WO2019054808A2 - Use of peptides as therapeutic agent for autoimmune diseases and bone diseases - Google Patents

Abstract

The present invention relates to a use of peptides as a therapeutic agent. It has been confirmed that the peptides of the present invention significantly inhibit the activity of T cells and the differentiation of T helper 17 cells (Th17 cells), which are associated with autoimmune diseases, and have remarkable effects of treating and ameliorating arthritis in an animal model of arthritis. Therefore, the peptides may be used as an active ingredient in therapeutic agents for various autoimmune diseases such as bone diseases, inflammatory diseases or rheumatoid arthritis

Description

Use of peptides as therapeutic agents for autoimmune diseases and bone diseases

The present invention relates to the use of a peptide as a therapeutic agent for bone diseases and autoimmune diseases and more specifically to a peptide comprising the amino acid sequence represented by the general formula 1 of the present invention and to a bone disease including osteoporosis of the peptide, Or rheumatoid arthritis, and the like.

Bone tissue is composed of various kinds of cells such as extracellular substance such as collagen and glycoprotein, osteoblast, osteoclast, and osteocyte. In particular, the balance between osteoblasts and osteoclasts is essential for a healthy skeletal system. In other words, bone metabolism and bone remodeling are important for maintaining homeostasis of bone in the balance between osteoblasts that make up the bone matrix and osteoclasts that absorb bone. Do.

Bone tissue is composed of various kinds of cells such as extracellular substance such as collagen and glycoprotein, osteoblast, osteoclast, and osteocyte. In addition, bone resorption by osteoclasts and new bone matrix formation and mineralization by osteoblasts are repeated metabolic processes, and bone formation due to osteoblast activity is greater than bone resorption due to osteoclast activity. The bone resurfacing is the process of removing old bone after growth and replacing it with a new bone. It is secreted from bone tissues such as hormone such as parathyroid hormone (PTH), calcitonin, estrogen, etc. and insulin-like growth factor I It regulates the activity balance of osteoblasts and osteoclasts through cytokines such as various growth factors and tumor necrosis factor-alpha (TNF-α), and maintains homeostasis. If the balance between osteoblasts and osteoclasts is broken, it will lead to diseases such as osteoporosis and arthritis.

In particular, when the balance between osteoblasts and osteoclasts is broken, excessive bone destruction by osteoclasts occurs, leading to diseases such as osteoporosis. These osteoclasts are specialized cells that are responsible for bone resorption in the bone metabolism process and are formed through differentiation programs from monocytes or macrophages, which are progenitor cells. In addition, since osteoclasts bind to bone through αvβ3 integrin or the like to form an acidic environment and secrete various collagenases and proteases to cause bone resorption, Suppression of cells can be an effective method of treating bone disease.

In addition, autoimmune diseases are caused by the immune system of the human body, which causes self-cells to attack their own cells. The human immune system basically recognizes microorganisms invading the human body and cancer cell development as external antigens, and has strong power to attack and remove them, but it does not attack against the self cells because of its self-tolerance. This is called the self-tolerance phenomenon of the human body. However, when self-tolerance of the immune system is destroyed, the body reacts to its own cell (or autoantigen), activates autoreactive T cells, and autoantibodies are constantly destroyed, causing inflammation and an immune response .

T cells and B cells are specific for the antigen in the immune system. T cells respond to specific antigens presented by antigen presenting cells. When the antigen presented in the antigen-presenting cells is recognized as 'non-self', the immune system Response, and when it is recognized as 'self', the immune response is neglected. When T cells are activated against an antigen, most of the B cells are activated one after another, and B cells turn into plasma cells, producing antibodies that specifically react to the recognized specific antigen. Therefore, when autoimmunity occurs, the T cell abnormally recognizes the autoantigen and activates the B cell, thus generating an autoantibody that reacts with the autoantigen. An immune response occurs.

