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New small peptides for inhibiting newborn blood vessels, and application thereof

An angiogenesis and peptide technology, which is applied to cardiovascular diseases, medical preparations containing active ingredients, peptides, etc., can solve problems such as complex spatial conformation, residual endotoxin, and large molecular weight

Inactive Publication Date: 2014-08-27
SHANGHAI FIRST PEOPLES HOSPITAL
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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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  • New small peptides for inhibiting newborn blood vessels, and application thereof
  • New small peptides for inhibiting newborn blood vessels, and application thereof
  • New small peptides for inhibiting newborn blood vessels, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0130] Synthesis of small peptide UK12 and derived peptides

[0131] The SYMPHONY 12-channel peptide synthesizer (Protein Technologies, USA) was used to calculate and prepare the required Fmoc protective amino acid solution, condensation reagent and cleavage reagent according to its software (Version.201 version). Edit the program, in which the resin swelling time is 30min; the deprotection time is 5min and 15min respectively; the condensation time is 30min; the cutting time is 2h. Start up and synthesize peptides according to the above procedures, and use high performance liquid chromatography (SHIMADZU) to purify the peptides to obtain purity> 95% white powdery peptide.

[0132] Take a small amount of the product small peptide UK12, carry out the purity identification by HPLC analysis and the molecular weight identification by ESI-MS.

[0133] The results showed that the purity identification was greater than 95%, the small peptide UK12 had a total of 12 amino acids, and the molec...

Embodiment 2U

[0135] Example 2 UK12 polypeptide inhibits VEGF-induced proliferation of vascular endothelial cells

[0136] (1) In vitro culture of human umbilical vein endothelial cells (HUVECs)

[0137] Primary HUVECs (purchased from ScienCell) were cultured at 37°C with 5% CO using ECM medium supplemented with ECGS (ScienCell) and 5% fetal bovine serum (ScienCell) 2 In the incubator. All in vitro cell experiments in the present invention use HUVECs from the 3rd to 8th generation.

[0138] (2) MTS method detects the inhibition of VEGF-induced vascular endothelial cell proliferation by UK12 polypeptide

[0139] The MTS cell proliferation quantitative detection method is a method that uses tetrazole and an electron coupling compound to produce water-soluble colored products under the action of the mitochondrial dehydrogenase of the metabolized cells, which is used as a detection signal to colorimetrically determine the proliferation of living cells.

[0140] The specific implementation method is as f...

Embodiment 3U

[0143] Example 3 UK12 polypeptide inhibits VEGF-induced migration of vascular endothelial cells

[0144] Vascular endothelial cell migration experiment adopts Transwell chamber (Corning) method.

[0145] The specific implementation method is as follows: After HUVECs grow close to fusion, they are starved overnight in a serum-free medium, and 0.25% trypsinization is performed to prepare a cell suspension. The polypeptide was mixed with a cell suspension containing 4×105 HUVECs to prepare a volume of 100 μL and a UK12 polypeptide concentration of 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM. After pretreatment in a 37°C incubator for 30 minutes, Join the upper room. 600 μL of serum-free medium containing 25ng / ml VEGF was added to the lower chamber as a chemokine. Continue to incubate the Tranwell chamber in a 37°C incubator for 24 hours, take out the upper Transwell chamber, wipe off the non-migrated cells on the polycarbonate membrane upper chamber with a cotton swab, and stain with hemat...

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Abstract

The invention relates to polypeptides for preventing and inhibiting angiogenesis, and an application thereof. The invention also relates to a preparation method of the polypeptides, an application of the polypeptides, and a medicinal composition containing the polypeptides. The polypeptides have the advantages of small molecular weight, permeation of various eye tissue barriers, good water solubility, and maintenance of a high concentration in neutral tears, aqueous humor and vitreous humor.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and relates to a new polypeptide with the effect of inhibiting neovascularization, which is called UK12 polypeptide. Specifically, the present invention relates to the amino acid sequence and preparation method of the UK12 polypeptide, as well as the effect of the polypeptide in inhibiting the proliferation, migration, and lumen formation of vascular endothelial cells in vitro and inhibiting retinal neovascularization in mice in vivo. Background technique [0002] The formation of new blood vessels is an extremely complex process, which includes: the expansion of existing blood vessels, the increase of vascular permeability, the degradation of the surrounding matrix, the activation and proliferation and migration of endothelial cells, and the formation of new capillary-like lumens. [0003] In the eye, about 2 / 3 of blinding diseases are related to pathological neovascularization, such as corneal neo...

Claims

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Application Information

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IPC IPC(8): C07K7/08C12N15/11A61K38/10A61P27/02A61P35/00A61P9/10A61P9/00A61P7/02A61P29/00A61P1/00A61P1/04A61P19/02A61P17/00A61P17/06A61P15/08
Inventor 苏莉
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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