KR101377797B1 - Composition for angiogenesis inhibiting comprising mini-PEGylated anti-flt-1 peptide - Google Patents

Abstract

The present invention can effectively treat diseases caused by abnormal or excessive angiogenesis, such as cancer, rheumatoid arthritis, and 8-amino-3, for the purpose of preventing or treating angiogenic diseases that can enhance the treatment convenience of patients. Angiogenesis-inhibiting composition containing anti-flt-1 peptide conjugated with 6-dioxaoctanoic acid (8-amino-3,6-dioxaoctanoic acid; mini-PEG) as an active ingredient, in particular angiogenesis for oral administration It relates to an inhibitor. The novel D-type mini PEG conjugated anti-flt-1 peptide targeting the vascular endothelial growth factor receptor (VEGFR) -1 (flt-1) according to the present invention targets the existing VEGF or VEGFR. Compared with the drug, it effectively inhibits the binding between vascular endothelial growth factor and its receptor and exhibits almost the same antiangiogenesis effect as parenteral administration. It can be used as a therapeutic agent that can be administered.

Description

Composition for angiogenesis inhibiting comprising mini-PEGylated anti-flt-1 peptide comprising mini-PEJ conjugated anti-ｆｌｔ-1 peptide as an active ingredient

The present invention relates to a pharmaceutical composition for the purpose of preventing or treating angiogenic diseases that can effectively treat diseases caused by abnormal or excessive angiogenesis, such as cancer and rheumatoid arthritis, and can enhance the convenience of treatment of patients. .

Angiogenesis, the process of forming new blood vessels from existing vessels, is known to accelerate many diseases, such as the growth and metastasis of solid cancers, rheumatism, diabetic retinopathy, and the development of various inflammatory diseases (Kohn, EC et. al. (1995) Proc. Natl. Acad. Sci. USA 92, 1307; Folkman, J. et al. (1987) Science 235, 442; Risau, W. (1997) Nature 386, 671). Angiogenesis plays an essential role in cancer growth and metastasis, in particular because it allows for a smooth supply of nutrients through the blood and provides a pathway for metastatic cancer (Hanahan, D. et al. (1996) Cell 86, 353; Skobe, M., et al. (1997) Nature Med. 3, 1222). Angiogenesis is therefore an essential step in cancer growth as well as cancer metastasis, an indicator of malignancy.

Vascular endothelial cell growth factor (VEGF) is one of the most potent angiogenic inducers with various biological activities through binding to two receptors (VEGFR1 and VEGFR2) that exist only on the surface of vascular endothelial cells. Compared with other factors involved in angiogenesis, vascular endothelial growth hormone is an evolutionary immobilized glycoprotein that plays a central role in both vasculogenesis and angiogenesis. In addition, it induces various cellular signaling and physiological responses essential for angiogenesis such as intracellular calcium inflow, activation of proteolytic enzymes, or enhanced vascular permeability (Thomas, KA (1996) J. Biol. Chem. 271, 603). ).

Thus, various drugs targeting VEGF are already being developed or developed, and are producing good clinical data in VEGF-dependent diseases occurring in humans such as cancer disease and macular degeneration. For example, bevacizumab, an anti-VEGF antibody suitable for humans, has been approved by the US FDA as the first anti-angiogenic drug for the treatment of metabolic colorectal cancer and is also effective in treating lung and kidney cell cancers. It is known. Pegaptanib, an anti-VEGF RNA aptamer, has also been shown to reduce vision loss when administered to patients with age-related macular degeneration (AMD). Synthetic ribozymes of VEGF-Trap and flt-1 (VEGF receptor 1), such as chimerized anti-KDR antibodies, which are targeted at It is undergoing stage 1 or 2 clinical trials.

Therefore, substances capable of effectively inhibiting the binding between vascular endothelial growth factor (VEGF) and its receptors (VEGFR) will play an important role in the study of signaling through each receptor, in particular abnormal or excessive angiogenesis and Various related diseases caused by angiogenesis may be used as an active ingredient of a composition for therapeutic development.

Accordingly, the present inventors have discovered that a novel D-type mini-PEG conjugated anti-flt-1 peptide targeting vascular endothelial growth factor receptor (VEGFR) -1 (flt-1) may be used to replace existing VEGF, PlGF or VEGFR. Compared with the targeted drug, it effectively inhibits the binding between vascular endothelial growth factor or placental growth factor and its receptor, and for the first time, oral administration shows almost the same antiangiogenic effect as parenteral administration. By completing the present invention, it was confirmed that the anti-flt-1 peptide consisting of six amino acids according to the present invention can be used as a therapeutic agent for diseases caused by angiogenesis.

Therefore, an object of the present invention is to effectively prevent or treat various diseases induced by angiogenesis, the peptide having the amino acid sequence of SEQ ID NO: 1 and mini-PEG (8-amino-3,6-dioxaoctanoic acid; mini-PEG It is to provide an effective angiogenesis inhibiting composition that can be administered orally containing a conjugate of) as an active ingredient.

In order to achieve the above object, the present invention is a combination of a peptide (peptide) and a mini-PEG (8-amino-3,6-dioxaoctanoic acid; mini-PEG) comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient It provides a composition for inhibiting angiogenesis.

In one embodiment of the invention, the peptide may be form (form).

In one embodiment of the invention, the peptide inhibits the binding of vascular endothelial growth factor (VEGF) or placental growth factor (PlGF) and vascular endothelial growth factor receptor (VEGFR) -1 (flt-1) or Can interfere.

In one embodiment of the invention, the composition may prevent or treat diseases caused by abnormal or excessive angiogenesis.

In one embodiment of the present invention, the disease is cancer, diabetic retinopathy, prematurity retinopathy, corneal graft rejection, neovascular glaucoma, melanoma, proliferative retinopathy, psoriasis, hemophiliac joints, atherosclerotic plaque Vascular proliferation, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcers, cirrhosis, glomerulonephritis, diabetic nephropathy, Malignant neurosis, thrombotic microangiopathy, organ transplant rejection, renal glomerulopathy, diabetes, inflammatory and neurodegenerative diseases.

In one embodiment of the invention, the composition may be an oral or injectable preparation.

In addition, in one embodiment of the present invention, the oral preparation may be selected from the group consisting of capsules, tablets, pills, suspensions, syrups and powders.

