WO2012000409A1

WO2012000409A1 - Peptides with activity of inhibiting angiogenesis and uses thereof

Info

Publication number: WO2012000409A1
Application number: PCT/CN2011/076348
Authority: WO
Inventors: 许迅; 苏莉; 赵卉
Original assignee: 上海市第一人民医院
Priority date: 2010-06-30
Filing date: 2011-06-25
Publication date: 2012-01-05
Also published as: CN102311485A; CN102311485B

Abstract

Provided are peptides derived from t-PA kringle 2 domain with activity of inhibiting angiogenesis and uses thereof. Also provided are method for the preparation of these peptides and pharmaceutical composition comprising these peptides.

Description

Peptide with inhibition of neovascular activity and application thereof

The present invention relates to the field of biomedicine, and more particularly to a novel polypeptide-polypeptide series having the function of inhibiting angiogenesis. The polypeptide inhibits proliferation, migration, lumen formation and inhibition of chicken embryo chorioallantoic membrane and mouse corneal neovascularization in vitro. The invention also relates to methods of making and using the polypeptides and pharmaceutical compositions comprising the polypeptides. Background technique

Angiogenesis refers to the process of forming new blood vessels by proliferation and migration of vascular endothelial cells on the basis of the original capillary network. Angiogenesis plays an important role in physiological processes such as embryonic development, injury repair, and is also a major cause of many neovascular diseases such as tumor growth and metastasis, proliferative diabetic retinopathy, retinopathy of prematurity, and rheumatoid arthritis. Pathological changes. Therefore, the research and application of neovascular inhibitors are important for some refractory neovascular related diseases.

Tissue-type plasminogen activator (I-I) is a serine protease containing multiple domains that converts inactive plasminogen to serine proteases in vivo. Active plasmin, which is involved in the fibrinolytic process of the body. t-PA consists of a type I fibronectin region, an epidermal growth factor-like region, two kringle domains and a carboxy terminal protein cleavage region.

The Kringle domain is a conserved structure consisting of approximately 80 amino acids and internally consisting of three pairs of disulfide bonds forming a bicyclic conformation. At present, it has been found that the kringle structure contained in various endogenous proteins has an effect of inhibiting neovascularization in vitro and in vivo, and therefore it is considered that the kringle structure may be an independent conserved structure and functional unit having an inhibitory effect on neovascularization. Foreign researchers have found that t-PA kringle l_2 (TKl-2) inhibits proliferation, adhesion and migration of vascular endothelial cells in vitro and inhibits neovascularization in vivo. In addition, t-PA kringle2 has an independent inhibitory effect on proliferation and migration of vascular endothelial cells, and is effective in inhibiting neovascularization in vivo. t-PA kringl e2, a macromolecular protein angiogenesis inhibitor, provides important information for the development of peptide-based neovascular inhibitors with a conserved amino acid sequence or structure that inhibits neovascularization.

When developing effective angiogenesis inhibitors, the specificity of ophthalmic medication should be fully considered.

First, there are multiple anatomical and functional barriers in the eye. The systemic administration is often unable to achieve sufficient drug concentration in the ocular tissue due to the blood-aqueous barrier and the blood-retinal barrier; local administration, such as intravitreal injection, greater than 76.5 kDa is theoretically difficult to penetrate. The retina acts on the retina and choroidal neovascularization. For ocular surface administration, the drug must penetrate the lipophilic corneal epithelial cell tight junction and the hydrophilic corneal stroma, so only the appropriate fat solubility, low molecular weight or energy Drugs that bind to transporters in ocular surface tissues (eg, amino acid transporters, oligopeptide transporters, etc.) can reach the anterior chamber.

Second, the extent to which the drug dissolves in hydrophilic tears, aqueous humor, and vitreous humor is positively correlated with its effectiveness.

Third, based on the above-mentioned main reasons, the bioavailability of ophthalmic drugs is very low; to increase it, the concentration of the drug can be increased. Compounds used to treat neovascularization of tumors are more toxic and side effects, and are not administered at high doses both systemically and locally. In addition, exogenous proteins with large molecular weight are also sensitive foreign sources, which can cause immune damage to ocular tissues such as uvea.

Fourth, although a series of relatively safe endogenous angiogenesis inhibitors have been confirmed, such as angiostatin, it is composed of plasminogen Kringle 1-4 (plasminogen Kringle 1-4). The composition can significantly inhibit the growth of vascular-dependent tumors, but due to its large molecular weight and complex spatial conformation, there are cumbersome recombination and purification processes and endotoxin residues in the preparation process.

Due to the above various conditions, the drugs currently used to treat ocular neovascularization are very limited, such as recombinant anti-human VEGF monoclonal antibody bevac i zumab (Avast in), recombinant anti-human VEGF monoclonal antibody fragment ranibi zumab (Lucent) Is), etc., but they are expensive and need to be administered repeatedly through the vitreous cavity. Risks such as bleeding, infection, and vascular embolism are difficult to avoid; and VEGF monoclonal antibodies block the normalization of VEGF during the process of blocking neovascularization. Physiological function, repeated use can lead to atrophy of retinal nerve tissue.

Compared with the widely studied protein angiogenesis inhibitors, peptide angiogenesis inhibitors have simple synthesis methods, easy chemical modification, low immunogenicity, good solubility, high bioavailability, and strong tissue penetration. The advantages of various medicines and low prices are outstanding advantages. However, there are currently no small molecule polypeptides with satisfactory effects from the t-PA Kringle domain.

Therefore, there is an urgent need in the art to develop an effective and safe small molecule neovascular inhibitor suitable for eye tissue. Summary of the invention

It is an object of the present invention to provide an effective and safe angiogenesis-inhibiting small molecule polypeptide suitable for eye tissue, as well as fragments, analogs and derivatives thereof.

Another object of the invention is to provide a process and use comprising the polypeptide. In a first aspect of the invention, a polypeptide represented by the following formula I, or a pharmaceutically acceptable salt thereof, is provided

[XaaO] - [Xaal]— [Xaa2]— [Xaa3] - [Xaa4] - [Xaa5] - [Xaa6] - [Xaa7] - [Xa a8] - [Xaa9] - [XaalO] - [Xaal 1] - [Xaal2] - [Xaal3] (I) In the formula,

XaaO is no, or 1-3 amino acids constitute a peptide;

Xaal is an amino acid selected from the group consisting of His or Arg;

Xaa2 is an amino acid selected from the group consisting of Val, Thr, Leu, He, Met or Ala;

Xaa3 is an amino acid selected from the group consisting of Leu, He, Val, Met or Ala;

Xaa4 is an amino acid selected from the group consisting of Lys or Arg;

Xaa5 is an amino acid selected from the group consisting of: Asn, Asp or Gin;

Xaa6 is an amino acid selected from the group consisting of Arg or Lys;

Xaa7 is an amino acid selected from the group consisting of Arg, Gin or Lys;

Xaa8 is an amino acid selected from the group consisting of Leu, He, Val, Met or Ala;

Xaa9 is an amino acid selected from the group consisting of Thr or Ser;

XaalO is an amino acid selected from the group consisting of Trp or Tyr;

Xaal l is an amino acid selected from the group consisting of Glu or Asp;

Xaal2 is an amino acid selected from the group consisting of Tyr or Phe;

Xaal3 is no, or 1-3 amino acids constitute a peptide;

And the polypeptide has an activity of inhibiting angiogenesis, and the polypeptide is 12-18 amino acids in length.

