Defibrase modified by carbowax

A technology of polyethylene glycol and defibrase, which is applied in the direction of enzymes, enzyme stabilization, hydrolytic enzymes, etc., can solve the problems of difficult purification of products, failure to satisfy defibrase, and low retention of biological activity of modified products, and achieve good stability , the effect of low immunogenicity

Inactive Publication Date: 2005-01-12
SHANGHAI INST OF PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method also has the disadvantages of multi-site modification of the product by activated PEG, low biological activity retention of the modified product and difficulty in purification of the modified product, which cannot meet the clinical application requirements of defibrase

Method used

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  • Defibrase modified by carbowax
  • Defibrase modified by carbowax
  • Defibrase modified by carbowax

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Selection of Modification Conditions of Defibrase by Methoxypolyethylene Glycol Succinimidyl Propionate 5000 (mPEG-SPA-5000)

[0032] 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 0.12mg of mPEG-SPA-5000 solid, dissolve, mix well, and 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.

[0033] 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 0.12mg of mPEG-SPA-5000 solid, dissolve, mix well, each take 0.3 ml was placed in four stoppered test tubes, and then reacted at 25°C for 5,...

Embodiment 2

[0037] Separation, purification and identification of modified products

[0038] Take 2ml of 1.1mg / ml defibrase solution, add phosphate buffer to make the pH of the solution 6.5, then add 8.5mg of mPEG-SPA-5000 solid, dissolve, mix well, react at 25°C for 30min, add 3g of glycine The solid terminated the reaction.

[0039] The above reaction solution was taken, concentrated to 2 ml with an ultrafiltration membrane with a cut-off molecular weight of 10,000, and separated on a column. The chromatographic conditions are as follows:

[0040] Chromatography medium: Superdex 75 pre grade

[0041] Column specification: 1.6×60cm

[0042] Mobile phase: 0.05M Na 2 HPO 4 -NaH 2 PO 4 , pH7.0

[0043] Flow rate: 0.5ml / min

[0044] Detection wavelength: 215nm

[0045] Collection: 3ml per tube

[0046] As a result, two elution peaks were obtained, which were defibrase modified with polyethylene glycol and defibrase respectively.

[0047] The above two eluted peaks were properly d...

Embodiment 3

[0048] Example 3 Determination of biological activity (specific activity) of the modified product (State Food and Drug Administration: National Drug Standard: Defibrase)

[0049] Use the defibrase standard substance as a control, measure the initial setting time of the standard substance and the sample coagulated fibrinogen, and then calculate the specific activity (U / mg) of the sample. The results are shown in Table 1.

[0050] Table 1 The biological activity of defibrase and polyethylene glycol modified defibrase

[0051] Sample specific activity (U / mg)

[0052] Defibrase 2500

[0053] PEG-modified Defibrase 1500

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Abstract

This invention discloses a de-plasmin decorated by PEG and its preparation method. Dibasic sodium phosphate then activated PEG or its dirivants and added into the solution of de-plasmin to be reacted, then chromatogram separation is carried cut to the reacted solution by a gel filtration chromatographic column to get the de-plasmin decorated by PEG which can maintain the bioactive of the de-plasmin and has rather lower immunogenicity and better stability than the un-decorated ones.

Description

technical field [0001] The invention relates to a defibrase, in particular to a polyethylene glycol-modified defibrase. Background technique [0002] The safety and effectiveness of clinical application of protein drugs has always been a focus of attention and research. Due to poor stability, high plasma clearance rate, short half-life in vivo, protein drugs are prone to antigen-antibody reactions, so they are greatly limited in clinical treatment. Therefore, researchers have adopted a variety of drug delivery techniques to improve the efficacy of protein drugs, including the use of different routes of administration (such as oral administration, nasal administration, transdermal absorption), different carriers (such as microspheres, liposomes, red blood cells, etc.) , monoclonal antibody), different polymer materials (such as polysaccharides), and different drug release technologies (such as controlled release and sustained release technology, prodrugs). At present, the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/43A61P7/02C12N9/00C12N9/64C12N9/96
CPCC12N9/6418A61P7/02A61P7/04
Inventor 冯军赵文杰
Owner SHANGHAI INST OF PHARMA IND
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