Anti-flt-1 peptide conjugated with mini-PEG (8-amino-3,6-dioxaoctanoic acid; mini-PEG) according to the present invention is more effective than conventional anti-flt-1 peptides. As an active ingredient of an angiogenesis inhibitor that can prevent the binding between growth factor and vascular endothelial growth factor receptor, the present inventors convert the existing L-type peptide to D-type and conjugate the mini-PEG to the modified D-type. In vitro and in vivo conditions have been shown to inhibit angiogenesis better than conventional L-type peptides. In addition, oral administration of the peptides according to the present invention in the animal model of arthritis exhibits the same antiangiogenic effect as in the parenteral administration, and through the action of reducing the exacerbation of arthritis symptoms, an excellent therapeutic effect of angiogenic diseases. While ensuring that oral administration is possible there is an effect that can increase the comfort of the patient.

1 is a graph showing the results of measuring the stability of the type D anti-flt-1 peptide according to the present invention.
Figure 2 is a photograph and a graph showing the effect of inhibiting capillary formation in vascular endothelial cells of the mini-PEG conjugated all-type D peptide according to the present invention.
Figure 3 is a photograph and graph showing the effect of inhibiting wound migration (migotaxis) and chemotaxis of the HEVEC of all-type D-peptide conjugated mini-PEG according to the present invention.
Figure 4 is a photograph and graph showing the effect of inhibiting angiogenesis induced by VEGF165 in vivo of the all-type D peptide conjugated mini-PEG according to the present invention.
Figure 5 is a photograph and a graph confirming the effect of arthritis treatment in a collagen-induced arthritis (CIA) animal model that appears when oral administration of the all-type D-peptide conjugated mini-PEG according to the present invention.
Figure 6 is a photograph and graph showing the growth inhibitory effect of colon tumor cells of all-type D-peptide conjugated mini-PEG according to the present invention.

The present invention is a combination of a peptide comprising the amino acid sequence of GNQWFI (Gly-Asn-Gln-Trp-Phe-Ile, SEQ ID NO: 1) and mini-PEG (8-amino-3,6-dioxaoctanoic acid; mini-PEG) It was first identified that the effect of inhibiting the angiogenesis, it is characterized by providing a composition for inhibiting angiogenesis containing a combination of the peptide of the SEQ ID NO: 1 and the mini PEG as an active ingredient.

Angiogenesis means that new capillaries are made from existing blood vessels. Strictly controlled phenomena rarely occur under normal physiological conditions, when embryos develop during fertilization, embryos heal in adults, and changes in reproductive system in the female reproductive cycle. In adults, capillary endothelial cells do not divide relatively well and the rate of division usually ranges from months to years. Angiogenesis occurs as a complex process by the interaction of various types of cells with soluble factors and extracellular matrix components, and its mechanism of action is not yet fully understood.

Looking at the general angiogenic process that has been discovered so far, angiogenesis is first started by an angiogenic factor secreted from cancer cells. This inducer binds to vascular endothelial receptors to stimulate endothelial cells, and stimulated endothelial cells undergo a variety of complexities such as proliferation, invasion, migration and differentiation, and capillary formation. Begin a series of processes. Invasion and migration of endothelial cells requires the activation of histolytic enzymes, which is very similar to cancer cell infiltration. Many promoters and inhibitors that regulate angiogenesis have been reported. Among them, vascular endothelial growth factor (VEGF) is the most well known angiogenesis promoter, and angiostatin and endostatin are angiogenesis inhibitors. Most known as

Vascular endothelial growth factor (VEGF) is secreted by almost all cells and is closely related to tumor permeability (Takahashi Y, Kitadai Y, Bucana CD et al., Cancer Res. 1995, 55 (18). ): 3964-3968). Vascular endothelial growth factor receptor (VEGFR) is generally present in vascular endothelial cells but is expressed in neurons, Kaposi's sarcoma, hematopoietic stem cells, etc., and VEGFR-1 (flt-1) and VEGFR-2 (Kdr / flk). -1) and VEGFR-3 (flt-4). When VEGF is bound to VEGFR, signal is transmitted through receptor dimerization with phosphorylation of receptor tyrosine residues. VEGFR-1 is involved in endothelial cell migration and VEGFR-2 is involved in endothelial cell growth and blood leakage effects. In addition to direct angiogenesis factors, non-specific factors such as aFGF, bFGF, TGF, EGF, platelet-derived endothelial cell growth factor (PDGF), angiogenin, IL-8, MIP, PF4, and GRO are known to be involved in angiogenesis (Moore BB , Arenberg DA, Addison CL et al., J Lab Clin Med. 1998, 132 (2): 97-103).

In recent years, research on the expression and mechanism of action of VEGF and the study of substances that can inhibit angiogenesis by inhibiting higher signaling processes that regulate it are active. Certain proteins, antibodies, and receptor phosphorylation inhibitors that can bind to VEGF receptors reduce the activity of angiogenesis-promoting factors, and are being developed as candidates for the treatment of diseases induced by excessive angiogenesis. Investigations have been actively conducted on mechanisms that induce apoptosis, inhibition of angiogenesis and indirect factors regulating endothelial cell survival factors, and methods of increasing the activity of angiogenesis inhibitors present in the body.

In addition, the placental growth factor (Placenta growth factor) PlGF is known as a homologue of VEGF, and cooperates with VEGF to contribute to neovascularization that triggers cancer development. However, unlike VEGF, it does not affect normal angiogenesis and is only involved in angiogenesis in diseased tissues, and thus, PlGF inhibitors may inhibit tumor growth without side effects of other antiangiogenic agents. In addition, PlGF has been shown to be an important substance causing rheumatoid arthritis ( Seung - Ah Yoo , Hyung - Ju Yoon , Hyun- Sook Kim meat al ., Arthritis & Rheumatism, 2009, 60 (2): 345-354), and have been evaluated as excellent molecular targets for the treatment of rheumatoid arthritis. PlGF inhibitor TB-403 (ThromobiGenics) is also known to be effective in blocking uncontrolled vascular proliferation, such as age-related macular disease (AMD) and diabetic retinopathy and prematurity retinopathy.

