Peptide for cartilage regeneration and use thereof

Abstract

The invention relates to a peptide with a cartilage regeneration effect and application thereof. Provided are a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 or Arg (R)-Tyr (Y)-Phe (F), a composition for cartilage regeneration comprising the peptide, and a pharmaceutical composition for preventing or treating cartilage diseases comprising the composition for cartilage regeneration as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a peptide for cartilage regeneration and its use. This patent application claims the priority of Korean Patent Application No. 10-2022-0171941, filed with the Korean Patent Office on December 9, 2022, the disclosure of which is incorporated herein by reference.

Background Art

[0002] Due to the characteristics of cartilage tissue, it is difficult to regenerate tissue by natural healing when damaged over a large area, so it is treated by surgical procedures such as artificial joints, arthroscopic chondroplasty, and microfracture. However, existing methods often leave scars due to incisions and result in low durability of fibrocartilage regeneration. Therefore, compared with the difficulty of the surgical method, there is a problem of low treatment effect.

[0003] Therefore, injections applied intra-articularly or compositions for restoring cartilage tissue using hydrogels and collagen have been developed. The surgical procedure is simple and the treatment effect is rapid (Korean Patent Publication No. 2013-0028012). However, although the method can temporarily relieve pain, it is not sufficient to induce cartilage tissue regeneration.

[0004] In addition, among treatment methods using cells, various treatment methods using autologous chondrocytes or stem cells have been developed, that is, a method of inducing cartilage tissue regeneration by transplanting in vitro cultured cells to the defect site (Korean Patent Publication No. 2013-0072983). However, in autologous chondrocyte therapeutic agents, when the injury area is large, treatment with only cultured cells collected from the patient is limited. And in stem cell therapeutic agents, there are the following problems: due to different collection sites, there are differences in cell number and differentiation ability, cell dedifferentiation during in vitro culture leads to changes in cell phenotype, the ratio of differentiation into chondrocytes after transplantation into the body is low, and gene expression related to cell hypertrophy induces apoptosis and vascular permeability of cells, resulting in calcification of chondrocytes.

[0005] In this technical background, there is currently a need to develop effective factors that can more effectively treat cartilage injury diseases by promoting chondrogenic differentiation or chondrogenesis of stem cells or chondrocytes, but it is still not perfect.

Summary of the Invention

[0006]

Technical Problem

[0007] To solve the above problems, the inventors of the present invention developed a novel peptide, and it was confirmed that the novel peptide can be effectively used for preventing or treating cartilage diseases and promoting cartilage regeneration by significantly increasing various cartilage components such as glycosaminoglycan, collagen, COMP, and aggrecan, thus completing the present invention.

[0008] One object of the present invention is to provide a peptide composed of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F).

[0009] Another object of the present invention is to provide a composition for cartilage regeneration, which comprises a peptide composed of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F) as an active ingredient.

[0010] Still another object of the present invention is to provide a pharmaceutical composition, which comprises the composition for cartilage regeneration as an active ingredient.

[0011] However, the technical problems to be achieved by the present invention are not limited to the above problems, and those skilled in the art can clearly understand other problems not mentioned through the following description.

[0012]

Technical Solution

[0013] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art of the present invention. Generally, the nomenclature used in this specification is well-known and commonly used in the art.

[0014] On the one hand, a peptide composed of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F) is provided.

[0015] The term "peptide" as used in this specification may refer to a linear molecule formed by binding multiple amino acid residues 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). As a result of efforts to develop a peptide having biologically effective activity, the present inventors identified a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F). Here, the biologically effective activity may refer to at least any one of the characteristics selected from (a) inducing the production of glycosaminoglycan; (b) inducing the production of COL2A1 (Collagen type II Alpha 1), COMP (Cartilage oligomeric matrix protein), COL11A1 (Collagen type XI Alpha1), or aggrecan; and (c) inducing the production of SOX5 (Sex determining region Y-Box Transcription Factor 5), SOX6 (Sex determining region Y-Box Transcription Factor 6), or SOX9 (Sex determining region Y-Box Transcription Factor 9) as regulatory factors. Therefore, the peptide can be used for cartilage regeneration.