In another preferred embodiment, Xaal3 is a peptide consisting of 1-3 amino acids; more preferably, the peptide is CDV, CD, or C.

In another preferred embodiment, XaaO is C or WC.

In another preferred embodiment, the polypeptide is 12-15 amino acids in length.

In another preferred embodiment, the polypeptide is selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 1;

(b) The amino acid sequence represented by SEQ ID NO: 1 is formed by substitution, deletion or addition of 1-5 (preferably 1-3, more preferably 1-2) amino acid residues, and has inhibition A polypeptide derived from (a) angiogenic function.

In another preferred embodiment, the derivative polypeptide retains 70% of the angiogenic activity of the indicated polypeptide of SEQ ID NO: 1.

In another preferred embodiment, the derivative polypeptide is 80% identical to SEQ ID NO: 1, preferably 90%; more preferably 95%.

The invention also provides dimeric and multimeric forms of the compounds of formula I which inhibit angiogenic function. In a second aspect of the invention, there is provided an isolated nucleic acid molecule encoding the above-described polypeptide of the invention.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising:

(a) the above polypeptide of the present invention or a pharmaceutically acceptable salt thereof; (b) a pharmaceutically acceptable carrier or excipient.

In another preferred embodiment, the composition is in the form of eye drops, injections (e.g., periocular and intraocular injections), ophthalmic gels or ophthalmic ointments.

In another preferred embodiment, the composition is a sustained release dosage form.

In a fourth aspect of the invention, there is provided the use of a polypeptide or a pharmaceutically acceptable salt of the invention for the preparation of a medicament for inhibiting angiogenesis or preventing diseases associated with angiogenesis.

In another preferred embodiment, the angiogenesis-related disease is selected from the group consisting of neovascular ophthalmopathy, tumor, ischemic heart disease, non-inflammatory cardiomyopathy, coronary arteriosclerosis, arteriosclerosis obliterans, arteries. Embolism, arterial thrombosis, Berger's disease, chronic inflammation, inflammatory bowel disease, ulcers, rheumatoid arthritis, scleroderma, psoriasis, infertility or sarcoma.

In another preferred embodiment, the neovascular ophthalmopathy comprises involving the choroid, the retina, the cornea or the iris, including age-related macular degeneration, proliferative diabetic retinopathy, retinal vein occlusive disease, retinopathy of prematurity, corneal infection, Neovascular glaucoma and the like.

In a fifth aspect of the invention, there is provided a method of inhibiting angiogenesis in a mammal comprising the steps of: administering to a subject in need thereof a polypeptide of the invention or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the object is a human.

In another preferred embodiment, the angiogenesis is angiogenesis associated with neovascular eye disease. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Limited to the length, no longer one by one. DRAWINGS

The following drawings are used to illustrate the specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the appended claims.

Figure 1 shows a schematic representation of the t-PA Kringle2 domain and the amino acid sequence of each small peptide.

Figure 2 shows that the TKII series polypeptide of the present invention inhibits VEGF-induced vascular endothelial cell proliferation. Among them, VEGF 10ng/ml can significantly induce the proliferation of HUVECs. TK 11-20, TK 11-30, and TK 11-10 had no significant inhibitory effect on VEGF-induced proliferation of HUVECs in the range of 10 μΜ (compared with VEGF group, P) 0.05). TKII-12 can effectively inhibit the proliferation of HUVECs induced by VEGF at concentrations of 100nM, 1 μM and 10 μM, and the inhibitory effect is gradually increased with the increase of TKII-12 concentration. (**P<0.01 compared with VEGF group). The TK II -12S polypeptide does not have the effect of inhibiting the proliferation of HUVECs induced by VEGF.

Figure 3 shows that the indole series polypeptide of the present invention inhibits VEGF-induced vascular endothelial cell migration. Among them, the A picture shows the hematoxylin staining of HUVECs cells in the lower chamber of the Transwel l chamber.

Panel B shows that VEGF 25 ng/ml can significantly induce HUVECs migration. TK 11-20 had no significant inhibitory effect on VEGF-induced HUVECs migration in the concentration range of 10 μM. TK 11-30 at high concentration (10 When μ M), it inhibited the migration of VEGF-induced HUVECs. TK 11-10 inhibited the migration of VEGF-induced HUVECs at a concentration of 1 μM and 10 μ Μ. TK II -12 can effectively inhibit VEGF-induced HUVECs migration at concentrations of 100 nM, 1 μM, and 10 μM, and the inhibitory effect is significantly enhanced with the increase of ΤΚΠ-12 concentration (* compared with VEGF group, corpse < 0.05, ** compared with the VEGF group, 0.01). The TKII-12S polypeptide does not have the effect of inhibiting the migration of VEGF-induced HUVECs.

Figure 4 shows that the ΤΚΠ series polypeptide of the present invention inhibits VEGF-induced vascular endothelial cell lumen formation. Among them, Figure A shows the formation of vascular endothelial cells lumen. The formation of luminal-like structures in the VEGF-free group was less, and the formation of luminal-like structures in the VEGF group was significantly increased, staggered into a network. There was no significant decrease in the formation of the lumen-like structure in the ΤΚΠ-20 group ΙΟ μ Μ concentration compared with the VEGF group. The luminal-like structure was formed in the VEGF-30, TKII-10, and TKII-12 groups at 10 μΜ. The ratio is significantly reduced.

Panel B shows that VEGF 15 ng/ml can significantly induce lumen formation in HUVECs. ΤΚΠ-20 polypeptide had no significant inhibitory effect on VEGF-induced lumen formation of HUVECs. ΤΚΠ-30, TKII-10, and TK II -12 peptides were effective at inhibiting VEGF-induced lumen formation of HUVECs at concentrations of 100 nM, 1 μM, and 10 μM, and the inhibitory effect was significantly enhanced with increasing concentration. ΤΚΠ-12 inhibited vascular endothelial cell formation in vascular endothelial cells more strongly than ΤΚΠ-30 and ΤΚΠ-10 (** compared with VEGF group, P<0.01). TKII-12S polypeptide could not inhibit VEGF-induced lumen formation in HUVECs.

Figure 5 shows that the indole-12 polypeptide of the present invention inhibits neovascularization of the chicken embryo chorioallantoic membrane. Among them, the A picture shows the capillary growth of the chicken chorioallantoic membrane. Capillary chorioallantoic membrane capillaries grew well in a diameter range around the PBS group; in the 10 ng group of ΤΚΠ-12 polypeptide, the number of capillaries in the chicken chorioallantoic membrane was reduced within a diameter range around the filter paper; ΤΚΠ-12 polypeptide 50 ng group, The number of capillaries in the chicken chorioallantoic membrane was significantly reduced within a diameter range around the filter paper, and some avascular regions appeared. TKII-12S polypeptide 50ng group, chicken embryo chorioallantoic capillary growth in a diameter range around the filter paper.