Accordingly, the present inventors have developed GNQWFI (Gly-Asn-Gln) through screening of positional scanning synthetic peptide libraries to develop a new therapeutic agent for the prevention or treatment of diseases caused by abnormal or excessive angiogenesis. A new anti-flt-1 hexapeptide with the amino acid sequence of -Trp-Phe-Ile) was found, which selectively binds to vascular endothelial cell growth factor receptor (VEGFR) 1 (flt-1) and associates with VEGF or PlGF. It was found that the interaction between flt-1, a VEGF receptor, is effectively inhibited, ultimately preventing angiogenesis.

Accordingly, the present invention provides an anti-flt-1 peptide that inhibits angiogenesis by binding to vascular endothelial growth factor receptors as well as vascular endothelial growth factor receptors. Various diseases caused by excessive or abnormal angiogenesis can be prevented or treated.

On the other hand, in the case of protein drugs containing peptides in general, there are inherent problems in the formulation and preparation of ingredients. For example, biopharmaceuticals have a disadvantage in that they are likely to cause an immune response upon delivery to a patient due to their size and structure. In addition, the bioavailability is particularly low, making it difficult to develop oral formulations. Bioavailability refers to the fraction of the administered drug that reaches systemic circulation. It refers to the fraction of the administered drug required for systemic circulation in a form that is not chemically changed, and consequently, the physiological effect of a certain amount of drug. .

In the case of peptide preparations, peptide bonds are easily broken down due to gastric secretion of the stomach upon oral administration, and can be digested, so that the half-life is extremely short, so that they cannot exhibit sufficient efficacy in vivo. In the case of insulin protein, which is widely used worldwide as a therapeutic agent for diabetes, the bioavailability of oral preparations is extremely low, which is inevitably produced as injections. As such, unlike synthetic drugs, the development of oral dosage forms remains the biggest challenge in drug development in the field of protein pharmaceuticals. To overcome these difficulties, pharmaceutical companies and researchers have found an absorption enhancer in the delivery of protein drugs. Or other situation is developing a separate delivery technology.

To this end, the present inventors have found that the problem of inhibiting angiogenesis and reducing half-life according to oral administration, which has been pointed out as a problem of anti-flt-1 peptides that had an effect of inhibiting angiogenesis by inhibiting binding between VEGF and its receptors In order to solve the problem, oral administration of the anti-flt-1 effect, which was exhibited by parenteral administration, continued unchanged even after oral administration, which can enhance the treatment convenience of patients and medical staff suffering from diseases caused by excessive angiogenesis. Efforts have been made to develop antiangiogenic inhibitors suitable for administration.

As a result, the inventors have found that mini-PEG (8-amino-3) has been added to hexapeptides having the amino acid sequence of GNQWFI (Gly-Asn-Gln-Trp-Phe-Ile, SEQ ID NO: 1), which itself has an antiangiogenic effect. , 6-dioxaoctanoic acid; mini-PEG) conjugated to develop a peptide conjugate, of course, if the six amino acids constituting the hexapeptide according to the invention is converted from the basic L-type to D-type, Compared with oral administration, it was confirmed that the anti-angiogenic effect was excellent.

Accordingly, the present invention provides an angiogenesis inhibitor containing a conjugate of a peptide comprising the amino acid sequence (GNQWFI) described in SEQ ID NO: 1 and a mini-PEG (8-amino-3,6-dioxaoctanoic acid; mini-PEG) as an active ingredient. It can provide an angiogenesis inhibitor for oral administration containing a composition for.

In addition, mini-PEG used in the present invention is a reagent widely used for cell fusion, and the present inventors attempted to prepare an angiogenesis inhibitor suitable for oral administration by conjugating mini-PEG to the peptide of SEQ ID NO: 1. PEGylation, which attaches mini-PEG to the amino group of the protein, reduces the total available amount of the amino group of the protein, thus reducing the total charge. It is believed that this is an efficient and easy way for the immunogen to have no immunogenicity. Since the living body does not respond to such excessive charges, the immunogenicity of the total formulation may be less.

In addition, the hydrophilicity of mini-PEG can also reduce immunogenicity. Once a mini-PEG substance binds to a protein, it attracts water molecules, and this hydration reduces the body's immune system's perception of the molecule as foreign. This also makes the protein more stable. In general, mini-PEG binding to proteins lowers protein aggregation, reduces protein uptake, and reduces antigen and immune epitopes, as they reduce plasma-removal rates by reducing metabolic degradation and receptor-mediated uptake through systemic circulation. Protection is formed. In addition, conjugation of the mini-PEG can prevent the drug from being absorbed into the body, and the targeted delivery of protein drugs is also possible because of the increased molecular size. Macromolecular delivery systems could be used to selectively deliver drugs to the site of action.

On the other hand, amino acids, which are the living materials that make up proteins of living organisms, and also the core basic materials of fine chemicals and medicines, are classified into L-type (left-handed) and D-type (right-handed) according to their optical activity. Natural amino acids exist in L form, and D corresponds to non-natural amino acids. Optical activity is a phenomenon in which the polarization rotates at a certain angle when the polarization is applied. The L- and D-types rotate in opposite directions, and the D-type is named right-handed. When it passes, it rotates like a right screw. When amino acids are found in nonliving organisms or synthesized in a laboratory, each type of amino acid appears in two chemically identical forms. Half is preferential type (D type) and half is left linear type (L type). But all of the amino acids in living organisms, like plants, animals, bacteria, fungi, and even viruses, are L-type.

In the case of the peptides previously produced as angiogenesis inhibitors, most of the peptides produced through peptide bonds of L-type amino acids which are natural amino acids. However, even though L-type amino acids are known to have specific physiological activity, whether the existing physiological activity will be retained or the physiological activity will disappear when the conversion to D-type, or will show a superior physiological activity, or completely different Whether to show the biological activity of the properties of those in the field of biopharmaceuticals is unpredictable until the actual use is confirmed.

Indeed, amino acids having the same molecular formula and molecular weight may have completely different properties of the two isomers, as L-type is used as a therapeutic agent and D-type is used as a pesticide. Of course, not all amino acids have different chemical properties as in this case, but the properties of amino acids according to L and D forms are unpredictable, and the specific aspects are different for each amino acid.

In particular, in the case of a peptide consisting of a combination of several amino acids, it can be said that the chemical properties of the peptide can be sufficiently changed depending on the type of the isomer of each constituent amino acid.

