USE OF PEPTIDES AS THERAPEUTIC AGENT FOR AUTOIMMUNE DISEASES AND BONE DISEASES

Abstract

The present invention relates to use of peptides as a therapeutic agent, wherein 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 disease, and have remarkable effects of treating and improving 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 disease, inflammatory disease or rheumatoid arthritis.

Claims

1. A synthetic peptide consisting of an amino acid sequence of Formula 1 below:
(X.sub.1-X.sub.2-X.sub.3).sub.n  [Formula 1] wherein, X.sub.1 to X.sub.3 each is as below: i) X.sub.1 is proline (P), each of X.sub.2 and X.sub.3 is serine (S); ii) X.sub.1 is proline (P), X.sub.2 is threonine (T), X.sub.3 is serine (S); iii) each of X.sub.1 and X.sub.3 is serine (S), X.sub.2 is proline (P), n is an integer from 2 to 10, and when n is 2, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 96, when n is 3, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 97, when n is 4, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 98, when n is 5, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 99, when n is 6, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 100, when n is 7, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 101, when n is 8, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 102, when n is 9, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 103, when n is 10, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 104.

2. The synthetic peptide according to claim 1, wherein an N- or C terminus of the peptide is bound to a protective group selected from the group consisting of an acetyl group, a fluorenylmethoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG).

3. A method for treating bone disease, inflammatory disease or autoimmune disease, comprising administering a synthetic peptide consisting of an amino acid sequence of Formula 1 below in a pharmaceutically effective dose to a subject with bone disease, inflammatory disease or autoimmune disease:
(X.sub.1-X.sub.2-X.sub.3).sub.n  [Formula 1] wherein, X.sub.1 to X.sub.3 each is as below: i) X.sub.1 is proline (P), each of X.sub.2 and X.sub.3 is serine (S); ii) X.sub.1 is proline (P), X.sub.2 is threonine (T), X.sub.3 is serine (S); iii) each of X.sub.1 and X.sub.3 is serine (S), X.sub.2 is proline (P), n is an integer from 2 to 10, and when n is 2, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 96, when n is 3, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 97, when n is 4, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 98, when n is 5, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 99, when n is 6, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 100, when n is 7, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 101, when n is 8, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 102, when n is 9, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 103, when n is 10, the amino acid sequence of Formula 1 is represented by SEQ ID NO: 104.

4. The method according to claim 3, wherein the bone disease is at least one selected from the group consisting of arthritis, osteoporosis, bone metastatic cancer, solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia caused by malignant tumor, multiple myeloma, primary bone tumor, periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease, and Paget's disease.

5. The method according to claim 3, wherein the inflammatory disease is selected from the group consisting of atopy, psoriasis, dermatitis, allergies, arthritis, rhinitis, otitis media, sore throat, tonsillitis, cystitis, nephritis, pelvic inflammatory, Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus erythematodes (SLE), asthma, edema, delayed allergy (Type IV allergy), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemic-reperfusion injury, vascular restenosis, glomerulonephritis, and gastrointestinal allergy.

6. The method according to claim 3, 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, Crohn's disease, Hashimoto's disease, Grave's disease, Goodpasture's syndrome, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, irritable bowel syndrome, myasthenia gravis, narcolepsy, vulgaris ulcer, pernicious anemia, primary biliary cirrhosis, ulcerative colitis, vasculitis, Wegener's granulomatosis, and psoriasis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0018] FIG. 1 is a diagram showing an active T cell population (%) by a synthetic peptide of the present invention.

[0019] FIG. 2 is a diagram showing an active T cell population (%) by a synthetic peptide of the present invention in a graph.

[0020] FIG. 3 is a diagram showing a T cell activation inhibition rate (%) by a synthetic peptide of the present invention.

[0021] FIG. 4 is a diagram showing a Th17 cell differentiation inhibitory effect by a synthetic peptide of the present invention.

[0022] FIG. 5 is a schematic diagram schematically showing a process of preparing a collagen-induced arthritis mouse model and a time of administration of peptides according to the present invention.

