Peptide having osteoclast differentiation inhibitory activity and use thereof

Abstract

The present application relates to a peptide having osteoclast differentiation inhibitory activity and a use thereof, and provides a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and a pharmaceutical composition for preventing or treating bone diseases comprising 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 and surrounds internal organs to protect the internal organs from external impacts. Also, as one of the key parts of the human body, it not only structurally supports muscles or organs but also stores calcium or other essential inorganic substances, such as phosphorus or magnesium, in the body. Therefore, the bones of adults after growth cessation are very dynamically and continuously regenerated until death, repeatedly removing old bones and replacing them with new bones through a process of formation and resorption to maintain balance, which is called bone remodeling. Bone turnover, which removes old bones and replaces them with new bones, is essential for restoring minor bone damage caused by growth and stress and properly maintaining bone function.

[0003] It is known that generally two types of cells are involved in bone remodeling. One of the two types of cells is osteoblasts that generate bone, and the other is osteoclasts that break down bone. Osteoblasts produce receptor activator of nuclear factor-κB ligand (RANKL) and its decoy receptor 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, if OPG binds to RANKL, the binding between RANKL and RANK is blocked. Therefore, the formation of osteoclasts is inhibited, and excessive bone resorption does not occur. The absorption or destruction of old bones is achieved by osteoclasts produced in blood cells (hematopoietic stem cells), which is used to form cavities on the bone to release a small amount of calcium through the bloodstream to maintain body functions. Osteoblasts generated in bone cells fill the cavities with collagen and cover them with hydroxyapatite of calcium and phosphorus to form strong new bones to rebuild the skeleton. In this case, when the bone resorption rate and bone formation rate are balanced, effective bone density can be maintained. When this balance is disrupted, many diseases may be caused. In particular, osteoporosis and diseases related to bone damage caused by bone metastasis of cancer cells are representative.

[0004] For osteoporosis and bone damage caused by bone metastasis of cancer cells as described above, therapeutic agents of the bisphosphonate series are used, for example, Fosamax (ingredient name: alendronate) and Actonel (ingredient name: risedronate). Most of these bisphosphonate preparations cause an effect of slowing down or stopping bone loss by disabling the function of osteoclasts that destroy bone and inducing apoptosis. However, there are currently reports of cases of osteonecrosis of the jaw, severe atrial fibrillation, weakening of bones or joints, or myoskeletal pain in patients taking bisphosphonates.

[0005] In this technical background, various studies have been conducted to discover new substances for the treatment of bone diseases (Korean Patent Publication No. 10-2016-0024463), but they are still not perfect.

Summary of the Invention

[0006]

Technical Problem

[0007] On the one hand, a peptide is provided, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] On the other hand, a pharmaceutical composition for preventing or treating bone diseases is provided, which comprises the peptide as an active ingredient.

[0009] Other objects and advantages of the present application will become clearer in combination with the appended claims, the accompanying drawings, and the following detailed description. Regarding the content not described in this specification, as long as it can be fully recognized and analogized by those of ordinary skill in the art to which the present application belongs or those of ordinary skill in similar technical fields, the relevant description thereof is omitted.

[0010]

Technical Solution

[0011] The various descriptions and embodiments disclosed in the present application can also be applied to various other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. And it cannot be determined that the scope of the present application is limited to the specific descriptions recorded below.

[0012] On the one hand, a peptide is provided, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] The term "peptide" as used in this specification may refer to a linear molecule formed by multiple amino acid residues bound to each other through peptide bonds. The peptide can be prepared by chemical synthesis methods well known in the art, particularly solid-phase synthesis techniques or liquid-phase synthesis techniques (US Registered Patent No. 5,516,891). The inventors have made efforts to develop peptides with biologically effective activities and have successfully identified peptides consisting of the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2. Here, the biologically effective activity may refer to at least any one selected from the following characteristics: (a) inhibiting osteoclast differentiation; (b) inhibiting nuclear factor of activated T cells cytoplasmic 1 (NFATc1) or c-Fos; (c) expression of tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), cathepsin K, or dendritic cell-specific transmembrane protein (DC-STAMP); and (d) expression of AtPase H+ transporting V0 subunit D2 (Atp6v0d2). Therefore, the peptide can be used to prevent or treat bone diseases by inhibiting osteoclast differentiation.

