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Conjugate of polyethylene glycol and tumor necrosis factor alpha or analogue of polyethylene glycol and tumor necrosis factor alpha and medical application of conjugate

A technology of tumor necrosis factor and polyethylene glycol, which is applied in the field of prevention or treatment of tumors or cancers, can solve the problems of poor clinical curative effect, short circulation time, and high organ toxicity of the blood circulation system, so as to prolong the half-life of biological activity and reduce toxicity. Side effects, effect of increasing water solubility

Active Publication Date: 2016-02-03
岳阳新华达制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The published experimental data of clinical treatment of early melanoma and malignant soft tissue sarcoma show that TNFα circulates in the human body for too short a time, and the organ toxicity of the blood circulation system is too high. Poor clinical efficacy in enteral injection form (BartlettDL et al., Aphase 1 trial of continuous hyperthermic peritoneal perfusion with tumor necrosis factor and Cisplatin in the treatment of peritoneal carcinomatosis, Cancer, 83:1251-1261.)

Method used

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  • Conjugate of polyethylene glycol and tumor necrosis factor alpha or analogue of polyethylene glycol and tumor necrosis factor alpha and medical application of conjugate
  • Conjugate of polyethylene glycol and tumor necrosis factor alpha or analogue of polyethylene glycol and tumor necrosis factor alpha and medical application of conjugate
  • Conjugate of polyethylene glycol and tumor necrosis factor alpha or analogue of polyethylene glycol and tumor necrosis factor alpha and medical application of conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Gene expression of TNFα protein

[0034] Using the total chemical synthesis method, the following TNFα gene was compiled into the special amino acid expression code of Escherichia coli (Pennica, D.Nedwin, G.E., Hayflick, J.S., atal, Nature, 1984, 312 (20 / 27), 724-729, Humantumournecrosisfactor :precursorstructure, expressionandhomologytolymphotoxin) into the expression plasmid pET28a (+) (pETSystemNovagenManual11thEdition, UserProtocolTB055Rev.C0611JN), and then according to the routine procedures in this method manual of Novagen Company (NovagenUserProtocolTB055Rev.C0611JN), the plasmid was penetrated into Escherichia coli) BL21 (DE3 BL21(DE3)sS (NovagenpETSystemManual11thEdition), then select a single colony from the agar plate of the Escherichia coli expressing the above plasmid, and put it into 5mL of sterile LB (Luria-Bertani) medium (purchased from Sinopharm Chemical Reagent Ltd.), cultured at 37°C according to the standard operation manual. Then gradua...

Embodiment 2

[0041] Gene expression of embodiment 2 TNFα protein analog (mutant)

[0042] Using the total chemical synthesis method, the following TNFαA21K, TNFαG31K, TNFαA21KG31K genes were compiled into specific amino acid expression codes of Escherichia coli, and inserted into the expression plasmid pET28a(+). When fully chemically synthesizing the TNFα gene code: 1) change the alanine code of the 21st sequence to a lysine code, 2) change the 31st glycine code to a lysine code, 3) change the 21st sequence to a lysine code Propanic acid code, the 31st glycine code is changed to lysine code at the same time, and then according to the procedure in Novagen Company's method manual (NovagenUserProtocolTB055Rev.C0611JN), implant pET28a (+) plasmid (pETSystemNovagenManual11thEdition, UserProtocolTB055Rev.C0611JN), you can Expression of the TNFα analogue mutant protein described above. The above-mentioned TNFα protein analogs are cultivated in Escherichia coli BL21 (DE3), BL21 (DE3) sS agar cul...

Embodiment 3

[0062] Example 3 Purification of TNFα protein and mutants thereof

[0063] The supernatant after the lysis buffer solution obtained in Examples 1 and 2 above contains a large amount of water-soluble expressed protein. The supernatant after breaking the bacteria was slowly passed through nickel ions (Ni-Sepharose) at a flow rate of 1.0 ml / min. TM 6FastFlow) affinity column (GE Healthcare Bioscience AB, Sweden), the TNFα protein was retained on the nickel ion column, and then the solution was eluted with a special nickel ion column, 0.6M imidazole histamine (imidazole) aqueous solution was eluted, and the initially purified (>95% ) TNFα protein. The resulting protein was passed through a fast-flow anion (SPSepharose TM FastFlow) resin exchange column (GE Healthcare Bioscience AB, Sweden), the protein is retained on this resin ion exchange column, and then eluted with phosphate buffer (pH8.5) containing 0.5M NaCl to obtain further highly purified (>99.5%) TNFα protein. The mu...

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Abstract

The invention relates to conjugate of polyethylene glycol and a tumor necrosis factor alpha or analogue of the polyethylene glycol and the tumor necrosis factor alpha and medical application of the conjugate, in particular to conjugate of polyethylene glycol-tumor necrosis factor alpha or analogue of the polyethylene glycol-tumor necrosis factor alpha, a preparation method of the conjugate and application of the conjugate serving as a therapeutic agent in preventing or treating tumor or cancer. The conjugate of polyethylene glycol-tumor necrosis factor alpha or analogue of the polyethylene glycol-tumor necrosis factor alpha can lower acute toxicity of medicine, reduce the intravenous dosing frequency, reduce the dosage of applied medicine, increase the anti-tumor therapeutic effect, solve the problem that efficacy half-life of the tumor necrosis factor alpha protein is short through parenteral medicine application, achieve the target of preventing and treating tumor or cancer and thus has beneficial medical prospect.

Description

technical field [0001] The present invention relates to a conjugate of polyethylene glycol-tumor necrosis factor alpha (TNF alpha) or polyethylene glycol-tumor necrosis factor alpha analogue, its preparation method, and its use as a therapeutic drug in preventing or treating tumor or cancer the use of. Background technique [0002] Tumor necrosis factor α (TNFα) is a multifunctional cytokine protein that can induce apoptosis of malignant tumor cells. Secreted by activated mononuclear macrophages, TNFα is the cytokine with the strongest anti-tumor activity found so far, and it has obvious tumoricidal and tumoristatic effects on a variety of malignant tumor cells (Carswell, EA et al., 1975, Anendotoxin induced serum factor that causes necrosisofumors. Proc. Natl. Acad. Sci. USA72; 3666-3670). [0003] TNFα was discovered by American scientists in the late 1970s. TNFα can transduce all cell physiology, individual physiology, pathology, and 杀灭恶性肿瘤细胞的药理学作用(AndrewsJS等人,1990.Char...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/525A61K38/19A61P35/00
CPCA61K38/00C07K14/525
Inventor 郭凌云唐建军刘群奇时小燕徐戎
Owner 岳阳新华达制药有限公司
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