CN109776656B

CN109776656B - Polypeptide TIN7N for inhibiting angiogenesis and application thereof

Info

Publication number: CN109776656B
Application number: CN201910030074.2A
Authority: CN
Inventors: 许元生; 宋燕; 张时群; 苏博雅; 汤宇晴
Original assignee: Link Health Group
Current assignee: Link Health Group
Priority date: 2019-01-11
Filing date: 2019-01-11
Publication date: 2022-03-11
Anticipated expiration: 2039-01-11
Also published as: CN109776656A

Abstract

The invention discloses a polypeptide TIN7N, the amino acid sequence of which is an amino acid sequence with more than 13/16 sequence consistency compared with SEQ ID NO. 1; and the application of the polypeptide TIN7N or the pharmaceutically acceptable salt thereof in preparing medicaments for treating and/or preventing diseases caused by angiogenesis; the invention also discloses a pharmaceutical composition for treating diseases caused by angiogenesis, which comprises the polypeptide TIN7N or pharmaceutically acceptable salt of the polypeptide TIN7N and a pharmaceutically acceptable carrier. Compared with the existing medicine for inhibiting angiogenesis, the medicine has the following advantages: (1) the polypeptide TIN7N is 16 amino acids long, and has the advantages of easy synthesis and low cost; (2) the polypeptide TIN7N has remarkable effect of inhibiting angiogenesis; (3) the polypeptide TIN7N has good solubility in water and can be easily administered by injection.

Description

Polypeptide TIN7N for inhibiting angiogenesis and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a polypeptide TIN7N for inhibiting angiogenesis and application thereof.

Background

Angiogenesis refers to the formation, development and growth of new blood vessels. In general, angiogenesis is tightly regulated, primarily by a delicate balance between factors that induce angiogenesis (e.g., vascular endothelial growth factor, VEGF, etc.) and inhibitory factors. When this balance is broken, pathological angiogenesis is usually caused. Angiogenesis is a dynamic, multi-step process, and various diseases are now known to be associated with angiogenesis disorders, such as cancer, ocular vascular abnormalities, infections, cardiovascular diseases and injuries, so that it is of great interest to study angiogenesis inhibitors. The main processes of angiogenesis include degradation of vascular basement membrane, activation, proliferation and migration of vascular endothelial cells, and remodeling of new blood vessels and vascular networks on the basis of the original blood vessels in a budding manner, which is mainly induced by soluble angiogenesis stimulating factors such as VEGF.

The formation of new blood capillaries is a key component in the development and spread of many diseases, including tumors, inflammatory diseases, and some ocular syndromes. The neovascular eye disease is an eye intractable disease and also an important cause of blindness of many eye diseases, such as neovascular corneal disease, retinal disease, iris disease, choroidal disease or vitreous disease, eye trauma and the like. Age-related macular degeneration (AMD) is an age-related disease in which irreversible vision loss or loss is caused by degeneration of the retinal pigment epithelium and the neural retina. There are about 3000 million patients with AMD worldwide, and about 50 million people per year are blinding. With the increasing economic development and aging of population in China, the incidence of AMD in China tends to increase year by year, and more than 500 million AMD patients in China become the third leading cause of blindness in China. AMD is a chronic progressive disease of the retina in the central region, and is clinically divided into two major categories, the collapsed (dry) and exudative (wet): the collapsed form of AMD often manifests as progressive loss of vision in both eyes, with pigmentary disturbances in the macular area, atrophy of choroidal capillaries and the formation of drusen. The exudative type is mainly characterized by the formation of Choroidal Neovascularization (CNV) and a series of pathological changes caused by exudation, hemorrhage, organization, scar and the like. 10% of AMD is neovascular leak (i.e., wet), but 90% of its blindness is caused. In recent years, basic and clinical studies on angiogenesis have been advanced, and in angiogenesis-related diseases such as neovascular corneal diseases, retinal diseases, iris diseases, choroidal diseases, vitreous diseases, and ocular trauma, the effects of controlling the development of diseases and improving clinical symptoms can be achieved by reducing the formation of new blood vessels.

The growth and metastasis of tumors depend on angiogenesis, which is one of the essential conditions for the growth, proliferation and metastasis of tumor cells. In the absence of angiogenesis, the tumor cell mass receives sufficient nutrients and oxygen from the surrounding environment in a diffuse manner, but the tumor volume rarely exceeds 1mm³～2mm³. In 1971, Folkman first proposed that "tumor growth and metastasis are vascular-dependent, and blocking tumor angiogenesis is an effective strategy for suppressing tumor growth", and studies conducted on the basis of this theory led anti-angiogenesis drugs to enter the clinic and to achieve better efficacy in tumor therapy.