[0014] In the peptide, in order to obtain chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., a wide range of biological activities), and reduced antigenicity, a protecting group may be bound to the N- or C-terminus at the end of the peptide. In one embodiment, the N-terminus of the peptide may be bound to any one of the protecting groups 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 a polyethylene glycol (PEG); and / or the C-terminus of the peptide may be bound to any one of the protecting groups selected from the group consisting of an amino group (-NH2), a tertiary alkyl group, and a hydrazino group (-NHNH2). Further, the peptide may optionally further include a targeting sequence, a tag, a labeled residue, and an amino acid sequence prepared for a specific purpose of increasing the half-life or peptide stability.

[0015] The peptide is artificially synthesized or non-naturally occurring or engineered, and the "non-naturally occurring or engineered" refers to a state generated by artificial modification, rather than the state of the existence itself produced in the natural state. Herein, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking the structures of multiple amino acids, or may include being engineered as above to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0016] The term "stability" used in this specification may refer not only to the in vivo stability of protecting the peptide from the attack of in vivo proteases, but also to storage stability (e.g., room temperature storage stability).

[0017] On the other hand, a pharmaceutical composition for preventing or treating bone diseases is provided, which includes a peptide composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0018] Among the terms or elements mentioned in the description of the peptide or composition, the same content as that already mentioned is as described above.

[0019] In this specification, the term "prevention" refers to all actions of inhibiting or delaying the occurrence of a disease by administering the composition.

[0020] In this specification, the term "treatment" refers to any form of treatment that provides an effect to an individual who has a disease or is likely to develop a disease, and the effects include improvement of the individual's condition (e.g., one or more symptoms), delay of disease progression, delay of symptom occurrence, or deceleration of symptom progression, etc. Therefore, the "treatment" and "prevention" do not represent that the symptoms are cured or completely eliminated.

[0021] The "individual" refers to the target of a disease to be treated. More specifically, it refers to humans or non-human primates, such as mammals such as mice, dogs, cats, horses, and cows.

[0022] The term "bone disease" in this specification, such as disorders, diseases or conditions related to RANKL-mediated signaling, may include not only disorders, diseases or conditions related to the regulation of bone formation and resorption, but also pathological conditions, which include bone loss such as osteopenia, osteoporosis, and osteolysis. The bone disease may be, for example, osteoporosis, osteogenesis imperfecta, osteomalacia, osteonecrosis, rickets, osteomyelitis, alveolar bone loss, Paget's disease of bone, hypercalcemia, primary hyperparathyroidism, multiple myeloma, bone loss in rheumatoid arthritis, bone loss caused by cancer, fibrous dysplasia of bone, osteodysplasia imperfecta, metabolic bone disease, or bone mass loss with age, but is not limited thereto.

[0023] The disadvantage of existing functional peptides is that even if they have effective biological activities, due to the size of the peptides themselves, they cannot be effectively introduced into target tissues or cells, or disappear in the body in a short time due to a short half-life. In contrast, the pharmaceutical composition according to an embodiment includes a peptide composed of about 10 or fewer amino acids as an active ingredient. Therefore, the skin permeability of the active ingredient is excellent. For example, when administered locally, an effective therapeutic effect on bone diseases can be obtained.

[0024] According to one embodiment, the peptide can not only inhibit the expression of NFATc1 and c-Fos, which are osteoclast differentiation transcription factors induced by RANKL, but also inhibit the expression of downstream signal transducers related to differentiation, such as TRAP, OSCAR, CTSK, DC-STAMP, and Atp6vOd2, to hinder the function of osteoclasts. Therefore, the peptide can be used as an active ingredient of 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).

