Orexin-A pegylation modifier and preparation method thereof

A PEGylation and polyethylene glycol technology, applied in the field of medicinal chemistry, can solve the problems of restricting the development and application of orexin-A, instability of orexin-A, etc., and achieve the effects of good product uniformity and simple separation and purification

Inactive Publication Date: 2014-05-21
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, orexin-A is very unstable, and the reported half-life in vivo is only ab

Method used

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  • Orexin-A pegylation modifier and preparation method thereof
  • Orexin-A pegylation modifier and preparation method thereof
  • Orexin-A pegylation modifier and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0017] Example 1

[0018] The preparation method of orexin-A pegylated modified substance includes the following steps:

[0019] (1) Weigh 10mg of orexin-A, dissolve it in 10ml of PB buffer (10mM, pH=6), then add monooxypolyethylene glycol propionaldehyde (molecular weight: 5000Da) at a molar ratio of 1:10 And mix well, put it at 25℃ to react for 6 hours and then terminate the reaction;

[0020] (2) Pass the reaction solution of step (1) through a SepHadex G-25 column for desalting, and load the protein collected after desalting on a QAE-SepHadex A-50 anion exchange column, eluting with 10mM PB buffer, The elution peaks are collected, concentrated and freeze-dried to obtain orexin-A pegylated modification.

Example Embodiment

[0021] Example 2

[0022] The preparation method of orexin-A pegylated modified substance includes the following steps:

[0023] (1) Weigh 10mg of orexin-A, dissolve it in 10ml of PB buffer (10mM, pH=7), then add monooxypolyethylene glycol propionaldehyde (molecular weight: 5000Da) at a molar ratio of 1:20 And mix well, put it at 25℃ to react for 5 hours and then terminate the reaction;

[0024] (2) Pass the reaction solution of step (1) through a SepHadex G-25 column for desalting, and load the protein collected after desalting on a QAE-SepHadex A-50 anion exchange column, eluting with 10mM PB buffer, The elution peaks are collected, concentrated and freeze-dried to obtain orexin-A pegylated modification.

Example Embodiment

[0025] Example 3

[0026] The preparation method of orexin-A pegylated modified substance includes the following steps:

[0027] (1) Weigh 10mg of orexin-A, dissolve it in 10ml of phosphate buffer (10mM, PH=8), then add monooxypolyethylene glycol propionaldehyde (molecular weight: 5000Da) at a molar ratio of 1:30 ) And mix well, and stop the reaction after 4 hours in the dark at 25°C;

[0028] (2) Pass the reaction solution of step (1) through SepHadex G-25 column for desalting, load the protein collected after desalting on QAE-SepHadex A-50 anion exchange column, and elute with 10 mM PB buffer , Collect the elution peaks, concentrate and freeze-dry to obtain orexin-A pegylated modification.

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PUM

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Abstract

The invention discloses an Orexin-A pegylation modifier which is connected with polyethylene glycol by an N-terminal amino covalent bond of Orexin-A. A preparation method of the Orexin-A pegylation modifier comprises the steps of enabling the Orexin-A and methoxy polyethylene glycol propionaldehyde or methoxy polyethylene glycol succinamide carbonate to undergo a reaction for 0.5-24 hours under the conditions that the pH is 4-9, and the temperature is 4-37 DEG C, wherein the molar ratio of the Orexin-A to the methoxy polyethylene glycol propionaldehyde or the methoxy polyethylene glycol succinamide carbonate is 1: 5-1: 50; then, adding small molecule amino acid to stop the reaction; desalting the product obtained in the first step by a Sephadex G-50 chromatographic column; after that, sampling the protein collected after desalting onto a QAE-Sephadex A-50 anion exchange column; and carrying out elution by a 10mM PB (phosphate buffer) solution, collecting the eluting peak, concentrating and carrying out freeze drying to obtain the Orexin-A pegylation modifier. The Orexin-A pegylation modifier is good in stability, long in half-life period and low in toxic and side effects.

Description

technical field [0001] The invention relates to a modified product of orexin-A modified with polyethylene glycol and a preparation method thereof, belonging to the field of medicinal chemistry. Background technique [0002] Protein and polypeptide drugs PEGylation technology refers to a popular technology for chemically modifying protein and polypeptide drugs by using polyethylene glycol derivatives. With the use of modern genetic engineering methods, people can obtain the required protein in large quantities, but because natural or recombinant protein drugs without any modification are easy to cause immune reactions, and their half-life in the body is short, the clinical effect is weakened. effect, which greatly limits their use. In contrast, partially PEG-modified proteins include physical conjugates and chemical modifications of PEG and protein and polypeptide drugs, which can change some properties of protein drugs, such as: Increase the solubility and stability of su...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/107A61K38/17A61K47/48A61P25/00A61P9/10
CPCA61K38/22A61K47/60C07K14/47A61K38/1709
Inventor 董海龙郭超吴畏
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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