B shows that compared with the PBS group, the number of capillaries in the diameter range of the 10 ng and 50 ng TKlI-12 polypeptide experimental papers is reduced, and the difference is statistically significant; TKII-12S polypeptide 50 ng group chicken embryo allantoic sac There was no significant reduction in the number of membrane capillaries. (** 0.01 compared with the PBS group; # means compared with the 10 ng group, P < 0.05)

Figure 6 shows that the ΤΚΠ-12 polypeptide of the present invention inhibits VEGF-induced corneal neovascularization in mice. Wherein, Figure AD represents implanted VEGF sustained release granule (A), VEGF + TKII-12 polypeptide 1 μg sustained release granule (B), VEGF + TKII-12 polypeptide 5 μg sustained release granule (C) and VEGF + Corneal neovascularization in mice 5 days after TK II - 12S peptide 5 μg sustained-release granules (D). In the VEGF group, the corneal neovascularization was dense and tortuous, and the corneal neovascularization of the VEGF + TKII-12 polypeptide 1 μg group was short and sparse. The VEGF + TKII-12 peptide group 5 showed no obvious large neovascular growth, VEGF + ΤΚΠ -12S polypeptide 5 The corneal neovascularization was vigorous and distorted.

Figure E-H represents VEGF group (E), VEGF + TKII-12 polypeptide 1 μg group (F), VEGF + TKII -12 polypeptide 5 μg group (G) and VEGF + TK II - 12S polypeptide 5 μg Group (H) mouse corneal pathology group Weaving inspection. In the VEGF group, the corneal stroma plate layer is loosely arranged, and a large number of neovascular lumens are present in the stroma, and red blood cells are filled therein. A small amount of neovascular lumen was seen in the corneal stroma of VEGF + TKII-12 polypeptide 1 μg group, and red blood cells were filled. In the VEGF + TKII-12 peptide group 5, no obvious neovascular lumen formation was observed, and there was no obvious edema in the corneal stroma. A large number of neovascular lumens were seen in the corneal stroma of VEGF + TKII-12S peptide 5 g group, and red blood cells were filled therein.

Figure 1 - κ represents the length of corneal neovascularization (1), neovascularization time (J), and neovascular area (K), respectively. Compared with VEGF group, lwg and 5 μ _§ ΤΚΠ-12 polypeptide group cornea The length of the new blood vessels, the number of hours and the area were significantly reduced, and the difference was statistically significant (** P < 0.01 compared with the VEGF group).

Through extensive and intensive research, the present inventors have for the first time prepared a small molecule polypeptide derived from the t-PA Kringle2 domain and having an angiogenesis-inhibiting function with a molecular weight of less than 5 kD (e.g., only about l-3 kD). Specifically, the inventors applied bioinformatics methods, based on homology analysis and biological characteristics analysis, designed several candidate sequences, synthesized by solid phase method, and then passed through chicken embryo chorioallantoic membrane Models, VEGF-induced endothelial cell proliferation, migration, lumen formation models, and mouse corneal neovascularization screening have resulted in a new class of small molecule polypeptides with prophylactic and therapeutic angiogenic functions.

The small peptide of the invention has small molecular weight and can penetrate various eye tissue barriers; has good water solubility and can maintain high concentration in neutral tears, aqueous humor and vitreous humor; high safety and low toxicity to biological tissues Eye bioavailability is high, which reduces the dose and reduces systemic side effects. On the basis of this, the present invention has been completed. Active polypeptide

In the present invention, the terms "polypeptide of the present invention", "ΤΚΠ-12 polypeptide", "ΤΚΠ-12 small peptide", "short peptide ΤΚΠ-12" or "peptide ΤΚΠ-12" are used interchangeably and refer to having blood vessels. A protein or polypeptide of a novel inhibitory activity peptide ΤΚΠ-12 amino acid sequence (SEQ ID NO: 1). Furthermore, the term also includes variant forms of the sequence of SEQ ID NO: 1 which have an angiogenic function. These variants include (but are not limited to): 1-5 (usually 1-4, preferably 1-3, more preferably 1-2, optimally 1) amino acid deletions, insertions And/or substitution, and addition or deletion of one or several (usually within 5, preferably within 3, more preferably within 2) amino acids at the C-terminus and/or N-terminus. For example, in the art, when substituted with amino acids of similar or similar properties, the function of the protein is generally not altered. As another example, the addition or deletion of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the structure and function of the protein. Furthermore, the term also encompasses polypeptides of the invention in both monomeric and multimeric forms. The term also includes both linear as well as non-linear polypeptides (such as cyclic peptides).

The invention also encompasses active fragments, derivatives and analogs of the ΤΚΠ-12 polypeptide. As used herein, The terms "fragment,""derivative," and "analog" refer to a polypeptide that substantially retains angiogenic function or activity. A polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusing a TKI I-12 polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) The amino acid sequence is fused to the polypeptide sequence to form a polypeptide (a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6Hi s). These fragments, derivatives and analogs are within the purview of those skilled in the art in light of the teachings herein.

A preferred class of reactive derivatives means that up to 5, preferably up to 3, more preferably up to 2, and optimally 1 amino acid are similar or similar amino acids to the amino acid sequence of Formula I. Substituting to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table I. A particularly preferred sequence of the derived polypeptide is shown in SE: 1 and in the examples.

The invention also provides analogs of the TKII-12 polypeptide. The difference between these analogs and the natural ΤΚΠ-12 polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof. Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, Υ-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

Modifications (usually without altering the primary structure) include: chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.

The polypeptide of the present invention can also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base. These salts include, but are not limited to, salts formed with: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, malay Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid. Other salts include: salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional "prodrugs". Coding sequence

The invention also relates to polynucleotides encoding a -12 polypeptide. A preferred coding sequence is cacgtgctgaagaaccgcaggctgacgtgggagtac (SEQ ID NO: 6), which encodes the amino acid sequence set forth in SEQ ID NO: 1.

The polynucleotide of the present invention may be in the form of sputum or RNA. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 6 or may be a degenerate variant. As used herein, as SEQ ID NO: 1, "degenerate variant" in the present invention refers to a polypeptide having the sequence of SEQ ID NO: 1, but with the corresponding coding region sequence of SEQ ID NO: 6. Differential nucleic acid sequences.

The full-length 12-nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombination method or a synthetic method. At present, it has been possible to obtain a DNA sequence encoding the polypeptide of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis. The DNA sequence can then be introduced into various existing purine molecules (e.g., vectors) and cells known in the art.

The invention also relates to vectors comprising the polynucleotides of the invention, and to host cells genetically engineered using the vector of the invention or the ΤΚΠ-12 polypeptide coding sequence.

In another aspect, the invention also includes a polypeptide encoding a ΤΚΠ-12 DNA or a fragment thereof. Heterogenic polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies. Preparation

The polypeptide of the invention may be a recombinant polypeptide or a synthetic polypeptide. The polypeptides of the invention may be chemically synthesized, or recombinant. Accordingly, the polypeptide of the present invention can be artificially synthesized by a conventional method or can be produced by a recombinant method.