[0023] FIG. 6 is a diagram showing an arthritis improvement effect of a peptide Pep1 of the present invention.

[0024] FIG. 7 is a diagram showing an arthritis improvement effect of a peptide Pep2 of the present invention.

[0025] FIG. 8 is a diagram showing an arthritis improvement effect of a peptide Pep4 of the present invention.

[0026] FIG. 9 is a diagram showing an arthritis improvement effect of a peptide Pep6 of the present invention.

DESCRIPTION OF EMBODIMENTS

[0027] Hereinafter, terms of the present invention will be defined as follows.

[0028] In the present invention, general one-letter or three-letter codes for naturally existing amino acids are used, and three-letter codes generally allowed for other amino acids, such as α-aminoisobutyric acid (Aib) and N-methylglycine (Sar) are also used. The amino acids mentioned herein as abbreviations are described according to the IUPAC-IUB nomenclature.

[0029] The “peptide” of the present invention refers to a polymer consisting of two or more amino acids linked by an amide bond (or peptide bond), and for the purposes of the present invention, refers to a peptide having a therapeutic effect on bone disease, inflammatory disease, and autoimmune disease.

[0030] The “stability” of the present invention means not only in-vivo stability that protects the peptides of the present invention from the attack of protein cleavage enzymes in vivo, but also storage stability (e.g., room-temperature storage stability).

[0031] The “prevention” of the present invention means all actions that inhibit disease or delay the onset of the disease by administration of a pharmaceutical composition according to the present invention.

[0032] The “treatment” of the present invention means all actions that improve or advantageously change symptoms of the disease by the administration of the pharmaceutical composition according to the present invention.

[0033] The “subject” of the present invention refers to a subject in need of treatment for diseases, and more particularly, refers to mammals such as human or non-human primates, mice, dogs, cats, horses and cattle.

[0034] The “improvement” of the present invention means all actions that at least reduce parameters associated with conditions to be treated, e.g., the degree of symptoms.

[0035] Hereinafter, the present invention will be described in more detail.

[0036] The present invention provides a peptide consisting of an amino acid sequence of Formula 1 represented by SEQ ID NO: 1 below and a pharmaceutical composition for preventing and treating bone disease, inflammatory disease, or autoimmune disease containing the peptide as an active ingredient:

(X.sub.1-X.sub.2-X.sub.3).sub.n  [Formula 1]

[0037] wherein, X.sub.1 to X.sub.3 each is any one selected from the group consisting of proline (P), serine (S), threonine (T), glutamine (Q), asparagine (N), and cysteine (C),

[0038] n is an integer from 1 to 10, and

[0039] the case where the amino acid sequence of Formula 1 above includes a PSP represented by SEQ ID NO: 2 and n=1 to 3 is excluded.

[0040] The peptide may be prepared as various peptides using Formula 1 above, which are all included in the present invention. In addition, the X.sub.1 to X.sub.3 each is preferably any one selected from the group consisting of proline (P), serine (S), and threonine (T), and more preferably proline (P) or serine (S), but the present invention is not limited thereto.

[0041] In addition, in Formula above, n is preferably an integer of 1 to 6, and more preferably an integer of 1 to 3.

[0042] The peptide of the present invention may be obtained by various methods well-known in the art. As an example, the peptide may be prepared by using polynucleotide recombination and a protein expression system or prepared by in-vitro synthesis through chemical synthesis such as peptide synthesis, cell-free protein synthesis, and the like.

[0043] In addition, in order to obtain better chemical stability, enhanced pharmacological properties (half-life, absorbency, titer, efficacy, etc.), modified specificity (e.g., a wide biological activity spectrum), and reduced antigenicity, a protective group may bind to an N- or C-terminus of the peptide. Preferably, the protective group may be an acetyl group, a fluorenylmethoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but may include any ingredient that may enhance the modification of the peptide, particularly the stability of the peptide, without limitation.