Similarly, in organ transplant recipients, organ transplantation after organ transplantation recognizes the tissue as 'non-self', resulting in rejection of organ transplantation that attempts to attack and remove the transplanted organ. In order to suppress this, various immunosuppressants have been used, such as inhibiting the activation of immune cells and inhibiting migration of immune cells to transplanted organs. However, the use of continuous immunosuppressants causes various side effects.

In addition, it has been recently shown that Th17 cells, a CD4 + T cell line, plays a key role in inflammation induction and progression of autoimmune diseases. The significance of such Th17 cells is that IL-17 secreted from these cells is directly associated with autoimmune diseases But it is getting bigger as it is revealed that there is a relationship.

In addition, Th17 cells induce RANKL and several inflammatory cytokines (Chabaud and Miossec, 2001; Connell and McInnes, 2006), which are known to further activate the inflammatory and joint destruction mechanisms. Therefore, since Th17 cells are recognized as a key tumor cell in the signal transduction process related to autoimmune diseases including osteoarthritis and bone diseases, candidate substances that effectively inhibit Th17 cell differentiation are required to be discovered.

In addition, rheumatoid arthritis is an inflammatory disease characterized by polyarthritis, an autoimmune phenomenon is known as a major mechanism. Symptoms include inflammation of synovial membrane tissue, macrophages, dendritic cells, T lymphocytes, and B lymphocytes, which migrate to synovial tissue, resulting in increased joint fluid and joint pain. When inflammation continues, hyperplasia of inflammatory synovial tissue destroys bones and cartilage, deforming the joint structure and causing movement disorder. Various studies have also shown that inflammatory cytokines in rheumatoid arthritis patients produce collagenase and protease in synovial fibroblasts and chondrocytes and these enzymes destroy collagen and proteoglycan And it is known to destroy cartilage.

Accordingly, the present inventors have made efforts to develop a new therapeutic agent for bone diseases and autoimmune diseases having an effective therapeutic effect while minimizing adverse effects as therapeutic agents. As a result, the present inventors have found that the peptide produced by the present invention is useful as a bone or osteoporosis- The present invention has been completed based on the finding that the compounds of the present invention can be effectively used for the treatment of autoimmune diseases including arthritis.

It is an object of the present invention to provide peptides for the prevention and treatment of bone diseases, inflammatory diseases and autoimmune diseases.

In order to achieve the above object,

A peptide composed of the amino acid sequence of the general formula 1 described in the following SEQ ID NO: 1, and a pharmaceutical composition for preventing and treating bone diseases, inflammatory diseases and autoimmune diseases containing the peptide as an active ingredient, A health food for the prevention and improvement of autoimmune diseases is provided.

[Formula 1]

(X ₁ -X ₂ -X ₃ ) _n

Wherein X ₁ to X ₃ are each independently selected from the group consisting of proline P, serine S, threonine T, glutamine Q, asparagine N, and cysteine C ), ≪ / RTI >

_n is an integer of 1 to 10, provided that the amino acid sequence of the general formula (1) includes the PSP represented by SEQ ID NO: 2 and _n = 1 to 3 at the same time.

The peptides of the present invention significantly inhibited T cell (T cell) activity and Th17 cell (T helper 17 cell) differentiation associated with autoimmune diseases, and it was confirmed that the peptides have a remarkable effect of treating and improving arthritis in an animal model of arthritis , The peptides may be used as active ingredients in various autoimmune disease therapeutic agents such as bone diseases, inflammatory diseases or rheumatoid arthritis.

1 is a graph showing the population (%) of active T cells by the synthetic peptides of the present invention.

Figure 2 is a graph showing the population (%) of active T cells by the synthetic peptide of the present invention.

Fig. 3 is a graph showing percent inhibition of T cell activation by the synthetic peptide of the present invention. Fig.

4 is a graph showing the inhibitory effect of Th17 cell differentiation on the synthetic peptide of the present invention.

