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Venomous snake thrombin sample enzyme modified by polyethylene glycol

A technology of polyethylene glycol and PEGylation, which is applied in the direction of enzymes, blood diseases, enzymes, etc., can solve the problems of no further disclosure of modification sites of modified products, low sample uniformity, and no disclosure of modifications, etc.

Inactive Publication Date: 2007-02-14
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Patent application CN1563367 discloses polyethylene glycol modified defibrase, only discloses the modification of polyethylene glycol to defibrase in this application, does not disclose to other several snake venom thrombin-like enzymes such as enkoloase, basil The polyethylene glycol modification of Aspergillus and Agruzin did not further disclose the specific modification site of the modified product, but only announced the single modification of the defibrase molecule. This single modification can be the N of the defibrase molecule. The terminal amino group, which may also be the amino group of one of the lysine residues, has the disadvantages of low sample uniformity and difficult quality control for drug development

Method used

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  • Venomous snake thrombin sample enzyme modified by polyethylene glycol
  • Venomous snake thrombin sample enzyme modified by polyethylene glycol
  • Venomous snake thrombin sample enzyme modified by polyethylene glycol

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Selection of Modification Conditions of Snake Venom Thrombin-Like Enzyme by Methoxypolyethylene Glycol Succinimidyl Propionate 20000 (mPEG-SPA-20000)

[0062] Choice of reaction temperature: Take 2ml of 0.8mg / ml defibrase solution, add 2ml of phosphate buffer to make the pH of the solution 6.5, then add 8.0mg of mPEG-SPA-20000 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 the PEG-modified defibrase could be obtained at these temperatures, and the modification rate was the highest at 25°C.

[0063] Choice of reaction time: Take 2ml of 0.8mg / ml defibrase solution, add 2ml of phosphate buffer to make the pH of the solution 6.5, then add 8.0mg of mPEG-SPA-20000 solid, dissolve, mix well, and take 0.3 for each ml was placed in four stoppered test tubes, an...

Embodiment 2

[0068] Isolation, Purification and Identification of PEG Modified Defibrase

[0069] Take 2ml of 1.1mg / ml defibrase solution, add about 5ml of phosphate buffer to make the pH of the solution 6.5, then add 12.2mg of mPEG-SPA-20000 solid, dissolve, mix well, and react at 25°C for 30min. The reaction was terminated by adding 3 g of glycine solid.

[0070] The above reaction solution was taken, dialyzed against 0.05 mol / L Tris-HCl buffer solution with pH 7.8, concentrated to 5 ml with an ultrafiltration membrane with a molecular weight cut-off of 10,000, and separated on a column. The chromatographic conditions are as follows:

[0071] Chromatography medium: SOURCE 30Q

[0072] Column volume: 5ml

[0073] Flow rate: 1.0ml / min

[0074]Column equilibration: equilibrate 5 times column volume with 0.05mol / L, pH 7.8 Tris-HCl (starting buffer)

[0075] Sample volume: 5ml

[0076] Elution: First use 3 times the column volume of starting buffer to elute the unadsorbed part, and then...

Embodiment 3

[0089] Isolation, Purification and Identification of Polyethylene Glycol Modified Ancholozyme

[0090] Take 2ml of 0.8mg / ml Ankelo enzyme solution, add about 5ml of phosphate buffer to make the pH of the solution 6.5, then add 9.1mg of mPEG-SPA-20000 solid, dissolve, mix well, and react at 25°C After 30 min, 3 g of glycine solid was added to terminate the reaction.

[0091] The above reaction solution was taken, dialyzed against 0.05mol / L, pH 8.3 Tris-HCl buffer solution, concentrated to 5ml with an ultrafiltration membrane with a molecular weight cut-off of 10000, and separated on a column. The chromatographic conditions are as follows:

[0092] Chromatography medium: SOURCE 30Q

[0093] Column volume: 5ml

[0094] Flow rate: 1.0ml / min

[0095] Column equilibration: Equilibrate 5 times column volume with 0.05mol / L, pH 8.3 Tris-HCl (starting buffer)

[0096] Sample volume: 5ml

[0097] Elution: First use 3 times the column volume of starting buffer to elute the unadsorbe...

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Abstract

The invention discloses reptilase sample decorated by polyethylene glycol. Its formula is mPEG-O-(CH2)n-T-NH-R whose base group definition can be seen in the specification. It can be used as anticoagulation thrombolysis medicine or nerve protection medicine. And it has lower immunogenicity than undecorated enzyme, higher enzyme activity and fiber reducing capability than polyethylene glycol decoration fiber reducing enzyme. Its preparation method is simple and easy.

Description

technical field [0001] The invention relates to a class of snake venom thrombin-like enzymes, in particular to a class of polyethylene glycol-modified snake venom thrombin-like enzymes. Background technique [0002] Snake venom thrombin-like enzyme (thrombin-like enzyme, TLE) refers to a class of protease extracted from snake venom, which is similar to physiological thrombin and can degrade plasma fibrinogen, so it is also called thrombin-like enzyme. So far, more than 30 kinds of snake venoms have been found to contain thrombin-like enzyme components, and more than 20 kinds have been separated and purified successively, some of which have been widely used in clinic as therapeutic drugs (He Haiping, Liang Ningsheng. Research on snake venom thrombin Overview. Snake Journal, 2000;12:73-77.). Among them, the most widely used thrombin-like enzymes at home and abroad are Ancrod from the venom of Agkistrodon rhodostoma in Malaysia and batroxobin from the venom of Bothrops atrox i...

Claims

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Application Information

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IPC IPC(8): C12N9/74A61K38/48A61P7/02A61P25/00
Inventor 冯军武霞赵文杰高勇张来芳张喜全张锡昌
Owner SHANGHAI INST OF PHARMA IND
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