WO2017111388A1 - Composition comprising periostin-derived peptide as active ingredient for promoting angiogenesis - Google Patents

Abstract

The present invention relates to a composition comprising a periostin-derived peptide as an active ingredient for promoting angiogenesis, a composition for preventing or treating ischemic disease, and a composition for wound healing. The periostin-derived peptide according to the present invention comprises at least ten amino acids, facilitating the preparation and mass production of peptides, and has excellent effects of promoting the migration of vascular endothelial progenitor cells, promoting angiogenesis, promoting vasculogenesis, promoting the reepithelialization of skin wounds, at low concentrations compared with when the whole proteins are used, and thus, the periostin-derived peptide can be usefully utilized as a composition for promoting angiogenesis and a composition for treating vascular diseases, especially, ischemic disease and wounds.

Description

Composition for promoting angiogenesis comprising periostin-derived peptide as an active ingredient

The present invention relates to a composition for promoting angiogenesis, a composition for preventing or treating ischemic diseases, and a composition for treating wounds, comprising a periostin-derived peptide as an active ingredient.

Angiogenesis is the process by which new blood vessels are made from existing blood vessels, and the process of migration of vascular endothelial cells that make up blood vessels and infiltration through extracellular matrix (ECM), an intercellular barrier. (invasion) process, the proliferation process of the cells constituting the blood vessels (tube formation) and the process (tube formation) is made. This series of reactions is known to be tightly regulated by the balance between angiogenesis factors and angiogenesis inhibitors. These neovascularization reactions are rarely suppressed under normal conditions and occur mainly when early embryonic development, wound healing and periodic female genital system changes. Except for this case, the replacement of blood vessels is slow, and most of the capillary endothelial cells in normal tissues can be considered to be stationary.

Vessel disease, particularly ischemic disease, is a term that refers to a disease that causes various forms of pathological abnormalities in the blood vessels that supply blood to each organ of the body, resulting in disorders of normal blood flow. . Blood supply through blood vessels is an essential phenomenon for wound healing and tissue regeneration. In particular, diseases such as arteriosclerosis, myocardial infarction and angina pectoris are caused by poor blood supply.

In addition, angiogenesis must be involved in the necessary wound healing process for the rejuvenation of wounded skin tissue. In the early stages of the wound, inflammatory reactions occur due to necrosis of cells and destruction of blood vessels, and after inflammatory reactions, such as kallikrein, thrombin, and plasmin, with devascularization of blood components, activation of platelets and coagulation It goes through a series of processes in which biological mediators are formed.

On the other hand, periostin is an extracellular matrix (ECM) of 90 kDa in size, overexpressed in various cancers of humans and is involved in tumor growth and metastasis. Periostin has been mainly studied in relation to cancer, and recently reported that overexpression of periostin in mesenchymal stem cells and application to the myocardial infarction model of rats are better than that of mesenchymal stem cells. (Cho YH, Biomaterials, 2012, 33 (5), 1376-85). However, since the expression of periostin is difficult compared to other proteins, a high cost is required in the manufacturing process, and there is a problem that it is difficult to be used commercially.

The present inventors continued to develop a novel angiogenesis-promoting composition that overcomes the above-mentioned problems. As a result, instead of using the entire protein of periostin, the inventors select a minimum unit of peptides having angiogenic activity, The present invention has been completed by confirming that it can be used to treat ischemic diseases or wounds.

An object of the present invention is to provide an angiogenesis composition comprising a periostin-derived peptide as an active ingredient or an angiogenesis-promoting method comprising administering the periostin-derived peptide to a subject.

Another object of the present invention is to provide a composition for the prevention or treatment of ischemic disease comprising periostin-derived peptide as an active ingredient or a method for preventing or treating ischemic disease comprising administering the periostin-derived peptide to a subject. .

Still another object of the present invention is to provide a wound treatment composition comprising periostin-derived peptide as an active ingredient or a wound treatment method comprising administering or applying the periostin-derived peptide to an individual.

In order to solve the above problems, the present invention provides a composition for promoting angiogenesis comprising a peptide comprising at least one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 4 as an active ingredient.

The present invention also provides a method for promoting angiogenesis comprising administering the peptide to a subject in need thereof.

