Peptides with osteoclast differentiation inhibitory activity and uses thereof

Abstract

The present application relates to a peptide having osteoclast differentiation inhibitory activity and its uses, and provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and a pharmaceutical composition for preventing or treating bone diseases, which contains the peptide as an active ingredient.

Description

[Technical Field]

[0001] The present application relates to a peptide having osteoclast differentiation inhibitory activity and uses thereof. [Background technology]

[0002] Bones support the body's soft tissues and weight, surround internal organs, and protect them from external shocks. They not only provide structural support for muscles and organs, but are also one of the essential components of the human body, storing calcium and other essential minerals, such as phosphorus and magnesium. Therefore, the bones of fully grown adults maintain balance through a dynamic and continuous process of bone resorption, in which old bone is removed and replaced with new bone, until death. This process is called bone remodeling. Bone turnover, in which old bone is removed and replaced with new bone, is essential for repairing micro-damage caused by growth and stress and maintaining proper bone function.

[0003] Meanwhile, two major types of cells are known to be involved in bone remodeling. One is the osteoblast, which produces bone, and the other is the osteoclast, which destroys bone. Osteoblasts produce RANKL (receptor activator of nuclear factor-κB ligand) and its decoy receptor, OPG (osteoprotegerin). When RANKL binds to RANK, a receptor on the surface of osteoclast progenitor cells, the osteoclast precursors mature into osteoclasts, causing bone resorption. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, inhibiting osteoclast formation and preventing excessive bone resorption. The resorption or destruction of old bone is caused by osteoclasts, which are generated from blood cells (hematopoietic stem cells), which create holes in the bone, releasing small amounts of calcium into the bloodstream for use in maintaining bodily functions. Meanwhile, osteoblasts, generated from bone cells, fill the holes with collagen, cover them with calcium and phosphorus deposits (hydroxyapatite), and create solid new bone to rebuild the skeleton. When the rate of osteoclasts and osteoblasts is balanced, effective bone density can be maintained. When this balance is disrupted, many diseases can occur, particularly osteoporosis and diseases related to bone damage caused by bone metastasis of cancer cells.

[0004] Bisphosphonates, such as Fosamax (alendronate) and Actonel (risedronate), are used to treat bone damage caused by osteoporosis and bone metastasis of cancer cells. These bisphosphonates slow or prevent bone loss by weakening the function and inducing the death of osteoclasts, which destroy most bone. However, cases of osteonecrosis of the jaw, severe atrial fibrillation, bone and joint weakness, and musculoskeletal pain have been reported in patients taking bisphosphonates.

[0005] Under these technical backgrounds, multifaceted research is being conducted to discover new substances for treating bone diseases (Korean Patent Publication No. 10-2016-0024463), but the reality is that there are still shortcomings. Summary of the Invention [Problem to be solved by the invention]

[0006] One embodiment is to provide a peptide consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0007] Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases, which comprises the peptide as an active ingredient.

[0008] Other objects and advantages of the present application will become more apparent from the following detailed description taken in conjunction with the claims and drawings. The contents not described in this specification are fully understood and can be inferred by those skilled in the art of the present application or a similar art, and therefore, the description thereof will be omitted. [Means for solving the problem]

[0009] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the specific descriptions described below are not intended to limit the scope of this application.

[0010] One embodiment provides a peptide consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0011] The term "peptide" as used herein refers to a linear molecule formed by linking amino acid residues together via peptide bonds. The peptide can be prepared by chemical synthesis methods known to those skilled in the art, particularly solid-phase synthesis or liquid-phase synthesis (U.S. Patent No. 5,516,891). The present inventors have made extensive efforts to develop peptides with biologically effective activity, and as a result have identified peptides consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] Here, the biologically effective activity may be any one or more of the following: (a) inhibition of osteoclast differentiation; (b) inhibition of the expression of NFATc1 (nuclear factor-activated T cell cytoplasmic 1) or c-Fos; (c) inhibition of the expression of TRAP (tartrate-resistant acid phosphatase), OSCAR (osteoclast-associated receptor), CTSK (cathepsin K), or DC-STAMP (dendritic cell-specific transmembrane protein); and (d) inhibition of the expression of Atp6v0d2 (ATPase H+ Transporting V0 Subunit D2). Therefore, the peptide can be used for the prevention or treatment of diseases through the inhibition of osteoclast differentiation.

