Polyethylene decorative nevin fibrinolytic enzyme and its preparation method

A technology of polyethylene glycol and plasmin, which is applied in biochemical equipment and methods, medical preparations without active ingredients, and medical preparations containing active ingredients, etc., can solve the problems of short half-life and poor stability, and achieve Good stability, low immunogenicity

Inactive Publication Date: 2008-02-20
SHANGHAI INST OF PHARMA IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The technical problem to be solved by the present invention is, in view of the shortcomings such as immunogenicity, short half-life and poor stability in the preparation of snake venom plasmin in the prior art, protein modification technology is used to improve the stability of plasmin, prolong its half-life, reduce its Immunogenicity, so as to improve the pharmacokinetic properties of the protein, to improve its tolerance and reduce the number of administrations, to better meet the needs of snake venom plasmin in clinical applications

Method used

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  • Polyethylene decorative nevin fibrinolytic enzyme and its preparation method
  • Polyethylene decorative nevin fibrinolytic enzyme and its preparation method
  • Polyethylene decorative nevin fibrinolytic enzyme and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Snake venom fibrinolytic enzyme modified by methoxypolyethylene glycol succinimidyl propionate 5000 (mPEG-SPA-5000)

[0044] The choice of reaction temperature: get 1.0mg / ml fibrinolytic enzyme solution (from the fibrinolytic enzyme of Agkistrodon venom of Changbai Mountain, hereinafter the same) 2ml, add 2ml phosphate buffer saline to make the pH value of solution be 7.6, then add mPEG- Dissolve 2.0 mg of SPA-5000 solid, mix well, take 0.3 ml each and place in 4 test tubes with stoppers, then place them 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 polyethylene glycol-modified plasmin could be obtained at these temperatures, and the modification rate was the highest at 25°C. See Table 1 for specific data.

[0045] Table 1: Effects of different temperatures on the modification rate of plasmin

[0046] temperature

4℃

10℃

25℃

...

Embodiment 2

[0054] Isolation, Purification and Identification of Polyethylene Glycol Modified Plasmin

[0055] Take 2ml of 1.0mg / ml plasmin solution, add about 5ml of phosphate buffer to make the pH of the solution 7.6, then add 2.0mg of mPEG-SPA-5000 solid, dissolve, mix well, and react at 25°C for 30min. The reaction was terminated by adding 3 g of glycine solid.

[0056] 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:

[0057] Chromatography medium: SOURCE 30Q

[0058] Column volume: 5ml

[0059] Flow rate: 1.0ml / min

[0060] Column equilibration: equilibrate 5 times column volume with 0.05mol / L, pH 7.8 Tris-HCl (starting buffer)

[0061] Sample volume: 5ml

[0062] Elution: First use 3 times the column volume of starting buffer to elute the unadsorbed par...

Embodiment 3

[0075] Determination of the biological potency (specific activity) retention rate of the modified product (see the State Food and Drug Administration for the method: National Drug Standard: Plasmin)

[0076] According to the titer determination method of plasmin in drug standard, measure the titer of plasmin and the plasmin modified with polyethylene glycol, then calculate the specific activity (U / mg) of sample. The results are shown in Table 4.

[0077] Table 4 Defibrase and the biological potency of the defibrase modified with polyethylene glycol

[0078] sample

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Abstract

The present invention discloses a fibrinolysim enzyme modifying with carbowax and the preparation method; the present invention is characterized in that the amino group of the fibrinolysim enzyme is modified with activatory carbowax or the ramification strand of the carbowax. The present invention of fibrinolsim enzyme modifying with carbowax can basically preserves the biological value of the fibrinolysim enzyme; meanwhile, the present invention has low immunogenic and greater stability as compared with the fibrinolysim enzyme which is unmodified; therefore, the pharmacokinetics of the fibrinolysim enzyme is improved and the tolerance is increased and the administration times is reduced. The present invention also discloses the application of fibrinolysim enzyme modifying with carbowax in the preparation of high-efficiency low-toxin drug of dissolving thrombus and nervous protective drug.

Description

technical field [0001] The invention relates to a snake venom fibrinolytic enzyme, its preparation method and its application in the preparation of high-efficiency and low toxicity thrombolytic drugs and neuroprotective drugs, in particular to a polyethylene glycol-modified snake venom fibrinolytic enzyme and its preparation The method and its application in the preparation of high-efficiency and low-toxicity thrombolytic drugs and neuroprotective drugs. Background technique [0002] Snake venom fibrinolytic enzyme refers to a class of protease extracted from snake venom, which can directly dissolve fibrin in vivo and in vitro by acting on the α chain or β chain of fibrin. So far, more than 30 kinds of snake venoms have been found to contain plasmin components, and more than 69 kinds have been isolated and purified successively, some of which have been used clinically as therapeutic drugs (Swenson S and Markland FS. Snakevenom fibrin(gen)olytic enzymes. Toxicon, 2005;45:102...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K38/48A61P7/02A61P25/00C12N11/08C12N9/68A61K47/60
Inventor 冯军赵文杰公伟
Owner SHANGHAI INST OF PHARMA IND
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