[0016] In the peptide, for the purpose of obtaining 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.

[0017] The peptide is artificially synthesized or non-naturally occurring or engineered. The term "non-naturally occurring or engineered" refers to a state generated by artificial modification, rather than the state of the existence itself produced in a 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 engineering as described above to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0018] 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).

[0019] On the other hand, a composition for cartilage regeneration is provided, which comprises a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F) as an active ingredient.

[0020] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0021] As used herein, the term "cartilage regeneration" may refer to improving cartilage tissue by repairing damaged cartilage tissue or inducing the generation of defective cartilage tissue. The "improvement" may refer to all actions that alleviate the condition or at least reduce treatment-related parameters such as the degree of symptoms.

[0022] The cartilage includes hyaline cartilage, fibrocartilage, or elastic cartilage, but is not limited thereto. For example, the cartilage may be at least one selected from the group consisting of articular cartilage, ear cartilage, nasal cartilage, elbow cartilage, meniscus cartilage, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage, and spinal cartilage.

[0023] The disadvantage of existing functional peptides is that, even if they have effective biological activities, they cannot be effectively introduced into target tissues or cells due to the size of the peptide itself, or they disappear from the body within a short time because of their short half-life. On the other hand, the composition for cartilage regeneration according to an embodiment includes a peptide composed of 10 or fewer amino acids as an active ingredient. Therefore, the skin permeability of the active ingredient is very excellent. For example, when applied topically, an effective cartilage regeneration effect can be obtained.

[0024] According to an embodiment, since the peptide can significantly increase the expression of glycosaminoglycan, COL2A1, COMP, COL11A1, aggrecan, which are cartilage-related substances, and SOX5, SOX6, or SOX9, which are regulatory factors, the peptide can be used as an active ingredient of a composition for cartilage regeneration (Orthop Res Rev., September 1, 2010; 2010(2):85-94.doi:10.2147 / ORR.S7194, JOSPT Volume 28Number 4, October 1998).

[0025] On the other hand, a pharmaceutical composition for preventing or treating cartilage diseases is provided, which includes a peptide composed of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F) as an active ingredient.

[0026] Among the terms or elements mentioned in the description of the peptide or composition, the same items as those already mentioned are as described above.

[0027] In this specification, the term "prevention" refers to all actions of inhibiting or delaying the occurrence of a disease by administering the composition.

[0028] In this specification, the term "treatment" refers to any form of treatment that provides an effect to an individual who has or is likely to have a disease, and the effects include improvement of the individual's condition (e.g., one or more symptoms), delay in disease progression, delay in symptom occurrence, or deceleration of symptom progression, etc. Therefore, the "treatment" and "prevention" do not represent that the symptoms are cured or completely eliminated.

[0029] The "individual" refers to the target for treating a disease, and more specifically, refers to a human or non-human primate, and mammals such as a mouse, a dog, a cat, a horse, and a cow.

[0030] In this specification, the term "cartilage disease" refers to all cartilage-related diseases that require cartilage differentiation or regeneration. The cartilage disease may be selected from at least one of the group consisting of cartilage injury, cartilage defect, degenerative disc disease, disc herniation, degenerative arthritis, fracture, muscle tissue injury, joint injury caused by non-union of fracture or trauma, osteomalacia, and chondromalacia.

[0031] The cartilage disease may occur in the temporomandibular joint, shoulder joint, elbow joint, wrist joint, finger joint, spinal joint, hip joint, knee joint, ankle joint, or toe joint.

[0032] The pharmaceutical composition may include a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier, but is not limited thereto.

[0033] The term "pharmaceutically effective amount" used in this specification refers to an amount sufficient to achieve the cartilage regeneration efficacy of the pharmaceutical composition.

[0034] 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.

[0035] The pharmaceutically acceptable carriers are those commonly used in the preparation of pharmaceutical formulations, including 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 not limited thereto. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th Edition, 1995).

[0036] 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 not limited thereto.

[0037] 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 not limited thereto.

[0038] 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 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.

[0039] The pharmaceutical composition can be formulated into 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 filling into multi-volume containers.

[0040] 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, extractant, powder, granule, lozenge, or capsule, and can further include powders and / or stabilizers.