[0013] The peptides may also have protecting groups attached to their N- or C-termini to achieve chemical stability, enhanced pharmacological properties (half-life, absorbency, potency, efficacy, etc.), altered specificity (e.g., a broader spectrum of biological activity), or reduced antigenicity. In one embodiment, the N-terminus of the peptide may be conjugated with any one protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG); and / or the C-terminus of the peptide may be conjugated with any one protecting group selected from the group consisting of an amino group (-NH), a tertiary alkyl group, and an azide (-NHNH). Optionally, the peptide may further comprise a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically designed to increase half-life or peptide stability.

[0014] The peptides are artificially synthesized or non-naturally occurring or engineered, and the term "non-naturally occurring or engineered" refers to a state in which the peptides are produced by artificial modification, rather than in the state in which they exist in nature. Here, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking multiple amino acid structures, or engineering the peptides to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity, as described above.

[0015] The term "stability" as used herein may refer not only to in vivo stability, which protects the peptide from attack by in vivo proteolytic enzymes, but also to storage stability (eg, storage stability at room temperature).

[0016] Another embodiment provides a pharmaceutical composition for preventing or treating bone diseases, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0017] Among the terms or elements mentioned in the description of the peptide or composition, the same as those already mentioned are as described above.

[0018] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition.

[0019] As used herein, the term "treatment" refers to any form of care that provides a benefit to an individual suffering from or susceptible to a disease, including improvement of the individual's condition (e.g., one or more symptoms), delay in disease progression, delay in symptom onset, or slowing of symptom progression, etc. Thus, the terms "treatment" and "prevention" are not intended to mean a cure or complete elimination of symptoms.

[0020] The term "individual" refers to a subject in need of treatment for a disease, and more specifically refers to mammals such as human or non-human primates, mice, dogs, cats, horses, and cows.

[0021] As used herein, the term "bone disease" refers to, for example, diseases, disorders, or conditions associated with RANKL-mediated signaling, including those involved in the regulation of bone formation and resorption, as well as pathological conditions associated with bone loss, including osteopenia, osteoporosis, and osteolysis. Examples of bone diseases include, but are not limited to, osteoporosis, osteogenesis imperfecta, osteomalacia, osteonecrosis, rickets, osteomyelitis, alveolar bone loss, Paget's disease of bone, hypercalcemia, primary hyperparathyroidism, myeloma, bone loss in rheumatoid arthritis, bone loss due to cancer, fibrous dysplasia, hypoplastic bone disease, metabolic bone disease, and age-related bone loss.

[0022] However, conventional functional peptides, despite their effective biological activity, have the disadvantages of being unable to be effectively delivered to target tissues or cells due to their large size, or being rapidly eliminated in the body due to their short half-life. In contrast, a pharmaceutical composition according to one embodiment contains a peptide consisting of approximately 10 or less amino acids as an active ingredient, which allows for excellent skin penetration of the active ingredient, and, for example, when administered topically, provides effective therapeutic effects for bone diseases.

[0023] According to one embodiment, the peptide not only inhibits the expression of NFATc1 and c-Fos, which are osteoclast differentiation transcription factors induced by RANKL, but also inhibits the expression of differentiation-related subordinate signaling factors TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2, thereby inhibiting osteoclast function. Therefore, the peptide can be used as an active ingredient in a pharmaceutical composition for treating bone diseases (J Bone Metab 2014;21:233-241 http: / / dx.doi.org / 10.11005 / jbm.2014.21.4.233 pISSN 2287-6375 eISSN 2287-7029).

[0024] The pharmaceutical composition may comprise, but is not limited to, a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier.

[0025] The term "pharmaceutical effective amount" as used herein means an amount sufficient to achieve the therapeutic efficacy of the pharmaceutical composition for bone disease.

[0026] The weight ratio between the peptide and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500. Examples of the weight ratio include, but are not limited to, 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2.

[0027] The pharmaceutically acceptable carriers are those commonly used in pharmaceutical preparations, and include, but are not limited to, lactose, dextrose, saccharose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0028] The pharmaceutical composition may further contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited to these.