Accordingly, the present inventors, according to one embodiment of the present invention, by chemically synthesizing 6 constituent amino acids of the peptide described in SEQ ID NO: 1 of the present specification having an anti-angiogenic effect from L to D form One seven different Form D anti-flt-1 peptides were prepared; gNQWFI (1D), GnQWFI (2D), GNqWFI (3D), GNQwFI (4D), GNQWfI (5D), GNQWFi (6D), gnqwfi (all) D-type (uppercase L-type, lowercase D-type amino acids ). They were then tested for their inhibitory effects using an ELISA-based binding assay. As a result, it was confirmed that 1D, 6D and all D peptides blocked binding to receptor flt-1 in a dose dependent manner compared to the original L-type peptide (see Example 1), and thus the inventors of the GNQWFI The anti-angiogenic effects of the hexapeptides converted from the L-type structure of all amino acids to D-type were confirmed.

Thus, according to one embodiment of the present invention, the present inventors tested the angiogenesis inhibitory activity of the peptides according to the present invention in vivo. After subcutaneous injection of several samples into the abdomen of the mouse, the amount of hemoglobin was measured to quantify the formation of the vasculature. In the case, lower hemoglobin was detected compared to the original L-type peptide, and thus an excellent angiogenesis inhibitory effect was confirmed. In addition, it was found that the D-type peptide according to the present invention to which mini-PEG was conjugated has an activity of inhibiting macrophage invasion as well as inhibition of neovascularization (see Example 2).

In addition, the present inventors conducted an experiment on whether the peptide according to the present invention would have a therapeutic effect when orally administered to an animal model suffering from angiogenesis-induced disease. Rheumatoid arthritis was selected as a target disease, and as a result of oral administration of the original L-type and the mini-PEG conjugated D-peptide according to the present invention to mice, compared to the original L-type peptide, toenail inflammation and the like have no obvious side effects. Significantly inhibiting rheumatoid arthritis treatment effect of mini-PEG-D-type peptide, such as significantly inhibited was confirmed (see Example 3).

According to another embodiment of the present invention, as a result of testing the activity of the peptides according to the present invention on the survival and proliferation of HUVECs, which are human endothelial cells in vitro, all amino acids are D-type peptides, mini-PEG It was confirmed that the peptide according to the present invention, in which all of the amino acids bound to D, significantly inhibited the formation of VEGF ₁₆₅ -induced tube-like structures. In addition, PlGF-induced tube formation was also found that peptides of the amino acid sequence according to the present invention in which all amino acids are D-type peptides and all amino acids combined with mini-PEG are D-types effectively inhibit their production. In addition, it was confirmed that the migration and chemotaxis of HUVECs due to PlGF and VEGF-induced wounds were also inhibited (see Example 4).

In addition, as a result of the activity test for cancer disease, a representative disease caused by abnormal and excessive angiogenesis of the peptides according to the present invention, the metastasis and growth inhibitory effect of colon tumor cells could be confirmed (see Example 5). .

Thus, based on the above experimental results, the present inventors orally administered PEGylated type D anti-flt-1 peptide to mice, in which mini-PEG was conjugated to type D anti-flt-1 peptide according to the present invention, It was confirmed that the worsening of symptoms of collagen-induced arthritis (CIA) mice was inhibited, the effect of inhibiting cancer cell growth was also confirmed, and the excellent angiogenesis inhibitory effect was found. The peptides according to the present invention were rheumatoid arthritis. In addition, it was determined that the present invention may be useful for treating angiogenesis-dependent diseases including cancer diseases.

Therefore, the present invention can provide a composition for inhibiting angiogenesis, which comprises a D-type peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient, and can provide a composition for inhibiting angiogenesis that is particularly oral. have.

In addition, the present invention can provide a composition for inhibiting angiogenesis comprising a conjugate comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a mini-PEG conjugated as an active ingredient, the peptide is characterized in that the D-type Can be.

In addition, it was confirmed that the peptides DL placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) -induced angiogenesis are excellently inhibited according to the present invention. Alternatively, the vascular endothelial growth factor (VEGF) and the vascular endothelial growth factor receptor (VEGFR) may inhibit or inhibit the binding.

Thus, the composition for inhibiting angiogenesis of the present invention can prevent or treat diseases caused by abnormal or excessive angiogenesis.

The angiogenesis-related diseases include, but are not limited to, cancer, diabetic retinopathy, prematurity retinopathy, corneal graft rejection, neovascular glaucoma, melanoma, proliferative retinopathy, psoriasis, hemophiliac joints, atherosclerotic plaques Capillary hyperplasia, keloids, wound granulation, vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune disease, Crohn's disease, restenosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcers, cirrhosis, glomerulonephritis, diabetic kidney Pathologies, malignant neurosis, thrombotic microangiopathy, organ transplant rejection, renal glomerulopathy, diabetes, inflammatory and neurodegenerative diseases and the like.

Accordingly, the peptides according to the present invention can provide excellent angiogenesis inhibitors with high in vivo stability and an increased half-life of 24 hours or more, and these angiogenesis inhibitors can prevent or treat various diseases induced by abnormal or excessive angiogenesis. Can be used for

The term treatment refers to reversing, alleviating, inhibiting, or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder, unless otherwise stated, As used herein, the term " treatment " refers to an act of treating when the treatment is defined as above. Thus, the treatment or therapy of angiogenic diseases in a mammal may comprise one or more of the following:

(1) inhibits the growth of angiogenic diseases, ie inhibits their development,

(2) preventing the spread of angiogenic diseases, ie preventing metastasis,

(3) relieves angiogenic diseases.

(4) prevent recurrence of angiogenic diseases, and

(5) to relieve symptoms of angiogenic diseases

In addition, the conjugate of the D-type peptide and mini-PEG described in SEQ ID NO: 1 according to the present invention may be a compound represented by the following formula (1).

[Formula 1]

In addition, the peptide according to the present invention may be included in a concentration of 1 ~ 500μM relative to the total volume of the composition, it can provide a composition for inhibiting angiogenesis comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient, The composition may be used as a pharmaceutical composition for inhibiting angiogenesis.

The composition for inhibiting angiogenesis according to the present invention may include a peptide comprising an amino acid sequence according to the present invention in a pharmaceutically effective amount alone or may include one or more pharmaceutically acceptable carriers, excipients or diluents.