[0025] The pharmaceutical composition may include a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier, but is not limited thereto.

[0026] The term "pharmaceutically effective amount" used in this specification refers to an amount sufficient to achieve the cartilage regeneration efficacy of the pharmaceutical composition.

[0027] The weight ratio between the peptide and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500. For example, the weight ratio may be 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.

[0028] The pharmaceutically acceptable carrier is commonly used in the preparation of preparations, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc., but is not limited thereto. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th Edition, 1995).

[0029] In addition to the above components, the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc., but is not limited thereto.

[0030] The pharmaceutical composition can be administered enterally or parenterally, preferably parenterally. For parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0031] The dosage of the pharmaceutical composition can be 0.0001 to 1000 μg (micrograms) per day, 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg, but is not limited thereto, and can be administered differently according to factors such as the formulation method, administration mode, patient's age, weight, gender, medical condition, diet, administration time, administration route, excretion rate, and reaction sensitivity.

[0032] The pharmaceutical composition can be formulated into a unit volume form by using pharmaceutically acceptable carriers and / or excipients according to methods easily implemented by those of ordinary skill in the technical field to which the present invention pertains, or can be prepared by injecting it into a multi-volume container.

[0033] The dosage form can be in the form of a solution, suspension, or emulsion in an oily or aqueous solvent, and can also be in the form of an ointment, cream, gel, transdermal absorbent, cataplasm, patch, paste, extract, powder, granule, lozenge, or capsule, and can further include a powder and / or a stabilizer.

[0034] The peptide can be included in nanobodies or nanoparticles to further improve skin penetration problems or stability problems. For example, the nanobodies can be prepared using a microfluidizer with lecithin as a raw material and can be included in lecithin particles. As the preparation method of the nanobodies, any known method can be used. The size of the nanobody particles is preferably 30 to 200 nm. When the size of the nanobody particles is less than 30 nm, skin penetration may be very fast, resulting in skin side effects. When the size of the nanobody particles is greater than 200 nm, it may not easily penetrate the skin, making it difficult to obtain the use effect of the nanobodies.

[0035] On the other hand, a method for preventing or treating bone diseases is provided, which includes the step of administering a composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient to an individual.

[0036] Among the terms or elements mentioned in the description of the peptide, composition, etc., those same as the items already mentioned are as described above.

[0037] As used herein, the terms "apply", "administer", and "coat" are used interchangeably and can refer to at least partially localizing a composition according to an embodiment at a desired site, or disposing a composition according to an embodiment within an individual by an administration route.

[0038] On the other hand, a cosmetic composition is provided, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0039] Among the terms or elements mentioned in the description of the peptide, composition, etc., those identical to the items already mentioned are as described above.

[0040] The cosmetic composition may comprise a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier, but is not limited thereto.

[0041]

Beneficial effects

[0042] Through the peptide according to one aspect, it is possible to inhibit osteoclast differentiation by blocking the differentiation transcription factor of osteoclasts induced by RANKL and the downstream signal transducers related to differentiation.

[0043] Through the peptide according to one aspect, it is possible to be applied to the prevention or treatment of bone diseases by inhibiting osteoclast differentiation.

Description of the drawings

[0044] Figure 1 The results of confirming the osteoclast differentiation level under a microscope after RAW264.7 cells were treated with both peptide-1 and receptor activator of nuclear factor-kappa B ligand (RANKL) to induce differentiation into osteoclasts are shown.

[0045] Figure 2 The results of confirming the osteoclast differentiation level under a microscope after RAW264.7 cells were treated with both peptide-2 and RANKL to induce differentiation into osteoclasts are shown.

[0046] Figure 3 To show the results of confirming the CCK-8 activity after treating RAW264.7 with peptide-2.