[0041] The peptide may be included in a nanobody or nanoparticle to further improve skin penetration problems or stability problems. For example, the nanobody can be prepared using a microfluidizer with lecithin as a raw material and can be included in lecithin particles. As a method for preparing the nanobody, any well-known method can be used. The size of the nanobody particles is preferably 30 to 200 nm. In the case where the size of the nanobody particles is less than 30 nm, skin penetration may be very fast, resulting in skin side effects. In the case where the size of the nanobody particles is greater than 200 nm, it may not be easy to penetrate the skin, making it difficult to obtain the usage effect of the nanobody.

[0042] In another aspect, there is provided a method for preventing or treating cartilage diseases, which includes the step of administering a therapeutically effective amount of a pharmaceutical composition to an individual, wherein the pharmaceutical composition includes a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F) as an active ingredient.

[0043] 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.

[0044] The terms "apply", "administer", and "coat" used in this specification can be used interchangeably and can refer to localizing at least part of the composition according to an embodiment at a desired site, or disposing the composition according to an embodiment within an individual through an administration route.

[0045] In another aspect, there is provided a cosmetic composition, which includes a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F) as an active ingredient.

[0046] 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.

[0047] The cosmetic composition may include a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier, but is not limited thereto.

[0048] In another aspect, there is provided a method for cartilage regeneration, which includes the step of administering a composition to an individual, wherein the composition includes a peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F) as an active ingredient.

[0049] 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.

[0050]

Beneficial effects

[0051] The peptide according to one aspect can exhibit excellent cartilage regeneration effects by significantly increasing various cartilage components such as glycosaminoglycans, collagen, COMP, and aggrecan.

[0052] The peptide according to one aspect can be applied to the prevention or treatment of cartilage diseases and promote cartilage regeneration by significantly increasing various cartilage components such as glycosaminoglycans, collagen, COMP, and aggrecan.

[0053]

Brief description of the drawings

[0054] Figure 1 Shows the results of confirming the CCK-8 activity after treating C28 / I2 cells with peptide-1.

[0055] Figure 2 Shows the results of confirming the CCK-8 activity after treating C28 / I2 cells with peptide-2.

[0056] Figure 3 Shows the results of confirming an increase in the production of glycosaminoglycans after treating C28 / I2 cells with peptide-1.

[0057] Figure 4 Shows the results of confirming an increase in the production of glycosaminoglycans after treating C28 / I2 cells with peptide-2.

[0058] Figure 5 Shows the results of confirming an increase in the expression of mRNA of ECM components after treating C28 / I2 cells with peptide-1, after 3 days of culture (a) and after 7 days of culture (b).

[0059] Figure 6 Shows the results of confirming an increase in the expression of mRNA of ECM components after treating C28 / I2 cells with peptide-2, after 3 days of culture (a) and after 7 days of culture (b).

[0060] Figure 7 Shows the results of confirming an increase in the mRNA expression of SOX9, which is an ECM regulator, after treating C28 / I2 cells with peptide-1.

[0061] Figure 8 Shows the results of confirming an increase in the mRNA expression of SOX9, which is an ECM regulator, after treating C28 / I2 cells with peptide-2.

[0062] Figure 9 Shows the results of confirming an increase in the expression of SOX5, SOX6, and SOX9 proteins, which are ECM regulators, after treating C28 / I2 cells with peptide-1.

[0063] Figure 10 The results showing an increase in the expression of SOX9 protein, which is an ECM regulator, were confirmed after treating C28 / I2 cells with Peptide-2.

Detailed Description of the Invention

[0064] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only for exemplarily describing the present invention, and the scope of the present invention is not limited to these examples.

[0065]

Example 1. Synthesis of Peptide

[0066] Peptide-1 or Peptide-2 described in Table 1 below was synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptide was 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.

[0067] [Table 1]

[0068] Name Amino acid sequence (N-terminal -> C-terminal) SEQ ID NO. Peptide-1 RYF - Peptide-2 TAVRVG 1

[0069]

Example 2. Confirmation of Cytotoxicity

[0070] The purpose was to analyze the cytotoxicity caused by adding Peptide-1 or Peptide-2 in human chondrocytes (C28 / I2, human chondrocyte cell line) using the CCK-8 assay.

