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Polyethylene glycol modified and recombined human interleukin and its preparation method

A technology of human interleukin and polyethylene glycol, applied in the field of interleukin, can solve the problems of easily causing immune response, short half-life, etc., and achieve the effects of simple and easy preparation method, reduced immunogenicity, and long in vivo half-life

Inactive Publication Date: 2005-08-10
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Recombinant human interleukin-2 has the functions of promoting the proliferation of T lymphocytes, activating lymphokine-activated killer cells (LAK cells), and promoting the production of β-interferon. It plays an important role in the generation and regulation of immune responses. It is an important cytokine, but its short half-life and repeated injections can easily cause immune reactions, which limit its clinical application

Method used

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  • Polyethylene glycol modified and recombined human interleukin and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0023] Preparation of Monomethoxy Polyethylene Glycol Succinimide Carbonate (SC-mPEG)

[0024] Weigh 14.4g of monomethoxypolyethylene glycol 12,000 and 0.56g of triphosgene, dissolve in 20ml of toluene and 12ml of dichloromethane, add 1ml of anhydrous triethylamine dropwise, and stir overnight.

[0025] The reaction solution was evacuated, most of the solvent was evaporated, and the obtained residue was redissolved in 8 ml of toluene and 10 ml of dichloromethane. Add 0.21 g of N-hydroxysuccinimide, take 0.3 ml of triethylamine, dilute with 3 ml of dichloromethane, add dropwise to the reaction solution, and continue the reaction to complete, filter the reaction solution and vacuumize, and dissolve the obtained residue in 50 °C in 60 ml of ethyl acetate, filtered, and the crystals obtained after cooling were the crude SC-PEG. The resulting product was repeatedly recrystallized from ethyl acetate. Measure the infrared spectrum of product, following characteristic peak is arrang...

Embodiment 2

[0027] Selection of Modification Conditions of Interleukin-2 by SC-PEG

[0028] Choice of reaction time:

[0029] Take 4 1.5ml Eppendorf tubes, add 0.5ml of interleukin-2 concentrate (2.0mg / ml) to each tube, then add an equal volume of 0.05M borax buffer solution with a pH value of 8.0, mix well, and then add 6.0mg of SC -PEG, stop the reaction after reacting for 15min, 30min, 1h, and 2h, and perform SDS-PAGE electrophoresis. The results showed that the modification rate was low when reacting for 15 minutes, most of the interleukin-2 was not modified, and the modification rate increased when reacting for 30 minutes, and the product was mainly modified interleukin-2. After reacting for 1 hour, a highly modified band The highly modified bands increased slightly after 2 hours of reaction compared with 1 hour of reaction.

[0030] Choice of reaction pH:

[0031] Take five 1.5ml Eppendorf tubes, add 0.5ml (2.0mg / ml) of interleukin-2 concentrated solution to each tube, and then a...

Embodiment 3

[0033] Separation, purification and identification of modified products

[0034] Take two 1.5ml Eppendorf tubes, add 0.5ml of interleukin-2 concentrate (2.0mg / ml) to each tube, then add an equal volume of 0.05M borax buffer solution with a pH of 7.5, then add 6.0mg of SC-mPEG solid, mix Mix well, react at 4°C for 30 min, and terminate the reaction.

[0035] Take the above reaction solution and put it on the column for separation respectively. Chromatographic conditions: Sephacryl S-200 (1cm×50cm), mobile phase is 50mM acetate buffer (pH6.0), flow rate: 0.3ml / min. Collect each elution peak.

[0036] The results showed that there were two elution peaks with a molecular weight greater than that of the standard interleukin-2, which were single-modified interleukin-2 and multi-modified interleukin-2.

[0037] The eluted peaks were appropriately concentrated and detected by SDS-PAGE electrophoresis, the separation gel was 15%, and the stacking gel was 5%. see results figure 1 , e...

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Abstract

A polyethanediol modified recombinant human interleukin used for preparing the antiviral and anticancer medicines features that one or more polyethanediol chains are linked to recombinant human interleukin for higher activity and stability.

Description

technical field [0001] The present invention relates to an interleukin, specifically a polyethylene glycol modified interleukin. Background technique [0002] The rapid development of biotechnology has promoted the production and application of protein (enzyme) drugs. However, since exogenous proteins are natural antigens and have strong immunogenicity, they will trigger the production of antibodies and be attacked by various proteases to lose their biological activity. There are also some drugs whose half-life is too short (especially small molecular proteins) to fully exert their biological functions. These have become key factors restricting the development of protein drugs. 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...

Claims

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Application Information

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IPC IPC(8): A61K38/20A61K47/60A61P31/12A61P35/00C07K14/54C07K17/02
Inventor 魏东芝宫兴文何明磊
Owner EAST CHINA UNIV OF SCI & TECH
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