Ciliary neurotrophic factors decorated by polyglycol polymer and preparation method thereof

A technology of ciliary neurotrophy and polyethylene glycol, which is applied in neuromuscular system diseases, medical preparations with non-active ingredients, medical preparations containing active ingredients, etc., and can solve the problem of unseen polymer chemical modifier modification. And other problems, to achieve the effect of long half-life in vivo circulation, good stability, and longer half-life in vivo circulation

Inactive Publication Date: 2008-05-28
BEIJING INST OF BIOLOGICAL PROD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no reports of polymer chem

Method used

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  • Ciliary neurotrophic factors decorated by polyglycol polymer and preparation method thereof
  • Ciliary neurotrophic factors decorated by polyglycol polymer and preparation method thereof
  • Ciliary neurotrophic factors decorated by polyglycol polymer and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Preparation of rhCNTF

[0049] According to the natural human CNTF gene sequence (from GENE BANK), two primers were designed: P1: GAC CAT ATG GCT TTC ACA GAG CAT TCA CCG; P2: GGT CAG CTC GGA TCC TTA ATC AGT GCT TGC CAC. Using human cDNA as a template (for the method, refer to translation by Jin Dongyan et al., "Extraction of Molecular Cloning Experiment Guide" (1996 edition), p. 463-469), add P1 and P2 primers, and add P1 and P2 primers in a reaction system of 50 μl {10× buffer, containing Mg 2+ 5.0μl, dNTP (2.5mmol / L) 1.0μl, P1 (30μmol / L) 1.0μl, P2 (30μmol / L) 1.0μl, Pfu DNA polymerase 1U, add ultrapure water to the total volume to 50.0μl}, After denaturation at 95°C for 30 seconds, denaturation at 93°C for 30 seconds, annealing at 55°C for 60 seconds, and extension at 68°C for 2 minutes, repeat the above cycle 30 times, and finally extend at 68°C for 8 minutes; after the reaction, quickly cool down to 4°C. The above PCR product was double-digested with NdeI ...

Embodiment 2

[0051] Example 2 Preparation of Modifiers and Selection of Modification Conditions

[0052] The polyethylene glycol-based polymers used are as follows:

[0053] SPA-mPEG, purchased from Beijing Kaizheng Bioengineering Development Co., Ltd., batch number: 20060101, molecular weight 20KD; SC-mPEG, purchased from Beijing Kaizheng Bioengineering Development Co., Ltd., batch number: 20051019, molecular weight 20KD; SMB-mPEG, Purchased from NEKTAR Company, batch number: PT-02E-13, molecular weight 20KD.

[0054] In the following examples, unless otherwise specified, the rhCNTF solution and the PEG solution used were prepared using 0.02M phosphate buffer (pH 7.2) as a solvent.

[0055] In this example, rhCNTF bound to a single molecule polyethylene glycol polymer is represented by rhCNTF-PEG1.

[0056] 1. Effect of reaction time on modification reaction

[0057] Take 1ml of rhCNTF (1.016mg / ml), add SPA-mPEG at a molar ratio of 20:1, and react at room temperature. Take out 0.2ml a...

Embodiment 3

[0072] Example 3 Preparation and purification of rhCNTF modified by SPA-mPEG

[0073] The rhCNTF prepared in Example 1 was dialyzed, the dialysate was 0.1 mol / L sodium tetraborate aqueous solution, and the pH value was 9.2. They were dialyzed at 4°C for 48 hours, and the medium was changed 4 times. After dialysis, samples were taken, and the concentration of rhCNTF protein was determined by the Lowry method. Then take 200ml of dialyzed rhCNTF (2mg / ml) and add SPA-mPEG at a molar ratio of 1:5. At 25°C, the pH value of the reaction system is controlled at 8. The reaction time is 2h, and 6N HCl is added to terminate the reaction. reaction.

[0074] In order to better separate unmodified rhCNTF, rhCNTF combined with single-molecule SPA-mPEG and rhCNTF combined with multi-molecule SPA-mPEG, QS-HighPerformance ion-exchange chromatography was used for research. Take 40ml of the solution after the above-mentioned terminated reaction, and put it on the column for separation. The chr...

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Abstract

The invention relates to a polyethylene glycol polymer-modified ciliary neurotrophic factor and a preparation method thereof. The method can just include the mixing reaction of polyethylene glycol polymer and ciliary neurotrophic factor. The polyethylene glycol polymer-modified ciliary neurotrophic factor related by the invention maintains the activity of the ciliary neurotrophic factor in the body, and the toxicity and side effects are significantly reduced, at the same time, the experimental results prove that the ciliary neurotrophic factor can obviously improve the pharmacokinetics property of the ciliary neurotrophic factor and greatly reduce the immunogenicity of the ciliary neurotrophic factor.

Description

technical field [0001] The invention relates to the biological field, in particular to ciliary neurotrophic factors modified by polyethylene glycol polymers and a preparation method thereof. Background technique [0002] Ciliary neurotrophic factor (Ciliary neurotrophic factor, CNTF) was first discovered in the choroid, ciliary body and iris of the chick embryo eyeball, and was named because it can promote the survival of chick embryo parasympathetic ciliary ganglion neurons in vitro. CNTF is a polypeptide hormone composed of about 200 amino acids, which is secreted by Schwann cells in the peripheral nerves of mature individuals, some subcellular populations in astrocytes and the central nervous system rather than by target tissues. CNTF was originally considered as a trophic factor in the culture of ciliary ganglion parasympathetic neurons, hence the name. It can promote the growth and differentiation of a variety of nerve cells and glial cells, including motor neurons, se...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K47/34A61K38/19A61P21/00A61P43/00A61K47/60
Inventor 张振龙姜江倪道明
Owner BEIJING INST OF BIOLOGICAL PROD
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