FIG. 5 is a schematic diagram schematically showing the preparation process of the collagen-induced arthritis mouse model and the administration time of the peptide according to the present invention.

6 is a graph showing the effect of the Pep1 peptide of the present invention on improving arthritis.

FIG. 7 is a graph showing the arthritis improving effect of the Pep2 peptide of the present invention. FIG.

FIG. 8 is a graph showing the arthritis improving effect of the Pep4 peptide of the present invention. FIG.

9 is a graph showing the arthritis improving effect of the Pep6 peptide of the present invention.

Hereinafter, terms of the present invention are defined as follows.

In the present invention, the usual 1-letter and 3-letter codes for naturally occurring amino acids are used, as well as those generally accepted for other amino acids such as Aib (alpha -aminoisobutyric acid), Sar (N-methylglycine) Three character codes are used. Also, the amino acids referred to herein by abbreviations have been described in accordance with the IUPAC-IUB nomenclature.

&Quot; Peptide " of the present invention means a polymer composed of two or more amino acids linked by an amide bond (or a peptide bond). For the purpose of the present invention, a peptide having an effect of treating bone diseases, inflammatory diseases, .

&Quot; Stability " of the present invention also means storage stability (e.g., room temperature storage stability) as well as in vivo stability that protects the peptide of the present invention from attack by a protein cleaving enzyme in vivo.

By " prophylactic " of the present invention is meant any action which inhibits the disease or delays its development by administration of the pharmaceutical composition according to the invention.

The term " treatment " of the present invention means any action that improves or alters the symptoms of the disease by administration of the pharmaceutical composition according to the present invention.

The term " individual " of the present invention means a subject in need of treatment of a disease, and more specifically refers to mammals such as primates, mice, dogs, cats, horses and cattle, which are human or non-human.

&Quot; Improvement " of the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated, for example.

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

The present invention

There is provided a peptide comprising the amino acid sequence of the general formula 1 described in the following SEQ ID NO: 1, and a pharmaceutical composition for preventing and treating bone diseases, inflammatory diseases or autoimmune diseases containing the peptide as an active ingredient:

[Formula 1]

(X ₁ -X ₂ -X ₃ ) _n

Wherein X ₁ to X ₃ are each independently selected from the group consisting of proline P, serine S, threonine T, glutamine Q, asparagine N, and cysteine C ), And _n is an integer of 1 to 10, provided that the amino acid sequence of the general formula (1) is the PSP represented by SEQ ID NO: 2 and _n = 1 to 3 at the same time.

The above peptides can be used to produce various peptides using the above general formula 1, all of which are included in the present invention. X ₁ to X ₃ are preferably any one selected from the group consisting of proline (P), serine (S) and threonine (T), and proline Serine (S) is more preferable, but is not limited thereto.

In the above general formula, _n is preferably an integer of 1 to 6, more preferably 1 to 3.

The peptides of the present invention can be obtained by a variety of methods well known in the art. As an example, it can be produced by polynucleotide recombination and protein expression system, by synthesis in vitro through chemical synthesis such as peptide synthesis, and by cell-free protein synthesis.

In addition, to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbency, potency, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), reduced antigenicity, - A protecting group may be attached at the terminal. Preferably, the protecting group may be an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or a polyethylene glycol (PEG), but it is preferable to modify the peptide, The ingredients may be included without limitation.

The above bone diseases include osteoarthritis, osteoporosis, bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumor, periodontal disease, Inflammatory alveolar bone disorder, inflammatory alveolar bone disorder, inflammatory bone resorption disease, Paget's disease, and the like, but is not limited thereto.

The inflammatory disease is selected from the group consisting of atopic, psoriasis, dermatitis, allergies, arthritis, rhinitis, otitis, sore throat, tonsillitis, cystitis, nephritis, pelvic inflammatory disease, Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus erythematodes (Including but not limited to asthma, edema, delayed allergy (type IV allergy), graft rejection, graft versus host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemia- Nephritis, and gastrointestinal allergy. However, the present invention is not limited thereto.

