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Preparation and use of galactosylated human serum albumin fused interferon

A technology of serum albumin and interferon, applied in the field of fusion interferon, can solve the problems of IFN not being able to target the liver and insufficient galactosylation, and achieve the effect of reducing treatment costs, reducing the degradation rate in the body, and reducing side effects

Inactive Publication Date: 2008-12-24
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In order to increase the targeting of IFN, in 1994, Zhong Yuguo, School of Pharmacy, West China University of Medical Sciences, etc. disclosed its national invention patent (CN1087093A) "liver-targeted galactose interferon conjugated conjugate", and in 2000 they disclosed another A national invention patent (CN99117484.4) "liver-targeted high-glucose-density galactosyl interferon conjugate", both of these two patents use common IFN as the galactosylation precursor, but due to common IFN There are only about 10 lysine residues, and the number of lysine residues available for glycosylation is even less, so when galactosylation is modified, the degree of galactosylation is not high enough to be effective target IFN to the liver

Method used

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  • Preparation and use of galactosylated human serum albumin fused interferon
  • Preparation and use of galactosylated human serum albumin fused interferon
  • Preparation and use of galactosylated human serum albumin fused interferon

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Preparation of GHSA-IFNα2b

[0042] The reaction equation is:

[0043]

[0044] Compound 1 (0.28g, 0.69mmol) was dissolved in 10mL of anhydrous methanol, sodium methoxide (3.7mg, 0.069mmol) was added, and stirred at 20-22°C for more than 24 hours to obtain IME-thiogalactoside (compound 2, produced The yield is about 56%) and by-products (deacetylation products of compound 1), compound 2 need not be isolated.

[0045] Take 6mL of the above reaction solution, rotate to evaporate, remove the solvent, add 4.5mL of 50mg~900mg HSA-IFN solution prepared with 0.1mol / L pH8.5 boric acid buffer, stir and react at 20-22°C for 4 hours, then use PD-10 After column purification, the dialysis was continued with distilled water, and the dialysate was changed every 4 hours until no sugar was detected in the dialyzed fluid to obtain GHSA-IFNα2b (compound 3).

Embodiment 2

[0046] Example 2: Analysis and identification of GHSA-IFNα2b

[0047] The protein concentration of GHSA-IFNα2b was determined by the Lowry method, the protein was quantified, subpackaged, freeze-dried, and stored at -30°C; the sugar concentration of GHSA-IFNα2b was measured by the phenol-sulfuric acid method, and the average sugar concentration of the prepared GHSA-IFNα2b was calculated. The density is 24.

[0048] Sugar density is controlled by the formula:

[0049] Sugar density=4.5+0.12×(IME-thiogalactoside / HSA-IFNα2b)

[0050] In Example 1, GHSA-IFNα2b with different sugar densities can be prepared by controlling the reaction molar ratio of IME-thiogalactoside and HSA-IFNα2b.

[0051] Proteins with standard molecular weights (attached figure 2 Middle B) and HSA-IFNα2b (attached figure 2 Middle C) is a control, and GHSA-IFNα2b is determined by SDS-PAGE (attached figure 2 The molecular weight in A) shows that half-GHSA-IFNα2b is a single band with a molecular weight ...

Embodiment 3

[0052] Example 3: Analysis of antiviral activity of GHSA-IFNα2b

[0053] According to the third appendix XC of Chinese Pharmacopoeia 2005, the biological activity assay of interferon (cytopathic inhibition method), the antiviral activity of HSA-IFN and GHSA-IFNα2b was compared and analyzed by WISH / VSV system, and the results are shown in the appendix image 3 As shown, with the increase of VSV titer, the cell viability gradually decreased. However, the cell survival rates of HSA-IFNα2b and GHSA-IFNα2b corresponding to the same VSV titer were almost the same, which indicated that there was little change in the antiviral activity of HSA-IFNα2b before and after galactosylation.

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Abstract

The invention relates to a preparation method and an application of a galactosyl proserum fused interferon, belonging to the targeted biological medicine technical field. The invention takes a proserum fused interferon as a galactosyl precursor, can provide enough sites for glycosylation, and then more effectively prepare the galactosyl proserum fused interferon with different sugar densities (between 7 and 35). Compared with the proserum fused interferon, the compound has obvious liver targeting character and the same pharmacological activity, is hopeful to be used for preparing a novel virus hepatitis targeted therapeutic medicine, obviously improves the medicine concentration of IFN in the liver, prolongs the biological half-life of the galactosyl proserum fused interferon, effectively reduces the dosage of the clinic IFN, eases the pain of patients due to repeated injection, reduces generation of side reaction, greatly reduces the treatment cost of the patients, improves the treatment level of virus hepatitis in China, promotes excellent health of Chinese people, and has good social benefit and significant economic benefit.

Description

technical field [0001] The invention relates to a fusion interferon modified by galactosylation and belongs to the technical field of targeted biological medicines. Background technique [0002] my country is a country with a large population, but also a country with a large number of liver diseases, and there are many patients with various liver diseases. The HBV carrier rate in my country is as high as 10% to 13%, and about 50% of them become chronic hepatitis B patients. Among these chronic hepatitis B patients, about 25% to 40% of patients will eventually die of liver cirrhosis or combined liver cancer; HCV infection The rate is 3.2%, about 42 million people. The chronicity rate of HCV infection is extremely high, up to 50% to 85%. After 20 years of infection, about 10% to 30% can develop into liver cirrhosis, and about 3% to 10% can evolve into hepatocellular carcinoma. [0003] At present, there is no specific drug for the treatment of viral hepatitis, and α-interferon...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K14/555C07K1/113A61K38/21A61P1/16A61P31/12
Inventor 张荣军蒋孟军蔡刚明周尧远张波顾晓波周杏琴曹国宪包建东
Owner JIANGSU INST OF NUCLEAR MEDICINE
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