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Chemically modified G-CSF

a technology of granulocyte colony and g-csf, which is applied in the direction of peptide/protein ingredients, immunoglobulins, peptides, etc., can solve the problems of reducing stability, publications have not disclosed an improvement in biological activity and pharmacokinetics, etc., to accelerate the recovery from neutropenia, prolong the pharmacological effect, and increase the number of neutrophils

Inactive Publication Date: 2007-09-20
KIRIN AMGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to a method for improving the function and stability of human G-CSF by binding polyethylene glycol to it. This can be achieved by covalently binding polyethylene glycol to the amino acid residues of human G-CSF through a terminal reactive group. The resulting human G-CSF has a molecular weight of from 500-20,000 and is preferably a polypeptide with a specific amino acid sequence. The polyethylene glycol used in the invention has a molecular weight of from 500-20,000 and is preferably activated. The chemical modification can be carried out under suitable conditions and the activated polyethylene glycol can be used in a certain ratio to human G-CSF. The technical effect of this invention is to improve the function and stability of human G-CSF for various applications.

Problems solved by technology

Furthermore, high hydrophobicity of the proteins reduces their stability.
However, these prior art publications have not disclosed an improvement in biological activity and pharmacokinetics, which may be expected as a result of the modification of human G-CSF by polyethylene glycol.

Method used

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Examples

Experimental program
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Effect test

example 1

Preparation of PEG (4,500) G-CSF

[0031] Recombinant human G-CSF (Japanese Patent Application Laying Open KOHYO No. 500636 / 88) having the following amino acid sequence (SEQ ID NO: 2) was used for the chemical modification according to the present invention:

(SEQ ID NO: 2)                                        MetThr Pro Leu Gly Pro Ala Ser Ser Leu Pro GlnSer Phe Leu Leu Lys Cys Leu Glu Gln Val ArgLys lle Gln Gly Asp Gly Ala Ala Leu Gln GluLys Leu Cys Ala Thr Tyr Lys Leu Cys His ProGlu Glu Leu Val Leu Leu Gly His Ser Leu GlyIle Pro Trp Ala Pro Leu Ser Ser Cys Pro SerGln Ala Leu Gln Leu Ala Gly Cys Leu Ser GlnLeu His Ser Gly Leu Phe Leu Tyr Gln Gly LeuLeu Gln Ala Leu Glu Gly Ile Ser Pro Glu LeuGly Pro Thr Leu Asp Thr Leu Gln Leu Asp ValAla Asp Phe Ala Thr Thr Ile Trp Gln Gln MetGlu Glu Leu Gly Met Ala Pro Ala Leu Gln ProThr Gln Gly Ala Met Pro Ala Phe Ala Ser AlaPhe Gln Arg Arg Ala Gly Gly Val Leu Val AlaSer His Leu Gln Ser Phe Leu Glu Val Ser TyrArg Val Leu Arg His Leu Ala Gln Pro

[...

example 2

Characterization of PEG (4,500) G-CSF

[0034] PEG (4,500) G-CSF prepared in EXAMPLE 1 was characterized by the number of unmodified amino groups and a molecular weight estimated by SDS-PAGE.

[0035] The number of the unmodified amino groups was determined by reacting them with 0.1% TNBS in 4% NaHCO.sub.3 followed by measurement of absorbance at 335 nm (Habeeb et al., Anal. Biochem., 14, pp. 328-336, (1966)).

[0036] The molecular weight of PEG (4,500) G-CSF was determined by SDS-PAGE (16% gel, CBB staining) according to a method of Laemli, Nature, 227, p. 680, 1970. Each lane on the gel was scanned by using a chromato-scanner (SHIMADZU CORPORATION: CS-930) after staining.

[0037] When a molar ratio of the activated PEG to the number of free amino groups of human G-CSF increased, the extent of the modification also increased. The product prepared in said molar ratio of 1 has in addition to a band corresponding to an intact human G-CSF (19K) another band with an apparent molecular weight...

example 3

Preparation of PEG (10,000) G-CSF

[0040] The same human G-CSF as used in EXAMPLE 1 was modified by an activated polyethylene glycol (an activated PEG 2; Seikagaku Kogyo K.K.) with a molecular weight of about 10,000 having the following formula:

which had been prepared by reacting polyethylene glycol monomethyl ether with cyanuric chloride.

