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Methoxy polyethylene glycol-lumbrukinase conjugate and its preparation method

A technology of methoxypolyethylene glycol and lumbrokinase coupling, which is applied in the field of peptide or protein modification complex and its preparation, can solve the problems of low oral bioavailability, unfavorable multiple administration, allergic reactions, etc., and achieve Easy to separate products, improve thrombolytic efficacy, and reduce immunogenicity

Inactive Publication Date: 2012-11-21
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as a thrombolytic drug, it is very important to have the characteristics of fast onset and strong effect, and Fan et al. (Fan Q, et al. Some features of intestinal absorption of intact fibrinolytic enzyme III-1. Biochim Biophya Acta, 2001 Jun 15; 1523 (3): 286-292) showed by immunohistochemical method that only 10-15% of the complete lumbrokinase EFE-III-1 was absorbed by intestinal epithelial cells in the enteric-coated oral capsule of the lumbrokinase, proving its Oral bioavailability is very low
[0004] In response to this problem, there are currently domestic researches on lumbrokinase injections, mainly through further extraction and purification of lumbrokinase to prepare freeze-dried powder injections. The application will inevitably cause allergic reactions. At the same time, allergies will induce the production of antibodies and affect the efficacy of the drug, which is not conducive to repeated administration.

Method used

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  • Methoxy polyethylene glycol-lumbrukinase conjugate and its preparation method
  • Methoxy polyethylene glycol-lumbrukinase conjugate and its preparation method
  • Methoxy polyethylene glycol-lumbrukinase conjugate and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1. Purify lumbrokinase crude product with protein purifier

[0034] 20 mg of lumbrokinase was dissolved in 0.1 M sodium phosphate (pH 7.4), prepared to a concentration of 10 mg / mL, and separated by molecular sieve using superdex75 gel column and Akta Purify 10 protein purifier. Obtain two partial peaks (peak A and peak B), do in vitro titer determination, keep the peak with in vitro activity (peak B), discard the peak with no activity or very little activity (peak A), and obtain the in vitro activity peak (peak A). The active peak B is separated by Hi Trap DEAE FF ion exchange column, and two parts of the peak (peak C and peak D) are separated, and then the in vitro titer is measured, and the peak with in vitro thrombolytic activity (peak C ) was collected, freeze-dried, and the finally obtained peak D with in vitro activity was considered to be the isolated and purified lumbrokinase monomer peak that we wanted. The peaks of the four parts separated by gel e...

Embodiment 2

[0035] Example 2.mPEG-NHS 10000 - Preparation of lumbrokinase conjugates

[0036] 20 mg of lumbrokinase obtained after separation and purification by the method of the above-mentioned Example 1 was dissolved in 5 mM pH sodium acetate buffer solution (pH 7.0), prepared to a concentration of 5 mg / mL, and 100 mg of mPEG-NHS was added 10000 (The molar ratio is lumbrokinase:mPEG-NHS 10000 =1:5), reacted under ice bath (0°C) for 2h, added glycine and stirred for 10min to terminate the reaction, and obtained mPEG-NHS 10000 - Lumbrokinase conjugates. The solutions before and after the reaction were detected by high performance liquid phase for comparison. The calculated modification rate is 61.5% (see attached Figure 7-8 ).

Embodiment 3

[0037] Example 3.mPEG-SC 5000 - Preparation of lumbrokinase conjugates

[0038] 5 mg of lumbrokinase obtained after separation and purification by the method of the above-mentioned Example 1 was prepared into a solution with a concentration of 10 mg / ml with 0.1 M sodium phosphate (pH7.4) solution; mPEG was added at a molar ratio of lumbrokinase: mPEG of 1:5 -SC 5000 , adjust the pH to 8.0 with sodium hydroxide, react at 25°C for 2h, add 0.5M glycine to terminate the reaction, and obtain mPEG-SC 5000 -lumbrokinase conjugate, the solution of this conjugate carries out SDS-PAGE electrophoresis, checks the result (see attached Figure 9 ).

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Abstract

The invention relates to a methoxy polyethylene glycol (mPEG)-lumbrukinase conjugate having a physiological activity, wherein the structure of the mPEG-lumbrukinase conjugate is mPEG-CH2-W-NH-Lumbrokinase, and the molecular weight of the mPEG is 5000-40000 Daltons. The in-vivo retention time of the mPEG-lumbrukinase conjugate is long, so the immunogenicity of the mPEG-lumbrukinase conjugate is reduced, and the thrombolytic efficacy of the mPEG-lumbrukinase conjugate is improved.

Description

technical field [0001] The invention relates to a methoxy polyethylene glycol (mPEG) lumbrokinase conjugate and a preparation method thereof. Specifically, the present invention relates to an mPEG-lumbrokinase conjugate prepared by reacting an amino group of lumbrokinase with a polyethylene glycol derivative with an active functional group and a preparation method thereof, belonging to a peptide or protein modification complex and the technical field of its preparation. Background technique [0002] The high morbidity, high mortality, and high disability of thrombotic diseases seriously threaten the survival of human beings, and thrombolytic drugs are the first choice for the treatment of this disease. Lumbrokinase is a kind of protease extracted from fresh earthworms. It has the fibrinase activity of directly dissolving fibrin, and has the plasminogen activation activity similar to urokinase, so it can not only dissolve old thrombus but also inhibit new thrombus. It is a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48A61K38/48A61K47/48A61P9/00A61P9/10A61P27/16
Inventor 高钟镐陈卫金明姬
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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