Peptides having osteoclast differentiation inhibitory activity and their uses

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 its use.

Background Art

[0002] Bone supports the soft tissues and body weight of the human body, surrounds internal organs, and protects the internal organs from external impacts. In addition to structurally supporting muscles and organs, it is also an important part of the human body that stores calcium and other essential inorganic substances in the body, namely substances such as phosphorus and magnesium. Therefore, the bones of an adult who has grown continuously repeat the generation and absorption process of removing old bones and replacing them with new bones until death, while maintaining a balance, and this is called bone remodeling. The turnover of bone, which removes old bone and replaces it with new bone, is essential for recovering the fine damage to bone caused by growth and stress and appropriately maintaining the function of bone.

[0003] On the other hand, it is known that two main types of cells are involved in bone regeneration. Of these two cells, one is the osteoblast, which generates 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 progenitor cells mature into osteoclasts, and bone resorption occurs. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, inhibiting osteoclast formation and preventing excessive bone resorption. The absorption or destruction of old bone is carried out by osteoclasts, which are produced from blood cells (hematopoietic stem cells). These osteoclasts bore holes in the bone, releasing small amounts of calcium into the bloodstream, which is used to maintain bodily functions. On the other hand, osteoblasts, produced from bone cells, fill these holes with collagen and cover calcium and phosphorus deposits (hydroxyapatite) to create new, hard bone and rebuild the skeleton. When the rates of osteoclasts and osteoblasts are in balance, effective bone density can be maintained. When this balance is disrupted, many diseases can occur, most notably osteoporosis and diseases involving bone damage due to bone metastasis from cancer cells.

[0004] For bone damage caused by bone porousness and bone metastasis of cancer cells, as described above, bisphosphonate drugs such as Fosamax (active ingredient: alendronate) and Actonel (active ingredient: risedronate) are used. These bisphosphonate preparations weaken the function of osteoclasts, which destroy most bone, inducing their death and thus delaying or suppressing bone loss. However, there have been reports of osteonecrosis of the jaw, severe atrial fibrillation, bone and joint weakness, and musculoskeletal pain occurring in patients taking bisphosphonates.

[0005] Against this technological backdrop, multifaceted research is underway to discover new materials for the treatment of bone diseases (Korean Published Patent No. 10-2016-0024463), but the situation remains inadequate. [Overview of the project] [Problems that the invention aims to solve]

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

[0007] Another embodiment is to provide a pharmaceutical composition for the prevention or treatment of bone disease, comprising the peptide as an active ingredient.

[0008] Other purposes and advantages of this application will be further clarified by the following detailed description, along with the claims and drawings. Any matters not described herein are readily apparent and inferable to those skilled in the art of this application or similar art, and are therefore omitted from this description. [Means for solving the problem]

[0009] Each description and embodiment disclosed in this application may also apply to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not considered to be limited by the specific descriptions described below.

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

[0011] As used herein, the term "peptide" may mean a linear molecule formed by the bonding of amino acid residues to one another via peptide bonds. Such peptides can be produced by chemical synthesis methods known to those skilled in the art, particularly by solid-phase or liquid-phase synthesis techniques (US Patent No. 5,516,891). The inventors, through diligent efforts to develop a biologically effective peptide, have identified a peptide consisting of the amino acid sequence of Sequence ID No. 1 or Sequence ID No. 2.

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

[0013] The peptide may also have a protecting group attached to its N or C terminus to acquire chemical stability, enhanced pharmacological properties (half-life, water absorption, potency, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), or reduced antigenicity. In one specific example, the N terminus of the peptide may have an acetyl group, a fluorenyl methoxycarbonyl group, or a fluorenyl methoxycarbonyl group. en The peptide is bonded to one protecting group selected from the group consisting of ylmethoxycarbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group, butoxycarbonyl group, allyloxycarbonyl group, and polyethylene glycol (PEG); and / or the C-terminus of the peptide is an amino group (-NH2), three class Alkyl group (tertiary alkyl group) and Hydrazide ( Hydor The peptide may be conjugated with any one protecting group selected from the group consisting of azide, -NHNH2). The peptide may also selectively further include a targeting sequence, a tag, labeled residues, or an amino acid sequence manufactured for a specific purpose to increase half-life or peptide stability.