At present, the therapeutic target of angiogenesis-related diseases is mainly VEGF, and common therapeutic drugs are mainly anti-VEGF antibodies (such as bevacizumab) or fusion proteins, chemotherapeutic drugs and the like. However, these drugs are expensive, and there is still a great demand for novel angiogenesis-inhibiting drugs in the medical field. The polypeptide is a candidate with great potential in the field of biological pharmacy, and has the advantages of simple structure, low immunogenicity, easy synthesis, low cost and the like. Therefore, the polypeptide capable of inhibiting angiogenesis has wide application prospect in the aspects of treating cancers, ocular vascular abnormalities, infections, cardiovascular diseases, injuries and the like.

Disclosure of Invention

Based on the above problems, the present invention aims to overcome the defects of the prior art and provide a polypeptide capable of inhibiting angiogenesis, which can be used for treating diseases related to angiogenesis.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following aspects:

in a first aspect, the invention provides a polypeptide TIN7N, the amino acid sequence of the polypeptide TIN7N being an amino acid sequence having sequence identity above 13/16 compared to SEQ ID No. 1. Preferably, the amino acid sequence of the polypeptide TIN7N is an amino acid sequence having 14/16 or 15/16 sequence identity compared to SEQ ID No. 1. It should be noted that the amino acid sequence of the polypeptide TIN7N of the present invention includes, but is not limited to, the amino acid sequence having sequence identity of 13/16 or more compared to SEQ ID No.1, and may also be the amino acid sequence having sequence identity of 9/16 to 12/16 compared to SEQ ID No.1, as long as the polypeptide has the function of inhibiting angiogenesis, and all of them fall within the scope of the present invention.

Preferably, the amino acid sequence of the polypeptide TIN7N is shown in SEQ ID NO. 1.

In a second aspect, the present invention provides the use of the polypeptide TIN7N as described above or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and/or prevention of a disease caused by angiogenesis.

Preferably, the salt is an acetate, hydrochloride, hydrobromide, sulphate, phosphate, nitrate, oxalate or tartrate salt. More preferably, the salt is an acetate salt.

Preferably, the disease caused by angiogenesis is an eye disease or a tumor.

Preferably, the ocular disease is neovascular corneal disease, retinal disease, iris disease, choroidal disease or vitreous disease. More preferably, the retinal disease is diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity or retinal artery occlusion. More preferably, the choroidal disease is wet age-related macular degeneration.

Preferably, the tumor is a primary tumor or a secondary tumor.

In a third aspect, the present invention provides a pharmaceutical composition for treating diseases caused by angiogenesis, the pharmaceutical composition comprising the polypeptide TIN7N as described above, or a pharmaceutically acceptable salt of the polypeptide TIN7N, and a pharmaceutically acceptable carrier. The disease caused by angiogenesis is an ocular disease or a tumor; preferably, the ocular disease is neovascular corneal disease, retinal disease, iris disease, choroidal disease, or vitreous disease; more preferably, the retinal disease is diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity or retinal artery occlusion; more preferably, the choroidal disease is wet age-related macular degeneration; preferably, the tumor is a primary tumor or a secondary tumor.

Preferably, the carrier is water or a colloidal solution.

Preferably, the colloidal solution is a hyaluronic acid gel.

Preferably, the pharmaceutical composition is administered by injection; more preferably, the administration by injection comprises intravitreal injection, subcutaneous injection, or intravenous injection.

Compared with the existing drugs for inhibiting angiogenesis, the polypeptide TIN7N has the following advantages:

(1) the polypeptide TIN7N is 16 amino acids long, and has the advantages of easy synthesis and low cost;

(2) in vitro experiments of cells, the polypeptide TIN7N can inhibit the angiogenesis and migration of human umbilical vein endothelial cells (HUVEC cells), and in a zebra fish angiogenesis model, the polypeptide TIN7N can inhibit the formation of eye neovascularization, so that the polypeptide TIN7N has a remarkable angiogenesis inhibiting effect;

(3) the polypeptide TIN7N has good solubility in water and can be easily administered by injection.