[0047] Figure 4 The results of evaluating tartrate-resistant acid phosphatase (TRAP) activity after RAW264.7 cells were treated with both peptide-1 and RANKL to induce differentiation into osteoclasts are shown.

[0048] Figure 5The results of evaluating TRAP activity after inducing the differentiation into osteoclasts by treating RAW264.7 with both peptide-2 and RANKL are shown.

[0049] Figure 6 The results of confirming the inhibition of mRNA expression of NFATc1 or c-Fos after inducing the differentiation into osteoclasts by treating RAW264.7 cells with both peptide-1 and RANKL are shown.

[0050] Figure 7 The results of confirming the inhibition of mRNA expression of NFATc1 or c-Fos at the time point 24 hours after (A) or 48 hours after (B) treating RAW264.7 cells with both peptide-1 and RANKL to induce the differentiation into osteoclasts are shown.

[0051] Figure 8 The results of confirming the inhibition of protein expression of NFATc1 after inducing the differentiation into osteoclasts by treating RAW264.7 cells with both peptide-1 and RANKL are shown.

[0052] Figure 9 The results of confirming the inhibition of protein expression of NFATc1 or c-Fos at the time point 24 hours after (A) or 48 hours after (B) treating RAW264.7 cells with both peptide-2 and RANKL to induce the differentiation into osteoclasts are shown.

[0053] Figure 10 The results of confirming the inhibition of mRNA expression of TRAP, OSCAR, CTSK, or DC-STAMP after inducing the differentiation into osteoclasts by treating RAW264.7 cells with both peptide-2 and RANKL are shown.

[0054] Figure 11 The results of confirming the inhibition of mRNA expression of TRAP, OSCAR, CTSK, DC-STAMP, or Atp6v0d2 at the time point 24 hours after (A) or 48 hours after (B) treating RAW264.7 with both peptide-2 and RANKL to induce the differentiation into osteoclasts are shown.

[0055] Figure 12 The results of confirming the inhibition of mRNA expression of c-Fos or NFATc1 at the time point 3 hours after (A) or 6 hours after (B) treating osteoclast precursors induced by RANKL with peptide-2 are shown.

[0056] Figure 13The results of confirming the inhibition of protein expression of c-Fos or NFATc1 at the time point 3 hours (A) or 6 hours (B) after treating RANKL-induced osteoclast precursors with Peptide-2 are shown.

[0057] Figure 14 The results of confirming the inhibition of mRNA expression of TRAP, OSCAR, CTSK, DC-STAMP, or Atp6v0d2 at the time point 3 hours (A) or 6 hours (B) after treating RANKL-induced osteoclast precursors with Peptide-2 are shown.

Detailed Description of the Invention

[0058] Hereinafter, more detailed description will be given by way of examples. However, these facts are only for providing exemplary illustration, and the scope of the present invention is not limited to these examples.

[0059]

Example 1. Synthesis of Peptides

[0060] An automatic peptide synthesizer (Milligen 9050, Millipore, USA) was used to synthesize peptides (Peptide-1 or Peptide-2) having the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2 described in Table 1 below, and the synthesized peptides were purified by C18 reversed-phase high performance liquid chromatography (HPLC) (Waters Associates, USA). ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co. USA) was used as the column.

[0061] [Table 1]

[0062] Name Amino acid sequence (N-terminus → C-terminus) SEQ ID NO Peptide-1 EGSNSRTI 1 Peptide-2 IIAPTGYYGN 2

[0063]

Example 2. Confirmation of the Effect of Inhibiting Differentiation into Osteoclasts

[0064] The purpose is to confirm the inhibitory effect of Peptide-1 or Peptide-2 treatment on osteoclast differentiation during the process of inducing the differentiation of osteoclast precursor cell line RAW264.7 cells into osteoclasts by treating with RANKL.

