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Polyethylene glycol modified L-asparaginyl amine enzyme

A technology of asparaginase and PEGylation, applied in hydrolytic enzymes, medical preparations containing active ingredients, peptide/protein components, etc., can solve the problem of separation of unmodified products, increase of aspart immunogenicity, and easy hydrolysis And other issues

Inactive Publication Date: 2012-10-10
SHANGHAI INST OF PHARMA IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since polyethylene glycol succinimide succinate belongs to the first generation of activated polyethylene glycol modifier, it has the disadvantages that the modified product is unstable in vivo and easy to hydrolyze
The succinic acid tail left on the protein after hydrolysis acts as a hapten but increases the immunogenicity of asparaginase (see Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Advanced Drug Delivery Reviews, 2002, 54: 459-476.); In addition, single-modification, double-modification and multi-modification products cannot be separated only by ultrafiltration. From the accompanying drawing 1 of the patent publication, it can be seen that the modified product is a mixture of various modifications. Obviously, using this The use of this modified mixture as a drug has obvious disadvantages

Method used

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  • Polyethylene glycol modified L-asparaginyl amine enzyme
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  • Polyethylene glycol modified L-asparaginyl amine enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Selection of Modification Conditions for L-Asparaginase by Methylbutyrate Methoxypolyethylene Glycol Succinimidyl Ester 5000 (mPEG-SMB-5000)

[0092] Selection of reaction pH value: Take 0.5ml of 1mg / ml L-asparaginase solution each, put them in 6 test tubes with stoppers, add 1ml of buffer solution with different pH values ​​respectively to make the pH value of the solution 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, then add 1.773 mg of mPEG-SMB-5000 solid, dissolve, mix well, and react at 25°C for 30 minutes to terminate the reaction. Compare the modification rate and determine the modification condition.

[0093] The results showed that the polyethylene glycol-modified L-asparaginase could be obtained under these conditions, and the modification rate was the highest when the pH value was 9.5.

[0094] Selection of reaction temperature: Take 2ml of 2mg / ml L-asparaginase solution, add 2ml of borax-sodium hydroxide buffer to make the pH of the solution 9.5, then add 14.2mg of mPEG-S...

Embodiment 2

[0101] Preparation of L-asparaginase modified by methylbutyrate methoxypolyethylene glycol succinimidyl ester 5000 (mPEG-SMB-5000)

[0102] Modification reaction:

[0103] Take 2ml of 2mg / ml L-asparaginase solution, add borax-sodium hydroxide buffer to make the pH of the solution 9.5, then add 7.092mg of mPEG-SMB-5000 solid, dissolve, mix well, and react at 25°C After 30 min, 100 mg of glycine was added to terminate the reaction.

[0104] Ion exchange chromatography:

[0105] Take the above reaction solution, with 0.05M Na 2 HPO 4 -NaH 2 PO 4 , pH8.0 buffer solution was ultrafiltered three times, fully balanced, and separated on the column. The chromatographic conditions are as follows:

[0106] Chromatography medium: Source 30 Q

[0107] Column specification: 5ml

[0108] mobile phase:

[0109] A 0.05M Na 2 HPO 4 -NaH 2 PO 4 , pH8.0

[0110] B 0.05M Na 2 HPO 4 -NaH 2 PO 4 , 0.4M NaCl, pH8.0

[0111] Flow rate: 5ml / min

[0112] Gradient: 1-15% 1 column vol...

Embodiment 3

[0135] Determination of biological activity (specific activity) of the modified product (see Chinese Pharmacopoeia 2005 edition: asparaginase)

[0136] Using the L-asparaginase standard as a control, the amount of catalytic substrate produced by the standard and the sample was determined, and then the specific activity (U / mg) of the sample was calculated. See Table 1.

[0137] Table 1: Biological activity of L-asparaginase and PEG-modified L-asparaginase

[0138] sample

[0139] The results show that the polyethylene glycol-modified L-asparaginase obtained by the invention has high biological activity.

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Abstract

The present invention discloses one kind of new compound, polyethylene glycol derivative modified L-asparaginase in the structure as shown. The present invention also provides the preparation of the polyethylene glycol derivative modified L-asparaginase and its medicine composition. The compound of the present invention has homogeneous component, high antitumor effect, high chemical stability, and low immunogenicity.

Description

technical field [0001] The present invention relates to modifying medicine with polyethylene glycol, in particular to protein modified with polyethylene glycol and its application. Background technique [0002] The L-asparaginase (L-ASP) involved in the present invention widely exists in the serum of microorganisms, plants and some rodents, and has strong antitumor effect. (Abuchow ski A, Theo V S, Palczuk N C, et al. Alteration of immunological properties of Bovine Serum Album in by covalent attachment of polyethylene glycol. J Biol Chem, 1977, 252: 3578). [0003] The active form of L-asparaginase is a homologous tetramer, each subunit consists of 326 amino acids. L-asparagine is an essential amino acid for the growth of certain tumor cells. For normal human cells, they have the ability to synthesize L-asparagine, while tumor cells lack asparagine synthetase and cannot synthesize L-gate by themselves. Asparagine, the cells need to ingest exogenous L-asparagine to survive...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/80A61K38/50A61P35/02
Inventor 冯军赵文杰公伟薛春佳翟宁
Owner SHANGHAI INST OF PHARMA IND CO LTD
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