[0071] Specifically, human chondrocytes were seeded in a 96-well plate at a density of 3×10 3 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was changed to a new one, and Peptide-1 or Peptide-2 was treated at different concentrations. Then, after 3 days, a CCK-8 (Dojindo, CCK-8 kit) solution was added at one-tenth of the culture medium volume and incubated for 2 hours. Samples of the culture medium were taken, and the CCK-8 activity was confirmed using a microplate reader at a wavelength of 450 nm. On the other hand, a test group without adding the peptide was used as a negative control (CON).

[0072] As a result, as Figure 1 and Figure 2 shown, it was found that neither Peptide-1 nor Peptide-2 showed toxicity in human chondrocytes.

[0073]

Example 3. Confirmation of the production effect of glycosaminoglycan

[0074] The purpose is to confirm the effect of increasing the production of glycosaminoglycan by adding Peptide-1 or Peptide-2 in human chondrocytes, so as to confirm the effect of the peptide on inducing chondrogenesis and promoting the production of extracellular matrix (ECM).

[0075] Specifically, human chondrocytes were seeded in a 96-well plate at a density of 3×10 3 cells / well and cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, the medium was changed to a new one, and Peptide-1 or Peptide-2 was treated at different concentrations. Subsequently, the medium was changed every 3 days, and Peptide-1 or Peptide-2 was treated at different concentrations. After 7 days, the medium was aspirated, and 60 μL of 3.7% formaldehyde was added to the 96-well plate for staining and fixed for 1 minute. After aspirating the 3.7% formaldehyde, 70 μL of Alcian blue staining solution (50 mL of 3% acetic acid + 0.5 g of 1% Alcian blue 8GX, pH 2.5) was added. After incubating at 37 °C for 24 hours, the staining solution was aspirated, washed three times with distilled water and dried, and observed under a microscope. On the other hand, the experimental group without adding the peptide was used as the negative control group (CON).

[0076] As a result, as Figure 3 and Figure 4 shown, it was found that both Peptide-1 and Peptide-2 could promote the production of glycosaminoglycan.

[0077]

Example 4. Confirmation of the effect of increased mRNA expression of ECM components

[0078] The purpose is to confirm whether the treatment of Peptide-1 or Peptide-2 will increase the mRNA expression of ECM components in human chondrocytes.

[0079] Specifically, human chondrocytes were seeded at a density of 8.9×10 4After seeding cells at a density of cells / well in a 6-well plate, they were cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was replaced with fresh medium, and the cells were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and the cells were treated with peptide-1 or peptide-2 at different concentrations. After 3 days and 7 days, the medium was aspirated, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit and a PCR premix (Intron, Korea), PCR was performed using the hCOL2A1, COMP, hCOL11A1, ACAN, and GAPDH primers shown in Table 2 below. On the other hand, an experimental group without the addition of the peptide was used as a negative control (CON), and an experimental group with the addition of TGF-β1 (20 ng / ml) was used as a positive control.

[0080] In Table 2 below, hCOL2A1 encodes α1 of type II collagen, hCOMP encodes cartilage oligomeric matrix protein, hCOL11A1 encodes α1 of type XI collagen, hACAN encodes aggrecan, and hGAPDH encodes glyceraldehyde-3-phosphate dehydrogenase.

[0081] [Table 2]

[0082]

[0083] As a result, as Figure 5 and Figure 6 shown, it was found that both peptide-1 and peptide-2 could increase the mRNA expression of COL2A1, COMP, COL11A1, and ACAN as components of the ECM.

[0084] [Example 5. Confirmation of the effect of increased mRNA expression of SOX9 as an ECM regulator]

[0085] The aim was to confirm whether the treatment with peptide-1 or peptide-2 would increase the mRNA expression of SOX9, an ECM regulator, in human chondrocytes.

[0086] Specifically, human chondrocytes were seeded at 8.9×10 4After inoculating the cells at a density of cells / well in a 6-well plate, they were cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was replaced with fresh medium, and the cells were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and the cells were treated with peptide-1 or peptide-2 at different concentrations. After 3 days, the medium was aspirated, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit and a PCR premix (Intron, Korea), PCR was performed using the primers shown in Table 3 below. On the other hand, a test group without the addition of the peptide was used as a negative control (CON), and a test group with the addition of TGF-β1 (20 ng / ml) was used as a positive control.