[0029] The pharmaceutical composition may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, it may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0030] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 to 1000 μg (μg), 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg per day, and may vary depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity of the patient.

[0031] The pharmaceutical composition may be prepared in a unit dose form or in a multi-dose container by formulating it with pharmaceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person skilled in the art to which the invention pertains.

[0032] The dosage form may be in the form of a solution, suspension or emulsion in an oily or aqueous medium, or in the form of an ointment, cream, gel, transdermal agent, cataplasm, patch, paste, extract, powder, granule, tablet or capsule, and may further comprise a dispersing agent and / or a stabilizing agent.

[0033] To further improve skin penetration or stability, the peptide can be incorporated into nanosomes or nanoparticles. For example, the nanosomes can be prepared using lecithin as a raw material using a microfluidizer, and then incorporated into lecithin particles. Any known method can be used to prepare the nanosomes. The nanosome particle size is preferably 30 to 200 nm. Nanosome particle sizes less than 30 nm can penetrate the skin too quickly, resulting in side effects. Nanosome particle sizes greater than 200 nm can penetrate the skin too quickly, making it difficult to achieve the benefits of using the nanosome structure.

[0034] Yet another embodiment provides a method for preventing or treating a bone disease, comprising administering to an individual a composition comprising, as an active ingredient, a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0035] Among the terms or elements mentioned in the description of the peptides, compositions, etc., the same as those already mentioned are as described above.

[0036] As used herein, the terms "apply," "administer," and "apply" are used interchangeably and refer to at least partially localizing a composition according to an embodiment at a desired site or placing a composition according to an embodiment within an individual by a route of administration.

[0037] Yet another embodiment provides a cosmetic composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0038] Among the terms or elements mentioned in the description of the peptides, compositions, etc., the same as those already mentioned are as described above.

[0039] The cosmetic composition may contain, but is not limited to, a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier. [Effects of the Invention]

[0040] The peptide according to one embodiment can inhibit osteoclast differentiation by inhibiting RANKL-induced osteoclast differentiation transcription factors and lower signaling factors associated with differentiation.

[0041] The peptide according to one embodiment can be applied to prevent or treat bone diseases by inhibiting osteoclast differentiation. [Brief explanation of the drawings]

[0042] [Figure 1] RAW264.7 cells were treated with Peptide-1 and RANKL to induce differentiation into osteoclasts, and the level of osteoclast differentiation was then examined under a microscope. [Figure 2] RAW264.7 cells were treated with RANKL together with Peptide-2 to induce differentiation into osteoclasts, and the differentiation level of osteoclasts was then confirmed under a microscope. [Figure 3] This shows the results of examining CCK-8 activity after treating RAW264.7 cells with Peptide-2. [Figure 4] RAW264.7 cells were treated with RANKL together with Peptide-1 to induce differentiation into osteoclasts, and then TRAP activity was assessed. [Figure 5] RAW264.7 cells were treated with RANKL together with Peptide-2 to induce differentiation into osteoclasts, and then TRAP activity was assessed. [Figure 6] RAW264.7 cells were treated with RANKL together with Peptide-1 to induce differentiation into osteoclasts, and then the inhibition of NFATc1 or c-Fos mRNA expression was confirmed. [Figure 7] RAW264.7 cells were treated with RANKL together with Peptide-2 to induce differentiation into osteoclasts, and the inhibition of NFATc1 or c-Fos mRNA expression was confirmed at (A) 24 hours or (B) 48 hours. [Figure 8] RAW264.7 cells were treated with RANKL together with Peptide-1 to induce differentiation into osteoclasts, and then the inhibition of NFATc1 protein expression was confirmed. [Figure 9] RAW264.7 cells were treated with RANKL together with Peptide-2 to induce differentiation into osteoclasts, and the inhibition of NFATc1 or c-Fos protein expression was confirmed (A) 24 hours later or (B) 48 hours later. [Figure 10] RAW264.7 cells were treated with RANKL together with Peptide-1 to induce differentiation into osteoclasts, and then the inhibition of TRAP, OSCAR, CTSK, or DC-STAMP mRNA expression was confirmed. [Figure 11] RAW264.7 cells were treated with RANKL together with peptide-2 to induce osteoclast differentiation, and then the inhibition of TRAP, OSCAR, CTSK, DC-STAMP, or Atp6v0d2 mRNA expression was confirmed at (A) 24 hours or (B) 48 hours. [Figure 12] RANKL-induced osteoclast precursor cells were treated with Peptide-2, and the inhibition of c-Fos or NFATc1 mRNA expression was confirmed (A) 3 hours later or (B) 6 hours later. [Figure 13] RANKL-induced osteoclast precursor cells were treated with Peptide-2, and the inhibition of c-Fos or NFATc1 protein expression was confirmed (A) 3 hours or (B) 6 hours after treatment. [Figure 14] RANKL-induced osteoclast precursor cells were treated with Peptide-2, and the inhibition of TRAP, OSCAR, CTSK, DC-STAMP, or Atp6v0d2 mRNA expression was confirmed (A) 3 hours or (B) 6 hours after treatment. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0044] Example 1. Synthesis of peptides Peptides (Peptide-1 or Peptide-2) having the amino acid sequences of SEQ ID NO: 1 or 2 listed in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA). The synthesized peptides were purified and separated using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was an ACQUITY UPLC BEH300 C18 (2.1 mm ∘ 100 mm, 1.7 μm, Waters Co., USA).

