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Novel polypeptide capable of inhibiting new vessels and application thereof

An angiogenesis and peptide technology, applied in the field of biomedicine, can solve problems such as high price, complex spatial conformation, and endotoxin residues

Inactive Publication Date: 2015-02-11
SHANGHAI FIRST PEOPLES HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Third, based on the above main reasons, the bioavailability of ophthalmic drugs is very low; to improve it, the concentration of administration can be increased
[0008] Fourth, although a series of relatively safe endogenous angiogenesis inhibitors have been confirmed successively, such as angiostatin, which consists of plasminogen Kringle domains 1-4 (plasminogen Kringle1-4), Can significantly inhibit the growth of blood vessel-dependent tumors, but due to its large molecular weight and complex spatial conformation, there are deficiencies in the preparation process such as cumbersome recombinant expression and purification processes and endotoxin residues
It is precisely because of the limitations of the above-mentioned conditions that the drugs currently used to treat ocular neovascularization are very limited, such as recombinant anti-human VEGF monoclonal antibody bevacizumab (Avastin), recombinant anti-human VEGF monoclonal antibody fragment ranibizumab (Lucentis), etc., but They are expensive and require repeated administration through the vitreous cavity, which may even cause risks such as vascular embolism

Method used

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  • Novel polypeptide capable of inhibiting new vessels and application thereof
  • Novel polypeptide capable of inhibiting new vessels and application thereof
  • Novel polypeptide capable of inhibiting new vessels and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0170] Synthesis and Purification of Small Peptide CP912

[0171] Using a commercially available SYMPHONY 12-channel peptide synthesizer (Protein Technologies, USA), the synthesized sequences were CP912 polypeptides shown in SEQ ID NO:1. The specific method is as follows:

[0172]Calculate and configure the reagents according to the software of the peptide synthesizer (Version.201), put 2-Chlorotrityl Chloride Resin resin (Tianjin Nankai Synthetic Technology Co., Ltd.) into the reaction tube, add DMF (15ml / g) (Dikma), shake 30min. Filter the solvent through the sand core, add 3-fold molar excess of Fmoc-L-H-OH (small peptide CP912) amino acid (Suzhou Tianma Pharmaceutical Group Fine Chemicals Co., Ltd.), and then add 10-fold molar excess of DIEA (Sinopharm Group Shanghai Chemical Reagent Company), and finally add DMF to dissolve, shake for 30min. Remove DMF, add 20% piperidine (Shanghai Chemical Reagent Company) DMF solution (15ml / g), 5min, remove DMF, add 20% piperidine DM...

Embodiment 2

[0182] Identification and preservation of small peptide CP912

[0183] Take a small amount of finished small peptide CP912, and do HPLC analysis for purity identification, and ESI-MS molecular weight identification. The conditions were as follows: electrospray ionization, detection in positive ion mode, nitrogen as sheath gas, auxiliary gas and sweep gas. Spray voltage 4.7kV: sheath gas 15arb, auxiliary gas 5arb, purge gas 0arb; capillary temperature: 275°C, capillary voltage 40V, lens voltage 120V. Full scan mass range: 300-2000amu. Secondary mass spectrometry was collected in data-dependent mode. The three peaks with the highest responsivity intensities in the full scan spectrum were used for secondary mass spectrometry analysis. High-purity helium was used as a collision gas with a collision energy of 35 eV.

[0184] The results showed that the small peptide CP912 molecular weight: 882.89DA, its structural information is correct, conforms to the theoretically designed ami...

Embodiment 3

[0187] Effect of Small Peptide CP912 on Proliferation of Human Umbilical Vein Endothelial Cells

[0188] Using the CCK-8 method, the specific method is as follows:

[0189] Human umbilical vein endothelial cells HUVECs (purchased from ScienCell) were seeded in 96-well plates at a concentration of 2×10 4 After the cells adhered to the wall, add serum-free medium ECM and incubate at 37°C for 24 hours; then add serum-free medium ECM to each well as a negative control, VEGF (20ng / ml) (purchased from R&D Company) as a positive control, VEGF (20ng / well)+Avastin (purchased from Roche), VEGF (20ng / well)+small peptide CP912 of different concentrations were used as treatment groups; after continuing to culture for 96 hours, 20 μl of CCK-8 solution was added to each well ( purchased from Dojindo Company); after incubation at 37°C for 4 hours, use a microplate reader (Molecular Device Company) to measure the absorbance of each well at 450 nm, and judge the proliferation activity of the c...

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Abstract

The invention relates to a novel polypeptide capable of inhibiting new vessels and an application thereof. The invention also relates to a polypeptide preparing method and an application thereof, and a pharmaceutical composition containing polypeptide. The polypeptide has a plurality of advantages that the molecular weight is little, polypeptide can penetrate through various ocular tissue barriers; water solubility is good, such that the polypeptides can maintain a high concentration in a neutral tear fluid, aqueous humor and vitreous humor; and the like.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a new class of small peptides for inhibiting neovascularization, and the small peptides are polypeptides derived from placenta growth factor (PlGF). The present invention also relates to the preparation method and application of the polypeptide and the pharmaceutical composition containing the polypeptide. Background technique [0002] The formation of neovascularization is an extremely complex process, which includes: dilation of existing blood vessels, increase of vascular permeability, degradation of perivascular matrix, activation of proliferation and migration of endothelial cells, and formation of new capillary-like lumens. [0003] In the eye, about two-thirds of blinding diseases are associated with pathological neovascularization, such as corneal neovascularization in herpetic stromal keratitis, choroidal neovascularization in age-related macular degeneration, and diabetic ret...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/08C07K14/00C12N15/11A61K38/08A61K38/10A61K38/16A61P27/02A61P35/00A61P9/10A61P17/06A61P15/08A61P19/02A61P29/00A61P7/02A61P1/00A61P13/12
Inventor 许迅柯潇郑颖罗德伦邬智刚
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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