The autoimmune disease may be selected from the group consisting of rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis, polymyositis, systemic angitis, mixed connective tissue disease, Crohn's disease, Hashimoto's disease, Grave's disease, Goodpasture's sydrome, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, irritable bowel syndrome, But are not limited to, those selected from the group consisting of myasthenia gravis, narcolepsy, imaginative pemphigus, malignant anemia, primary biliary cirrhosis, ulcerative colitis, vasculitis, Wegener's granulomatosis and psoriasis.

In addition, polynucleotides encoding the peptides of the present invention are also included in the present invention, since the same therapeutic effects can be obtained by using the polynucleotides encoding the peptides of the present invention.

In a specific embodiment of the present invention, the present inventors prepared various peptides using the above general formula 1 [(X ₁ -X ₂ -X ₃ ) _n ] (see Table 1).

As a result of confirming the inhibitory effect of the peptides on the T cell activity, the present inventors found that the peptides of Table 1 significantly inhibited T cell activity by an average of 15 to 25%, and inhibited the T cell activity of arbitrarily selected peptides The effect is shown in Figs. 1 to 3 (see Fig. 3 also in Fig. 1).

In addition, the present inventors confirmed that the synthetic peptides prepared in Example 1 were effective in inhibiting Th17 cell differentiation, and as a result, it was confirmed that the peptides of the present invention significantly inhibited Th17 cell differentiation (see FIG. 4) .

In addition, the present inventors confirmed the therapeutic effects of the peptides of the present invention after producing an animal model of rheumatoid arthritis (see FIG. 5). As a result, they showed significant arthritis-improving effects and particularly similar effects to the positive control MTX (See Figs. 6 to 9).

Therefore, the peptides of the present invention significantly inhibit T cell (T cell) activity and Th17 cell (T helper 17 cell) differentiation associated with autoimmune diseases, and have remarkable arthritis treatment and improvement effect in an arthritic animal model The peptides can be effectively used as an active ingredient in various autoimmune disease therapeutic agents such as bone diseases, inflammatory diseases or rheumatoid arthritis.

Alternatively, the peptides of the invention or polynucleotides encoding them can be delivered to a pharmaceutically acceptable carrier such as a colloidal suspension, powder, saline, lipid, liposome, microspheres, or nanospheric particles. They may be associated with or associated with carriers and may be in the form of lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, And can be delivered in vivo using known delivery systems.

In addition, pharmaceutically acceptable carriers may be formulated with pharmaceutically acceptable carriers such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, in addition to the above components, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be further included.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically) depending on the intended method, The severity and severity of the disease, the form of the drug, the route of administration and the time of administration, may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other bone diseases, inflammatory diseases, or autoimmune disease therapeutic agents, and can be administered separately, simultaneously with a conventional bone disease, inflammatory disease or autoimmune disease therapeutic agent , Or sequentially, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, body weight, absorbency of the active ingredient, inactivity, excretion rate, disease type, The severity of obesity, sex, weight, age, and the like.

The present invention also provides a health food for preventing or ameliorating bone diseases, inflammatory diseases or autoimmune diseases comprising the peptide of the present invention or a polynucleotide encoding the same as an active ingredient.

The health food may be used either simultaneously with or separately from the medicament for treatment before or after the onset of the disease for the prevention or improvement of the disease.

In the health food of the present invention, the active ingredient may be directly added to the food or may be used together with other food or food ingredients, and suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention may be added in an amount of preferably not more than 15% by weight, preferably not more than 10% by weight, based on the raw material. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The health food of the present invention may contain other ingredients as an essential ingredient without any particular limitations other than those containing the active ingredient. For example, various flavoring agents or natural carbohydrates may be added as an additional ingredient, such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. (Such as taurine, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (such as saccharin and aspartame) are advantageously used as flavorings other than those described above . The ratio of the natural carbohydrate can be appropriately determined by a person skilled in the art.