[0065] 【2-1. TRAP Staining and Cytotoxicity Evaluation】

[0066] RAW264.7 cells were seeded at 1.7×10 3The cells were seeded at a density of [X] cells / well in a 96-well plate and cultured in DMEM for 24 hours. After these 24 hours, they were treated with RANKL, Peptide-1, or Peptide-2 at different concentrations and induced to differentiate into osteoclasts for 4 days. Then, a TRAP staining was performed using an acid phosphatase kit from Sigma aldrich. After adding a fixing buffer thereto, the reaction was carried out for 30 seconds and washed with distilled water. 200 μl of the staining solution was added to each well and the reaction was carried out at 37 °C for 30 minutes, followed by washing 3 times with distilled water. After drying for 1 day, the staining results were confirmed under a microscope.

[0067] Furthermore, to further confirm the cytotoxicity of Peptide-2, RAW264.7 cells cultured in DMEM for 24 hours in the same manner as the above method were treated with the said peptide at different concentrations. Then, after adding 1 / 10 volume of CCK-8 (Dojindo, CCK-8 kit) of the culture medium to dilute it 3 days later, it was cultured for 2 hours. Then, the culture medium was sampled and the CCK-8 activity at a wavelength of 450 nm was confirmed using a microplate reader.

[0068] As a result, it was confirmed that as Figure 1 and 2 shown, compared with the control group in which osteoclast differentiation was induced by RANKL treatment and the TRAP expression increased, the TRAP expression in the groups treated with Peptide-1 or Peptide-2 was significantly reduced. And, as Figure 3 shown, Peptide-2 did not show cytotoxicity.

[0069] 【2-2. Evaluation of TRAP Activity】

[0070] RAW264.7 cells cultured in DMEM for 24 hours in the same manner as the above method were treated with RANKL and Peptide-1 or Peptide-2 at different concentrations and induced to differentiate into osteoclasts for 4 days. Then, after adding 100 μL of the TRAP activity solution [15 ml of TRAP buffer (0.1 sodium citrate + 50 μM sodium tartrate, pH 5.0) and 4-nitrophenylphosphate disodium hexahydrate (sigma, 1 tablet mix)] thereto, it was incubated at 37 °C for 1 hour, and 10 μl of 2N NaOH was added. Then, after measuring the absorbance at a wavelength of 450 nm using a microplate reader, it was quantified.

[0071] As a result, it was confirmed that as Figure 4 and Figure 5 shown, the TRAP activity in the groups treated with Peptide-1 or Peptide-2 was inhibited, and this inhibitory effect showed a concentration-dependent trend.

[0072]

Example 3. Confirmation of the effect of inhibiting osteoclast differentiation transcription factors

[0073] Among the various mechanisms that regulate osteoclast differentiation, the activation of the transcription factor nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) induced by RANKL is known to be essential for osteoclast differentiation. Therefore, it was planned to confirm the inhibitory effect of peptide-1 or peptide-2 on osteoclast differentiation transcription factors during the induction of osteoclast differentiation by treating the osteoclast precursor cell line RAW264.7 cells with RANKL.

[0074] Specifically, RAW264.7 cells were inoculated into a 96-well plate at a density of 1.7×10 3 cells / well and cultured in DMEM for 24 hours. After 24 hours, RANKL and peptide-1 or peptide-2 were used to treat them differently, and osteoclasts were induced to differentiate for 4 days. After 24 hours or 48 hours, the culture medium was aspirated, the cells were recovered, 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 below.

[0075] [Table 2]

[0076]

[0077] In addition, RAW264.7 cells cultured in DMEM for 24 hours in the same manner as the above method were induced to differentiate into osteoclasts. After 24 hours or 48 hours, the culture medium was aspirated, the cells were harvested, lysates were prepared, and then immunoblotting was performed. As the antibodies for detection, sc-166940 (Santa Cruz, USA) was used for c-Fos, and sc-7294 (Santa Cruz, USA) was used for NFATc1.