The present invention also provides a pharmaceutical composition for the prevention or treatment of ischemic disease comprising the peptide as an active ingredient.

In another aspect, the present invention provides a food composition for preventing or improving ischemic disease comprising the peptide as an active ingredient.

The present invention also provides a method for preventing or treating ischemic disease, comprising administering the peptide to a subject in need thereof.

The present invention also provides a pharmaceutical composition for treating wounds comprising the peptide as an active ingredient.

In addition, the present invention provides a skin external preparation for wound treatment comprising the peptide as an active ingredient.

The present invention also provides a method for treating wounds comprising administering or applying the peptides to a subject in need thereof.

Periostin-derived peptides according to the present invention are composed of at least ten amino acids, which facilitates the production and mass production of peptides, and promotes the migration of vascular endothelial progenitor cells, promotes angiogenesis, and promotes angiogenesis at low concentrations compared to using whole proteins. The re-epithelialization promoting effect of the skin wound is excellent, it can be usefully used as a composition for promoting angiogenesis, vascular diseases, in particular ischemic diseases and wound treatment.

FIG. 1 is a diagram showing a vector map and a selected peptide site for preparing a peptide used for screening a site having angiogenic function in periostin first FAS I domain.

Figure 2 shows the results confirming the effect of the periostin-derived peptides of various lengths on the movement of vascular endothelial progenitor cells.

Figure 3 is a diagram showing the results confirming the effect of the peptide consisting of 136 amino acids to 151 amino acids of periostin on the movement of vascular endothelial progenitor cells.

Figure 4 is a view showing the results of confirming the effect of the peptide consisting of the 136 th amino acid to 151 th amino acid of periostin on the vessel formation.

Figure 5 shows the results confirming the effect of the peptides of various lengths including some of the 136 th amino acid 151 amino acids of periostin on the movement of vascular endothelial progenitor cells.

Figure 6 is a view showing the results confirming the effect of the peptides of various lengths including some of the amino acids 136 to 151 of periostin on the formation of vasculature.

7 and 8 illustrate the results of confirming the effect of the peptide consisting of 142 th amino acid to 151 th amino acid of periostin in the wound animal model to the wound treatment.

The present invention provides a composition for promoting angiogenesis comprising a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

The present invention also provides a method for promoting angiogenesis comprising administering the peptide to a subject in need thereof.

Hereinafter, the content of the present invention will be described in more detail.

In the present invention, the amino acid sequence represented by SEQ ID NO: 1 is located at 142 th to 151 th positions of the periostin protein (NCBI GenBank Accession No. NP006466.2), and is located within the first FAS I domain of the periostin protein. The amino acid sequence is as follows and variants thereof are included within the scope of the present invention.

* SEQ ID NO 1: [N terminal-W-D-N-L-D-S-D-I-R-R-C terminal]

In the present invention, the term "peptide" means a polymer consisting of amino acids linked by amide bonds, and the amino acids may be L-form or D-form.

In the present invention, the term "amino acid" is a concept comprising natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a similar manner to natural amino acids. Natural amino acids are amino acids encoded by the genetic code, and amino acid analogs are compounds having α carbon bonded to hydrogen, carboxyl, amino and R groups, such as natural amino acids, modified R groups (e.g., norleucine) or modified polypeptides. It may have a skeleton. An amino acid mimetic means a compound that has a structure that differs from the general chemical structure of an amino acid, but which acts in a similar manner to a natural amino acid.

Amino acid sequences used in the present invention are described as follows according to the IUPAC-IUB nomenclature.

Arginine: R, Histidine: H. Lysine: K, Aspartic acid: D, Glutamix acid: E, Serine: S, Threonine: T, Asparagine: N, Glutamine: Q, Cysteine: C, Glycine: G, Proline: P, Alanine: A, Isoleucine: I, Leucine: L, Methionine: M, Phenylalanine: F, Tryptophan: W, Tyrosine: Y, Valine: V.

In the present invention, the peptide as an active ingredient is a peptide in which other amino acids, peptides, etc. are fused to the N or C terminus of the amino acid sequence represented by SEQ ID NO: 1 and include all peptides showing angiogenic activity.