[0045] [Table 1]

[0046] Example 2. Confirmation of the inhibitory effect on osteoclast differentiation In the process of inducing differentiation into osteoclasts by treating RAW264.7 cells, an osteoclast precursor cell line, with RANKL, we attempted to confirm the inhibitory effect of Peptide-1 or Peptide-2 treatment on osteoclast differentiation.

[0047] 2-1.TRAP staining and cytotoxicity assessment 1.7x10 RAW264.7 cells 3 The cells were seeded into a 96-well plate at a density of 100 cells / well and cultured in DMEM for 24 hours. After 24 hours, they were treated with RANKL and Peptide-1 or Peptide-2 at different concentrations to induce osteoclast differentiation for 4 days. TRAP staining was then performed using a Sigma-Aldrich acid phosphatase kit. Fixation buffer was added to the wells, incubated for 30 seconds, and washed with distilled water. 200 μl of staining solution was added per well, incubated at 37°C for 30 minutes, and washed three times with distilled water. After drying for one day, the staining results were examined under a microscope.

[0048] To further confirm the cytotoxicity of Peptide-2, RAW264.7 cells were cultured in DMEM for 24 hours in the same manner as described above and treated with the peptide at various concentrations. After 3 days, CCK-8 (Dojindo, CCK-8 kit) solution was added at 1 / 10 the volume of the culture medium, followed by dilution and incubation for 2 hours. The culture medium was then sampled and CCK-8 activity was measured using a microplate reader at 450 nm.

[0049] As a result, as shown in Figures 1 and 2, it was confirmed that TRAP expression was significantly reduced in the groups treated with Peptide-1 or Peptide-2 compared to the control group, in which osteoclast differentiation was induced by RANKL treatment and TRAP expression increased. Furthermore, as shown in Figure 3, it was found that Peptide-2 did not exhibit cytotoxicity.

[0050] 2-2. Evaluation of TRAP activity RAW264.7 cells were cultured in DMEM for 24 hours as described above and treated with RANKL and Peptide-1 or Peptide-2 at various concentrations for 4 days to induce osteoclast differentiation. Then, 100 μl of TRAP activation solution [15 ml of TRAP buffer (0.1% sodium citrate + 50 μM sodium tartrate, pH 5.0) and 4-nitrophenly phosphate disodium salt hexahydrate (Sigma, 1 tablet mix)] was added and incubated at 37°C for 1 hour. Then, 10 μl of 2N NaOH was added. Absorbance at 405 nm was measured using a microplate reader and quantified.

[0051] As a result, as shown in Figures 4 and 5, it was confirmed that TRAP activity was inhibited in the groups treated with Peptide-1 or Peptide-2, and such an inhibitory effect tended to be concentration-dependent.

