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A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol

A technology of colony-stimulating factor and polyethylene glycol, which is applied in the direction of extracellular fluid diseases, drug combinations, medical preparations of non-active ingredients, etc., can solve the problem of low specific activity of conjugates, and achieve high specific activity level, Effect of improved pharmacokinetic parameters

Inactive Publication Date: 2013-06-05
BIOCAD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0028] 1. The specific activity of the obtained conjugate is low (68% of the activity of unmodified G-CSF);

Method used

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  • A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol
  • A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol
  • A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1: Preparation of recombinant human G-CSF (rhG-CSF, filgrastim)

[0069] rhG-CSF was isolated and purified as described in patent RU2278870.

Embodiment 2

[0070] Example 2: Preparation of PEGylated rhG-CSF

[0071] Add 50 mL of 1M sodium cyanoborohydride solution to 2300 mL of buffer solution (50 mM sodium acetate, pH 5.0±0.2), containing 2500 mg of purified rhG-CSF, prepared according to the method in Example 1. The mixture was stirred, followed by the addition of 10 g of solid mPEG-ButyrALD (methoxy-PEG-butyraldehyde) with an average molecular weight of 30 kDa. The coupling reaction mixture was stirred at (20±2)°C for 22 hours. After various time intervals, 50 μl samples were taken from the reaction mixture and the kinetics of PEG-G-CSF conjugate formation were analyzed using SDS-PAGE on a 12.5% ​​gel under reducing conditions. To this end, add 17 microliters of buffer solution containing 125mM Tris-HC1, pH6.8, 20% glycerol, 3% SDS, 15% 2-mercaptoethanol, 0.005% bromophenol blue to the sample to be selected, in a boiling water bath Heat for 3 minutes. Load a volume of 5 microliters of sample into each well. SDS-PAGE was pe...

Embodiment 3

[0072] Example 3: Purification of single PEG-G-CSF conjugates

[0073] The diluted reaction mixture prepared according to Example 2 was applied to a column filled with 300 mL of CM-agarose and equilibrated with 10 mM sodium acetate buffer (buffer A) at pH 4.8 at a flow rate of 10 mL / min. After the material was applied to the column, it was eluted with 1500 mL buffer A to remove unbound material. Mono-PEG-G-CSF was eluted from the column using a linear gradient of 0-0.2 M NaCl in buffer A (6000 mL) at a flow rate of 5 mL / min. Fractions (50 mL) were collected for absorbance detection at wavelengths of 280 and 260 nm. Fractions containing protein were analyzed by RP-HPLC and SDS-PAGE as described in Example 2. Fractions containing monoPEGylated G-CSF with a purity greater than 95% were dialyzed against 10 volumes of 1.6 mM sodium acetate buffer, pH 4.0 ± 0.2. Subsequent filter sterilization was performed and samples were stored at 4±2°C.

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Abstract

The present invention relates to pharmaceuticals and medicine, namely, to new physiologically active conjugates of granulocyte colony-stimulating factor (G-CSF) with the general formula (I) where: n - integers from 681 to 1 000; m - integer > 4; NaH-G-CSF - natural or recombinant polypetide, having the activity of G-CSF. The invention is also related to medicines containing the claimed conjugate of formula (I), pharmaceutical compositions, the use of conjugate of formula (I) for drugs and medicines with granulocyte colony stimulating factor as an active ingredient, approaches to prevent and / or treat neutropenia, the container that comprises pharmaceutical composition.

Description

technical field [0001] The present invention relates to pharmaceutical preparations and drugs, namely, to novel physiologically active conjugates of granulocyte colony-stimulating factor (G-CSF), especially to novel conjugates of G-CSF and polyethylene glycol , which are suitable for medical use, for example, for the treatment of leukopenia, mainly in patients receiving myelosuppressive chemotherapy, in patients receiving hematopoietic stem cell transplantation, in patients with chronic neutropenia, and in patients with AIDS and other infections Various types of neutropenia in patients. Background technique [0002] G-CSF is a hematopoietic growth factor that stimulates the proliferation, differentiation and maturation of granulocytes (Metcalf, 1992). The intervention of exogenous G-CSF leads to a rapid, specific, and dose-dependent increase in peripheral blood neutrophils (Welte et al., 1985; Hartung et al., 1995). [0003] The human G-CSF gene was cloned and expressed in...

Claims

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Application Information

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IPC IPC(8): C07K14/535A61K38/18A61K47/48A61P7/00
CPCC07K14/535A61K47/48215A61K38/193A61K47/60A61P7/00A61K38/19A61K47/50
Inventor 大菊阿纳·伟尼阿迷诺夫娜·茄尔锘夫斯卡娅德迷特例·瓦连吉诺维奇·莫罗佐夫列夫·阿列克三渡罗维奇·杰尼琐夫耶列娜·吉尔吉耶夫娜·橹邓郭耶列娜·列欧尼多夫娜·莫罗唑娃
Owner BIOCAD
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