In addition to the above, the health food of the present invention may further contain various additives such as various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. These components can be used independently or in combination, and the proportion of such additives can also be appropriately selected by those skilled in the art.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

Example 1 Production of Peptide

Various peptides were prepared based on the following [Formula 1]. Then, each peptide synthesized by high performance liquid chromatography (SHIMADZU Prominence HPLC) was purified and the column was used Shiseido capcell pak C18 column (4.6 × 50 mm). The mass of each of the synthesized peptides was confirmed using a mass spectrometer (HP 1100 series LC / MSD).

[Formula 1]

(X ₁ -X ₂ -X ₃ ) _n

Wherein X ₁ to X ₃ are each independently selected from the group consisting of proline P, serine S, threonine T, glutamine Q, asparagine N, and cysteine C ), ≪ / RTI >

_n is an integer of 1 to 10, provided that the amino acid sequence of the general formula 1 is the PSP represented by SEQ ID NO: 2, and _n = 1 to 3 are excluded.

In addition, the peptides synthesized by the above method are listed in Table 1 below.

<Experimental Example 1> Confirmation of T cell activity inhibitory effect

In order to confirm the T cell activity inhibitory effect of the synthetic peptide prepared in Example 1 above, ex vivo activity inhibition experiments were carried out using T cells extracted from the lymph nodes of mice.

Specifically, to induce T cell activation, CD3 antibody was coated on a 96-well plate and incubated overnight at 4 ° C to prepare 96 wells with CD3 antibody. Then, the naive T cells extracted from the mouse were seeded at a density of 1 × 10 ⁵ / well on a 96-well plate, treated with each of the synthetic peptides prepared in Example 1 for 18 hours, and then subjected to flow cytometry to obtain active T The population of cells was confirmed. For this purpose, the same number of cells were collected from each cultured group and washed with PBS. Rabbit anti-mouse CD4, which is a helper T cell marker, and rabbit anti-mouse CD69 antibody, which is an activation marker of T cells, staining procedure. After washing with PBS, CD4 + CD69 + T cell population was analyzed.

As a result, it was confirmed that the peptides synthesized in Example 1 significantly inhibited T cell activity by an average of 15 to 25%.

Among the peptides synthesized in Example 1, the inhibitory effects of the peptides shown in Table 2 on T cell activity are shown in FIGS. 1 to 3.

Specifically, it was confirmed that the activation T cells were increased to 74.8% in the CD3 antibody-activated group and decreased to 55 to 62% in the group treated with each synthetic peptide compared with the group without activation (0.98%) . In addition, it was confirmed that the inhibitory rate of active T cells of each synthetic peptide was about 15% to 25% as in the case of the peptide synthesized in Example 1.

Thus, it was confirmed that the synthetic peptide of the present invention can be effectively used for the treatment of autoimmune diseases by significantly inhibiting T cell activity (FIGS. 1 to 3).

Peptide name	Amino acid sequence
Pep1	PSSPSS
Pep2	SSPSSP
Pep3	PTPPTP
Pep4	PTSPTS
Pep5	STPSTP
Pep6	STSSTS
Pep7	SPSSPS

<Experimental Example 2> Confirmation of inhibitory effect of Th 17 cell differentiation

In order to confirm the efficacy of the synthetic peptides prepared in Example 1 to inhibit Th17 cell differentiation, naive CD4 + T cells extracted from mouse lymph nodes were incubated with 20 ng / ml of IL-6 and TGF-beta 5 ng / ml to induce differentiation into Th17 cells.

At the same time, the three peptides (Pep2, Pep3, Pep4) shown in Table 2 of <Experimental Example 1> were treated at a concentration of 10 to 1000 ng / ml. After incubation for 3 days, CD4 + IL-17 + T cell populations were analyzed.