[0052] Example 3. Confirmation of the inhibitory effect of transcription factors on osteoclast differentiation Among the various mechanisms regulating osteoclast differentiation, the transcription factor NFATc1 (Nuclear factor of activated T-cells, cytoplasmic 1), which is induced by RANKL, is known to be essential for osteoclast differentiation. Therefore, we attempted to investigate the inhibitory effect of Peptide-1 or Peptide-2 on the osteoclast differentiation transcription factor during the treatment of RAW264.7 cells, an osteoclast precursor cell line, with RANKL to induce osteoclast differentiation.

[0053] Specifically, 1.7x10 RAW264.7 cells 3 The cells were seeded into 96-well plates at a density of 100 cells / well and cultured in DMEM for 24 hours. After 24 hours, the cells were treated with RANKL and Peptide-1 or Peptide-2 at various concentrations to induce osteoclast differentiation for 4 days. After 24 or 48 hours, the medium was removed by suction, the cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and RT-PCR was performed using the c-Fos and NFATc1 primers listed in Table 2 below.

[0054] [Table 2]

[0055] In addition, RAW264.7 cells were cultured in DMEM for 24 hours and then induced to differentiate into osteoclasts using the same method as described above. After 24 or 48 hours, the medium was removed by suction, the cells were harvested, and lysates were prepared and analyzed by Western blotting. Antibodies used for detection were sc-166940 (Santa Cruz, USA) for c-Fos and sc-7294 (Santa Cruz, USA) for NFATc1.

[0056] As a result, as shown in Figures 6 to 9, it was confirmed that Peptide-1 and Peptide-2 each inhibited the expression of osteoclast differentiation transcription factors such as NFATc1 and c-Fos induced by RANKL.

[0057] Example 4. Confirmation of the inhibitory effect of downstream signaling factors associated with osteoclast differentiation In the process of inducing osteoclast differentiation by treating RAW264.7 cells, an osteoclast precursor cell line, with RANKL, we attempted to confirm the inhibitory effect of Peptide-1 or Peptide-2 on downstream signaling factors related to osteoclast differentiation.

[0058] Specifically, 1.7x10 RAW264.7 cells 3 The cells were seeded into 96-well plates at a density of 100 cells / well and cultured in DMEM for 24 hours. After 24 hours, the cells were treated with RANKL and Peptide-1 or Peptide-2 at various concentrations to induce osteoclast differentiation for 4 days. After 24 or 48 hours, the medium was removed by suction, the cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and RT-PCR was performed using the TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2 primers shown in Table 3 below.

[0059] [Table 3]

[0060] As a result, as shown in Figures 10 and 11, Peptide-1 and Peptide-2, respectively, It was confirmed that it inhibited the expression of downstream signaling molecules associated with osteoclast differentiation, such as TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2d, induced by RANKL.

[0061] Example 5. Confirmation of the inhibitory effect on osteoclast precursor cell differentiation We attempted to confirm the inhibitory effect of Peptide-2 treatment on osteoclast precursor differentiation in macrophages whose differentiation into osteoclasts was induced by RANKL.

[0062] 5-1. Confirmation of the inhibitory effect of differentiation transcription factors 1.7x10 RAW264.7 cells 3 Cells were seeded into 96-well plates at a density of 100 cells / well and cultured in DMEM for 24 hours. After 24 hours, the cells were treated with various concentrations of RANKL and Peptide-2 to induce osteoclast differentiation. After 72 hours, the medium was removed and the cells were treated again with RANKL and Peptide-2. After 3 or 6 hours, the cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea) and RT-PCR was performed using the c-Fos and NFATc1 primers listed in Table 2. RAW264.7 cells were cultured in DMEM for 24 hours and then induced to differentiate into osteoclasts using the same method as described above. After 3 days, the medium was removed and the cells were treated again with RANKL and Peptide-2. After 3 or 6 hours, the cells were harvested and lysates were prepared for Western blotting. The antibodies used for detection were sc-166940 (Santa Cruz, USA) for c-Fos and sc-7294 (Santa Cruz, USA) for NFATc1.

[0063] As a result, as shown in FIGS. 12 and 13, it was confirmed that Peptide-2 inhibits the expression of differentiation transcription factors of osteoclast precursor cells, such as c-Fos and NFATc1.