Drawings

FIG. 1 is a photomicrograph of the inhibition of human HUVEC cell vascularization by PBS, TIN7N (100. mu.g/ml), bevacizumab (100. mu.g/ml);

FIG. 2 is a bar graph of the results of PBS, TIN7N, bevacizumab inhibiting angiogenesis in human HUVEC cells;

FIG. 3 is a photograph of a Transwell showing that human HUVEC cells were inhibited by PBS, TIN7N (100. mu.g/ml), and bevacizumab (100. mu.g/ml);

FIG. 4 is a Transwell bar graph of PBS, TIN7N, bevacizumab inhibiting human HUVEC cells;

FIG. 5 is a photograph of ocular vessels of zebrafish treated with normal saline, TIN7N (1 ng/tail), bevacizumab (250 ng/tail) for ocular vascular proliferation model;

FIG. 6 is a statistical result diagram of the area of the blood vessels of the eyes of a zebra fish eye vascular proliferation model treated by physiological saline, TIN7N and bevacizumab;

fig. 7 is a pathological image of a zebrafish eye vascular proliferation model treated by physiological saline, TIN7N and bevacizumab.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. It should be noted that many changes and modifications can be made by those skilled in the art without departing from the inventive concept, which falls within the scope of the invention. Unless otherwise specified, the experimental methods in this application are all conventional methods. Unless otherwise specified, the concentrations of the reagents in the present application are mass concentrations.

EXAMPLE 1 Synthesis of the polypeptide TIN7N

The polypeptide TIN7N (SEQ ID NO:1) is synthesized by a conventional solid phase process, and the purity of the synthesized peptide is more than 98%.

Example 2 human umbilical vein endothelial cell (HUVEC cells) vascularization

Human umbilical vein endothelial cells (HUVEC, Human umbilical vein endovenous cells) are primarily cultured, 3 mu g/ml, 10 mu g/ml, 30 mu g/ml, 100 mu g/ml, 300 mu g/ml and 1000 mu g/ml of TIN7N are added for treatment, the cells are cultured in Matrigel, after 4 to 6 hours, the angiogenesis condition is observed and counted, PBS is used as a negative control, and 100 mu g/ml of bevacizumab is used as a positive control.

The results are shown in fig. 1 and 2, from which it can be seen that TIN7N has a significant inhibitory effect on HUVEC vascularization compared to the negative control PBS (.: p < 0.05;: p <0.01) and is dose-effect significant.

Example 3 Transwell assay for cell migration ability

The cells treated with different drugs in example 2 were collected and counted at 1X 10⁵The individual cells were resuspended in serum-free medium and added to the upper chamber of a Transwell cell culture plate, and 600. mu.l of complete medium was added to the lower chamber. 5% CO at 37 ℃₂After incubation for 12-48 hours in the environment, taking out the chamber, wiping the cells in the upper chamber with a cotton swab, fixing the cells for 20mins with 4% paraformaldehyde, washing the cells once with PBS, dyeing the cells for 10mins with crystal violet, washing the cells once with PBS, observing whether the cells pass through the small holes under a microscope, stopping other experimental groups if the cells pass through the small holes, and photographing for statistics.

As can be seen from fig. 3 and 4, TIN7N inhibited HUVEC migration in a dose-dependent manner (×: p < 0.01).

Example 4 Effect on Zebra Fish eye vascular proliferation model

Treating Fli-1 strain transgenic vascular fluorescent zebra fish 1 day (1dpf) after fertilization by using cobalt chloride hexahydrate, and establishing a zebra fish eye vascular proliferation model. The eye vascular proliferation model zebra fish is randomly divided into 11 groups, 30 tails of each group are placed in a six-well plate, TIN7N (the dose is 1, 3, 10 and 30 ng/tail dose) and a positive control bevacizumab 250 ng/tail are injected separately, and a normal control group (normal zebra fish, physiological saline injection) and a model control group (physiological saline injection) are set at the same time. After incubation in an incubator at 28 ℃ for 5 days, 10 zebra fish in each group were randomly picked up, observed in a fluorescence microscope for eye blood vessels, photographed and stored. Image analysis is carried out by using advanced image processing software Nikon NIS-Elements D3.10, the blood vessel area (S) of the eyes of the zebra fish is calculated, and the inhibition effect of TIN7N on the cobalt chloride-induced zebra fish eye blood vessel proliferation is respectively evaluated according to the statistical significance of the blood vessel area of the eyes. The statistical treatment result is expressed by mean + -SE, and the inhibition effect on the zebra fish eye vascular proliferation is calculated according to the following formula:

statistical analysis was performed using analysis of variance and Dunnett's T-test, with p <0.05 indicating significant differences.