The pharmaceutically effective amount as used herein refers to an amount sufficient for the physiologically active component to be administered to an animal or a human to exhibit a desired physiological or pharmacological activity. However, the pharmaceutically effective amount may be appropriately changed depending on the age, body weight, health condition, sex, administration route and treatment period of the subject to be administered.

Also, the pharmaceutically acceptable hereinabove refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. In particular, the compositions according to the invention may be formulated as oral or injectable preparations, which are in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like. Can be.

Oral formulations for oral administration may be formulated as capsules, tablets (such as ingested tablets or buccal tablets), pills, suspensions, syrups, powders, troches, elixirs, suspensions, and wafers, and these formulations may be In addition to active ingredients, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and suspending agents (e.g. silica, talc, stearic acid and their magnesium or calcium salts and / or polyethylene glycols) ) May be included. The tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, A disintegrating agent such as sodium salt, an absorbent, a coloring agent, a flavoring agent and / or a sweetening agent. The formulations may be prepared by conventional mixing, granulating or coating methods.

In addition, the composition of the present invention may further include auxiliaries such as preservatives, hydrating agents, emulsifiers, salts or buffers for controlling osmotic pressure and other therapeutically useful substances, and may be formulated according to conventional methods.

The composition according to the present invention can also be administered through parenteral routes including transdermal, subcutaneous, intravenous or muscle, but especially when administered orally, the angiogenesis inhibitory effect is superior to other angiogenesis inhibitors, thereby providing convenience to patients and medical staff. Oral administration is readily possible for The dosage of the active ingredient may be appropriately selected depending on several factors such as the age, sex, weight of the patient and the severity of the patient. In addition, the composition of the present invention can be administered in parallel with known compounds that can enhance the desired effect.

The route of administration of the pharmaceutical composition according to the present invention may be administered to humans and animals orally or parenterally, such as intravenous, subcutaneous, intranasal or intraperitoneal. Oral administration also includes sublingual application. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.

In the composition of the present invention, the total effective amount of a peptide comprising an amino acid according to the present invention can be administered to a patient in a single dose, and the multiple treatment method in which multiple doses are administered for a long time ( fractionated treatment protocol). The composition of the present invention may be administered several times a day at an effective dose of 100 μg to 3,000 mg on a single administration basis on an adult basis although the content of the active ingredient may be varied depending on the degree of the disease. However, the effective concentration of the peptide may be determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate, as well as the route and frequency of treatment of the drug. . Therefore, in view of the above, one of ordinary skill in the art can determine an appropriate effective dosage according to a specific use of the peptide as an angiogenesis inhibitor or an agent for treating or preventing angiogenesis-related diseases, and according to the present invention, Is not particularly limited to the formulation, route of administration and method of administration as long as the effect of the present invention is exhibited.

In addition, the present invention can provide a composition for food that can improve or prevent the symptoms of the disease caused by angiogenesis, the composition for food according to the invention is to improve or prevent disease symptoms due to abnormal or excessive angiogenesis It can be easily utilized as an effective food, such as a main raw material, a subsidiary material, a food additive, a functional food or a beverage.

Herein, the term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be directly eaten through a certain processing step, , Food additives, functional foods and beverages.

Foods to which the food composition according to the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. In addition, in the present invention, the food may include special nutritive foods (e.g., crude oil, spirits, baby food, etc.), meat products, fish meat products, tofu, mackerel, noodles (Such as soy sauce, soybean paste, kochujang, mixed potatoes), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, milk products (eg, fermented milk, cheese, But are not limited to, pickled foods (various kinds of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages and the like) and natural seasonings (e.g. The food, beverage or food additive may be prepared by a conventional production method.

In addition, the functional food is a biological defense rhythm control, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the food for a specific purpose. It means a food that is designed and processed to fully express the body regulatory function related to the living body, specifically, it may be a health functional food. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In addition, in the present invention, the beverage is a collective term for drinking thirst or for enjoying a taste, and includes a functional beverage. The beverage is an essential ingredient in the indicated proportions, and the composition for improving or preventing the symptoms of angiogenesis-related diseases is not particularly limited. Other beverages include various flavors or natural carbohydrates, such as ordinary drinks. It may contain as.

Furthermore, in addition to the foregoing, foods containing a food composition for improving or preventing the symptoms of angiogenesis-related diseases of the present invention may be flavored with various nutrients, vitamins, minerals (electrolytes), synthetic flavors, and natural flavoring agents. , Colorants and fillers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. It may contain, the components can be used independently or in combination.

In the food containing the food composition of the present invention, the amount of the composition according to the present invention may be 0.001% by weight to 90% by weight, preferably 0.1% by weight to 40% by weight, And may be contained in a ratio of 0.001 g to 2 g, preferably 0.01 g to 0.1 g, based on 100 ml in the case of beverage. However, in the case of long-term intake for health and hygiene purposes or for health control purposes May be less than the above range, and since the active ingredient has no problem in terms of safety, it can be used in an amount of more than the above range, so it is not limited to the above range.

Therefore, the present invention can provide an agent for inhibiting angiogenesis for preventing or treating a disease caused by angiogenesis, which contains the composition for inhibiting angiogenesis of the present invention as an active ingredient. It can provide an angiogenesis inhibition method comprising the step of administering to a mammal other than a person whose neoplasia lasts abnormally.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

< Reference Example >

term- flt -One Hexapeptide and  dental Isoform ( isoform ) synthesis

The original anti-flt-1 hexapeptide, GNQWFI (L-form; Gly-Asn-Gln-Trp-Phe-Ile) and its isoform (D-form) designed in small sizes Synthesized in the same manner as previously reported (Bae DG et al., Clin Cancer Res 2005; 11: 2651-61, Yoo SA et al., Arthritis Rheum, 2009; 60: 345-54), followed by isoforms of GNQWFI (all D-form) conjugated mini-PEG, that is, 8-amino-3,6-dioxaoctanoic acid represented by the chemical formula of FIG. 1D to increase its stability. In addition, these peptides were synthesized by Peptron (Peptron Inc., Daejon, Korea).

Immunohistochemistry ( Immunohistochemistry )

Immunohistochemical staining of ankle joints, tumor tissues and matrigel in mice with arthritis, an animal model used to confirm the in vivo activity of the peptides according to the invention Was performed using rabbit anti-vWF antibody (Abcam, Cambridge, MA) and retro anti-mouse MOMA-2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The amount was confirmed.