A preferred method is to use liquid phase synthesis techniques or solid phase synthesis techniques such as Boc solid phase method, Fmoc solid phase method or a combination of both methods. The solid phase synthesis can quickly obtain samples, and the appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the peptide of interest. For example, a preferred solid phase support in the Fmoc system is a Wang resin linked to a C-terminal amino acid in the peptide, a Wang resin structure is polystyrene, and an arm between the amino acids is 4-decyloxybenzyl alcohol; using 25% hexahydropyridine /dimethylformamide was treated at room temperature for 20 minutes to remove the Fmoc protecting group and extended from the C-terminus to the N-terminus according to the given amino acid sequence. After the completion of the synthesis, the synthesized related peptide was cleaved from the resin with trifluoroacetic acid containing 4% p-methylphenol, and the protecting group was removed. The resin was removed by filtration and diethyl ether was precipitated to obtain a crude peptide. After the solution of the obtained product was lyophilized, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. When solid phase synthesis is carried out using the Boc system, it is preferred that the resin is a PAM resin to which a C-terminal amino acid in the peptide is attached, the PAM resin structure is polystyrene, and the arm between the amino acid is 4-hydroxymethyl phenylacetamide; In the system, the protecting group Boc was removed with TFA/dichloromethane (DCM) in a deprotection, neutralization, coupling cycle and neutralized with diisopropylethylamine (DIEA) / dichloromethane. After completion of the condensation of the peptide chain, the peptide chain was cleaved from the resin by treatment with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0 ° C for 1 hour while removing the protecting group. The peptide was extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), and the solution was lyophilized and further purified by molecular sieve S-print hadd G10 or Tsk-40f, and then purified by high pressure liquid phase to obtain the desired peptide. Each of the amino acid residues can be coupled using various coupling agents and coupling methods known in the art of peptide chemistry, for example, dicyclohexylcarbodiimide (DCC), hydroxybenzotriazole (HOBt) or 1 can be used. 1,3,3-tetraurea hexafluorophosphate (HBTU) was directly coupled. For the synthesized short peptide, the purity and structure can be confirmed by reversed phase high performance liquid chromatography and mass spectrometry.

In a preferred embodiment, the polypeptide of the present invention 12 is prepared by solid phase synthesis according to the sequence thereof, and purified by high performance liquid chromatography to obtain a high-purity peptide freeze-dried powder, which is stored in -2 CTC.

Another method is to produce a polypeptide of the invention using recombinant techniques. The polynucleotide of the present invention can be used to express or produce a recombinant -12 polypeptide by conventional recombinant DNA techniques. Generally there are the following steps:

(1) using a polynucleotide (or variant) encoding a ΤΚΠ-12 polypeptide of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

(2) cultivating the host cell in a suitable medium;

(3) Separating and purifying the recombinant polypeptide from the culture medium or the cells. The recombinant polypeptide can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant polypeptide can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Since the polypeptide of the present invention is short, it is conceivable to connect a plurality of polypeptides in series, to obtain an expression product in a multimeric form after recombinant expression, and then to form a desired small peptide by enzymatic cleavage or the like. Pharmaceutical composition and method of administration

In another aspect, the present invention provides a pharmaceutical composition comprising (a) a safe and effective amount of a polypeptide of the present invention or a pharmaceutically acceptable salt thereof; and (b) a pharmaceutically acceptable carrier or excipient . The amount of the polypeptide of the present invention is usually from 10 μg to 100 mg / dose, preferably from 100 to 1000 μg / dose.

For the purposes of the present invention, an effective dose is from about 0.01 mg/kg to 50 mg/kg, preferably from 0.05 mg/kg to 10 mg/kg body weight of the polypeptide of the invention. In addition, the polypeptides of the invention may be used alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent. The term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Scences (Mack Pub. Co., N. J. 1991). Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.

The pharmaceutically acceptable carrier in the therapeutic composition may contain a liquid such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

In general, the therapeutic compositions can be formulated as injectables, such as liquid solutions or suspensions; solid forms suitable for liquid carrier preparation prior to injection.

Once formulated into a composition of the invention, it can be administered by conventional routes including, but not limited to, ocular, periocular, intraocular, intramuscular, intravenous, subcutaneous, intradermal or topical administration. . The subject to be prevented or treated may be an animal; especially a human.

When the pharmaceutical composition of the present invention is used for actual treatment, a pharmaceutical composition of various dosage forms may be employed depending on the use. Preferably, eye drops, injections, ophthalmic gels and eye ointments are exemplified. These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrating agents, binders, lubricants, diluents, buffers, isotonicity (i sotoni c it i es preservatives, wetting agents, emulsifiers, dispersants, stabilizers and co-solvents, and the formulation process can be carried out in a customary manner depending on the dosage form.

For example, the preparation of eye drops can be carried out by dissolving short peptide ΤΚΠ-12 or a pharmaceutically acceptable salt thereof together with a basic substance in sterile water (a surfactant is dissolved in sterile water) to adjust the osmotic pressure and The pH is in a physiological state, and a suitable pharmaceutical additive such as a preservative, a stabilizer, a buffer, an isotonic agent, an antioxidant, and a tackifier may be optionally added, and then completely dissolved.

The pharmaceutical compositions of the invention may also be administered in the form of sustained release agents. For example, the short peptide -12 or its salt can be plunged into a pill or microcapsule in which the sustained release polymer is used as a carrier, and then the pill or microcapsule is implanted into the tissue to be treated by surgery or injection. Further, the short peptide ΤΚΠ-12 or a salt thereof can also be applied by inserting a drug-coated intraocular lens. Examples of the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydroxymethacrylate (polyhydrometaacrylate polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, A lactic acid-glycolic acid copolymer or the like is preferably exemplified by a biodegradable polymer such as a lactic acid polymer and a lactic acid-glycolic acid copolymer.

When the pharmaceutical composition of the present invention is used for actual treatment, the dose of the short peptide 12 or its pharmaceutically acceptable salt as an active ingredient may be according to the weight, age, sex of each patient to be treated. , the degree of symptoms is reasonably determined. For example, when the eye drops are partially instilled, the concentration is usually about 0.1 to 10% by weight, preferably 1 to 5% by weight, and may be administered 2 to 6 times per day, 1-2 drops each time. Industrial applicability

A pharmaceutical composition containing the polypeptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient has a remarkable inhibitory activity against angiogenesis. It has been confirmed by in vitro and in vivo experiments that the polypeptide of the present invention can inhibit not only the proliferation, migration, and lumen formation of human umbilical vein endothelial cells, but also the angiogenesis of the chicken chorioallantoic membrane and the corneal neovascularization in mice. The main advantages of the invention include:

(a) the polypeptide of the present invention has a small molecular weight and is permeable to the ocular tissue barrier;

(b) Good water solubility, maintaining a high concentration in neutral tears, aqueous humor and vitreous humor;

(c) high safety, low toxic side effects on biological tissues; and high bioavailability of topical application of the eye, which can reduce the dose, thereby reducing systemic side effects;

(d) can be prepared by solid phase synthesis, with high purity, high yield and low cost;

(e) The polypeptide of the present invention has good stability.