[0044] The bone disease is preferably at least one selected from the group consisting of arthritis, osteoporosis, bone metastatic cancer, solid cancer bone metastasis, musculoskeletal complications due to solid cancer bone metastasis, hypercalcemia caused by malignant tumor, multiple myeloma, primary bone tumor, periodontal disease, inflammatory alveolar bone resorption disease, inflammatory bone resorption disease, and Paget's disease, but is not limited thereto.

[0045] The inflammatory disease is preferably selected from the group consisting of atopy, psoriasis, dermatitis, allergies, arthritis, rhinitis, otitis media, sore throat, tonsillitis, cystitis, nephritis, pelvic inflammatory, Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus erythematodes (SLE), asthma, edema, delayed allergy (Type IV allergy), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemic-reperfusion injury, vascular restenosis, glomerulonephritis, and gastrointestinal allergy, but is not limited thereto.

[0046] The autoimmune disease is preferably 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 syndrome, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, irritable bowel syndrome, myasthenia gravis, narcolepsy, vulgaris ulcer, pernicious anemia, primary biliary cirrhosis, ulcerative colitis, vasculitis, Wegener's granulomatosis, and psoriasis, but is not limited thereto.

[0047] In addition, since the same therapeutic effect may be exhibited even by using polynucleotides encoding the peptide of the present invention, it is obvious that the polynucleotides encoding the peptide of the present invention are also included in the present invention.

[0048] In a specific embodiment of the present invention, the present inventors prepared various peptides using Formula 1 above [(X.sub.1-X.sub.2-X.sub.3).sub.n] (see Table 1).

[0049] In addition, the present inventors confirmed a T cell activity inhibitory effect of the peptides, and as a result, the peptides of Table 1 significantly inhibit the T cell activity by an average of 15% to 25%, and the T cell activity inhibitory effect of peptides randomly selected among the peptides was shown in FIGS. 1 and 3 (see FIGS. 1 to 3).

[0050] In addition, the present inventors confirmed that a synthetic peptide prepared in Example 1 inhibits Th17 cell differentiation, and as a result, confirmed that the peptides of the present invention significantly inhibit the Th17 cell differentiation (see FIG. 4).

[0051] Further, the present inventors prepared a rheumatoid arthritis animal model (see FIG. 5) and then confirmed a therapeutic effect of the peptides of the present invention, and as a result, confirmed that the peptides showed a significant arthritis improvement effect, particularly, a similar effect compared to a positive control group MTX (see FIGS. 6 to 9).

[0052] Therefore, since 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 improving arthritis in an arthritis animal model, the peptides may be used as an active ingredient in therapeutic agents for bone disease, inflammatory disease or various autoimmune diseases such as rheumatoid arthritis.

[0053] On the other hand, the peptides of the present invention or polynucleotides encoding the same may be carried in pharmaceutically acceptable carriers such as colloidal suspensions, powders, saline, lipids, liposomes, microspheres, or nano spherical particles. These peptides or polynucleotides may form a complex with a vehicle or be associated with the vehicle and may be carried in vivo by using vehicle systems known in the art, such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids.

[0054] Besides, the pharmaceutically acceptable carrier may include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, which are generally used in preparation, but is not limited thereto. Further, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the ingredients.

[0055] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., applied intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically) according to a desired method, and a dose thereof varies depending on the condition and weight of a patient, a degree of disease, a drug form, and route and time of administration, but may be appropriately selected by those skilled in the art.

[0056] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective dose. In the present invention, the “pharmaceutically effective dose” refers to an amount sufficient to treat the diseases at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose level may be determined according to elements including the type and severity of disease of a patient, activity of a drug, sensitivity to a drug, a time of administration, a route of administration and an emission rate, duration of treatment, and simultaneously used drugs, and other elements well-known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents for bone disease, inflammatory disease, or autoimmune disease, and administered simultaneously, separately, or sequentially with conventional therapeutic agents for bone disease, inflammatory disease, or autoimmune disease, and may be administered singly or multiply. It is important to administer an amount capable of obtaining a maximum effect with a minimal amount without side-effects by considering all the elements, and this may be easily determined by those skilled in the art.

