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Glucagon-like peptide-1 analogue monomer, glucagon-like peptide-1 analogue dimer, and application of glucagon-like peptide-1 analogue monomer and glucagon-like peptide-1 analogue dimer

A technology for glucagon and analogues, applied in the direction of glucagon, hormone peptides, biochemical equipment and methods, etc., can solve the problems of complex and harsh purification conditions, complex tertiary structure, and limited clinical application, and achieve therapeutic Better effect, simple process, and simple peptide structure

Active Publication Date: 2020-11-17
GUANGDONG PHARMA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in vivo, GLP-1(7-37) and GLP-1(7-36)-NH 2 The DPP-4 enzyme that is widely distributed in organs and tissues cuts off the two amino acids His-Ala at the N-terminus, and becomes inactive GLP-1(9-37) and GLP-1(9-36)-NH 2 , due to its small molecular weight of about 3kDa, it is easily cleared by the kidneys and excreted from the body. The above two main metabolic pathways make the half-life of GLP-1 less than 2min, which greatly limits its clinical application
However, the above-mentioned HSA and IgG have relatively large molecular weights, and the tertiary structure of the protein is complex. The correct renaturation of the protein is the focus, difficulty and challenge of the technology. In particular, HSA has 35 cysteines, and its tertiary structure has 17 pairs. Disulfide bond and a free cysteine ​​Cys34, the correct renaturation of the protein makes the purification conditions complicated and harsh, which is difficult and the yield is low

Method used

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  • Glucagon-like peptide-1 analogue monomer, glucagon-like peptide-1 analogue dimer, and application of glucagon-like peptide-1 analogue monomer and glucagon-like peptide-1 analogue dimer
  • Glucagon-like peptide-1 analogue monomer, glucagon-like peptide-1 analogue dimer, and application of glucagon-like peptide-1 analogue monomer and glucagon-like peptide-1 analogue dimer
  • Glucagon-like peptide-1 analogue monomer, glucagon-like peptide-1 analogue dimer, and application of glucagon-like peptide-1 analogue monomer and glucagon-like peptide-1 analogue dimer

Examples

Experimental program
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Effect test

Embodiment 1

[0076] According to the metabolism characteristics of GLP-1 in vivo, the present invention has modified it from the following aspects: under the premise of ensuring the biological activity of GLP-1, the key amino acid at the action site of the mutated protease DPP-4 is replaced by Gly Ala at position 30 and Arg at position 30, the mutant mGLP-1 of GLP-1 is obtained; mGLP-1 is tandemly fused with the serum albumin binding domain ABD to increase molecular weight and reduce renal clearance, while the fusion protein passes through ABD Non-covalently binds to serum albumin HSA to form a complex, utilizes the FcRn-mediated recycling mechanism, and evades lysosome and glomerular filtration.

[0077] In the present invention, in order to facilitate the purification of the expressed fusion protein, a His purification tag is designed at the N-terminus; in order to release the active fusion protein, the restriction site Linker-1 of endogenous protease thrombin and DPP-4 is designed to Re...

Embodiment 2

[0090] Using the glucagon-like peptide-1 analog monomer in Example 1 to construct a glucagon-like peptide-1 analog dimer, the specific process includes the following:

[0091] 1. pET30a-m(GLP-1) 2 -Construction of ABD recombinant plasmid

[0092] pET30a-m(GLP-1) 2 -The construction of the ABD recombinant plasmid comprises four steps:

[0093] 1. SOE-PCR amplification (mGLP-1) 2 -ABD fusion gene

[0094] (mGLP-1) 2 - The ABD fusion gene has 507bp, and the full sequence of the fusion gene was obtained by three-step SOE-PCR using 3 pairs of overlapping complementary primers.

[0095] (1) The first step SOE-PCR

[0096] According to (mGLP-1) 2 -The amino acid sequence of the ABD fusion protein, using Escherichia coli preferred codons, designing overlapping complementary primers P1 and P2, the primer sequences are as follows:

[0097] Upstream primer P1 (SEQ ID NO.3)

[0098]

[0099] Downstream primer P2 (SEQ ID NO.4)

[0100] 5'-AACGTCGGAAGTGAAGGTACCTTCACCGTGAGCGGAGC...

Embodiment 3

[0147] 3.1. Glucagon-like peptide-1 monomer m (GLP-1) 2 -ABD and its dimer [m(GLP-1) 2 -ABD] 2 Pharmacokinetics in SD rats

[0148] SD rats with a body weight of about 300 g (provided by Suibei Experimental Animal Farm, Baiyun District, Guangzhou City) were randomly divided into two groups (m(GLP-1) 2 -ABD group, [m(GLP-1) 2 -ABD] 2 group), 8 rats in each group, half male and half male. The concentration of 100nmol / mL (m(GLP-1) 2 -ABD solution, [m(GLP-1) 2 -ABD] 2 Solution (prepared and obtained in Example 2). The dose of 30nmol / kg was subcutaneously injected into the rats of each group respectively. Blood was collected from the orbital venous plexus, and blood samples were collected 0 h before administration and 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 36, and 48 h after administration, and placed in EDTA-treated EP tube. 4°C, 4000g, centrifuge at low temperature for 10min, take the supernatant, quickly freeze it in liquid nitrogen and store it at -80°C for later use, ...

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Abstract

The invention discloses a glucagon-like peptide-1 analogue monomer. The amino acid sequence of the monomer is shown as SEQ ID NO.1, and a fusion protein obtained by fusing two-molecule mutant glucagon-like peptides-1 in series connection with a human albumin binding domain ABD is named as (mGLP-1) 2-ABD and is represented by the following general formula: 6His-Linker-1-mGLP-1-Linker-2-mGLP-1-Linker-2-ABD-Linker-2-Cys. The invention also discloses a glucagon-like peptide-1 analogue dimer prepared from the monomer, and an application of the glucagon-like peptide-1 analogue monomer and pharmaceutically acceptable salt thereof or the glucagon-like peptide-1 analogue dimer and pharmaceutically acceptable salt thereof to preparation of medicines with efficacy of preventing and treating non-insulin-dependent diabetes and complications thereof.

Description

technical field [0001] The invention belongs to the technical field of diabetes drugs, in particular to a glucagon-like peptide-1 analogue monomer and its dimer and its application, especially to a long-acting glucagon-like peptide-1 analogue with extended half-life It also relates to the preparation method of the above-mentioned monomer and its dimer and the preparation of the above-mentioned monomer and its dimer with the effect of preventing and / or treating non-insulin-dependent diabetes mellitus and its complications application in medicine. Background technique [0002] According to the latest global overview of diabetes (IDF Diabetes Atlas) (9th edition) released by the International Diabetes Federation (IDF), in 2019, about 463 million adults around the world suffer from diabetes, with a crude prevalence rate of 9.3%; it is estimated that by 2030 and In 2045, the number of diabetic patients will reach 578.4 million and 700.2 million, respectively, and the crude preva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K38/26A61K47/64A61P3/10A61P3/04A61P3/00C12R1/19
CPCC07K14/605C07K14/315C12N15/70A61P3/10A61P3/04A61P3/00C07K2319/21C07K2319/50A61K38/00
Inventor 桑延霞李占潮张军军
Owner GUANGDONG PHARMA UNIV
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