Statistical analysis

Data are expressed as mean ± standard deviation, and comparison of numerical data between groups was performed by Mann-Whitney paired or unpaired U test. In addition, when P value was less than 0.05, it was evaluated as statistically significant.

< Example  1>

Mini- according to the present invention PEG end Bonded  D type Of peptide  Stability analysis

The inventors have identified seven different D-type anti-flt-1 peptides chemically synthesized: gNQWFI (1D), GnQWFI (2D), GNqWFI (3D), GNQwFI (4D), GNQWfI (5D), GNQWFi (6D ), gnqwfi (all D-type) were prepared (in lowercase letters represent D-type amino acids), and ELISA-based binding assays were used to examine their inhibitory effects.

First, blood from animal models (16-week-old male Sprague Dawley rats) was collected and put into sterile centribugation tubes and clotted at 4 ° C. Then, centrifuged twice at 4 ° C., 1500xg for 10 minutes, and 20000xg for 20 minutes. The supernatant (serum) was filtered on a sterile filter (0.22 μm; Millipore, Billerica, Mass.), Aliquoted in small amounts until use, and stored at −70 ° C.

To assess the stability of each peptide in the serum, anti-flt-1 hexapeptide and modified D-type peptide (100 μg in 50 μg PBS) were previously described (Yoo SA et al., J Immunol, 2005; 174: 5846-). Incubated with 50 μl filtered let serum at 37 ° C. for the time specified in 55). Cultured samples were fractionated by C18 reverse phase HPLC according to linear gradients of acetonitrile (0.1% trifluoroacetate in H2O for equilibration, 0.1% trifluoroacetate in acetonitrile for elution). The amount of peptide was measured through each peak height and area for cultured serum, uncultured peptide, serum cultured without peptide (Yoo SA et al., J Immunol, 2005; 174: 5846-55).

All peaks (fractions) were collected for clarification of peaks isolated from C18 reversed phase HPLC profiles, lyophilizes under reduced pressure, and the method described in the previous literature (Yoo SA et al., J Immunol, 2005; 174: 5846-55). The mass spectrometry was analyzed as well. Original anti-flt-1 hexapeptide (100pM) and its D-type derivative (100pM), lyophilized samples under reduced pressure from column peaks were analyzed by 4700 proteomics analyzer MALDI-TOF / TOF (TOF / TOF; Applied Biosystems) All mass spectrometric spectra were recorded in positive reflector mode.

As a result, as in FIG. 1A, we found that the 2D, 3D, 4D, and 5D anti-flt-1 peptides significantly lost their inhibitory ability, while the original L for 1D, 6D and all D peptides. Compared with the type peptide, it was confirmed that there is an activity of inhibiting binding with the receptor flt-1 in a dose-dependent manner.

In addition, the inventors confirmed the type of D-type peptide that increases the stability in the serum by C18 reversed phase HPLC. As shown in FIG. 1B, the half-life of the all-D peptide was 2 days or more. Both the -flt-1 peptide and the 6D peptide were 30 minutes or less, indicating that only one amino acid modification was insufficient to increase the serum stability of the anti-flt-1 peptide. In FIG. 1C, the molecular weights of the peaks b were all identical in the original all D peptide, since the peak b shown in the HPLC showed exactly the same molecular weight as the peptide (100 pM) loaded in the mass spectrometer.

Therefore, in consideration of both the stability and the inhibitory ability of the modified peptides synthesized by the inventors, it was confirmed that the all (D) peptide has an excellent increased stability without a large loss of activity, even in the in vivo system It was found to be the most appropriate treatment to be applied. Therefore, the present inventors proceeded with the following experiments by selecting only the all D anti-flt-1 hexapeptide, and additionally conjugated mini-PEG (mini-PEG-all D) to in vivo stability. Follow-up measures were applied to further strengthen the In addition, the chemical structure of the mini-PEG-all (all) D peptide is shown in Figure 1D.

< Example  2>

Form D anti-according to the present invention flt -One Of peptide VEGF ₁₆₅ Of angiogenesis inhibitory effects

We tested whether mini-PEG-all D samples could block angiogenesis more effectively in in vivo conditions.

First, the present inventors have conducted neovascularization of L-type, all-D, mini-PEG-all-D anti-flt-1 induced VEGF ₁₆₅ in mice in which Matrigel plugs were placed. Compared to Anti-flt-1 peptide L, all D, mini-PEG-all D and reversed in Matrigel (500 μl) containing VEGF ₁₆₅ (500 ng / ml) and heparin (9 units / ml) Samples with or without the reverse peptide were injected subcutaneously in 7-week-old C57BL / 6 mice, respectively (Kong JS et al., Arthritis Rheum, 2010; 62: 179-90).

After 14 days, the skin of the mice was reversed and exposed to Matrigel plugs. After photographing and taking note of quantitative differences, hemoglobin levels were measured according to the Drabkin's method using a Drabkin reagent kit 525 (Sigma). Hemoglobin concentration was calculated from a known assay of the amount of hemoglobin, matrigel plugs were embedded with paraffin and stained with H & E. The migrated cells were counted under an optical microscope with 200 × magnification.

New vessels formed in vivo were filled with H & E stained, abundant original red blood cells, indicating the formation of functional vasculature through Matrigel. To quantify angiogenesis in these samples we measured the hemoglobin contents of Matrigel. As in FIG. 4A, the average hemoglobin amount in mini-PEG-all D treated Matrigel (n = 15) was Matrigel with all D (n = 12), L-type peptide (n = Much lower than that of Matrigel, including 9). In addition, the stained sections showed significantly higher levels of cells to which all D-PEG-treated Matrigels migrated than those of Matrigels including all D and reverse peptides. Showed low.

In addition, the inventors confirmed whether the effect of the addition of all the D-mini-PEG peptide will vary depending on the type of cells. As a result, the frequency of anti-vWF (+) cells typified by endothelial cells, as shown in FIG. 4C, was significantly higher in the gel treated with the mini-PEG-all (D) peptide compared to the gel treated with the reverse peptide. Decreased. The number of cells positive for the anti-MOMA antibody (specific antibody for detecting macrophages) showed a similar pattern as in FIG. 4D, indicating that mini-PEG-all (D) peptides were not only able to produce angiogenesis, It proves that it inhibits phagocytic infiltration.

