Preparation method of human vascular endothelial inhibitory peptide

A vascular endothelial and inhibitory peptide technology, applied in the field of anti-tumor applications, can solve problems such as residues, drug resistance, insensitivity, etc., and achieve the effects of low racemization, good product quality and high purity

Inactive Publication Date: 2013-07-03
HARBIN PHARMA GROUP BIOLOGICAL ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Tumor is a multi-gene and multi-factor disease. Traditional tumor treatment includes surgery, chemotherapy, radiotherapy, etc., mainly targeting tumor cells directly. There are postoperative residues, drug delivery and penetration problems, and chemotherapy, radiotherapy Disadvantages such as insensitivity and drug resistance

Method used

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  • Preparation method of human vascular endothelial inhibitory peptide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] (1) Swell Fmoc-Asp(OtBu)-Wang with DMF for 30 minutes, remove DMF, add 20% PIP / DMF solution, stir for 5 minutes, wash with DMF, and wash twice.

[0016] (2) Then add 20% PIP / DMF solution, stir and react for 25 minutes, wash with DMF twice, and the ninhydrin test should be positive.

[0017] (3) Weigh Fmoc-Gly-OH (3-5 times the number of moles of amino groups contained in the resin) and equimolar 1-hydroxy-benzo-triazole (HOBt) to dissolve in DMF, and cool in an ice bath for 30 minutes; Take an equimolar amount of DIC, dilute it with DCM, and pre-cool it in a water bath at 0°C to -10°C, add the cooled DIC solution to the DMF solution of Fmoc-Gly-OH, continue to cool and stir for more than 60 minutes, and then add it to The condensation reaction was carried out in the reaction column, and after the reaction (no color detected by ninhydrin chromogenic method) was washed with DMF for 3 times to complete a peptide grafting reaction.

[0018] (4) Repeat the steps of removing...

Embodiment 2

[0023] (1) Preparative HPLC was used for separation and purification, and the mobile phase system was 0.1% TFA-acetonitrile; the preparative liquid chromatography system used 10μm reversed-phase C18 (77mm*250mm) packing, the UV detector was set at 280nm, and the flow rate was 90ml / min .

[0024] (2) Using gradient elution, take the crude product solution and put it on the chromatographic column, start the mobile phase elution, collect the spectrum, observe the change of the absorbance, collect the main peak and use the analytical liquid phase to test the purity, combine the main peak solution, at 30-40 Concentrate under reduced pressure at ℃ to obtain a concentrated solution after purification of the anti-cancer polypeptide. The purity is about 90%, and the refined yield is 60%. The next step of purification is carried out.

[0025] (3) High performance liquid chromatography was used for desalting, and the mobile phase system was 0.2% acetic acid-acetonitrile. Take the concen...

Embodiment 3

[0028] Take the CS8023 acetate aqueous solution, filter it with a 0.45 μm filter membrane, transfer it to a freeze-drying tray, and freeze-dry it. The freeze-drying conditions (which can be adjusted according to different specifications of the freeze-drying machine) are shown in Table 1.

[0029] temperature time (hours) Remark -40℃ 4 pre-freeze room temperature 72 Vacuum room temperature out of the box

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Abstract

The invention provides a solid-phase synthesis process of a human vascular endothelial inhibitory peptide: H-His-Ser-His-Arg-Asp-Phe-Gln-Pro-Val-Leu-His-Leu-Val-Ala-Leu-Asn-Ser-Pro-Leu-Ser-Gly-Gly-Met-Arg-Gly-Asp-Arg-Gly-Arg. The process includes: taking Fmoc-Asp(OtBu)-Wang resin as a raw material and putting it in a polypeptide synthesis reactor, using N, N-dimethylformamide (DMF) as a solvent, taking 20% piperidine (PIP) as an alpha-amino deprotection agent, adopting N, N-diisopropyl carbodiimide (DIC) as a condensation agent, and introducing protected amino acid (Fmoc-AA) in order to perform a programmed reaction; then carrying out cracking, precipitation by anhydrous ether, and oxidation to obtain a crude product; and subjecting the crude product to purification, desalination and freeze-drying so as to obtain a refined end product.

Description

technical field [0001] The invention relates to polypeptide drugs in the field of biotechnology and pharmacy, its preparation method and anti-tumor application. Background technique [0002] Tumor is a multi-gene and multi-factor disease. Traditional tumor treatment includes surgery, chemotherapy, radiotherapy, etc., mainly targeting tumor cells directly. There are postoperative residues, drug delivery and penetration problems, and chemotherapy, radiotherapy Insensitivity, drug resistance and other shortcomings. The growth of tumors is dependent on blood vessels. When the tumor diameter is less than 2 mm, tumor cells absorb nutrients through diffusion, which is called "pre-vascular"; when the tumor grows further, new capillaries are needed to provide nutrients. called the "vascular phase". Angiogenesis is closely related to the occurrence, development and metastasis of tumors. Therefore, in the early 1970s, Folkman proposed anti-angiotherapy therapy for tumors. This idea w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C07K1/06C07K1/04
CPCY02P20/55
Inventor 徐岩李郑武王忠高晶李金花宋紫玉曹翊婕李会成王丽娜黄宇红李曲亮程广伟
Owner HARBIN PHARMA GROUP BIOLOGICAL ENG
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