[0064] 5-2. Confirmation of the inhibitory effect of downstream signaling factors related to differentiation RAW264.7 cells were cultured in DMEM for 24 hours in the same manner as described above and then treated with RANKL and Peptide-2 at various concentrations to induce osteoclast differentiation. After 72 hours, the medium was removed and RANKL and Peptide-2 were added again. After 3 or 6 hours, the cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and RT-PCR was performed using the TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2 primers listed in Table 3.

[0065] As a result, as shown in FIG. 14, it was confirmed that Peptide-2 inhibited the expression of downstream signaling molecules associated with osteoclast differentiation, such as TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2.

[0066] In summary, the above experimental results demonstrate that Peptide-1 and Peptide-2 according to one embodiment can be applied to the treatment of bone diseases by attenuating the differentiation and / or function of osteoclasts.

[0067] Dosage Form Example 1. Production of Peptide Nanosomes 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water with thorough stirring. The resulting solution was mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil, and then the mixture was adjusted to a total volume of 1 L with distilled water. The mixture was then emulsified using high pressure in a microfluidizer to produce peptide nanosomes with a size of approximately 100 nm.

[0068] Dosage form example 2. Pharmaceutical preparations

[0069] 2-1. Powder manufacturing The following ingredients are mixed and packed into an airtight cloth to produce a powder. The present invention Peptide 20mg Lactose 100mg Talc 10mg

[0070] 2-2. Tablet manufacturing The following ingredients are mixed and compressed into tablets by a conventional tablet manufacturing method. The present invention Peptide 10mg Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0071] 2-3. Capsule manufacturing The following ingredients are mixed and filled into gelatin capsules according to a conventional capsule manufacturing method to produce capsules. The present invention Peptide 10mg Crystalline cellulose 3mg Lactose 14.8mg Magnesium stearate 0.2mg

[0072] 2-4. Manufacturing of injections The following ingredients are prepared per ampoule (2 ml) using the usual injection manufacturing method. The present invention Peptide 10mg Mannitol 180mg Sterile distilled water for injection 2974mg Na2HPO4·2H2O 26mg

[0073] 2-5. Liquid drug manufacturing Using the usual method for producing liquid preparations, add each ingredient to purified water and dissolve it, then mix the ingredients listed below, add purified water to bring the total volume to 100 ml, and fill into a brown bottle and sterilize to prepare the liquid. The present invention Peptide 10mg Isomerized sugar 10g Mannitol 5g Purified water (appropriate amount)

[0074] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

2. The peptide according to claim 1, wherein the N-terminus of the peptide is bonded to one protecting group selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG).

3. The C-terminus of the peptide is an amino group (-NH₂). 2 ), tertiary alkyl group and hydrazide (-NHNH) 2 The peptide according to claim 1, which is bonded to any one protecting group selected from the group consisting of ).

4. The peptide according to claim 1, wherein the peptide exhibits one or more of the following characteristics: (a) Inhibition of osteoclast differentiation; (b) Inhibition of NFATc1 (nuclear factor-activated T cells cytoplasmic 1) or c-Fos expression; (c) Inhibition of the expression of TRAP (tartrate-resistant acid phosphatase), OSCAR (osteoclast-associated receptor), CTSK (cathepsin K), or DC-STAMP (dendritic cell-specific transmembrane preotein); and (d) Inhibition of Atp6v0d2 (ATPase H+ Transporting V0 Subunit D2) expression.

5. A pharmaceutical composition for the prevention or treatment of bone disease, comprising the peptide described in any one of claims 1 to 4 as an active ingredient.

6. The pharmaceutical composition according to claim 5, further comprising a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 5, wherein the peptide is formulated into a nanosome.

8. The pharmaceutically active composition according to claim 5, wherein the bone disease is osteoporosis, osteogenesis imperfecta, osteomalacia, osteonecrosis, rickets, osteomyelitis, alveolar bone loss, Paget's disease, hypercalcemia, primary hyperparathyroidism, myeloma, bone loss in rheumatoid arthritis, bone loss due to cancer, fibrous dysplasia, dysplasia of bone, metabolic bone disease, or loss of bone mass due to aging.

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