[0014] The peptides are artificially synthesized, non-naturally occurring, or engineered, where “non-naturally occurring or engineered” means a state produced by artificial modification rather than the state of existence that occurs naturally. Here, such artificial modification may include artificially synthesizing an amino acid sequence by mimicking multiple amino acid structures, or being engineered to acquire chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity, as described above.

[0015] As used herein, the term "stability" may refer not only to in vivo stability, which protects the peptide from attack by endogenous protein-cleaving enzymes, but also to storage stability (e.g., room temperature storage stability).

[0016] Another embodiment provides a pharmaceutical composition for the prevention or treatment of bone disease, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

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

[0018] In this specification, the term "prevention" means all actions that suppress or delay the onset of a disease by administering the composition.

[0019] In this specification, the term “treatment” means any form of treatment that provides an effect to an individual suffering from or at risk of developing a disease, including improvement of the individual’s condition (e.g., one or more symptoms), delay of disease progression, delay of symptom onset, or slowing of symptom progression. Accordingly, “treatment” and “prevention” are not intended to mean a cure or complete elimination of symptoms.

[0020] The term "individual" refers to an entity requiring treatment for a disease, and more specifically, to mammals such as humans or non-human primates, mice, dogs, cats, horses, and cattle.

[0021] In this specification, the term “bone disease” includes, for example, diseases, illnesses, or conditions related to signaling via RANKL, and may include not only diseases, illnesses, or conditions related to the regulation of bone formation and resorption, but also pathological conditions involving bone loss, including bone loss, osteoporosis, and osteolysis. The bone disease includes, but is not limited to, 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 age-related bone loss.

[0022] On the other hand, conventional functional peptides, despite their effective biological activity, suffer from disadvantages such as not being effectively absorbed into target tissues or cells due to the size of the peptide itself, or being eliminated from the body in a short period due to their short half-life. In contrast, the pharmaceutical composition according to one example contains a peptide consisting of approximately 10 amino acids or less as the active ingredient, resulting in excellent skin penetration of the active ingredient, and for example, when administered topically, an effective therapeutic effect on bone diseases can be obtained.

[0023] According to one example, the peptide not only inhibits the expression of NFATc1 and c-Fos, which are differentiation transcription factors of osteoclasts induced by RANKL, but also inhibits the expression of TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2, which are sub-signaling factors related to differentiation, thereby inhibiting osteoclast function. Therefore, the peptide can be utilized as an active ingredient in pharmaceutical compositions for the treatment of 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 also includes, but is not limited to, a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier.

[0025] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the therapeutic efficacy of the pharmaceutical composition for treating bone diseases.

[0026] The weight ratio between the peptide and the pharmaceutically acceptable carrier is, for example, also 500:1 to 1:500. As an example, the weight ratio is 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, but is not limited thereto.

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

[0028] In addition to the above components, the pharmaceutical composition may further include, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc.

[0029] The aforementioned pharmaceutical composition may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local administration, transdermal administration, etc., but is not limited to these.

[0030] The dosage of the aforementioned pharmaceutical composition may be 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, but is not limited to these values ​​and can be administered in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0031] The pharmaceutical composition may be manufactured in unit dose form or contained in multi-dose containers by formulating it with pharmaceutically acceptable carriers and / or excipients using a method readily available to a person with ordinary skill 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 oil or aqueous medium, or in the form of an ointment, cream, gel, transdermal agent, poultice, patch, paste, extract, powder, granule, tablet or capsule, and may further contain a dispersant and / or stabilizer.

[0033] The peptide may be contained in nanosomes or nanoparticles to further improve skin penetration or stability issues. For example, the nanosomes may be produced by a microfluidizer using lecithin as a raw material and contained within lecithin particles. Any known method for producing the nanosomes may be used. The size of the nanosome particles is preferably 30 to 200 nm. If the size of the nanosome particles is less than 30 nm, skin penetration proceeds very quickly, causing skin side effects, and if it exceeds 200 nm, skin penetration is not easy, making it difficult to obtain the benefits of using the nanosome structure.