[0078] As a result, it was confirmed that, as Figures 6 to 9 shown, peptide-1 and peptide-2 inhibited the expression of osteoclast differentiation transcription factors such as NFATc1 and c-Fos induced by RANKL, respectively.

[0079]

Example 4. Confirmation of the inhibitory effect on downstream signal transducers related to osteoclast differentiation

[0080] During the process of inducing the differentiation of osteoclast precursor cell line RAW264.7 cells into osteoclasts by RANKL treatment, confirm the inhibitory effect on downstream signal transducers related to osteoclast differentiation caused by peptide-1 or peptide-02 treatment.

[0081] Specifically, inoculate RAW264.7 cells at a density of 1.7×10 3 cells / well in a 96-well plate and culture in DMEM for 24 hours. After 24 hours from this point, treat this with RANKL and peptide-1 or peptide-2 at different concentrations and induce differentiation into osteoclasts for 4 days. After 24 hours or 48 hours from this point, aspirate the culture medium, recover the cells and isolate RNA. Use a cDNA synthesis kit & PCR pre-mix (Intron, Korea) to synthesize cDNA and then perform RT-PCR using the TRAP, OSCAR, CTSK, DC-STAMP, and Atp6vOd2 primers shown in Table 3 below.

[0082] [Table 3]

[0083]

[0084] As a result, it was confirmed that, as Figure 10 and Figure 11 shown, peptide-1 and peptide-2 respectively inhibited the expression of downstream signal transducers related to osteoclast differentiation such as TRAP, OSCAR, CTSK, DC-STAMP, and Atp6vOd2d induced by RANKL.

[0085]

Example 5. Confirm the inhibitory effect on the differentiation of osteoclast precursors

[0086] The purpose is to confirm the inhibitory effect on the differentiation of osteoclast precursors caused by peptide-2 treatment in macrophages induced to differentiate into osteoclasts by RANKL.

[0087] 【5-1. Confirm the inhibitory effect on differentiation transcription factors】

[0088] Inoculate RAW264.7 cells at a density of 1.7×10 3The cells were seeded at a density of [cells per well] in a 96-well plate and cultured in DMEM for 24 hours. After 24 hours, they were treated with RANKL and Peptide-2 at different concentrations and induced to differentiate into osteoclasts. After 72 hours, the culture medium was aspirated, and then they were treated with RANKL and Peptide-2 again. After 3 hours or 6 hours, the cells were harvested to isolate RNA. cDNA was synthesized using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), and RT-PCR was performed using the c-Fos and NFATc1 primers shown in Table 2. Also, RAW264.7 cells cultured in DMEM for 24 hours in the same manner as the above method were induced to differentiate into osteoclasts. After 3 days, the culture medium was aspirated, and then they were treated with RANKL and Peptide-2. After 3 hours or 6 hours, the cells were harvested and lysates were prepared, and then immunoblotting was performed. As the antibodies for detection, sc-166940 (Santa Cruz, USA) was used for c-Fos, and sc-7294 (Santa Cruz, USA) was used for NFATc1.

[0089] As a result, it was confirmed that, as shown in Figure 12 and Figure 13 , Peptide-2 inhibited the expression of the differentiation transcription factors of osteoclast precursors, c-Fos and NFATc1.

[0090] [5-2. Confirmation of the inhibitory effect on downstream signal transducers related to differentiation]

[0091] RAW264.7 cells cultured in DMEM for 24 hours in the same manner as the above method were treated with RANKL and Peptide-2 at different concentrations and induced to differentiate into osteoclasts. After 72 hours, the culture medium was aspirated, and then they were treated with RANKL and Peptide-2 again. After 3 hours or 6 hours, the cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), and RT-PCR was performed using the TRAP, OSCAR, CTSK, DC-STAMP, and Atp6vOd2 primers shown in Table 3.