Therefore, the polypeptide of the present invention is expected to be developed into a medicament for the treatment of neovascular eye diseases and related neovascular diseases such as tumor neovascularization. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions. Example 1

Peptide synthesis

Based on the amino acid sequence of human t-PA kringle2, t-PA kringle2 was divided into four peptides according to the t-PA kringle2 spatial conformation, and the disulfide bond was used as the boundary. They were named as ΤΚΠ-20, ΤΚΙΙ-30, and ΤΚΠ. - 10, ΤΚΠ-12, located in the amino acid sequence of t-PA, 215~235, 237~266, 268~277, 279~290, molecular weights are 2062.16, 3285·84, 1155.24 , 1614.88.

The four ΤΚΠ polypeptides shown in SEQ ID NOS: 1-4 were synthesized using a commercially available SYMI 3⁄40NY polypeptide synthesizer (Fig. 1).

TK 11-12: HVLKNRRLTWEY (SEQ ID NO: 1)

TK II -10: RNPDGDAKPW (SEQ ID NO: 2)

TK 11-20: YFGNGSAYRGTHSLTESGAS (SEQ ID NO: 3)

TK 11-30: LPWNSMILIGKVYTAQNPSAQALGLGKHNY (SEQ ID NO: 4)

Furthermore, in order to further confirm the correlation between the inhibitory activity and the sequence, a control polypeptide TKII-12S was synthesized based on the amino acid composition of the ΤΚΠ-12 polypeptide: KRYLTHNVRWLE (SEQ ID NO: 5)

The procedure is as follows: The desired Fmoc protected amino acid solution, condensation reagent and cleavage reagent were calculated and prepared according to the software (Version.201 version) using a SYMPHONY type 12-channel polypeptide synthesizer (American Protein Technologies). Editing procedure, in which the resin swelling time is 30 min; deprotection twice, the time is 5 min and 15 min respectively; the condensation time is 30 min; the cutting time is 2 h. The peptide was synthesized according to the above procedure, and the polypeptide was purified by high performance liquid chromatography (SHIMADZU) to obtain a white powdery polypeptide having a purity of >95% (120 mg each of each polypeptide), and lyophilized for use.

The human t-PA kringle2 consists of 82 amino acid residues and is located in positions 215 to 296 of the t-PA amino acid sequence. It contains six cysteines, forming a three-pair disulfide bond, which constitutes a bicyclic conformation. Example 2 Inhibition of VEGF-induced proliferation of vascular endothelial cells by a series of peptides

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

Primary HUVECs (purchased from ScienCell) using ECCM medium to add ECGS (ScienCell) Division) and 5% fetal bovine serum (ScienCell), cultured in an incubator containing 5% CO ₂ at 37 °C. The third to eighth generation HUVECs cells were used in all in vitro cell experiments in the present invention.

(2) Detection by MTS method ΤΚΠ series of peptides inhibit VEGF-induced proliferation of HUVECs

The MTS cell proliferation quantitative detection method is a method for producing a living cell proliferation by colorimetric determination of a water-soluble colored product by using a tetrazolium and an electron-conjugated compound under the action of a metabolically proliferating cell mitochondrial dehydrogenase.

The specific method is as follows: After the HUVECs grow close to the fusion, pass through, and inoculate the 96-well plate at a density of 3.5×107 ml, and culture for 24 hours in a 37°C, 5%C0 ₂ incubator for each well, and replace the serum-free ECM culture. Base, cells starved overnight. The 96-well plate medium was aspirated, and each group was added with 50 μL of serum-free medium containing 1 Μ, 10 nM, 100 Μ Μ, 1 μ Μ, 10 μ Μ ΤΚ Π series of peptide drugs, pretreated at 37 ° C for 30 min. Thereafter, serum-free medium containing VEGF (R&D) was added to each well to give a final concentration of VEGF of 10 ng/ml. A blank control group (no VEGF-free peptide group) and a VEGF control group (no TK II polypeptide group) were set, and 5 parallel wells were set for each experimental group. 37, 5%C0 ₂ incubator for 24h, add 20 μ LMTS solution (Promega) to each well, and act at 37 °C for l~4h. value.

Results: Compared with the blank control group (no VEGF non-purine peptide group), the 0D value of each hole in the VEGF group increased significantly, and the difference was statistically significant, 0.01, indicating that 10 ng/ml VEGF could effectively stimulate the proliferation of HUVECs.

Compared with the VEGF group, the TK 11-20, TK 11-30, and ΤΚΠ-10 polypeptides showed no significant change in the 0D value of each well in the concentration range of 1ηΜ~10 μ M. The difference was not statistically significant, Ρ>0.05 In the TKII-12 group, 100 Μ, 1 μ Μ, 10 μ Μ, the 0D value of each well was significantly decreased, and the difference was statistically significant, 0.01), indicating that the ΤΚΠ-12 polypeptide was at a concentration of 100 nM, 1 μM, 10 μM. It can effectively inhibit the proliferation of HUVECs induced by VEGF, and the inhibitory effect gradually increases with the increase of the concentration of TK II -12 polypeptide.

Compared with VEGF group, ΤΚΠ - 12 peptide 1 μ Μ, 10 μ Μ group inhibited the proliferation of HUVECs more than

50%, thus suggesting that the 有效-12 polypeptide inhibits VEGF-induced HUVECs proliferation by a half effective amount (ED ₅ ) between 100 nM and 1 μM (about 0.16 mg/L to L 6 mg/L) (Fig. 2).

Studies have shown that the half-effective amount of a wide range of protein-based angiogenesis inhibitor Angiostatin inhibits vascular endothelial cell proliferation is about 80 nM (about 3.04 mg / L), because its molecular weight is large, so its mass concentration is much larger than the polypeptide of the present invention, indicating The polypeptide of the present invention achieves the same inhibitory effect as Angiostatin at a lower concentration, and embodies the advantages of the polypeptide angiogenesis inhibitor. Compared with the VEGF group, the random sequence TKII-12S polypeptide which disrupted the amino acid sequence synthesis of the ΤΚΠ-12 polypeptide had no significant change in the 0D value of each well within the range of 1ηΜ~10 μΜ, and the difference was not statistically significant. >0.05), indicating that ΤΚΠ-12 polypeptide inhibits the proliferation of HUVECs induced by VEGF in a sequence-dependent manner.