[0057] Specifically, the effective dose of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, and weight of a patient, absorbance of an active ingredient in vivo, an inactivation rate, an excretion rate, a disease type, and drugs to be used in combination, and may be increased or decreased according to a route of administration, the severity of obesity, sex, weight, age, and the like.

[0058] Further, the present invention provides health foods for preventing and improving bone disease, inflammatory disease or autoimmune disease containing the peptide of the present invention or polynucleotides encoding the same as an active ingredient.

[0059] The health foods may be used simultaneously or separately with a drug for treatment before or after the onset of the corresponding disease in order to prevent or improve the disease.

[0060] In the health foods of the present invention, the active ingredient may be added to the foods as it is or used with other foods or food ingredients, and may be appropriately used according to a general method. The mixing amount of the active ingredients may be suitably determined according to the purpose of use thereof (prevention or improvement). In general, in preparation of foods or beverages, the composition of the present invention may be added preferably in an amount of 15 wt % or less, more preferably 10 wt % or less with respect to raw materials. However, in the case of long-term ingestion for the purpose of health and hygiene or health regulation, the amount may be used below the above range.

[0061] The health foods of the present invention may contain other ingredients as essential ingredients without particular limitation, in addition to the active ingredients. For example, like general beverages, various flavoring agents or natural carbohydrates may be contained as an additional ingredient. Examples of the above-mentioned natural carbohydrates may include monosaccharides, such as glucose, fructose, and the like; disaccharides, such as maltose, sucrose, and the like; and general sugars, such as polysaccharides such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, erythritol. In addition to those described above, as the flavoring agent, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The ratio of the natural carbohydrates may be appropriately determined by the selection of those skilled in the art.

[0062] In addition, the health foods of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acid, a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonic acid agent used in a carbonated drink, and the like. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0063] Hereinafter, the present invention will be described in detail by Examples and Test Examples.

[0064] However, the following Examples and Test Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples and Test Examples.

[0065] <Example 1> Preparation of Peptides Various peptides were prepared based on the following Formula 1. Subsequently, each of the synthesized peptides was purified and separated using high performance liquid chromatography (SHIMADZU Prominence HPLC), and a column used is a Shiseido capcell pak C18 column (4.6×50 mm). In addition, the mass of each synthesized peptide was confirmed by using a mass spectrometer (HP 1100 series LC/MSD).

(X.sub.1-X.sub.2-X.sub.3).sub.n  [Formula 1]

[0066] wherein, X.sub.1 to X.sub.3 each is any one selected from the group consisting of proline (P), serine (S), threonine (T), glutamine (Q), asparagine (N), and cysteine (C),

[0067] n is an integer from 1 to 10, and

[0068] the case where the amino acid sequence of Formula 1 above includes a PSP represented by SEQ ID NO: 2 and n=1 to 3 is excluded.

[0069] In addition, the peptides synthesized by the method were listed in Table 1 below.

