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A small peptide for inhibiting neovascularization and its application

An angiogenesis and application technology, applied in the field of biomedicine, can solve the problems of low bioavailability, residual endotoxin and limited use of ophthalmic drugs

Inactive Publication Date: 2017-05-17
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A small peptide for inhibiting neovascularization and its application
  • A small peptide for inhibiting neovascularization and its application
  • A small peptide for inhibiting neovascularization and its application

Examples

Experimental program
Comparison scheme
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preparation example

[0126] Synthesis of Small Peptide ZY7 and Derivative Peptides

[0127] Using a commercially available SYMPHONY type 12-channel polypeptide synthesizer (Protein Technologies, USA), according to the operation manual of the synthesizer, the Fmoc solid-phase method was used to synthesize the ZY7 polypeptide shown in SEQ ID NO: 2, and the ZY7 polypeptide shown in SEQ ID NO: 2, respectively. NO.: Derivative polypeptides ZY7-1 to ZY7-6 shown in 3-6.

[0128]After the synthesis is complete, cut the polypeptide from the resin (cutting solution (10 / g): TFA (J.T.Baker) 94.5%, water 2.5%, EDT (ALDRICH) 2.5%, TIS (ALDRICH) 1%; cutting time: 120min). The lysate was dried as much as possible with nitrogen (Shanghai Biou Gas Industry Co., Ltd.), washed six times with ether (Shanghai Shiyi Chemical Reagent Co., Ltd.), and evaporated to dryness at room temperature.

[0129] Use HPLC (SHIMADZU high performance liquid chromatograph model: preparative type, analytical type, software: Class-VP.Sev...

Embodiment 1

[0144] Effect of Small Peptide ZY7 on Proliferation of Human Umbilical Vein Endothelial Cells

[0145] Using the MTS method, the specific method is as follows:

[0146] Primary human umbilical vein endothelial cells HUVECs (purchased from ScienCell) were inoculated in 96-well plates at a concentration of 2×10 4 After the cells adhered to the wall, serum-free culture medium ECM was added and cultured at 37°C for 24 hours; then, serum-free culture medium ECM was added to each well as a negative control, VEGF (100ng / ml) (purchased from Sigma) as a positive control, VEGF (100ng / well) + different concentrations of small peptide ZY7 were used as treatment groups; after continuing to culture for 24 hours, 20 μl of MTS solution (purchased from Promega) was added to each well; after incubation at 37°C for 4 hours, microplate reader was used to (Bio-Rad Company) measured the absorbance of each hole at 490nm, judged the proliferation activity of the cells according to OD490, and finally...

Embodiment 2

[0149] Effects of Small Peptide ZY7 on Lumen Formation Activity of Human Umbilical Vein Endothelial Cells

[0150] Using the Matrigel method, the specific method is as follows:

[0151] Add 50 μl / well of Matrigel matrigel (purchased from BD Company) to a 96-well plate, and incubate at 37° C. for 30 minutes. After it was solidified, primary human umbilical vein endothelial cells HUVECs were inoculated on the surface of Matrigel at a concentration of 8×10 6 / ml; and in each well, add serum-free culture agent ECM as negative control, VEGF (100ng / ml) (purchased from Sigma company) as positive control, VEGF (100ng / ml) + different concentrations of small peptide ZY7 as treatment group, and continued to culture at 37°C. 6 hours after the treatment, the cells in the well plate were photographed randomly at 3 fields of view under a 200-fold microscope, and the sum of the largest diameters of the lumens formed therein was calculated using the software Image-ProPlus Program5.1 (Media C...

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Abstract

The invention relates to novel small peptides inhibiting new vessels and application. The invention also relates to a preparation method of the polypeptide, application and pharmaceutical composition containing the polypeptide. The polypeptide possesses a plurality of advantages, such as small molecular weight, capability of permeating various ocular tissue barriers, good water solubility, capability of possessing a relatively high concentration in neutral tear, 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 human Neuropilin-1. 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 retinopathy O...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C07K7/08C12N15/12A61K38/08A61K38/10A61P27/02A61P35/00A61P9/10A61P9/00A61P7/02A61P29/00A61P1/00A61P19/02A61P17/06A61P17/00A61P15/08
Inventor 郑颖许迅
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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