Conclusion: ΤΚΠ-12 polypeptide can effectively inhibit VEGF-induced proliferation of vascular endothelial cells, and There is good dose dependence and sequence dependence. ΤΚΠ-20, ΤΚΠ-30, and ΤΚΠ-10 polypeptides did not significantly inhibit VEGF-induced vascular endothelial cell proliferation. Example 3 ΤΚΠ series polypeptide inhibits VEGF-induced vascular endothelial cell migration

The vascular endothelial cell migration experiment was carried out by Transwell chamber (Corning) method. The specific method was as follows: After the HUVECs cells grew close to the fusion, the serum-free medium was starved overnight, and 0.25% trypsin was digested to prepare a cell suspension. The ΤΚΠ series polypeptide was mixed with a cell suspension containing ⁴ ×10 ⁵ HUVECs to prepare an upper chamber liquid having a volume of Ιθθμΐ^ ΤΚΠ, and the polypeptide concentrations were 1ηΜ, 10 ηΜ, 100 ηΜ, 1μΜ, ΙΟμΜ, 37°C incubator After pretreatment for 30 min, it was added to the upper chamber. A serum-free medium containing 25 ng/ml VEGF was added to the lower chamber as a chemokine. The Tranwell chamber was further cultured for 24 hours in a 37 ° C incubator, and the Transwell upper chamber was removed. The cotton swab was wiped off the polycarbonate membrane. The cells that had not migrated were stained with hematoxylin. The number of cells that migrated to the lower surface of the polycarbonate membrane was observed under a microscope. Five fields of view were taken from each cell membrane, and the average number of cells in the five fields was compared.

Results: Under the same conditions, the blank control group (no VEGF-free peptide group) had an average number of migrated cells per field of 8.6±4.3; in the VEGF group (no sputum polypeptide group), the average number of migrated cells per field was 49. 17± 12.43, the difference was statistically significant (Z6 ^ method, corpse <0.01), indicating that 25 ng / ml VEGF can effectively induce HUVECs migration.

The average number of migrated cells per field of ΤΚΠ-20 peptide 1ηΜ, 10ηΜ, 100ηΜ, 1μΜ, 10μΜ groups was 50.00±7.58, 50.60±4.83, 52.00±7.11, 49.20±6.80, 50.40±6.31, respectively. There was no significant decrease in the number of HUVECs cell migration compared with the group, and the difference was not statistically significant (biliary, Α0·05).

The average number of migrated cells per field of ΤΚΠ-30 peptide 1ηΜ, 10ηΜ, 100ηΜ, 1μΜ, 10μΜ groups was 49.40±4.51, 50.00 + 8.19, 49.00±7.28, 43.00±3.39, 36.00±2.55, respectively. Compared with the VEGF group, the migration of HUVECs in the 10μΜ concentration group was significantly reduced, and the difference was statistically significant (Z6^ method, P<0.05).

The average number of migrated cells per field of ΤΚΠ-10 peptide 1ηΜ, 10ηΜ, 100ηΜ, 1μΜ, 10μΜ group was 47.60±3.72, 49.40±5.73, 49.60±5.18, 38.80±3.27, 22.60±2.70, ΤΚΠ-10 polypeptide. Compared with the VEGF group, the migration of HUVECs was significantly reduced in the ΙμΜ and 10μΜ concentrations, the difference was statistically significant, P<0.05)

The average number of migrated cells per field of ΤΚΠ-12 peptide 1ηΜ, 10ηΜ, 100ηΜ, 1μΜ, 10μΜ groups was 50.80±9.26, 49.30±15.31, 33.00±7.85, 23.70±6.08, 14.56±3.68, ΤΚΠ-12 polypeptideΙΟΟηΜ, Compared with the VEGF group, the migration of HUVECs was significantly reduced in the ΙμΜ and 10μΜ concentrations, the difference was statistically significant, P<0.05)

Compared with the VEGF group, TKII-12S polypeptide 10μΜ groups no significant changes in the number of migrating cells (49.80 5.22 persons), the difference was not statistically significant (Ζ6 ^ method, P> 0.05) _a The above results indicate that: ΤΚΠ -30, ΤΚ II -10, ΤΚΠ-12 polypeptide can inhibit VEGF-induced HUVECs migration, in which ΤΚΠ-12 polypeptide has a significant inhibition of cell migration at a lower concentration (ΙΟΟηΜ), and with ΤΚΠ- 12 The increase in the concentration of the polypeptide inhibited the migration. Compared with the VEGF group, the Μ-12 polypeptide 1μΜ and ΙΟμΜ groups inhibited the migration of HUVECs by more than 50%, suggesting that the half effective amount of TK II - 12 polypeptide to inhibit VEGF-induced HUVEC s migration is between 10 OnM and 1 μΜ.

Conclusion: ΤΚΠ-12 polypeptide can effectively inhibit VEGF-induced vascular endothelial cell migration in a dose- and sequence-dependent manner. The ΤΚΠ-30 and ΤΚΠ-10 polypeptides inhibit VEGF-induced vascular endothelial cell migration at higher concentrations. The ΤΚΠ-20 polypeptide does not inhibit VEGF-induced vascular endothelial cell migration. Example 4 Inhibition of VEGF-induced vascular endothelial cell lumen formation by ΤΚΠ series polypeptides

Vascular endothelial cell lumen formation assay was performed using Matrigel (BD) in combination with VEGF to induce lumen formation.

The specific implementation method is as follows: No growth factor Matrigel was pre-coated with a pre-cooled 96-well plate, 50 μl per well, and polymerized at 37 ° C for 30 min. After the HUVECs grew close to the fusion, the serum-free medium was starved overnight, and 0.25% trypsin was digested to prepare a cell suspension. Different concentrations of ΤΚΠ series polypeptide solution (1ηΜ, 10 nM, 100 ηΜ, 1μΜ, 10μΜ) were mixed with cell suspension containing 3×10 ⁴ HUVECs, pretreated for 30 min in 37°C incubator, and then added to each group. A serum-free medium containing VEGF (R&D) was used to give a final concentration of VEGF of 15 ng/ml. A blank control group (no VEGF-free TK II polypeptide group) and a VEGF control group (no TK II polypeptide group) were added, and the above cell suspension was added to a 96-well plate covered with Matrigel, and 5 parallel holes were set for each experimental group. The culture was continued for 6 hours in a 37 ° C incubator. The formation of the cell lumen was observed under an inverted microscope, and four fields of view were taken for each well. The lumen formation length of each group was compared using NIH Imagejl.32 image analysis software.

Results: Under the same conditions, the blank control group (no VEGF non-purine peptide group) vascular endothelial cells arranged to form a small lumen-like structure, the vascular group-like structure of the VEGF group was significantly increased, staggered into a network, the two groups of tubes The difference in lumen formation length was statistically significant, 0.01), indicating that 15 ng/ml VEGF can effectively induce lumen formation in HUVECs.

相对-20 polypeptide 1ηΜ~10 μ Μ The relative length of the lumen-like structure in each concentration group was not significantly reduced compared with the VEGF group, the difference was not statistically significant, P>0.0) _a

The relative length of the luminal-like structure of ΤΚΠ-30, ΤΚΠ-10, ΤΚΠ-12 peptides 1ηΜ, ΙΟηΜ was not significantly reduced compared with the VEGF group. The formation of the tube-like structure in each group was compared with the VEGF group at the concentration of 100ηΜ~10μΜ. Significantly reduced, the difference was statistically significant (Z6 ^ method, 0.01), and the inhibition of each group gradually increased with the increase of peptide concentration. Among the three peptides of ΤΚΠ-30, TK 11-10, and ΤΚΠ-12, TKII-12 inhibited the formation of endothelial cells more strongly than ΤΚΠ-30 and ΤΚΠ-10 polypeptides.

