Polyethyleneglycol modified aprotinin and preparation thereof

A technology of polyethylene glycol and aprotinin, which is applied in the field of chemically modified aprotinin and its preparation, can solve the problems of short half-life and achieve the effects of improving specific activity, low immunogenicity and good stability

Inactive Publication Date: 2009-04-22
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] The technical problem to be solved by the present invention is: in view of the defects of immunogenicity and short half-life in the preparation of aprotinin in the prior art, protein modification technology is used to reduce its immunogenicity and prolong its half-life, thereby improving the pharmacokinetics of the protein characteristics, to improve its tolerance and reduce the number of administrations, to better meet the needs of aprotinin in clinical applications

Method used

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  • Polyethyleneglycol modified aprotinin and preparation thereof
  • Polyethyleneglycol modified aprotinin and preparation thereof
  • Polyethyleneglycol modified aprotinin and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Aprotinin modified with methoxypolyethylene glycol succinimidyl propionate 5000 (mPEG-SPA-5000)

[0060] Choice of reaction temperature: Take 2ml of 1.0mg / ml aprotinin solution, add 2ml of phosphate buffer to make the pH of the solution 7.0, then add 4.0mg of mPEG-SPA-5000 solid, dissolve, mix well, take 0.3 for each ml was placed in 4 test tubes with stoppers, and then placed at 4°C, 10°C, 25°C and 37°C for 30 minutes to stop the reaction. Compare the modification rate and determine the modification condition. The results showed that polyethylene glycol-modified aprotinin could be obtained at these temperatures, and the modification rate was the highest at 25°C. See Table 1 for specific data.

[0061] Table 1: Effects of different temperatures on the modification rate of aprotinin

[0062] temperature 4℃ 10℃ 25℃ 37℃ Modification rate (%) 28 24 46 38

[0063] Choice of reaction time: Take 2ml of 1.0mg / ml aprotinin solution, add 2ml of phosp...

Embodiment 2

[0070] Isolation, Purification and Identification of Polyethylene Glycol Modified Aprotinin

[0071] Take 10ml of 1.0mg / ml aprotinin solution, add about 5ml of phosphate buffer to make the pH of the solution 7.0, then add 16mg of mPEG-SPA-5000 solid, dissolve, mix well, react at 25°C for 30min, add 3 g of glycine solids terminated the reaction.

[0072] The above reaction solution was taken, dialyzed against 0.05 mol / L, Tris-HCl buffer solution with pH 6.0, concentrated to 5 ml with an ultrafiltration membrane with a molecular weight cut-off of 3000, and separated on a column. The chromatographic conditions are as follows:

[0073] Chromatography medium: SOURCE 30S

[0074] Column volume: 5ml

[0075] Flow rate: 3.0ml / min

[0076] Column equilibration: equilibrate 5 times column volume with 0.05mol / L, Tris-HCl (starting buffer) with pH 6.0

[0077] Sample volume: 5ml

[0078] Elution: First use 3 times the column volume of starting buffer to elute the unadsorbed part, an...

Embodiment 3

[0083] Determination of biological potency of aprotinin and polyethylene glycol-modified aprotinin (method see Chinese Pharmacopoeia 2005 Part II: aprotinin)

[0084] According to the titer determination method of aprotinin in Pharmacopoeia, the titer of aprotinin and polyethylene glycol-modified aprotinin was determined, and then the specific activity (U / mg) of the sample was calculated. The titer test showed that the specific activity of the polyethylene glycol-modified aprotinin not only did not decrease, but also increased nearly 1-fold. The specific results are shown in Table 4.

[0085] Table 4 Aprotinin and polyethylene glycol-modified aprotinin biopotency assay results

[0086]

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Abstract

The invention discloses a polyethylene glycol-modified aprotinin and a preparation method thereof. The polyethylene glycol-modified aprotinin is characterized in that an amino group of aprotinin is connected with a chain of polyethylene glycol or a derivative thereof. The polyethylene glycol-modified aprotinin can improve the specific activity of the aprotinin, dramatically lowers the immunogenicity of the aprotinin and improves the stability of the aprotinin, thereby improving pharmacokinetic properties of the aprotinin and the tolerance, and reducing drug delivery times.

Description

technical field [0001] The invention relates to a chemically modified aprotinin and its preparation method and use, in particular to a polyethylene glycol modified aprotinin and its preparation method, and also includes its use in hemostasis and anti-inflammation. Background technique [0002] Aprotinin (aprotinin) is a basic polypeptide extracted from bovine lung. It is a non-specific protease inhibitor and can inhibit a variety of proteases containing serine active centers. Aprotinin consists of 58 amino acids, and its amino acid composition is: RPDFCLEPPY TGPCKARIIR YFYNAKAGLCQTFVYGGCRA KRNNFKSAED CMRTCGGA. A large number of clinical studies have shown that large doses of aprotinin can significantly reduce bleeding after extracorporeal circulation, and it is mainly used clinically to prevent and treat acute bleeding caused by various fibrinolysis and various shocks, acute pancreatitis and pancreatic necrosis. In recent years, it has been widely used in various operations...

Claims

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Application Information

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IPC IPC(8): C12N9/96A61K38/57A61P7/04A61P29/00
Inventor 冯军张喜全张来芳赵伟朱鹏吕达娜赵珍潘娟
Owner SHANGHAI INST OF PHARMA IND
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