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

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

[0036] As used herein, the terms “apply,” “administer,” and “coat” are interchangeable and mean at least partially localizing the composition according to one embodiment to a desired site, or placing the composition according to one embodiment into an individual via an administration route.

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

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

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

[0040] According to one embodiment of the peptide, osteoclast differentiation can be inhibited by inhibiting the differentiation transcription factors and sub-signaling factors associated with differentiation of osteoclasts induced by RANKL.

[0041] According to one embodiment of the peptide, it can be applied to prevent or treat bone diseases by inhibiting the differentiation of osteoclasts. [Brief explanation of the drawing]

[0042] [Figure 1] This is the result of treating RAW264.7 cells with RANKL along with Peptide-1 to induce differentiation into osteoclasts, and then confirming the level of osteoclast differentiation via microscope. [Figure 2] This is the result of treating RAW264.7 cells with RANKL along with Peptide-2 to induce differentiation into osteoclasts, and then confirming the level of osteoclast differentiation via microscopy. [Figure 3] This is the result of checking CCK-8 activity after treating RAW264.7 cells with Peptide-2. [Figure 4] This study evaluates TRAP activity after inducing osteoclast differentiation by treating RAW264.7 cells with RANKL along with Peptide-1. [Figure 5] This study evaluates TRAP activity after inducing osteoclast differentiation by treating RAW264.7 cells with RANKL along with Peptide-2. [Figure 6] This study involved treating RAW264.7 cells with RANKL along with Peptide-1 to induce differentiation into osteoclasts, followed by confirmation of inhibition of NFATc1 or c-Fos mRNA expression. [Figure 7] The results show that after inducing osteoclast differentiation by treating RAW264.7 cells with RANKL together with Peptide-2, inhibition of NFATc1 or c-Fos mRNA expression was confirmed at (A) 24 hours or (B) 48 hours. [Figure 8] This study shows the results of inducing osteoclast differentiation in RAW264.7 cells by treating them with RANKL along with Peptide-1, followed by confirmation of inhibition of NFATc1 protein expression. [Figure 9] The results show that after treating RAW264.7 cells with RANKL along with Peptide-2 to induce differentiation into osteoclasts, inhibition of NFATc1 or c-Fos protein expression was confirmed at (A) 24 hours or (B) 48 hours. [Figure 10] This study involved treating RAW264.7 cells with RANKL along with Peptide-1 to induce osteoclast differentiation, followed by confirmation of inhibition of TRAP, OSCAR, CTSK, or DC-STAMP mRNA expression. [Figure 11] The results show that after inducing differentiation into osteoclasts by treating RAW264.7 cells with RANKL together with Peptide-2, inhibition of mRNA expression of TRAP, OSCAR, CTSK, DC-STAMP, or Atp6v0d2 was confirmed at (A) 24 hours or (B) 48 hours. [Figure 12] The results show that after treating osteoclast precursor cells induced by RANKL with Peptide-2, inhibition of c-Fos or NFATc1 mRNA expression was confirmed at (A) 3 hours or (B) 6 hours. [Figure 13] The results show that after treating osteoclast precursor cells induced by RANKL with Peptide-2, inhibition of c-Fos or NFATc1 protein expression was confirmed at (A) 3 hours or (B) 6 hours. [Figure 14] The results show that after treating osteoclast precursor cells induced by RANKL with Peptide-2, inhibition of mRNA expression of TRAP, OSCAR, CTSK, DC-STAMP, or Atp6v0d2 was confirmed at (A) 3 hours or (B) 6 hours. [Modes for carrying out the invention]

[0043] The present invention will be described in more detail below based on 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 having the amino acid sequences of SEQ ID NO: 1 or 2 (Peptide-1 or Peptide-2) listed in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and these synthesized peptides were separated into pure molecules using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm x 100 mm, 1.7 μm, Waters Co., USA).