As a result, as shown in FIG. 4, the group which induced Th17 differentiation showed a tendency to increase about 2.5 times (3.14%) in the group inducing Th17 differentiation compared to the group that did not induce differentiation into Th17 cells (1.2% In the groups treated with Pep4 peptides, it was confirmed that the proportion of Th17 cells was decreased similarly to the group not inducing differentiation (Fig. 4).

<Experimental Example 3> Effect of a collagen-induced arthritis (CIA) mouse model on the treatment effect of rheumatoid arthritis

<3-1> Production of mouse model of rheumatoid arthritis

A rheumatoid arthritis mouse model was prepared by referring to the known literature (Nat Protoc. 2007; 2 (5): 1269-75) in order to confirm the effect of the peptide prepared in Example 1 for improving rheumatoid arthritis.

Specifically, the CIA mouse model is an autoimmune disease arthritis model similar to human rheumatoid arthritis, and is the most commonly used mouse model in rheumatoid arthritis animal experiments. CIA mice were first emulsified by mixing 1: 1 of bovine type II collagen (Bovine type II collagen, Chondrex, USA) and Freund's complete adjuvant (Chondrex, USA) 50 μl of collagen solution was injected intradermally into the tail of DBA / 1J mouse at 6 weeks of age to perform primary immunization. After the first immunization, bovine type 2 collagen and Freund's incomplete adjuvant (Chondrex, USA) were mixed at a ratio of 1: 1 and emulsified. Then, 50 μl of the emulsified collagen solution was added to the mouse tail To induce secondary immunization (boosting). After the second immunization, each peptide was administered intraperitoneally three times a week from the next day, and the effect of the peptide on the treatment of rheumatoid arthritis was observed. The administered peptides were selected as the four peptides (Pep1, Pep2, Pep4, Pep6) shown in Table 2 (Fig. 5).

<3-2> Confirmation of therapeutic effect using rheumatoid arthritis mouse model

In order to examine the progress of rheumatoid arthritis progression according to the peptide treatment of the present invention, the degree of progression of rheumatoid arthritis with time was evaluated by evaluating the rheumatoid arthritis progression index.

Two observers who were unaware of the specific experimental conditions assessed the progression of arthritis three times a week. At this time, the arthritic progression index was expressed as 0 to 16 points (total of four legs) in total by evaluating the arthritic progression index from 0 to 4 points for each leg according to Rossoliniec et al. The severity of arthritis was quantified by calculating the average value of the results evaluated by the observer.

score		Symptom
0 point	No edema or swelling
1 point	Mild swelling and erythema limited to foot or ankle joints were observed
2 points	Mild swelling and eruption from ankle joint to foot
3 points	Moderate swelling and redness from ankle joint to footbone were observed
4 points	Edema and redness from the ankle to the entire leg, stiff joints were observed.

As a result, as shown in FIGS. 6 to 9, it can be seen that the arthritic score is significantly increased in the CIA-induced mouse (Vehicle control) compared with the normal mouse group, Pep1, Pep2, Pep4, and Pep6) were intraperitoneally administered, respectively. In addition, it was confirmed that similar efficacy was shown to the positive control MTX (Figs. 6 to 9).

Thus, it has been confirmed that the peptide of the present invention can be used as a therapeutic agent for various bone diseases and autoimmune diseases including arthritis.