TABLE-US-00001 TABLE 1 SEQ Synthetic ID No. peptide NO: 1 PPS 3 2 PPT 4 3 PPQ 5 4 PPN 6 5 PSS 7 6 PST 8 7 PSQ 9 8 PSN 10 9 PSC 11 10 PTP 12 11 PTS 13 12 PTT 14 13 PTQ 15 14 PTN 16 15 PTC 17 16 PQP 18 17 PQS 19 18 PQT 20 19 PQN 21 20 PQC 22 21 PNP 23 22 PNS 24 23 PNT 25 24 PNQ 26 25 PNN 27 26 PNC 28 27 PCP 29 28 PCS 30 29 PCT 31 30 PCQ 32 31 PCN 33 32 PCC 34 33 SSP 35 34 SPS 36 35 SPT 37 36 SPQ 38 37 SPN 39 38 STP 40 39 STS 41 40 TPP 42 41 TPS 43 42 TSP 44 43 TSS 45 44 PPSPPS 46 45 PPTPPT 47 46 PPQPPQ 48 47 PPNPPN 49 48 PSSPSS 50 49 PSTPST 51 50 PSQPSQ 52 51 PSNPSN 53 52 PSCPSC 54 53 PTPPTP 55 54 PTSPTS 56 55 PTTPTT 57 56 SSPSSP 58 57 SPSSPS 59 58 STPSTP 60 59 STSSTS 61 60 PSSPSSPSS 62 61 PTPPTPPTP 63 62 PTSPTSPTS 64 63 SSPSSPSSP 65 64 SPSSPSSPS 66 65 STPSTPSTP 67 66 STSSTSSTS 68 67 PPSPPSPPSPPS 69 68 PPTPPTPPTPPT 70 69 PPQPPQPPQPPQ 71 70 PSSPSSPSSPSSPSS 72 71 PTPPTPPTPPTPPTP 73 72 PTSPTSPTSPTSPTS 74 73 SSPSSPSSPSSPSSP 75 74 SPSSPSSPSSPSSPS 76 75 STPSTPSTPSTPSTP 77 76 STSSTSSTSSTSSTS 78 77 PSSPSSPSSPSSPSSP 79 SS 78 PTPPTPPTPPTPPTPP 80 TP 79 PTSPTSPTSPTSPTSP 81 TS 80 SSPSSPSSPSSPSSPS 82 SP 81 SPSSPSSPSSPSSPSS 83 PS 82 STPSTPSTPSTPSTPS 84 TP 83 STSSTSSTSSTSSTSS 85 TS 84 PSSPSSPSSPSSPSSP 86 SSPSS 85 PTPPTPPTPPTPPTPP 87 TPPTP 86 PTSPTSPTSPTSPTSP 88 TSPTS

[0070] <Test Example 1> Confirmation of T Cell Activity Inhibitory Effect

[0071] In order to confirm the T cell activity inhibitory effect of the synthetic peptide prepared in Example 1, an ex vivo activity inhibition test was performed using T cells extracted from the lymph nodes of a mouse.

[0072] Specifically, at first, in order to induce activation of T cells, CD3 antibodies were coated on a 96 well plate and incubated overnight at 4° C. to prepare 96 wells attached with the CD3 antibodies. Thereafter, naive T cells extracted from the mouse were seeded in the 96 well plate by 1×10.sup.5/well, treated with each of the synthetic peptides prepared in Example 1 and incubated for 18 hours, and then the population of the active T cells was confirmed by flow cytometry. To this end, the same number of cells were collected from each incubated group and washed with PBS, and then the collected cells were subjected to staining by using rabbit anti-mouse CD4 as a helper T cell marker and a rabbit anti-mouse CD69 antibody as a T-cell activation marker. The cells were washed with PBS and then CD4+CD69+ T cell population was analyzed.

[0073] 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%.

[0074] In addition, among the peptides synthesized in Example 1, the T cell activity inhibitory effect of the peptides shown in Table 2 was shown in FIGS. 1 to 3.

[0075] Specifically, it was confirmed that compared to a group that did not induce activation (0.98%), active T cells increased by 74.8% in a group activated with the CD3 antibody, and decreased by 55% to 62% in a group treated with each synthetic peptide. In addition, it was confirmed that the active T cell inhibition rate of each synthetic peptide was about 15% up to 25%, similarly to the peptides synthesized in Example 1.

[0076] Therefore, it was confirmed that the synthetic peptides of the present invention may be used to treat autoimmune diseases by significantly inhibiting the T cell activity (FIGS. 1 to 3).

TABLE-US-00002 TABLE 2 Name of peptide Amino acid sequence SEQ ID NO: Pep1 PSSPSS 89 Pep2 SSPSSP 90 Pep3 PTPPTP 91 Pep4 PTSPTS 92 Pep5 STPSTP 93 Pep6 STSSTS 94 Pep? SPSSPS 95

[0077] <Test Example 2> Confirmation of Th 17 Cell Differentiation Inhibitory Effect

[0078] In order to confirm the efficacy of the synthetic peptides prepared in Example 1 to inhibit Th17 cell differentiation, naive CD4.sup.+ T cells extracted from lymph nodes of the mouse were treated with IL-6 20 ng/ml and TGF-beta 5 ng/ml together with TCR activation to induce differentiation into Th17 cells.