Overall, the data showed that the inventors found that all three anti-flt-1 acts to reduce angiogenesis in vivo and furthermore, mini-PEG-all D Peptides were found to have better anti-angiogenic activity than other peptides.

< Example  3>

Form D anti-according to the present invention flt -One Of peptide  Analysis of Arthritis Treatment Effect by Oral Administration

Therapeutic effect analysis experiments on arthritis-induced animal models of D-type peptides according to the present invention have been reported (Yoo SA et al., J Immunol, 2005; 174: 5846-55., Yoo SA et al., Arthritis Rheum, 2009; 60). : 345-54., Kong JS et al., Arthritis Rheum, 2010; 62: 179-90), and borrowed accordingly.

Male DBA / 1 mice, 7-8 weeks old (The Jackson Laboratory, Bar Harbor, ME), were immunized with bovine type II collagen (CII; Chondrex, Redmond, WA) and were first immunized (primary). Three weeks after immunization, L- or PEG-all D-type peptides (100 μg) of anti-flt-1 peptide (100 μg) were administered to the mice every other day for 4 weeks and control mice (control mice). ) Only vehicle was administered. The incidence and severity of arthritis was confirmed by visual inspection as in the literature. Mice were sacrificed on day 49 for histologis analysis and paws and ankles (except for hind paws with immunization promoters) were collected for inflammation, synovial hyperplasia in the joints. (synovial hyperplasia) and degree of joint destruction were assessed according to standard scoring protocols (0 = none, 1 = weekly, 2 = mild, 3 = moderate, 4 = severe.

On the other hand, in general, when oral administration of peptides, bioavailability is significantly reduced due to their lack of visceral digestion and difficulty in enzymatic degradation. In consideration of this point, the present inventors confirmed that the biological activity of arthritis animal model (in vivo) remains intact when orally administered the peptide according to the present invention, which is D-type and mini-PEGylated (PEGylated), unlike L-type. Was intended.

As a result, as shown in FIG. 5A, oral administration of type L (100 μg) did not significantly inhibit the exacerbation of collagen-induced arthritis (when administered every other day for 3 to 4 weeks after initial immunization). . However, the oral administration of mini-PEG-all (D) peptide (100 μg) every other day inhibited the exacerbation of arthritis in mice, which was similar to the subcutaneous administration. Joints of the control group were filled with inflammatory cells and spindle-shaped fibroblast-like synoviocytes via H & E staining, whereas mini-PEG-ol all) The joints of mice administered D were less affected (see FIG. 5B).

When histological severity was assessed according to standard scoring protocols, the toenails and ankles of mice administered mini-PEG-all (D) peptide (100 μg) had a high degree of inflammation, hyperplasticity of bone cells and bone destruction. It was found to be insignificant compared to that of the mice treated only (vehicle) (see FIG. 5C).

In addition, immunohistochemical staining of synovial cells of the joints of vWF and MOMA antigens revealed that macrophage infiltration and neovascularization in mice treated with vehicle were very severe (see FIG. 5D), mini-PEG-ol (all ) Angiogenesis and macrophage infiltration were only moderate in the joints of mice treated with D peptide.

Through these experimental results, the inventors, as can be seen in Figures 1 to 4, all D-mini-PEG peptides, unlike the L form effectively inhibits the exacerbation of arthritis on in vivo (in vivo), and oral administration Even when it can be seen that there is an excellent effect stably inhibiting angiogenesis stably.

< Example  4>

sign In vitro  Form D anti-according to the present invention flt -One Of peptide  Angiogenesis Inhibitory Activity Assay

The inventors have previously reported the evaluation of VEGF binding inhibitory activity to the flt-1 receptor (VEGFR1) in vitro of the anti-flt-1 peptide and modified D-type peptides according to the present invention. ELISA was used in the same manner (Bae DG et al., Clin Cancer Res 2005; 11: 2651-61). In addition, human umbilical vein endothelial cells (HUVECs) were selected as endothelial cells to be used in this experiment.

HUVECs can be obtained from normal-term umbilical cords through collagenase digestion, as previously reported (Kong JS et al., Arthritis Rheum, 2010; 62: 179-90). veins) and grown and maintained in an environment under M199 medium containing 20% fecal calf serum (GES) in gelatin-coated dishes.

In order to analyze the activity of peptides according to the present invention (proliferation and survival analysis of HUVECs, capillary formation assays, etc.), the method shown in previously reported literature (Kong JS et al., Arthritis Rheum, 2010; 62: 179-90) Utilized. Thus, the proliferation and survival of HUVECs were measured by [ ³ H] -thymidine incorporation assay and MTT assay, capillary tube formation assay was also performed, and endothelial chemotaxis (chemotaxis) and wound migration (wounding migration) degree was confirmed.

As a result of confirming the effect of mini-PEG-all (D) peptide on the survival and proliferation of HUVEC, as shown in Figure 2A, L-type, all (all), mini-PEG-all (D) peptide 100 After addition of μg to HUVEC, no effect was found for 24 hours when the cell viability was examined through an MTT assay that ruled out the possibility of non-specific cytotoxicity due to peptides. In addition, the increase in [3H] -thymidine incorporation with VEGF ₁₆₅ into HUVEC was not reduced due to all three anti-flt-1 peptide derivatives (see FIG. 2B). This means that these peptides do not affect the proliferation of HUVECs.

Conjugation of PlGF or VEGF to its receptor flt-1, on the other hand, is an important step in stimulating tube formation, migration and chemotaxis of HUVECs. We therefore tested whether mini-PEG-all (D) samples could regulate endothelial tube formation, migration and chemotaxis. As a result, as in FIG. 2C, the formation of tube-like structures induced by VEGF ₁₆₅ was significantly reduced due to the mini-PEG-all D peptide. In addition, PIGF-induced tube formation, as in FIG. 2D, was also completely blocked due to both all D and mini-PEG-all D anti-flt-1 peptides. In addition, all D and mini-PEG-all D peptides inhibited the reduction of migration in response to wounding due to VEGF ₁₆₅ or PIGF-induced wounds, as in FIG. 3A. The chemotaxis of HUVECs stimulated with VEGF ₁₆₅ or PIGF in Boyden chambers was completely delayed due to the mini-PEG-all D peptide as shown in FIG. 3B.

