Pegylated exenatide ramification and use thereof

A technology of PEGylation and exenatide, which is applied in the direction of peptides, specific peptides, hormone peptides, etc., can solve the problems of protein activity loss, etc., and achieve the effects of prolonging half-life, reducing gastrointestinal emptying, and increasing the number of β cells

Inactive Publication Date: 2012-12-19
无锡和邦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the disadvantage is that the sulfhydryl group of cysteine ​​is usually in the active center of the protein, and the modification will cause a large loss of protein activity

Method used

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  • Pegylated exenatide ramification and use thereof
  • Pegylated exenatide ramification and use thereof
  • Pegylated exenatide ramification and use thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0037] Example 1 Preparation of Pegylated Exenatide Derivatives

[0038] Exenatide compound peptides are produced by reacting covalent polyethylene glycol derivatives with thioether bonds with PEG-maleimide. Taking monomethoxypolyethylene glycol-maleimide with a molecular weight of 40,000 Daltons as an example, accurately weigh 10 mg (0.002 mmol) of Exendin-4-Cys with a length of 40 amino acids, dissolve in 5 mL of pH 7.0 50 mM phosphate buffered saline. 121 mg of polyethylene glycol maleimide (mPEG-MAL:Exendin-4-Cys molar ratio = 1.3:1) was added to the exenatide derivative solution. The reaction was carried out at room temperature for 90 min to obtain a crude Exendin-4-Cys-PEG solution. PEGylated exenatide is purified by ion exchange, electrophoresis or high performance liquid phase assay.

Embodiment 2

[0039] Example 2 Purification and determination of pegylated exenatide derivatives

[0040] Taking the monomethoxypolyethylene glycol-maleimide-linked exenatide compound with an average molecular weight of 40,000 Daltons as an example, MacroCap SP (GE) was selected as the purification medium, and the column volume was 10 mL. 20mM NaAc buffer pH4.0 was used as buffer A, and 20mM NaAc buffer pH4.0 containing 1M NaCl was used as buffer B. Equilibrate the chromatographic column with buffer A first, and then load the sample after 5-fold dilution of buffer A. After sample loading is complete, wash 5 column volumes with Buffer A. Buffer B gradient elution, the gradient is 15%B+85%A, 25%B+75%A, 80%B+20%A, flow rate: 4mL / min, detection wavelength: 220nm detection. See the specific chromatogram figure 1 . The chromatogram shows that peak 1 is a breakthrough peak, peak 2 is an elution peak of 15%B+85%A, peak 3 is an elution peak of 25%B+75%A, and peak 4 is an elution peak of 80%B+20%...

Embodiment 3

[0042] Example 3 In Vitro Biological Activity Research of Pegylated Exenatide Derivatives

[0043]Taking monomethoxypolyethylene glycol-maleimide-linked Exendin-4-Cys with an average molecular weight of 20,000, 30,000, or 40,000 Daltons as an example, the in vitro biological activity was measured using a cell reporter system. The cell line is the HEK-293 cell line (GLP-1R / HEK293) that can stably express the GLP-1 receptor. This cell line has been preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, and the preservation number is CGMCC No.5909 (patent Application number 201210117317.4, title of invention: a cell line for measuring the biological activity of GLP-1 and its functional analogs and its application).

[0044] The assay process is as follows: first, the cell lines were diluted with DMEM / F12 medium containing low concentration serum, and plated in 96-well plates at 30,000 cells / 100 μL / well. 37°C, 5% CO 2 Condit...

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Abstract

The invention provides a pegylated exenatide ramification (Exendin-4-Cys-PEG (polyethylene glycol)) and the use of the pegylated exenatide ramification, belonging to the technical field of the preparation and the application of a polypeptide drug. A preparation method of the pegylated exenatide ramification provided by the invention comprises the following steps of: introducing a cysteine at the C-end of exenatide, and coupling maleic acylamino polyethylene glycol, so that the pegylated exenatide ramification can be formed. The pegylated exenatide ramification provided by the invention has slow clearance rate in the blood circulation and prolonged half-life period, and can be used for preparing the related illness treatment drugs for reducing the blood sugar, reducing the ingestion, reducing the gastrointestinal emptying, increasing the beta cell quantity or reducing the gastrointestinal motility.

Description

technical field [0001] The present invention relates to a compound, in particular to pegylated exenatide derivatives and uses thereof, that is, a cysteine ​​molecule is introduced at the C-terminal of exenatide, and mPEG-maleyl As a PEGylation reagent, imine can add the sulfhydryl group of the cysteine ​​molecule introduced at the C-terminal of exenatide to the double bond of mPEG-maleimide to realize the site-specific modification of the protein, so that the modified The exenatide compound slows down the clearance rate in the blood circulation, improves the stability, and prolongs the half-life. It belongs to the technical field of polypeptide drug preparation and application. Background technique [0002] Glucagon-like peptide-1 (Glucogan like Peptide-1, GLP-1) acts on the glucagon-like peptide-1 receptor (Glucogan like Peptide-1 Receptor, GLP-1R) of pancreatic beta cells to promote insulin gene The transcription, synthesis and secretion of insulin, and can stimulate the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/575C07K1/16A61K38/22A61K47/48A61P3/10
Inventor 杨建良陆晶孟如杰庄少颖
Owner 无锡和邦生物科技有限公司
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