Compared with the VEGF group, the random peptide TKII-12S polypeptide ΙΟ μ Μ group has a relatively long lumen formation of endothelial cells. There was no significant change in the degree, and the difference was not statistically significant, P>0.05)

Conclusion: ΤΚΠ-30, TK 11-10, ΤΚΠ-12 peptide can effectively inhibit VEGF-induced vascular endothelial cell lumen formation in a dose-dependent manner. Among the three polypeptides, ΤΚΠ-12 has the strongest inhibitory effect on endothelial cell lumen formation, and its inhibition is sequence dependent. The ΤΚΠ-20 polypeptide does not inhibit the vascular endothelial cell formation induced by VEGF. Example 5 TK II -12 polypeptide inhibits chicken embryo chorioallantoic membrane neovascularization in vivo

In vitro cell experiments showed that TK Π-12 polypeptide can completely and effectively inhibit the proliferation, migration and lumen formation of vascular endothelial cells during angiogenesis in four small peptides derived from t-PA Kringle2. In this example, the chicken embryo chorioallantoic membrane experiment and the mouse corneal neovascularization experiment were used to further confirm the effect of the ΤΚΠ-12 polypeptide on inhibiting neovascularization in vivo.

Experimental method of chicken chorioallantoic membrane neovascularization: The fertilized eggs of l~2d after birth were disinfected and incubated in an incubator at 37 °C and humidity 60~70% for 5 days. Then open a small window on the eggshell to observe the position of the heart and the direction of the blood vessels. Place the filter paper containing PBS, 10ng ΤΚΠ-12 polypeptide, 50ng ΤΚΠ-12 polypeptide, 50ng TKII-12S polypeptide on the urinary sac membrane. Between the blood vessels, close the eggshell opening. After continuing to incubate for 2 days in the incubator, peel off the eggshell, observe the growth of capillary blood vessels within the diameter of a filter paper around the filter paper, and take a picture to count the number of capillaries.

Results: The urinary vesicle capillaries grew well in a diameter range around the PBS experimental group; ΤΚΠ-12 peptide 10 ng experimental group, the number of capillaries in a diameter range around the filter paper was reduced; ΤΚΠ-12 polypeptide 50 ng experiment In the group, the number of capillaries in the urinary vesicle membrane was significantly reduced in a diameter range around the filter paper, and some avascular regions appeared. Compared with the PBS group, the 10 ng and 50 ng ΤΚΠ-12 polypeptide experimental group had a diameter of the urinary vesicle membrane around the filter paper. The number of blood vessels was reduced, and the difference was statistically significant, 0.01). Compared with the 10 ng group, the number of capillaries in the diameter of the 50 ng TKII-12 polypeptide experimental group was small, and the difference was statistically significant. Law, 0.05). In the 50 ng experimental group of TKII-12S peptide, the capillaries of the urinary vesicles grew well in a diameter range around the filter paper. The number of capillaries was not significantly reduced compared with the PBS group, and the difference was not statistically significant, P>0.05) _a ( Figure 5)

The above studies showed that ΤΚΠ-12 polypeptide can significantly inhibit the growth of chicken embryo chorioallantoic capillary, and with the increase of ΤΚΠ-12 polypeptide dose, the inhibition of neovascularization is enhanced, with good dose-dependent and sequence-dependent. Example 6 TK II -12 polypeptide inhibits VEGF-induced corneal neovascularization in mice

In this example, a mouse corneal microcapsule model was used to further verify the effect of the ΤΚΠ-12 polypeptide on the inhibition of neovascularization in vivo.

(1) Preparation of sustained-release granules: Physiology of 12% PolyHEMA ethanol solution and powder containing sucralfate The saline solution was mixed in an equal volume to prepare a blank slow-release granule having a volume of about 0.35 匪 X 0.35 匪 X 0.2 。. And 160 ng of VEGF and different doses of ΤΚΠ-12 polypeptide (1μ _§ , 5μ _§ ) and ΤΚ II -12S polypeptide (5μ _§ ) were added to the above particles to prepare blank particles, VEGF particles, VEGF+ TK II -12 polypeptide particles, VEGF + 5 g ΤΚΠ-12 polypeptide particles, VEGF + 5 g ΤΚΠ -12S polypeptide granules, stored in a refrigerator at 4 ° C.

(2) Establishment of mouse corneal microcapsule model and neovascularization inhibition experiment: Male C57BL/6 mice, 5 to 6 weeks old, weighing about 20 g. For each experimental mouse, the right eye was used as the experimental eye. According to the random number table method, 50 mice were randomly divided into a blank control group, a VEGF group, a low concentration group of ΤΚΠ-12 polypeptide (group g) and a high concentration group ( 5μ _§ group), TKII-12S polypeptide group (5μ _§ group), 10 in each group. Mice were anesthetized with 0.5% pentobarbital intraperitoneal injection, and 0.4% oxybuprocaine hydrochloride eye drops were applied topically. Under a stereo microscope, a 23G injection needle was used to separate a pocket of about 0.5mm×0.5mm in the corneal stroma of the mouse, and the sustained release particles containing VEGF and polypeptide were implanted into the corneal stroma pocket. The distance between the angle and the limbus is 0.6~0.8mm. After the operation, 0.5% chlortetracycline eye ointment is applied to prevent infection and reduce irritation. All operations are performed by the same surgeon.

(3) Quantitative observation of corneal neovascularization: The length of the neovascularization from the angle of the limbus toward the sustained release granules was measured 5 days after surgery. The area of the new blood vessel is calculated based on the longest blood vessel with a small continuous curvature and toward the growth of the sustained release particles. The formula is: neovascular area (mm ² ) = 0.5X3.14X longest neovascular length (mm) X neovascular involvement of the corneal circumference clock point X0.4 (mm).

(4) Histopathological observation: Each group of animals was sacrificed by excessive anesthesia 5 days after surgery. The eyeballs were removed under aseptic conditions, and 10% neutral formaldehyde was fixed for pathological examination. Specimens were embedded in paraffin, sliced at 3 μm, stained with HE, and observed under light microscope.

(5) Results: There was no growth of neovascularization in the cornea of the blank control group 5 days after surgery, so the effect of the surgery itself on corneal neovascularization could be eliminated. In the VEGF group, the corneal neovascularization from the hornbeam to the slow-release granules grows like a brush, twisted and dilated, and the blood vessels grow densely; the spasm of the ΤΚΠ-12 polypeptide group shows the growth of sparse and short neovascularization; ΤΚΠ-12 polypeptide 5 g There was no obvious growth of new blood vessels at the corner of the sclera; the corneal cells of the 5 g group of TKII-12S polypeptide showed obvious neovascular growth, and the blood vessels were oriented toward the sustained-release granules, which were distorted and expanded.

