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Polypeptide conjugates and methods of use

A technology of conjugates and peptide linkers, applied in the field of disease prevention and/or treatment, can solve the problems of short half-life, low efficacy, frequent dosing frequency, etc.

Active Publication Date: 2022-07-08
BEIJING QL BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing treatments for metabolic diseases face problems such as short half-lives and / or low efficacy
[0005] In addition, existing GLP-1 compounds are mainly administered by injection, and patients may fear self-administration if the frequency of administration is too frequent

Method used

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  • Polypeptide conjugates and methods of use
  • Polypeptide conjugates and methods of use
  • Polypeptide conjugates and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0499] Example 1: Recombinant expression and purification of GLP-1 protein

[0500] The GLP-1 proteins listed in Table 1 were produced using a BL21(DE3) derived strain, produced by a bacterial E. coli expression system. DNA encoding the GLP-1 precursor was codon optimized for E. coli expression, synthesized de novo and subcloned into a PET-derived expression vector (Novagen). Amino acid substitutions are accomplished by modifying the corresponding genetic code. Overexpression of GLP-1 precursor was induced with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) when the cell density reached an OD600 of 2.0 in Terrific broth (TB) medium. Cells were harvested after protein induction for 20-22 hours at 37°C. Cells were harvested and lysed by a cell crusher (900 bar, twice) in 20 mM Tris pH 8.0, 0.15 M NaCl buffer. Soluble fractions containing GLP-1 protein were collected by centrifugation (8,000 xg, 30 min). After removal of the tag by protease, the protein was purified by reverse...

Embodiment 2

[0501] Example 2: Incorporation of non-protein amino acids into recombinant proteins

[0502] The N-terminal His-Aib-Glu-Gly tetrapeptide or His-Aib dipeptide was dissolved in an organic solvent and added to a solution of GLP-1 protein dissolved in an organic solvent. The reaction was stirred at room temperature for 3 hours. Piperidine was then added to the reaction solution to remove the Fmoc protecting group.

Embodiment 3

[0503] Example 3: Preparation of GLP-1 Compounds with CRM

[0504] To the NaOH solution of GLP-1 protein was added dropwise a solution of CRM reagent (ie HOOC-(CH2)16-CO-gGlu-2XADO) in organic solvent. The reaction was stirred at room temperature for 1 h. Subsequently, the product was applied to reverse phase chromatography. This gave the compounds listed in Table 1 as shown above.

[0505] Detect and characterize conjugated GLP-GLP-conjugated GLP- 1 protein.

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Abstract

The present application provides a polypeptide conjugate comprising a GLP-1 receptor agonist and a peptide linker, and a pharmaceutical composition comprising the same. Also provided are methods of using such substances in the treatment of disease.

Description

[0001] Field of Invention [0002] The present invention relates to polypeptide conjugates, pharmaceutical compositions thereof and methods of using such substances to prevent and / or treat diseases. Background technique [0003] The major biologically active fragment of glucagon-like peptide-1 (GLP-1) is a 30 or 31 amino acid peptide fragment (amino acids 7-36 or 7-37). The original GLP-1 product GLP-1 stimulates insulin synthesis and secretion and has been shown to prevent hyperglycemia in diabetes, especially type 2 diabetes. However, endogenous GLP-1 only has a half-life of roughly 2 minutes, which results in fasting plasma levels of GLP-1 of only 0-15 pmol / L. [0004] Metabolic disorders are often associated with insulin resistance, visceral obesity, atherogenic dyslipidemia, etc., which pose a significant and escalating public health and clinical challenge worldwide. However, existing treatments for metabolic diseases face problems such as short half-lives and / or low e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
CPCA61K38/00C07K14/605C07K2319/31C12N15/70A61K47/65A61P3/04A61P3/06A61P3/10A61K38/26
Inventor 张媛媛吴心乐邹海霞翟鹏金耀光吴博陈旭郭威赵新宇王作斌曾伶俐
Owner BEIJING QL BIOPHARMACEUTICAL CO LTD