[0092] As a result, it was confirmed that, as shown in Figure 14 , Peptide-2 inhibited the expression of downstream signal transducers related to the differentiation of osteoclasts, such as TRAP, OSCAR, CTSK, DC-STAMP, and Atp6vOd2.

[0093] Based on the above experimental results, it can be seen that Peptide-1 and Peptide-2 according to one embodiment can respectively cause the differentiation and / or functional weakening of osteoclasts, and thus can be applied to the treatment of bone diseases.

[0094]

Formulation Example 1. Preparation of Peptide Nanobody

[0095] 50 mg of the peptide of Example 1 was dissolved by stirring well with 500 ml of distilled water. After mixing the complex solution with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol and a small amount of oil phase, the volume was adjusted to 1 L with distilled water, and then emulsified under high pressure using a high-pressure microfluidizer to prepare peptide nanobodies with a size of about 100 nm.

[0096]

Formulation Example 2. Pharmaceutical Preparation

[0097] 【2-1. Preparation of Powder】

[0098] The following components were mixed and filled into a sealed bag to prepare a powder.

[0099] 20 mg of the peptide of the present invention

[0100] 100 mg of lactose

[0101] 10 mg of talc

[0102] 【2-2. Preparation of Lozenge】

[0103] The following components were mixed and then compressed into lozenges according to the usual method for preparing lozenges.

[0104] 10 mg of the peptide of the present invention

[0105] 100 mg of corn starch

[0106] 100 mg of lactose

[0107] 2 mg of magnesium stearate

[0108] 【2-3. Preparation of Capsule】

[0109] According to the usual method for preparing capsules, the following components were mixed and filled into gelatin capsules to prepare capsules.

[0110] 10 mg of the peptide of the present invention

[0111] 3 mg of crystalline cellulose

[0112] 14.8 mg of lactose

[0113] 0.2 mg of magnesium stearate

[0114] 【2-4. Preparation of Injection】

[0115] According to the usual method for preparing injections, each ampoule (2 ml) was prepared with the following component contents.

[0116] 10 mg of the peptide of the present invention

[0117] 180 mg of mannitol

[0118] 2974 mg of sterile distilled water for injection

[0119] 26 mg of Na2HPO4·2H2O

[0120]

Preparation of liquid preparation

[0121] According to the usual method for preparing a liquid preparation, each component is added and dissolved in pure water, and the following components are mixed, and pure water is added to adjust the total volume to 100 ml, and then it is filled into a brown bottle and prepared into a liquid preparation after sterilization.

[0122] 10 mg of the peptide of the present invention

[0123] 10 g of isomerized sugar

[0124] 5 g of mannitol

[0125] An appropriate amount of pure water

[0126] The description of the present invention is only for providing examples, and those of ordinary skill in the art should be able to understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood as exemplary in all aspects and not restrictive.

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 bound to any one of the protecting groups selected from the group consisting of acetyl, fluorenylmethyloxycarbonyl, formyl, palmitoyl, myristoyl, stearoyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol.

3. The peptide according to claim 1, wherein, The C-terminus of the peptide is bound to any one of the protecting groups selected from the group consisting of amino, tertiary alkyl, and hydrazino.

4. The peptide according to claim 1, wherein, The peptide exhibits at least any one of the following properties: (a) Inhibiting the differentiation of osteoclasts; (b) Inhibiting the expression of nuclear factor of activated T-cells 1 or c-Fos; (c) Inhibiting the expression of tartrate-resistant acid phosphatase, osteoclast-associated receptor, cathepsin K, or dendritic cell-specific transmembrane protein; and (d) Inhibiting the expression of ATPase H+ transporting V0 subunit D2.

5. A pharmaceutical composition for preventing or treating bone diseases, comprising the peptide according to 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 prepared in the form of a nanobody.

8. The pharmaceutical composition according to claim 5, wherein, The bone diseases are 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, invisible bone disease, metabolic bone disease, or bone mass loss with aging.

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