[0079] At the same time, three peptides Pep2, Pep3, and Pep4 disclosed in Table 2 of Test Example 1 were treated at a concentration of 10 ng/ml to 1000 ng/ml, respectively. Then, after incubation for 3 days, CD4+IL-17+ T cell population was analyzed.

[0080] As a result, as shown in FIG. 4, there was a tendency of about 2.5-fold increase in a group that induced Th17 differentiation (3.14%), compared to a group that did not induce differentiation into Th17 cells (1.2%), and in a group treated with peptides Pep2, Pep3, and Pep4, it was confirmed that a ratio of Th17 cells was reduced similarly to the group that did not induce differentiation (FIG. 3).

[0081] <Test Example 3> Confirmation of Rheumatoid Arthritis Treatment Effect Using Collagen-Induced Arthritis (CIA) Mouse Model

[0082] <3-1> Preparation of Rheumatoid Arthritis Mouse Model

[0083] In order to confirm a rheumatoid arthritis improvement effect of the peptides prepared in Example 1, a rheumatoid arthritis mouse model was prepared with reference to a known literature (Nat Protoc. 2007; 2 (5): 1269-75.).

[0084] Specifically, a CIA mouse model is a mouse model most commonly used in a rheumatoid arthritis animal test as an autoimmune disease arthritis model having characteristics similar to human rheumatoid arthritis. In the CIA mouse model, bovine type II collagen (Chondrex, USA) was mixed and emulsified with a Freund's complete adjuvant (Chondrex, USA) at 1:1, and then first immunization was performed by injecting 50 μl of the emulsified collagen solution intradermally into a 6-week-old DBA/1J mouse tail. On 2 weeks after the first immunization, bovine type II collagen was mixed and emulsified with a Freund's incomplete adjuvant (Chondrex, USA) at 1:1, and then second immunization (boosting) was performed by injecting 50 μl of the emulsified collagen solution induced) intradermally into the mouse tail. After the second immunization, each peptide was intraperitoneally administered 3 times a week from the following day to observe a therapeutic effect of the peptides for rheumatoid arthritis. The administered peptides were selected as four peptides Pep1, Pep2, Pep4, and Pep6 shown in Table 2 (FIG. 5).

[0085] <3-2> Confirmation of Treatment Effect Using Rheumatoid Arthritis Mouse Model

[0086] In order to examine the progression of rheumatoid arthritis according to the peptide treatment of the present invention, the severity of rheumatoid arthritis over time was evaluated and measured by a rheumatoid arthritis progress index.

[0087] Two observers, who did not know specific test conditions, evaluated the progression of arthritis three times a week. At this time, the arthritis progress index was evaluated as Points 0 to 4 per leg in accordance with the arthritis progress evaluation criteria by Rossoliniec, etc. in Table 2 below and shown to total Points 0 to 16 (sum of four legs). Thereafter, an average value of the results evaluated by the two observers was calculated to quantify the severity of arthritis.

TABLE-US-00003 TABLE 3 Score Symptoms Point 0 There was no edema or swelling Point 1 Mild edema and redness confined to the foot or ankle joint were observed. Point 2 Mild swelling and redness across the tarsal bone at the ankle joint were observed. Point 3 Moderate swelling and redness across the tarsal bone at the ankle joint were observed. Point 4 There were edema and redness throughout the leg from the ankle, and joint stiffness was observed.

[0088] As a result, as shown in FIGS. 6 to 9, it can be seen that arthritis scores are significantly increased in CIA-induced mice (Vehicle control; PBS) as compared to a normal mouse group, and it was confirmed that the efficacy of arthritis improvement was shown in a group intraperitoneally administered with four peptides Pep1, Pep2, Pep4, and Pep6, respectively. In addition, it was confirmed that the similar efficacy was shown compared to the positive control group MTX (FIGS. 6 to 9).

[0089] Therefore, it was confirmed that the peptides of the present invention may be used as therapeutic agents for various bone diseases including arthritis and autoimmune diseases.