In the present invention, the peptide as an active ingredient may be a peptide in which several tens to several amino acids, preferably 1 to 10 amino acids are added to the N terminus of the amino acid sequence represented by SEQ ID NO: 1, and more preferably, a sequence. It may consist of the amino acid sequence of any one of Nos. 2 to 4. In other words, the peptide of the present invention may include the 142 th to 151 th amino acid sequences based on the known periostin amino acid sequence, and may further include a plurality of consecutive amino acids located before the 142 th. .

The amino acid sequence represented by SEQ ID NO: 2 to 4 is as follows.

* SEQ ID NO 2: [N-terminal-E-A-W-D-N-L-D-S-D-I-R-R-C terminal]

* SEQ ID NO 3: [N terminus-S-N-E-A-W-D-N-L-D-S-D-I-R-R-C terminus]

* SEQ ID NO 4: [N-terminal-A-P-S-N-E-A-W-D-N-L-D-S-D-I-R-R-C terminal]

In the present invention, the peptide as an active ingredient may be a peptide in which several amino acids are added to the C terminus of the amino acid sequence represented by SEQ ID NO: 1 based on the known amino acid sequence of periostin. In other words, the peptide of the present invention may include a 142 th to 151 th amino acid sequence based on the amino acid sequence of the known periostin, and may further include a plurality of consecutive amino acids located after the 151 th portion. .

In the present invention, the peptide, which is an active ingredient, may be in a form in which the N- or C-terminal is modified or protected by various organic groups to protect from protein cleavage enzymes in vivo and increase stability. That is, the C terminus of the peptide is not particularly limited as long as it can be modified to increase stability, but may be preferably modified with a hydroxy group (-OH) or an amino group (-NH ₂ ). In addition, as long as the N-terminal of the peptide can be modified to increase the stability, there is no particular limitation, but preferably an acetyl (Acetyl), fluorenyl methoxy carbonyl (Fmoc) group, formyl (Formyl) group , Palmitoyl (Palmitoyl) group, Myristyl (Myristyl) group, stearyl (Stearyl) group and polyethylene glycol (PEG) may be modified with a group selected from the group consisting of.

Peptides of the present invention can be synthesized by methods well known in the art, for example, automated peptide synthesizers, and can be produced by genetic engineering techniques, but is not limited thereto.

Peptides comprising the amino acid sequence represented by SEQ ID NO: 1 of the present invention may be encoded by the nucleotide sequence represented by SEQ ID NO: 5 and variants that can function functionally the same are included within the scope of the present invention.

* SEQ ID NO 5: tgggacaact tggattctga tatccgtaga

Conventional angiogenic activity of periostin protein has been reported, but since 90 kDa sized periostin protein is difficult to control and purify expression, high cost is required for mass production, and its use is limited. On the contrary, the periostin-derived peptide according to the present invention is a peptide containing ten amino acids located in the 142 th to 151 th positions of periostin as the minimum unit, and has a short length, which makes mass production easy and commercially available. have. In one embodiment of the present invention, a periostin-derived peptide was prepared by removing 10 amino acids from the C-terminal side of the first FAS I domain of periostin, and 137 of periostin through the screen for promoting the migration of vascular endothelial progenitor cells. It was confirmed that the movement promoting activity of vascular endothelial progenitor cells appeared in the peptide including the site after the fourth amino acid. In addition, in one embodiment of the present invention, further screening was performed to identify the minimum unit of the peptide including the 137th amino acid of periostin, and the peptide comprising the 136th to 151th consecutive amino acid sequences of periostin is preferred. Preferably, peptides containing the 142th to 151th consecutive amino acid sequences of periostin have been found to have an excellent effect of promoting vascular endothelial progenitor cells, promoting angiogenesis, promoting angiogenesis, and promoting re-epithelialization of skin wounds. Peptides from which two or more amino acids were removed from the short or the 151 th amino acid forward were significantly reduced in their effects on vascular endothelial progenitor cell migration and angiogenesis.

Therefore, the present invention provides a composition for promoting angiogenesis comprising a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient, and a method for promoting angiogenesis comprising administering the peptide to an individual in need thereof. .

In addition, the present invention provides a disease caused by inhibiting angiogenesis, including the peptide as an active ingredient, or a disease that can be cured by promoting angiogenesis, preferably ischemic disease or a wound treatment composition.

