Glucagon-like peptide-2 analog dimer and its preparation method and application

An analog and dimer technology, which is applied to the glucagon-like peptide-2 analog dimer and the fields of its preparation and application, and can solve the problems of short half-life and the like

Active Publication Date: 2018-01-12
天津天诚新药评价有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, many studies have adopted GLP-2 analog fusion protein technology to solve the retention time of GLP-2 analogs in the body (CN200680015213.5; CN200380101375.7; CN200680047664.7; CN200780041490.8; CN200510039265.3; CN200910126363.9 ), however, most of them change the structure of GLP-2 through mutations of amino acid residues such as substitution and deletion, which can resist the degradation of DPP-IV to a certain extent, but there is still a long way to go from the ideal clinical goal
[0007] Therefore, there is currently a need for methods to address the short in vivo half-life of GLP-2

Method used

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  • Glucagon-like peptide-2 analog dimer and its preparation method and application
  • Glucagon-like peptide-2 analog dimer and its preparation method and application
  • Glucagon-like peptide-2 analog dimer and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1: Solid Phase Synthesis of GLP-2 Analog Dimers

[0085] Using the solid-phase polypeptide synthesis method of the Fmoc strategy, the CS 336X instrument produced by CSBio Company was used to synthesize the polypeptide of the present invention. The method of synthesis was carried out according to the manufacturer's instruction manual.

[0086] The prepared polypeptide was purified using an HPLC C18 semi-preparative column (purchased from Thermal Company), and the mobile phase was acetonitrile. The polypeptide lyophilized powder is obtained by desalting and freeze-drying, which is the polypeptide lyophilized powder of the GLP-2 analog monomer of the amino acid sequence shown in SEQ ID NO:2.

[0087] Then, the lyophilized polypeptide powder of the GLP-2 analog monomer with the amino acid sequence shown in SEQ ID NO: 2 was mixed with 7 g / L ammonium bicarbonate (purchased by Shanghai Jier Company) overnight to form inter-monomer disulfide bonds A GLP-2 analog dim...

Embodiment 2

[0090] Example 2: GLP-2 analog dimer (formed by SEQ ID NO: 2 and SEQ ID NO: 2) in human serum Stability determination

[0091] In this embodiment, the polypeptide used is as follows: GLP-2 analogue dimer (SEQ2 / 2) formed by SEQ ID NO:2 and SEQ ID NO:2.

[0092] Two blood samples were taken from volunteers with a vacuum blood collection needle (BD Biosciences, Franklin Lakes, NJ), and then immediately centrifuged in a centrifuge at 13,000 rpm for 20 minutes, and the upper layer of serum was taken for later use.

[0093] Take the above-mentioned GLP-2 analog dimer polypeptide and 0.5 mg of GLP-2 purchased on the market and dissolve them in 0.5 mL of normal saline, respectively, and add them to serum after fully dissolving, and label the serum as GLP-2 group and SEQ2 / 2 experimental groups. After incubating at 37°C for 0, 0.5, 1, 2.5, 5, 10, 15 and 24 h, 50 μL of the serum mixture was taken respectively, and the serum samples were detected with the GLP-2 ELISA kit (purchased ...

Embodiment 3

[0095] Example 3: GLP-2 analog dimer (formed by SEQ ID NO:3 and SEQ ID NO:3) in human serum Stability determination

[0096] In this embodiment, the polypeptide used is as follows: SEQ ID NO:3 and the GLP-2 analog dimer of SEQ ID NO:3 (SEQ3 / 3).

[0097] Two blood samples were taken from volunteers with a vacuum blood collection needle (BD Biosciences, Franklin Lakes, NJ), and then immediately centrifuged in a centrifuge at 13,000 rpm for 20 minutes, and the upper layer of serum was taken for later use.

[0098] Take the above-mentioned GLP-2 analog dimer polypeptide and 0.5 mg of GLP-2 purchased on the market and dissolve them in 0.5 mL of normal saline, respectively, and add them to the serum after fully dissolving, and label the serum as GLP-2 group and SEQ3 / 3 experimental group. After incubating at 37°C for 0, 0.5, 1, 2.5, 5, 10, 15 and 24 h, 50 μL of the serum mixture was taken respectively, and the serum samples were detected with the GLP-2 ELISA kit (purchased from...

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Abstract

The present invention provides a glucagon-like peptide-2 analogue dimer, a preparation method and an application thereof, wherein two identical or different GLP-2 analogue monomers form a disulfide bond through cysteines on the monomers so as to prepare the dimer. The dimer preparation method comprises: adopting an Fmoc solid phase polypeptide synthesis method to synthesize a GLP-2 analogue monomer, and making the synthesized GLP-2 analogue monomers form a disulfide bond between the monomers. The application is an application of the GLP-2 analogue dimer in preparation of drugs for treatment of gastrointestinal related diseases. According to the present invention, with the GLP-2 analogue dimer formed from the GLP-2 analogue monomer, the problem of the short half-life of the GLP-2 is overcome, and the half-life of the GLP-2 analogue dimer can achieve more than 8-96 h in vivo, and is significantly prolonged compared with the half-life of the GLP-2 administered separately so as to substantially and easily achieve clinical promotion and application.

Description

technical field [0001] The present invention relates to a glucagon-like peptide-2 (Glucagon-like peptide-2) analogue dimer and its preparation method and application, specifically, the modified glucagon-like peptide-2 The medical use of analogues in the prevention or treatment of gastrointestinal related diseases, recovery of intestinal mucosal damage, promotion of small intestine growth, promotion of transplanted small intestine absorption function and recovery of ultrastructure belongs to the field of medical technology. Background technique [0002] Glucagon-like peptide-2 (hereinafter referred to as: GLP-2) is a single-chain polypeptide composed of 33 amino acid residues ( 1 HADGSFSDEMNTILDNLAARDFINWLIQTKITD 33 ), mainly secreted by intestinal specific tissues and L cells, encoded and expressed by proglucagon gene, highly expressed in pancreas, intestinal tract and brain tissue, and is degraded by prohormone converting enzyme one of the products. It is co-secreted wit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/605C07K19/00C07K1/04A61K38/26A61P1/00
CPCA61K38/00C07K14/605
Inventor 王玉丽郑学敏龚珉徐为人汤立达邹美香吴疆
Owner 天津天诚新药评价有限公司
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