Histological examination revealed that the vascular layer of the VEGF group was loosely arranged, and a large number of neovascular lumens were found in the matrix, and red blood cells were filled therein. A small amount of neovascular lumen was seen in the corneal stroma of the ΤΚΠ-12 polypeptide group 1. There was no obvious neovascularization in the ΤΚΠ-12 peptide group 5, and there was no obvious edema in the corneal stroma. A large number of neovascular lumens were seen in the corneal stroma of the TKII-12S polypeptide group 5, in which red blood cell filling was observed. Compared with VEGF group, the length of vascular length, time point and neovascular area of ΤΚΠ-12 peptide group were statistically significant, 0.01); ΤΚΠ-12 polypeptide 5μ _§ group compared with VEGF group, the longest blood vessel There were statistically significant differences in length, number of hours, and area of neovascularization, 0.01); and the longest vessel length, hour point, and new ratio of the ΤΚΠ-12 polypeptide 5 g group compared with the 1 μ _§ group. The difference in area of blood vessels was statistically significant, 0.01,). Compared with the VEGF group, the longest vessel length, the number of hours, and the area of neovascularization in the TKII-12S peptide 5μ _§ group were not statistically significant (LS, ^0.01). (Figure 6)

The above results showed that ΤΚΠ-12 polypeptide can significantly inhibit the growth of VEGF-induced corneal neovascularization in mice, and the inhibition of angiogenesis is enhanced with the increase of peptide dosage, indicating that ΤΚΠ-12 has a good inhibitory effect on neovascularization in vivo.

Conclusion: t-PAKringle2-derived small molecule peptide ΤΚΠ-12 can inhibit the proliferation, migration and lumen formation of vascular endothelial cells during angiogenesis in vitro, and can effectively inhibit the growth of new blood vessels in vivo.

Note: The experimental data in the above study was expressed as χ±, and statistical analysis was performed using the SPSS13.0 statistical software package. One-way analysis of variance was used to compare the differences between the experimental groups and the VEGF group. The least-significant difference (LSD) was used to compare the two groups. The difference was statistically significant at P < 0.05. Example 7

Preparation of eye drops

The following components were mixed using conventional techniques to prepare 1% eye drops, which were formulated as follows:

TKII-12 polypeptide 10 mg

Hydroxypropyl methylcellulose 0.03g

Sterile water added to 10 ml

Adjust the osmotic pressure to 3000sm and pH to 6.8-7.1.

After 4 weeks of trial by 4 volunteers, 3 times a day, 1 drop/eye each time. The results show that the eye drops can inhibit angiogenesis in the eye. Example 8

Preparation and activity of derived peptides

The following several derived polypeptides were prepared, and the inhibitory effect of each of the 12-12-derived polypeptides on VEGF-induced proliferation of vascular endothelial cells HUVECs was determined as shown in Example 2.

The derivative polypeptide 1 sequence is identical to SEQ ID NO 1, wherein the second Val is replaced by Thr. The derivative polypeptide 2 has the same sequence as SEQ ID NO 1, wherein the third Leu is replaced by lie; the derived polypeptide 3 sequence is the same as SEQ ID NO 1, wherein the fifth The Asn is replaced by Gin; the derivatized polypeptide 4 sequence is identical to SEQ ID NO 1, wherein the 5th Asn is replaced by Asp; the derivatized polypeptide 5 sequence is identical to SEQ ID NO 1, wherein the 7th Arg is replaced by Lys; the derived polypeptide 6 sequence is identical to SEQ ID NO 1, wherein the 7th Arg is replaced by Gin; the derivative polypeptide 7 sequence is the same as SEQ ID NO 1, wherein Cys is added before the 1st position of the N terminus; Derived polypeptide 8: The sequence is identical to SEQ ID NO: 1, wherein the CDV tripeptide is added after the 12th position of the C-terminus.

The results showed that HUVECs cell proliferation was significantly inhibited in the treatment group (??) of the above-derived polypeptide 1-8. In summary, the indole 12 and its derived polypeptides of the present invention all show good inhibition of pathological neovascularization of mouse cornea and inhibition of proliferation, migration and lumen formation of vascular endothelial cells in vitro. Therefore, it has broad application prospects. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made to the present invention, and the scope of the invention is defined by the scope of the appended claims.

Claims

Rights request

A polypeptide represented by the following formula I, or a pharmaceutically acceptable salt thereof

[XaaO] - [Xaal]— [Xaa2]— [Xaa3] - [Xaa4] - [Xaa5] - [Xaa6] - [Xaa7] - [Xa a8] - [Xaa9] - [XaalO] - [Xaal 1] - [Xaal2] - [Xaal3] (I)

In the formula,

XaaO is no, or 1-3 amino acids constitute a peptide;

Xaal is an amino acid selected from the group consisting of Hi s or Arg;

Xaa3 is an amino acid selected from the group consisting of Leu, H e , Val, Met or Ala;

Xaa4 is an amino acid selected from the group consisting of Lys or Arg;

Xaa5 is an amino acid selected from the group consisting of Asn, Asp or Gin;

Xaa6 is an amino acid selected from the group consisting of Arg or Lys;

Xaa7 is an amino acid selected from the group consisting of Arg, Gin or Lys;

Xaa8 is an amino acid selected from the group consisting of Leu, H e , Val, Met or Ala;

Xaa9 is an amino acid selected from the group consisting of Thr or Ser;

XaalO is an amino acid selected from the group consisting of Trp or Tyr;

Xaal l is an amino acid selected from the group consisting of Glu or Asp;

Xaal2 is an amino acid selected from the group consisting of Tyr or Phe;

Xaal3 is no, or 1-3 amino acids constitute a peptide;

The polypeptide according to claim 1, wherein Xaal3 is a peptide consisting of 1-3 amino acids.

The polypeptide according to claim 1, wherein XaaO is C or WC.

The polypeptide according to claim 1, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ ID NO: 1;

(b) A polypeptide derived from (a) which is formed by substitution, deletion or addition of the amino acid sequence of SEQ ID NO: 1 by 1-5 amino acid residues and which inhibits angiogenic function.

5. An isolated nucleic acid molecule, which encodes the polypeptide of claim 1.

6. A pharmaceutical composition characterized in that it comprises:

(a) the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof;

(b) a pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition according to claim 6, wherein the composition is in the form of an eye drop, an injection, an ophthalmic gel or an ophthalmic ointment.

The use of the polypeptide or the pharmaceutically acceptable salt according to claim 1, which is for the preparation of a medicament for inhibiting angiogenesis or preventing diseases associated with angiogenesis.

9. The use according to claim 8, wherein the angiogenesis-related disease is selected from the group consisting of neovascular ophthalmopathy, tumor, ischemic heart disease, non-inflammatory cardiomyopathy, coronary artery. Hardening, occlusive arteriosclerosis, arterial embolism, arterial thrombosis, Berger's disease, chronic inflammation, inflammatory bowel disease, ulcers, rheumatoid arthritis, scleroderma, psoriasis, infertility and sarcomatosis.

A method for inhibiting angiogenesis in a mammal, comprising the steps of: administering a polypeptide of the present invention or a pharmaceutically acceptable salt thereof to a subject in need thereof.