The composition comprises a pharmaceutical composition or a food composition.

The composition may be provided in the form of an external preparation for skin for wound treatment.

The present invention also provides a method for preventing or treating ischemic disease, or a method for treating a wound, comprising administering the peptide to a subject in need thereof.

In the present invention, the term “angiogenesis” refers to the process by which blood vessels are newly formed, i.e., new blood vessels are generated into cells, tissues or organs, and when new blood vessels are produced and in existing blood vessels This includes all cases in which blood vessels extend.

In the present invention, the term "ischemic disease" refers to a problem in blood supply due to narrowing or contraction of blood vessels supplying blood to body organs, tissues, or sites due to various blood vessel abnormalities. Ischemic necrosis, ischemic cerebrovascular disease, ischemic kidney disease, ischemic pulmonary disease, limb ischemic disease, ischemic heart disease, stroke, cerebral infarction, myocardial infarction, ischemic heart failure and obstructive atherosclerosis Including but not limited to.

In the present invention, the term "wound" is intended to encompass all damage to the living body, and means damage to skin tissue such as surgical incisions, burns, lacerations, and includes wounds.

When the composition of the present invention is provided in the form of a pharmaceutical composition, it may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions in addition to the active ingredient. Carriers, excipients and diluents which may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions according to the present invention may be used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods, respectively. Can be. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like in the active ingredient. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, etc. may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention may further contain at least one known active ingredient having a periostin-derived peptide and having an angiogenic or wound healing effect.

In the present invention, the term "administration" means providing a subject with a composition of the present invention in any suitable manner.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. However, upon oral administration, since the protein is digested, it is desirable to formulate oral compositions to coat or protect the active agent from degradation in the stomach.

Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight of the individual, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. For preferred effects, the compositions of the present invention can be administered at 0.001 to 1000 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

When the composition of the present invention is provided as a food additive, the active ingredient may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient in the food composition may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverages, the active ingredient of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less with respect to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for health control, it may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the food composition of the present invention may include a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is usually selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The composition of the present invention is a skin external preparation formulation that can be applied to the skin, prepared in the form of a skin external preparation such as cream, gel, patch, spray, ointment, warning, lotion, linen, pasta or cataplasma. Can be used, but is not limited thereto.

In addition, the compositions of the present invention can be administered in a manner that is applied to the skin for the purpose of wound healing.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example  One. Periostin  For screening sites with angiogenic function in the first FAS I domain Peptide  refine

The first FAS I domain (hereinafter referred to as Periostin D1), which has the best angiogenic function among the periostin proteins, is selected and 10 amino acids from the C terminus to find the amino acid sequence position with angiogenic function in the domain. A total of 14 different length peptides were purified by removing amino acids.

More specifically, Escheria coli expression plasma construct (Escheria coli expression plasma construct) by using a site-selective mutation technique to express it in BL21 Escherichia coli, after the endotoxin test and endotoxin removal process according to a known method Clean peptides were purified. The vector map and peptide sites used in the experiment are shown in FIG. 1.

Example  2. Vascular endothelial progenitor cells  Specific to facilitate movement Peptide  Site identification

Of the 14 peptides purified in Example 1, in order to identify a specific site that promotes angiogenesis, Example 1 in human-derived endothelial progenitor cells (EPCs), which are cells existing in human blood vessels Each of 14 peptides or periostin (hereinafter, referred to as Periostin FL) purified from the peptides were treated and cell migration was measured.

More specifically, EPCs were cultured in Endothelial Cell Growth Medium-2, Clonetics Co., San Diego, USA (EGM-2) medium, and the cultured EPCs were 96-well chemotactic chambers (Neuro Probe, Inc., Gaithersburg, At the top of MD), the cells were dispensed at a concentration of 5000 cells per well. The chamber was treated with a polycarbonate filter having pores coated with 20 μg / ml rat-tail collagen, and each protein was placed at the bottom of the chamber at a concentration of 10 μg / ml. Incubated at 37 ° C. for 10 hours. The positive control was treated with VEGF (10 ng / mL), and the negative control was not treated with anything. After incubation, hematoxylin staining was used to measure the degree of cell migration. The results are shown in FIG.