[0041] The human G-CSF was incubated with the activated PEG 2 at 5 times the molar amount of free amino groups of the human G-CSF in 0.25M sodium borate buffer solution (pH 10.0) for 1 hr at room temperature. The resulting product was applied to Sephadex G25 which had been equilibrated with 10 mM NH4 HCO3 for buffer-exchange, and then to DEAE ion-exchange chromatography to separate the PEG-modified human G-CSF from an unreacted human G-CSF and reagent. The estimation of a molecular weight of the product by SDS-PAGE as in EXAMPLE 2 has revealed that its average molecular weight is about 45K with distributed among 30K (10%), 40K (70%) and 66K (20...

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Abstract

The present invention provides a chemically-modified protein prepared by binding polyethylene glycol to a polypeptide characterized by being the product of expression by a host cell of an exogenous DNA sequence and substantially having the following amino acid sequence: (Met)nThr Pro Leu Gly Pro Ala Ser Ser Leu Pro GlnSer Phe Leu Leu Lys Cys Leu Glu Gln Val ArgLys Ile Gln Gly Asp Gly Ala Ala Leu Gln GluLys Leu Cys Ala Thr Tyr Lys Leu Cys His ProGlu Glu Leu Val Leu Leu Gly His Ser Leu GlyIle Pro Trp Ala Pro Leu Ser Ser Cys Pro SerGln Ala Leu Gln Leu Ala Gly Cys Leu Ser GlnLeu His Ser Gly Leu Phe Leu Tyr Gln Gly LeuLeu Gln Ala Leu Glu Gly Ile Ser Pro Glu LeuGly Pro Thr Leu Asp Thr Leu Gln Leu Asp ValAla Asp Phe Ala Thr Thr Ile Trp Gln Gln MetGlu Glu Leu Gly Met Ala Pro Ala Leu Gln ProThr Gln Gly Ala Met Pro Ala Phe Ala Ser AlaPhe Gln Arg Arg Ala Gly Gly Val Leu Val AlaSer His Leu Gln Ser Phe Leu Glu Val Ser TyrArg Val Leu Arg His Leu Ala Gln Pro(n = 0 (SEQ ID NO: 1) or 1 (SEQ ID NO: 2)) The chemically-modified protein according to the present invention has a neutrophils-increasing activity much more lasted than that of the intact human G-CSF, enabling fewer numbers of administration with a lower dose.

Description

REFERENCE TO RELATED APPLICATIONS [0001] This application is a Continuation of U.S. patent application Ser. No. 11 / 342,519, filed Jan. 30, 2006, which in turn is a Continuation of U.S. patent application Ser. No. 10 / 436,784, filed May 12, 2003, which is a Divisional application of U.S. patent application Ser. No. 09 / 921,114, filed Aug. 2, 2001, which is a Continuation of U.S. patent application Ser. No. 09 / 518,896 filed Mar. 6, 2000, which is a Continuation of U.S. patent application Ser. No. 08 / 957,719 filed Oct. 27, 1997, which is a Continuation of U.S. patent application Ser. No. 07 / 983,620 filed Nov. 30, 1992, which is a Continuation of U.S. patent application Ser. No. 07 / 566,451 filed Oct. 1, 1990, which is the U.S. National Stage of PCT / JP89 / 01292.TECHNICAL FIELD [0002] The present invention relates to a chemical modification of granulocyte colony-stimulating factor (G-CSF), by which the chemical and / or physiological properties of G-CSF can be changed. BACKGROUND ART [0003] Hu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/475A61K38/00A61K47/48C07K14/535C07K17/08
CPCA61K38/00A61K47/48215Y10S930/145C07K17/08C07K14/535A61K47/60
Inventor ISHIKAWA, RIKAOKADA, YUJIKAKITANI, MAKOTO
Owner KIRIN AMGEN
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