutant series polypeptide is used for preparation of pharmaceutical compositions for treating diabetes and treating and / or preventing obesity.

Description

technical field [0001] The present invention relates to the field of medicines related to diabetes, in particular, the present invention relates to a novel glucagon-like peptide-1 (GLP-1) mutant polypeptide, which has prolonged half-life of GLP-1 in vivo. The invention also relates to the preparation method and application of the polypeptide. Background technique [0002] Diabetes mellitus is a metabolic disorder syndrome characterized by chronic hyperglycemia associated with genetic factors and multiple environmental factors. Because diabetes is also accompanied by many complications, it has become the third largest health killer after malignant tumors and cardiovascular diseases. In 1984, Glucagon-like peptide-1 (GLP-1) was discovered, which is a 30-amino acid incretin that promotes insulin secretion and biosynthesis, inhibits pancreatic The secretion of glucagon promotes the proliferation of islet cells, inhibits the apoptosis of islet cells, and preserves the sensitivi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K19/00A61K38/26A61P3/10A61P3/04
Inventor 付刚龚珉徐为人王玉丽汤立达任晓文
Owner TIANJIN INSTITUTE OF PHARMA RESEARCH
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