As shown in FIG. 2, it was confirmed that the movement promoting effect of vascular endothelial progenitor cells in the peptide including the sequence region after the 137th amino acid (50 a.a) was superior to the positive control group.

Through the above experiment, the vascular endothelial progenitor cell migration-promoting activity was once again confirmed by treating the peptides containing the 137th amino acid of periostin, which is estimated to start the period for promoting the movement of vascular endothelial progenitor cells. The results are shown in FIG.

As shown in FIG. 3, the peptide consisting of the 136th to 151th amino acids of periostin (SEQ ID NO: 4) is a vascular endothelial at a relatively low concentration compared to the peptide consisting of the first FAS I domain (D1) region of periostin. It was confirmed that there is an effect of promoting the movement of progenitor cells. In particular, the degree of mobility of the peptides consisting of the 136th to 151th amino acids of periostin at 0.1 μg / ml was similar to that of 1 μg / ml of the peptide consisting of the first FAS I domain (D1) region of periostin. A peptide consisting of Austin's 136th to 151th amino acids promotes similar levels of vascular endothelial progenitor cell migration at a concentration 10 times lower than that of Periostin's first FAS I domain (D1). It confirmed that it was shown.

Example  3. Periostin  origin Peptide  Confirmation of promoting tube formation

In order to confirm whether the periostin-derived peptide of Example 2 promotes angiogenesis as well as movement of vascular endothelial progenitor cells, the following experiment was performed.

First, 50 μl of Matrigel (Matrigel, BD Bioscience, USA) was put into a 96-well plate and hardened to prevent bubbles. EGM-2 containing 2% fetal bovine serum (FBS) was treated with concentrations of periostin-derived peptides showing the effect of promoting the migration of vascular endothelial progenitor cells in a medium containing 1% FBS by concentration. After suspending EPCs cultured in the medium, the cells were inoculated at a concentration of 1 × 10 ⁴ cells per well and incubated at 37 ° C. VEGF was treated in the positive control group and nothing was treated in the negative control group. Comparative groups were treated with either periostin total protein (2 μg / mL) or the first FAS I domain (D1) of periostin (2 μg / mL), respectively. Calcein AM staining was performed after 10 hours, and the degree of angiogenesis was observed through a microscope. The results are shown in FIG.

As shown in Figure 4, in the treatment group of peptides (SEQ ID NO: 4) consisting of 136 amino acids to 151 amino acids of periostin, angiogenesis is promoted in spite of a relatively lower concentration than periostin protein. It was confirmed that the activity is excellent.

Example  4. Periostin  origin Peptides  To configure the minimum length Peptide  Screening Vascular endothelial progenitor cells  Movement promoting activity)

Since the shorter the length of the peptide can increase the production efficiency, in order to determine whether the sequence can be minimized within the interval between 136 and 151 amino acids of periostin, vascular endothelial progenitor cell migration in the same manner as in Example 2 Screening was performed to find the promoting active site. The peptide used a total of 11 peptides, including five different peptides from which the two amino acids were removed from the 135th amino acid to the rear and five different peptides from which the two amino acids were removed from the 151st amino acid to the front. Treatment was at a concentration of μg / mL. VEGF was treated in the positive control group and nothing was treated in the negative control group. The control group was treated with the total periostin protein or the first FAS I domain (D1) of periostin at a concentration of 10 μg / mL, respectively. The results are shown in FIG.

As shown in FIG. 5, when two amino acids are removed from the 135th amino acid to the rear, the activating activity of vascular endothelial progenitor cells is maintained from the 142th amino acid to the 151th amino acid peptide. And when the two or more amino acids are removed from the 151th forward anterior it was confirmed that the mobility of vascular endothelial progenitor cells is significantly reduced. Therefore, through the above experiment, the minimum length of the peptide excellent in promoting the movement of vascular endothelial progenitor cells is composed of 10 amino acids from 142 th amino acid to 151 th amino acid of periostin, which is 20 times lower than the total protein of periostin Nevertheless, it showed a remarkably excellent effect.