Claims (15)

1. A peptide consisting of the amino acid sequence of the general formula 1 described in SEQ ID NO: 1 below:

   [Formula 1]

   (X ₁ -X ₂ -X ₃ ) _n

   Wherein X ₁ to X ₃ are each independently selected from the group consisting of proline P, serine S, threonine T, glutamine Q, asparagine N, and cysteine C ), &Lt; / RTI &gt;

   _n is an integer of 1 to 10, provided that the amino acid sequence of the general formula 1 is the PSP represented by SEQ ID NO: 2, and _n = 1 to 3 are excluded.
2. The peptide according to claim 1, wherein X ₁ to X ₃ are each selected from the group consisting of proline (P), serine (S) and threonine (T).
3. The peptide according to claim 1, wherein X ₁ to X ₃ are each proline (P) or serine (S).
4. The peptide according to claim 1, wherein _n is an integer of 1 to 6.
5. The peptide according to claim 1, wherein _n is an integer of 1 to 3.
6. The peptide according to claim 1, wherein the N- or C-terminal of the peptide is selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group and a polyethylene glycol (PEG) Wherein said peptide is coupled to a selected protecting group.
7. A pharmaceutical composition for the prevention and treatment of a bone disease, inflammatory disease or autoimmune disease comprising as an active ingredient a peptide consisting of the amino acid sequence of the general formula 1 described in SEQ ID NO: 1 below,

   [Formula 1]

   (X ₁ -X ₂ -X ₃ ) _n

   Wherein X ₁ to X ₃ are each independently selected from the group consisting of proline P, serine S, threonine T, glutamine Q, asparagine N, and cysteine C ), &Lt; / RTI &gt;

   _n is an integer of 1 to 10, provided that the amino acid sequence of the general formula 1 is the PSP represented by SEQ ID NO: 2, and _n = 1 to 3 are excluded.
8. 8. The method of claim 7, wherein the bone disease is selected from the group consisting of arthritis, osteoporosis, bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignancy, multiple myeloma, A bone tumor, periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease, Paget's disease, and the like.
9. 8. The method of claim 7, wherein the inflammatory disease is selected from the group consisting of atopic, psoriasis, dermatitis, allergies, arthritis, rhinitis, otitis, sore throat, tonsillitis, cystitis, nephritis, pelvic inflammatory disease, Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus lupus erythematodes, SLE), asthma, edema, delayed allergy (type IV allergy), graft rejection, graft versus host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemia-reperfusion injury , Vascular restenosis, glomerulonephritis, and gastrointestinal allergy. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
10. 8. The method of claim 7, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis, polymyositis, systemic angitis, mixed connective tissue disease mixed connective tissue disease, Crohn's disease, Hashimoto's disease, Grave's disease, Goodpasture's sydrome, Guillain-Barre syndrome, idiopathic thrombocytopenia Characterized in that it is selected from the group consisting of purpura, irritable bowel syndrome, myasthenia gravis, narcolepsy, pemphigus pemphigus, malignant anemia, primary biliary cirrhosis, ulcerative colitis, vasculitis, Wegener's granulomatosis and psoriasis &Lt; / RTI &gt;
11. A health food for preventing or ameliorating a bone disease, an inflammatory disease or an autoimmune disease comprising as an active ingredient a peptide consisting of the amino acid sequence of the general formula 1 described in SEQ ID NO: 1 below,

    [Formula 1]

    (X ₁ -X ₂ -X ₃ ) _n

    Wherein X ₁ to X ₃ are each selected from the group consisting of proline, serine, threonine, glutamine, asparagine and cysteine,

    _n is an integer of 1 to 10, provided that the amino acid sequence of the general formula 1 is the PSP represented by SEQ ID NO: 2, and _n = 1 to 3 are excluded.
12. 1. A method for treating bone diseases, inflammatory diseases or autoimmune diseases, comprising administering a pharmaceutically effective amount of the peptide of claim 1 to a subject suffering from bone disease, inflammatory disease or autoimmune disease.
13. A method for preventing bone diseases, inflammatory diseases, or autoimmune diseases, comprising administering to a subject a pharmaceutically effective amount of the peptide of claim 1.
14. The use of the peptide of claim 1 for use as a pharmaceutical composition for the prevention and treatment of bone diseases, inflammatory diseases or autoimmune diseases.
15. The use of the peptide of claim 1 for use as a health food for preventing and ameliorating bone diseases, inflammatory diseases or autoimmune diseases.

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