Overall, the data showed that the present inventors confirmed that tube formation, migration and chemotaxis of endothelial cells in vitro was inhibited through mini-PEGylated all D anti-flt-1 peptide.

< Example  5>

Mini- according to the present invention PEG All D type Of peptide  Colorectal cancer treatment effect analysis

In order to analyze the therapeutic effect of mini-PEG conjugated type D peptides in colorectal cancer, Bae DG et al., J Biol Chem, 2000; 275: 13588-96., Bae DG Et al., Clin Cancer Res, 2005; 11: 2651-61), subcutaneously injecting CT-26 colorectal cancer cell lines to induce colorectal cancer in animal models, and tumor mass was measured 6 weeks thereafter. After fixation in% paraformaldehyde and embedded with paraffin, changes in tumor cells were observed.

As a result, when treated with the mini-PEG-ol (D) peptide as shown in Figure 6A, it was found that the tumor size was significantly reduced compared to the case of the control group mice, as shown in Figure 6B It was also confirmed that the weight of was sharply reduced. In addition, as shown in FIG. 6C, it was found that anti-vWF antibody (+) and anti-MOMA antibody (+) cells were hardly found in cancer cells of mice to which the peptides according to the present invention were administered. Therefore, the present inventors were able to confirm that the mini-PEG-ol (D) peptide according to the present invention has an effect of slowing metastasis including growth of tumor cells.

< Example  6>

Within the joint tissue PEG All D type Of peptide  Location analysis

To analyze the distribution and location of mini-PEG-ol D-type peptides in joint tissues, mice injected with arthritis with collagen were injected subcutaneously with Cy5-labeled mini-PEG-ol D-type peptides (500 ug). Blood was collected from the mice at 0, 1, 4 and 24 hours after peptide injection. In addition, after all the blood samples were killed, joint tissues, liver, spleen, lungs and kidneys were separated, and each tissue was observed using a Maestro in vivo fluorescence imaging system (CRI Inc, Woburn, MA). At this time, a normal mouse which did not induce arthritis was used as a control.

As a result, as shown in FIG. 7, the blood collected by time after injection of the Cy5-labeled peptide was observed. As a result, the Cy5 signal in serum showed the strongest signal at 1 hour and then until 4 hours. It was shown to persist (see FIG. 7A). In addition, as a result of observing Cy5 signal of each tissue of the mouse, strong signals were observed in kidney, spleen, liver and lung at 24 hours after peptide injection (see FIG. 7B). Perhaps this result suggests that the injected peptide spreads through the blood to each tissue in the body.

In addition, Cy5-labeled peptides were found to accumulate in the articular tissues of arthritic mice, which is in good agreement with the Arthritis Symptom Index analysis. In other words, it can be seen that the peptide of the present invention accumulates with inflammatory cells expressing the flt-1 receptor in a place where arthritis symptoms are serious (see FIG. 7C).

< Example  7>

mini- PEG All D type Peptides and flt -1 binding analysis

QCM (quartz crystal microbalance; stanford research systems, Sunnyvale, CA) was used to measure the binding of flt-1 and mini-PEG-ol D-type peptides in situ, and the Sauerbrey equation The degree of binding was analyzed.

Where f _o represents the oscillation frequency (5 MHz) of the unloaded crystal, A represents the active area (1.267 cm ² ) between the electrodes, p _q represents the density of the oscillator (2.648 g / cm 3) , u _q represents the shear modulus (2.947 x 10 ¹¹ g / cm / s ² ) relative to the vibrator.

The gold-coated vibrator crystals were first washed by UV irradiation for 20 minutes and placed in a 150 ul flow cell, the flow rate being a syringe pump (WPI, Sarasota, FL with low pressure liquid chromatography injection valve and 250 ul injection loop). ) To adjust. Each sample was then loaded into a 250 ul injection loop and flowed at a flow rate of 10 ul / min. The flow rate cell was washed with buffer solution after loading was complete, 10 ug / ml thiol-modified mini-PEG-ol D anti-flt-1 peptide was attached to the gold surface of the vibrator crystals and blocked using 0.1% BSA. . Thereafter, 500 ng / ml of sflt-1 was reacted by flowing on a mini-PEG-ol D anti-flt-1 peptide attached to the gold surface to analyze their specific binding degree. At this time, as a control, 0.1% BSA was first adsorbed onto the vibrator crystal to which the mini-PEG-ol D peptide was not attached, and then sflt-1 was used.

As a result, as shown in Figure 7d, the adsorption of slft-1 on the mini-PEG-ol D peptide appeared to induce a frequency shift of -4.3 Hz, the binding of the mini-PEG-ol D peptide and slft-1 Was found to be due to specific binding.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Attach an electronic file to a sequence list

Claims (7)

Form D peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 and 8-amino-3,6-dioxaoctanoic acid (8-amino-3,6-dioxaoctanoic acid; mini-PEG) Composition for inhibiting angiogenesis, containing the conjugate as an active ingredient. delete The method of claim 1,
The peptide composition for inhibiting angiogenesis, characterized in that it inhibits or interferes with the binding of vascular endothelial cell growth factor (VEGR) and vascular endothelial cell growth factor receptor (VEGFR). The method of claim 1,
The composition is an angiogenesis inhibiting composition, characterized in that it can prevent or treat diseases caused by abnormal or excessive angiogenesis. 5. The method of claim 4,
The disease may include cancer, diabetic retinopathy, prematurity retinopathy, corneal graft rejection, neovascular glaucoma, melanoma, proliferative retinopathy, psoriasis, hemophiliac joints, capillary hyperplasia in atherosclerotic plaques, keloids, wound granulation, Vascular adhesion, rheumatoid arthritis, osteoarthritis, autoimmune disease, Crohn's disease, restenosis, atherosclerosis, intestinal adhesion, cat scratch disease, ulcers, cirrhosis, glomerulonephritis, diabetic nephropathy, malignant neurosis, thrombotic microangiopathy, Organ transplant rejection, renal glomerulopathy, diabetes, inflammation and neurodegenerative diseases selected from the group consisting of diseases for inhibiting angiogenesis. The method of claim 1,
The composition is an oral preparation or composition for inhibiting angiogenesis, characterized in that the preparation for injection. The method according to claim 6,
The oral preparation is an angiogenesis inhibiting composition, characterized in that selected from the group consisting of capsules, tablets, pills, suspensions, syrups and powders.

Images