Example  5. Periostin  origin Peptides  To configure the minimum length Peptide  Screening (Angiogenic Activity)

In order to verify the results of Example 4, screening was performed to find an angiogenesis-promoting active site in the same manner as in Example 3, using a total of 11 peptides used in Example 4. The results are shown in FIG.

As shown in FIG. 6, when two amino acids are removed from the 135th amino acid to the rear, the angiogenesis-promoting activity is excellent from the peptide consisting of the sequence from the 142th amino acid to the 151th amino acid, and has a shorter length and the 151th amino acid. From the removal of two or more amino acids in the anterior it was confirmed that the degree of vessel formation is relatively low. Therefore, through the above experiment, it was confirmed that the minimum length of the peptide excellent in angiogenic activity is composed of 10 amino acids from 142 th amino acid to 151 th amino acid of periostin.

Example  6. Periostin  origin Peptide  Check the effect of wound healing

In order to confirm the wound healing effect of the peptide consisting of the 142 th amino acid to 151 th amino acid of periostin selected through Examples 4 and 5 as described below.

As experimental animals, 7-week-old C57 / BL6 mice were used, and each mouse was respiratory anesthesia with 2% concentration of Enflurane, and the back-waist hair was removed using an electric clipper. . The exposed area was sterilized with 70% ethanol, wounded with an 8 mm biopsy punch, and the wound was rounded off with scissors to avoid damage to the muscle layer. The wound site was treated with HBSS (Hank's balanced salt solution) containing a peptide consisting of 142 amino acids to 151 amino acids of 0.5 μM periostin (SEQ ID NO: 1), and the control group was treated only with HBSS. The wound site was visually observed on the 3rd, 6th, 9th and 12th days, and the wound area was measured. The results are shown in FIGS. 7 and 8.

As shown in FIG. 7 and FIG. 8, it was confirmed that the wound healing effect is excellent, such as rapid re-epithelialization of the wound site by the peptide consisting of the 142 th amino acid to the 151 th amino acid of periostin. Therefore, through the experiment, it was confirmed that the peptide consisting of 10 amino acids from 142 th amino acid to 151 th amino acid of periostin is excellent in promoting re-epithelialization and angiogenesis of the skin wound.

Through the above experiments, the periostin-derived peptides according to the present invention consist of at least ten amino acids, which facilitate the production and mass production of peptides, promote the migration of vascular endothelial progenitor cells, promote angiogenesis, promote angiogenesis, and rehabilitate skin wounds. It was confirmed that the epithelialization promoting effect can be usefully used as a composition for promoting angiogenesis and a composition for treating vascular diseases, particularly ischemic diseases and wounds.

Hereinafter, the formulation examples of the pharmaceutical composition and the food composition of the present invention will be described, but are not intended to limit the present invention only to be described in detail.

Formulation example  1. Preparation of pharmaceutical composition

1-1. Powder  Produce

20 mg of periostin-derived peptide

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

Periostin Derived Peptide 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

1-3. Preparation of Capsules

Periostin Derived Peptide 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

1-4. Preparation of Injectables

Periostin Derived Peptide 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄₂ H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

1-5. Liquid  Produce

20 mg of periostin-derived peptide

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

Formulation example  2. Preparation of Food Composition

2-1. Manufacture of health food

Periostin Derived Peptide 100 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium Phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Claims (8)

Composition for promoting angiogenesis comprising a peptide containing the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. According to claim 1, wherein the peptide is characterized in that 1 to 10 amino acids are further added to the N terminal of the amino acid sequence represented by SEQ ID NO: 1, composition for promoting angiogenesis. The composition for promoting angiogenesis according to claim 2, wherein the peptide comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 4. Pharmaceutical composition for the prevention or treatment of ischemic disease comprising a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. According to claim 4, wherein the ischemic disease ischemic necrosis, ischemic cerebrovascular disease, ischemic kidney disease, ischemic lung disease, limb ischemic disease, ischemic heart disease, stroke, cerebral infarction, myocardial infarction, ischemic heart failure and obstructive arteriosclerosis A pharmaceutical composition for the prevention or treatment of ischemic diseases, characterized in that selected from the group consisting of. Food composition for the prevention or improvement of ischemic disease comprising a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. A pharmaceutical composition for treating wounds comprising a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient. Skin external preparation for wound treatment comprising a peptide containing the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

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