Preparation method and application of polyethylene glycol-polysialic acid block copolymers

A technology of block copolymer and polysialic acid, which is applied in the preparation method of peptides, chemical instruments and methods, medical preparations of non-active ingredients, etc., can solve the problem of PEG not easy to degrade, polysialic acid with small molecular weight, and PEG degradation Slow and other problems, to achieve the effect of good biodegradability, good immunogenicity, and strong hydration ability

Inactive Publication Date: 2010-07-28
山东弥美生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PEG degrades very slowly in the body, and large molecular weight PEG is not easily degraded, and it is easy to accumulate in tissues in the body, such as in the bladder
In addition, although it has been described that various methods of linking PSA to proteins are feasible, and some of them have reached clinical trials, it has also been shown that polysialic acid can achieve the same effect as PEG when applied to protein modification, but currently produced by fermentation The molecular weight of polysialic acid is less than 60000
Polysialic acid is an acidic polysaccharide with a strong negative charge, causing it to form a thinner hydration layer on the protein surface, weakening its masking effect

Method used

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  • Preparation method and application of polyethylene glycol-polysialic acid block copolymers
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  • Preparation method and application of polyethylene glycol-polysialic acid block copolymers

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1: Preparation of activated polysialic acid PSH-SH

[0038] Dissolve 80 mg of purified polysialic acid in 8 mL of phosphate buffer (pH7.4, 20 mM), pre-cool at 4 ° C, and then add cystamine dihydrochloride (polysialic acid: cystamine dihydrochloride=1 : 100 molar ratio), while adding dropwise a small amount of 0.5M sodium hydroxide to adjust the pH to 7.4. Stir magnetically at 4 °C for 2 days. Dithiothreitol was then added to a final concentration of 50 mM, and reacted at 4° C. for 1 h. The reaction solution was ultrafiltered three times with a 10KDa ultrafiltration tube (3000g, 30min), the buffer system was changed to PBS (pH7.4), and the concentrated product was activated polysialic acid PSH-SH, whose terminal functional group was thiol.

Embodiment 2

[0039] Embodiment 2: Preparation of PEG-PSA block copolymer and its application in modifying SOD enzyme

[0040] Dissolve an appropriate amount of MAL-PEG-NHS in DMSO, add excess PSA-SH newly prepared in Example 1, react at room temperature for 30 minutes, dialyze the organic solvent with a 7K dialysis membrane, and freeze-dry to obtain NHS-PEG-PSA. Dissolve SOD enzyme (2mg / mL) in phosphate buffer (pH7.0~9.0), pre-cool at 4°C for 30min, add NHS-PEG-PSA (NHS-PEG-PSA:SOD=10~20:1 molar ratio ), stirred magnetically at 4°C for 1 h. The reaction solution was ultrafiltered three times with a 10KDa ultrafiltration tube (3000g, 30min), concentrated and freeze-dried to obtain the product SOD-PEG-PSA.

Embodiment 3

[0041] Example 3: MAL-PEG-NHS is first connected with SOD enzyme to form SOD-PEG-MAL, and then connected with activated polysialic acid PSH-SH

[0042] Dissolve SOD enzyme (2mg / mL) in phosphate buffer (pH7.0~9.0), pre-cool at 4°C for 30min, add MAL-PEG-NHS (MAL-PEG-NHS:SOD=10~20:1 molar ratio ), stirred magnetically at 4°C for 1 h. The reaction solution was ultrafiltered three times with a 10KDa ultrafiltration tube (3000g, 30min), and concentrated to obtain SOD-PEG-MAL.

[0043] Then the above freshly prepared reaction solution was pre-cooled at 4°C for 30 min, and then a certain amount of PSA-SH freshly prepared in Example 1 was added (PSA-SH:SOD-PEG-MAL molar ratio was 15-20:1). Phosphate buffer was added to make the reaction solution at an appropriate concentration, and the reaction was carried out at 4°C for 30 min. The reaction solution was ultrafiltered three times with a 10KDa ultrafiltration tube (3000g, 30min), concentrated and freeze-dried to obtain the product SO...

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Abstract

The invention provides a preparation method and application of polyethylene glycol-polysialic acid block copolymers, which belong to the technical field of polymer materials. The copolymers are formed by connecting activated polysialic acid with different-base bifunctional polyethylene glycol, and are expressed as X-PEG-F-PSA, wherein X is one of active groups of different-base bifunctional PEG and is used to be connected with the active groups on protein or polypeptide; F is another active group of the different-base bifunctional PEG and is used to be connected with activated polysialic acid PSA; the block copolymers X-PEG-F-PSA are connected with Protein so as to form Protein-PEG-PSA products. The products can be widely used to modify amino-containing biological functional molecules, particularly protein or polypeptide and other macromolecules, and by utilizing the strong hydration performance of PEG and the immunogenicity of PSA, low-molecular-weight PEG and PSA are connected to modify various protein or polypeptide medicaments in order to improve the dissolvability, stability and immunogenicity of the medicaments, prolong the half-life period of the medicaments and improve therapeutic effects..

Description

technical field [0001] The invention relates to a preparation method of polyethylene glycol-polysialic acid block copolymer and its application in medicine preparation. Other molecules are also mentioned, especially macromolecules such as proteins and polypeptides, and the invention also relates to pharmaceutical compositions comprising said block copolymers. It belongs to the technical field of polymer materials. Background technique [0002] At present, there are many kinds of drugs, which can be generally divided into two categories according to their molecular weight: one is low molecular weight (less than 1000), most of which are chemically synthesized drugs, biofermentation synthesized drugs and some natural drugs, For example, penicillin, pantothenic acid, paclitaxel, and 5-fluorocytosine all belong to this category; the other category is macromolecular drugs, most of which are protein and polypeptide drugs produced by modern biotechnology. However, both small molec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08G65/48C08B37/00C08H1/00C07K1/107A61K47/48A61K38/00A61K47/60
Inventor 吴剑荣詹晓北林怡郑志永
Owner 山东弥美生物科技股份有限公司
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