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Polyethylene glycol-interferon coupler and its preparation method

A technology of polyethylene glycol and interferon, applied in the direction of interferon, cytokines/lymphokines/interferon, chemical instruments and methods, etc., can solve the problems of increasing interferon, half-life extension is not obvious, and it is broken. Achieve high antiviral activity and long retention time

Inactive Publication Date: 2007-05-23
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are existing literature reports on PEG-modified interferon, but there are several problems in some. The first is that the biological activity of interferon is reduced after PEG is combined with interferon, and the dosage of interferon must be increased in use; - In the case of interferon conjugates, the linker used may be hydrolyzed and broken in the body, resulting in an insignificant extension of the half-life

Method used

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  • Polyethylene glycol-interferon coupler and its preparation method
  • Polyethylene glycol-interferon coupler and its preparation method
  • Polyethylene glycol-interferon coupler and its preparation method

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Embodiment 1

[0023] Embodiment 1. Preparation of polyethylene glycol-interferon alpha-2a

[0024] Dissolve interferon α-2a with 5mM sodium acetate buffer solution pH4.0, and prepare a solution of 8mg / ml; add mPEG-NHS (Mw=10000) according to the ratio of interferon: PEG is 1: 10 , adjust the pH to 85 with sodium hydroxide, react at 25°C for 5 hours, add 0.5ml of 0.5M glycine to terminate the reaction; after 5 minutes, dilute the reaction solution with 6 times the volume of 20mM pH4.0 sodium acetate buffer; The diluted reaction solution was applied to the (Waterman CM-52) column, washed with 6 times the volume of sodium acetate buffer at pH 4.0, and then eluted with sodium acetate buffer containing 05M sodium chloride to collect polyethylene glycol-containing The eluate of alcohol-interferon α-2a was detected by SDS-PAGE electrophoresis. The polyethylene glycol-interferon alpha-2a was further purified with Superdex200, and the eluent was sodium phosphate buffer (containing 0.15M NaCl) at pH...

Embodiment 2

[0025] Embodiment 2. Preparation of polyethylene glycol-composite interferon

[0026] Composite interferon was dissolved in sodium acetate buffer solution of 5mM pH4.0, and was formulated into a solution of 1 mg / ml; mPEG-NHS (Mw=5000, purchased from Sigama company, the same below), adjust the pH to 8.0 with sodium hydroxide, react at 4°C for 24 hours, add 0.2ml of 0.5M glycine to terminate the reaction; after 5 minutes, buffer with 10 times the volume of 20mM sodium acetate pH4.0 Dilute the reaction solution with liquid; apply the diluted reaction solution to a carboxymethyl cellulose column (Waterman CM-52), wash the column with 6 times the volume of sodium acetate buffer at pH 4.0, and wash the column with 0.4M sodium chloride Elute with sodium acetate buffer, collect the eluate containing polyethylene glycol-composite interferon, and detect it by SDS-PAGE electrophoresis. The polyethylene glycol-composite interferon was further purified with Superdex 200, and the eluent wa...

Embodiment 3

[0027] Embodiment 3. Preparation of polyethylene glycol-interferon alpha-2b

[0028] Dissolve interferon α-2b with 5mM sodium acetate buffer solution pH4.5, and prepare a solution of 3 mg / ml; add mPEG-NHS (Mw=20000) at a ratio of 1:15 for interferon:PEG , adjust the pH to 7.5 with sodium hydroxide, react at 25°C for 12 hours, add 0.5ml of 0.5M glycine to terminate the reaction; after 5 minutes, dilute the reaction solution with 5 times the volume of 20mM pH4.0 sodium acetate buffer; The diluted reaction solution was applied to a carboxymethylcellulose column (Waterman CM-52), washed with 6 times the volume of sodium acetate buffer at pH 4.5, and then eluted with sodium acetate buffer containing 0.35M sodium chloride , collect the eluate containing polyethylene glycol-interferon α-2b, and detect it by SDS-PAGE electrophoresis. The polyethylene glycol-interferon α-2b was further purified with Superdex 200, and the eluent was sodium phosphate buffer (containing 0.15M NaCl) at pH...

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Abstract

This invention relates to a polyethylene glycol and interferon conjugate with physiological activity, which is a molecular weight of 5000~30000 Dalton of linear polyethylene glycol-modified interferon, as shown in the right formula, wherein mPEG is the polyethylene glycol chain and interferon is interferon. The PEG-interferon has high antiviral activity and long in vivo retention time, and the plasma half-life is 10~60 hours.

Description

field of invention [0001] The invention relates to a PEG-interferon conjugate chemically modified with polyethylene glycol (PEG) for interferon and a preparation method thereof. Background technique [0002] Many natural and recombinant proteins have medicinal properties and, if purified and formulated, can be administered parenterally for a variety of medical indications. However, proteins administered by parenteral routes are likely to be immunogenic, and usually have a short plasma half-life. Therefore, it is difficult for a patient to achieve a therapeutically effective plasma concentration of a pharmaceutical protein. [0003] These problems can be overcome by combining proteins with high molecular weight polymers such as polyethylene glycol. Davis et al. disclosed in US Patent No. 4,179,337 a technical method for combining polyethylene glycol with proteins (enzymes and insulin) to obtain conjugates with physiological activity. Katre et al. in US Pat. Nos. 4,766,106 a...

Claims

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Application Information

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IPC IPC(8): C07K17/08C07K14/555C07K14/56A61K38/21A61K47/48A61P31/12A61K47/60
Inventor 苏志国马光辉雷建都郑春扬李兴奇翟艳琴
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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