[0045] [Table 1]

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

[0047] 2-1. TRAP staining and evaluation of cytotoxicity RAW264.7 cells 1.7 x 10 3 After seeding 96-well plates at a cell / well density, cells were cultured in DMEM for 24 hours. After 24 hours, RANKL and Peptide-1 or Peptide-2 were treated at different concentrations to induce differentiation into osteoclasts for 4 days. Subsequently, TRAP staining was performed using Sigma Aldrich's acid phosphatase kit. Fixation buffer was added, the mixture was allowed to react for 30 seconds, and then washed with distilled water. 200 μl of staining solution was added per well, and the mixture was allowed to react at 37°C for 30 minutes, followed by three washes with distilled water. After drying for one day, the staining results were examined under a microscope.

[0048] Furthermore, to confirm the cytotoxicity of Peptide-2, RAW264.7 cells cultured in DMEM for 24 hours using the same method as described above were treated with the peptide at different concentrations. Then, after 3 days, CCK-8 (Dojindo, CCK-8kit) solution was added to the culture medium at a volume of 1 / 10 to dilute it, and the mixture was incubated for 2 hours. Subsequently, the culture medium was sampled and CCK-8 activity was confirmed at a wavelength of 450 nm using a microplate reader.

[0049] As a result, as shown in Figures 1 and 2, we 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 and TRAP expression increased by RANKL treatment. Furthermore, as shown in Figure 3, Peptide-2 was found not to exhibit cytotoxicity.

[0050] 2-2. Evaluation of TRAP activity RAW264.7 cells, cultured in DMEM for 24 hours using the same method as described above, were treated with RANKL and Peptide-1 or Peptide-2 at different concentrations to induce differentiation into osteoclasts for 4 days. Subsequently, 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, followed by incubation at 37°C for 1 hour, and treatment with 10 μl of 2N NaOH. After that, the absorbance at a wavelength of 405 nm was measured using a microplate reader and quantified.

[0051] As a result, as shown in Figures 4 and 5, we confirmed that TRAP activity was inhibited in the groups treated with Peptide-1 or Peptide-2, and such inhibitory effects showed a concentration-dependent trend.

[0052] Example 3. Confirmation of the effect of inhibiting transcription factors in osteoclast differentiation. Among the various mechanisms that regulate osteoclast differentiation, NFATc1 (Nuclear factor of activated T-cells, cytoplasmic 1), a transcription factor induced by RANKL, is known to be essential for osteoclast differentiation. Therefore, we attempted to confirm the inhibitory effect of Peptide-1 or Peptide-2 on the transcription factor of osteoclast differentiation by treating RAW264.7 cells, an osteoclast progenitor cell line, with RANKL to induce differentiation into osteoclasts.

[0053] Specifically, RAW264.7 cells are divided into 1.7 x 10⁶ cells. 3 Cells were seeded into 96-well plates at a cell / well density and cultured in DMEM for 24 hours. After 24 hours, they were treated with RANKL and Peptide-1 or Peptide-2 at varying concentrations to induce differentiation into osteoclasts for 4 days. After 24 or 48 hours, the culture medium was suctioned, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), RT-PCR was performed using c-Fos and NFATc1 primers as shown in Table 2 below.

[0054] [Table 2]

[0055] Furthermore, RAW264.7 cells cultured in DMEM for 24 hours using the same method as described above were subjected to differentiation into osteoclasts. After 24 or 48 hours, the culture medium was suctioned, the cells were harvested, lysates were prepared, and Western blotting was performed. For detection, sc-166940 (Santa Cruz, USA) was used against c-Fos, and sc-7294 (Santa Cruz, USA) was used against NFATc1.

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

[0057] Example 4. Confirmation of inhibitory effects on lower signaling molecules associated with osteoclast differentiation. We attempted to confirm the inhibitory effect of Peptide-1 or Peptide-2 on lower signaling pathways associated with osteoclast differentiation during the process of inducing osteoclast differentiation in RAW264.7 cells, an osteoclast progenitor cell line, by treating them with RANKL.

