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Modified erythropoietin to which water-soluble long-chain molecule is added

An erythropoietin, water-soluble technology, applied in the directions of extracellular fluid diseases, medical preparations with non-active ingredients, and medical preparations containing active ingredients, etc., to achieve the effect of sustained drug effect and reduced pain

Active Publication Date: 2012-06-13
NIPPON ZENYAKU KOGYO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since EPO-like physiologically active proteins have species-specific amino acid sequences, administration of human-derived EPO preparations to animals other than humans may result in the expression of anti-EPO antibodies

Method used

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  • Modified erythropoietin to which water-soluble long-chain molecule is added
  • Modified erythropoietin to which water-soluble long-chain molecule is added
  • Modified erythropoietin to which water-soluble long-chain molecule is added

Examples

Experimental program
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Effect test

Embodiment

[0098] The present invention is illustrated in detail by the following examples, but the present invention is not limited by these examples. In the case of listing with a product name, unless otherwise specified, follow the instructions in the attached manual.

[0099] Synthesis of cat-derived erythropoietin was carried out according to the method described in JP-A-2007-89578, and the steps are reproduced as follows.

preparation example 1

[0100] (Preparation Example 1) Microinject retroviral vectors into chicken embryos and artificially hatch

[0101] Transgenic chickens expressing cat-derived erythropoietin were prepared by microinjecting a transcription virus vector into chicken embryos for expression of cat-derived erythropoietin. Both microinjection and artificial incubation were performed under sterile conditions. The outer side of chicken fertilized eggs (manufactured by Shiroyama Breeder Farm) was sterilized with a disinfectant solution (manufactured by Showa Furanki Co., Ltd.) and ethanol. Set the incubator P-008 (B) type (manufactured by Showa Furanki Co., Ltd.) to an environment of 38°C and a humidity of 50-60%. The eggs were turned 90° for incubation.

[0102] About 55 hours after the start of hatching, the eggs were taken out from the incubator, and the diameter was cut at the tip using a mini-router (manufactured by PROXXON) equipped with a diamond knife (tip diameter 20mm, shaft diameter 2.35mm)...

preparation example 2

[0104] (Preparation Example 2) Purification of cat-derived erythropoietin from egg white

[0105] Eggs of chicken individuals whose activity of cat-derived erythropoietin was confirmed in the egg white were recovered, and cat-derived erythropoietin was recovered and purified from the egg white using the column described later.

[0106] All samples loaded onto the column were syringe-filtered with Millex-HX (manufactured by Millipore Corporation, Japan) with a pore size of 0.45 μm before use. In the case where filtration is difficult, after pre-filtering with Puradisc25 (manufactured by WATT MANN) with a pore size of 2 µm, it is filtered through Millex.

[0107] During the purification process, the cat-derived erythropoietin content was measured using Biacore 3000 (manufactured by GE HealthCare Japan Co., Ltd. BIACORE). Anti-human erythropoietin monoclonal antibody (manufactured by R&D Systems) NHS was immobilized on the sensor chip CM5 research grade (manufactured by GE Healt...

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Abstract

Disclosed is a pharmaceutical composition which contains erythropoietin as an active ingredient and exerts a hematopoietic effect that lasts for 7 days or more when administered to a human and / or an animal. Specifically disclosed are: a pharmaceutical composition that contains erythropoietin, to which two or more water-soluble long-chain molecules are added, in an amount of not less than 50% of the total erythropoietin; a pharmaceutical composition that contains erythropoietin to which a water-soluble long-chain molecule is added, said water-soluble long-chain molecule having a molecular weight of not less than 30 kDa; and a pharmaceutical composition that contains erythropoietin to which a water-soluble long-chain molecule is added, said water-soluble long-chain molecule having a branched chain.

Description

technical field [0001] The present invention relates to pharmaceutical compositions containing erythropoietin supplemented with water-soluble long-chain molecules. Background technique [0002] In order to improve anemia, the hormone that promotes hematopoiesis, that is, erythropoietin (sometimes referred to as EPO), is produced by gene recombination, and its actual effect as a drug for treating anemia is improved. It is known that adding a water-soluble long-chain molecule polyethylene glycol (sometimes referred to as PEG) to human-derived EPO prevents the metabolism of EPO in the liver and prolongs its life in the blood (Patent Document 1). By prolonging the residence time in the blood, the advantages of prolonging the duration of the drug effect and reducing the frequency of administration are realized. Therefore, not only EPO, but other protein preparations modified with PEG have attracted attention as next-generation drugs, among which Some of them have been practicall...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/22A61K9/06A61K9/08A61K47/10A61K47/18A61K47/34A61P7/06A61P43/00
CPCA61K38/22A61K47/48215A61K38/1816A61K9/0019A61K47/60A61P7/06A61P43/00A61K9/06A61K9/08
Inventor 谷敍孝藤井俊秀渡边裕幸前田博文
Owner NIPPON ZENYAKU KOGYO CO LTD
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