Another 10 zebra fish were taken from each test group, eye tissues were fixed with 4% paraformaldehyde, and H & E stained sections were obtained by dehydration, embedding, sectioning and staining for histopathological examination.

The fluorescence observation results are shown in fig. 5 and fig. 6, the positive control bevacizumab group had a zebra fish eye blood vessel area significantly smaller than that of the model control group (p <0.001), and had an inhibitory effect on eye blood vessel proliferation of 94%, indicating that bevacizumab had a significant inhibitory effect on zebra fish eye blood vessel proliferation. The inhibition effect of TIN7N on ocular angiogenesis at doses of 1, 3, 10 and 30 ng/tail was 91%, 48%, 58% and 57%, respectively, which were significantly different from the model control group (p < 0.001). The results show that under the condition of being far lower than the concentration of bevacizumab (250 ng/tail), TIN7N has a remarkable inhibiting effect on the ocular vascular proliferation of zebra fish.

The pathological result is shown in fig. 7, the pathological examination of zebra fish eye tissues in the model control group shows that the pigment epithelium layer is thinner than that in the normal control group, and the structures of three layers of retinas of the pigment epithelium layer, the vision cone layer and the outer nuclear layer are fuzzy and disordered, which indicates that the model is successfully established. The positive control medicament bevacizumab group zebra fish eye histopathology examination shows that the pigment epithelial layer thickness returns to normal, and the pigment epithelial layer, the visual cone layer and the outer nuclear layer three-layer retina structure are clear, which shows that bevacizumab has obvious improvement effect on the zebra fish retina structure of ocular vascular hyperplasia. The histopathological examination of the eyes of the zebra fish with the TIN7N of the dose of 1 ng/tail shows that the thickness of the pigment epithelium layer is recovered to be normal, the structures of the three layers of the retinas of the pigment epithelium layer, the visual cone layer and the outer nuclear layer are clear, and the histopathological examination of the eyes of the zebra fish with the TIN7N of the dose of 3 ng/tail shows that the thickness of the pigment epithelium layer and the structures of the three layers of the pigment epithelium layer, the visual cone layer and the outer nuclear layer are recovered compared with the model control group, which indicates that the TIN7N has obvious improvement on the damaged retinas.

The results of the above examples show that the polypeptide TIN7N provided by the present invention can inhibit the angiogenesis and migration of HUVEC cells in vitro, and can inhibit the formation of ocular neovasculature in a zebra fish angiogenesis model, therefore, the polypeptide TIN7N has a significant effect of inhibiting angiogenesis, can be used for treating angiogenesis-related diseases, such as ocular diseases or tumors, etc., and has a very significant clinical application value.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Guangzhou Zhicheng medical science and technology Limited

<120> polypeptide TIN7N for inhibiting angiogenesis and application thereof

<130> 2019

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> PRT

<213> Artificial sequence

<400> 1

Gly Glu Ser Leu Thr Ile Asn Cys Val Phe Thr Asp Ser Ser Cys Gly

1 5 10 15

Claims

1. The polypeptide TIN7N, wherein the amino acid sequence of the polypeptide TIN7N is shown as SEQ ID NO. 1.

2. Use of the polypeptide TIN7N or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a medicament for the treatment and/or prevention of a disease caused by angiogenesis.

3. Use according to claim 2, characterized in that the salt is an acetate, hydrochloride, hydrobromide, sulphate, phosphate, nitrate, oxalate or tartrate salt.

4. Use according to claim 2, characterized in that the salt is an acetate salt.

5. The use according to claim 2, wherein the disease caused by angiogenesis is an ocular disease or a tumor.

6. The use according to claim 5, wherein said ocular disease is neovascular corneal disease, retinal disease, iris disease, choroidal disease or vitreous disease.

7. Use according to claim 6, wherein the retinal disease is diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity or retinal artery occlusion.

8. Use according to claim 6, wherein the choroidal disease is wet age-related macular degeneration.

9. Use according to claim 5, wherein the tumour is a primary tumour or a secondary tumour.

10. A pharmaceutical composition for treating diseases caused by angiogenesis, which comprises the polypeptide TIN7N of claim 1, or a pharmaceutically acceptable salt of the polypeptide TIN7N, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the carrier is water or a colloidal solution.

12. The pharmaceutical composition of claim 11, wherein the colloidal solution is a hyaluronic acid gel.