[0058] Specifically, RAW264.7 cells are divided into 1.7 x 10⁶ cells. 3 Cells were seeded into 96-well plates at a cell / well density and cultured in DMEM for 24 hours. After 24 hours, they were treated with RANKL and Peptide-1 or Peptide-2 at varying concentrations to induce differentiation into osteoclasts for 4 days. After 24 or 48 hours, the culture medium was suctioned, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), RT-PCR was performed using TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2 primers as 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 We confirmed that RANKL inhibits the expression of subtransmitters associated with osteoclast differentiation, such as TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2d.

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

[0062] 5-1. Confirmation of differentiation transcription factor inhibitory effect RAW264.7 cells 1.7 x 10 3 Cells were seeded into 96-well plates at a cell / well density and cultured in DMEM for 24 hours. After 24 hours, RANKL and Peptide-2 were added at varying concentrations to induce differentiation into osteoclasts. After 72 hours, the culture medium was suctioned and then treated again with RANKL and Peptide-2. After 3 or 6 hours, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), RT-PCR was performed using the c-Fos and NFATc1 primers shown in Table 2. In addition, RAW264.7 cells cultured in DMEM for 24 hours in the same manner as described above were subjected to differentiation into osteoclasts. After 3 days, the culture medium was suctioned and then treated again with RANKL and Peptide-2. Subsequently, after 3 or 6 hours, the cells were harvested to prepare lysates, and then Western blotting was performed. For detection, sc-166940 (Santa Cruz, USA) was used against c-Fos, and sc-7294 (Santa Cruz, USA) was used against NFATc1.

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

[0064] 5-2. Confirmation of inhibitory effects on differentiation-related lower signaling molecules RAW264.7 cells cultured in DMEM for 24 hours using the same method as described above were treated with RANKL and Peptide-2 at different concentrations to induce differentiation into osteoclasts. After 72 hours, the culture medium was suctioned and then treated again with RANKL and Peptide-2. After 3 or 6 hours, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), RT-PCR was performed using TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2 primers as shown in Table 3.

[0065] As a result, as shown in Figure 14, we confirmed that Peptide-2 inhibits the expression of subtransmitters associated with osteoclast differentiation, such as TRAP, OSCAR, CTSK, DC-STAMP, and Atp6v0d2.

[0066] In summary, the experimental results described above show that Peptide-1 and Peptide-2, as used in one example, can be applied to the treatment of bone diseases by weakening 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 by thorough stirring. The mixture was then mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil. After adjusting the volume with distilled water to a total volume of 1 L, the mixture was emulsified using a microfluidizer under high pressure to produce peptide nanosomes with a size of approximately 100 nm.

[0068] Dosage Form Examples 2. Pharmaceutical Preparations

[0069] 2-1. Manufacturing of powdered medicines The following ingredients are mixed and filled into an airtight cloth to produce the powder. This invention Peptide 20mg Lactose 100mg Talc 10mg

[0070] 2-2. Manufacturing of Tablets After mixing the following ingredients, tablets are manufactured by compressing them using a standard tablet manufacturing method. This invention Peptide 10mg Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0071] 2-3. Manufacturing of Capsules The following ingredients are mixed using a standard capsule manufacturing method and then filled into gelatin capsules to produce capsules. This invention Peptide 10mg Crystalline cellulose 3 mg Lactose 14.8mg Magnesium stearate 0.2 mg

[0072] 2-4. Manufacturing of injectable drugs The following ingredients are manufactured per ampoule (2 ml) using a standard method for manufacturing injectable drugs. This invention Peptide 10mg Mannitol 180mg Sterile distilled water for injection 2974 mg Na2HPO4·2H2O 26mg

[0073] 2-5. Manufacturing of liquid formulations The liquid preparation is prepared by dissolving each component in purified water using a standard liquid preparation method, mixing the following components, adding purified water to adjust the total volume to 100 ml, and then filling it into a brown bottle and sterilizing it. This invention Peptide 10mg Isomerized sugar 10g Mannitol 5g Purified water (appropriate amount)

[0074] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the embodiments described above should be understood to be illustrative in all respects and 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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