[0087] In Table 3 below, SOX9 refers to (sex determining region Y)-box 9.

[0088] [Table 3]

[0089]

[0090] As a result, as Figure 7 and Figure 8 shown, it was found that both peptide-1 and peptide-2 could induce an increase in the mRNA expression of SOX9, an ECM regulator.

[0091]

Example 6. Confirmation of the effect of increased expression of SOX5, SOX6, and SOX9 proteins as ECM regulators

[0092] The purpose was to confirm whether the treatment with peptide-1 or peptide-2 would increase the expression of SOX5, SOX6, and SOX9 proteins as ECM regulators in human chondrocytes.

[0093] Specifically, after inoculating human chondrocytes at a density of 8.9×10 4 cells / well in a 6-well plate, they were cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was replaced with fresh medium, and the cells were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and the cells were treated with peptide-1 or peptide-2 at different concentrations. After 7 days, the medium was aspirated, the cells were harvested, lysates were prepared, and Western blotting was performed. On the other hand, a test group without the addition of the peptide was used as a negative control (CON), and a test group with the addition of TGF-β1 (20 ng / ml) was used as a positive control.

[0094] As detection antibodies, sc-293215 (Santa Cruz, USA) was used for SOX5, sc-393314 (Santa Cruz, USA) was used for SOX6, and 82630S (Cell Signaling, USA) was used for SOX9.

[0095] As a result, as Figure 9 and Figure 10 shown, it was found that peptide-1 could increase the expression of SOX5, SOX6, and SOX9 proteins, which are ECM regulators, and peptide-2 could increase the expression of SOX9 protein, which is an ECM regulator.

[0096] Based on the above test results, it was found that both peptide-1 and peptide-2 according to one embodiment had the effect of inducing cartilage regeneration.

[0097]

Formulation Example 1. Preparation of Peptide Nanobody

[0098] 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.

[0099]

Formulation Example 2. Pharmaceutical Preparation

[0100] 【2-1. Preparation of Powder】

[0101] The following components were mixed and filled into a sealed bag to prepare a powder.

[0102] 20 mg of the peptide of the present invention

[0103] 100 mg of lactose

[0104] 10 mg of talc

[0105] 【2-2. Preparation of Troche】

[0106] The following components were mixed and then pressed into a troche according to the usual method for preparing troches.

[0107] 10 mg of the peptide of the present invention

[0108] 100 mg of corn starch

[0109] 100 mg of lactose

[0110] 2 mg of magnesium stearate

[0111] 【2-3. Preparation of Capsules】

[0112] According to the usual method for preparing capsules, the following components are mixed, filled into gelatin capsules, and capsules are prepared.

[0113] 10 mg of the peptide of the present invention

[0114] 3 mg of crystalline cellulose

[0115] 14.8 mg of lactose

[0116] 0.2 mg of magnesium stearate

[0117] 【2-4. Preparation of Injectables】

[0118] According to the usual method for preparing injectables, each ampoule (2 ml) is prepared with the following component contents.

[0119] 10 mg of the peptide of the present invention

[0120] 180 mg of mannitol

[0121] 2974 mg of sterile distilled water for injection

[0122] 26 mg of Na2HPO4·2H2O

[0123] 【2-5. Preparation of Liquid Preparations】

[0124] According to the usual method for preparing liquid preparations, each component is added and dissolved in pure water, and the following components are mixed, adjusted to a total volume of 100 ml with the addition of pure water, and then filled into brown bottles and sterilized to prepare liquid preparations.

[0125] 10 mg of the peptide of the present invention

[0126] 10 g of isomerized sugar

[0127] 5 g of mannitol

[0128] An appropriate amount of pure water

[0129] As described above, specific parts of the present invention have been described in detail, but it is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. A peptide consisting of the amino acid sequence shown by SEQ ID NO: 1 or Arg(R)-Tyr(Y)-Phe(F).

2. The peptide according to claim 1, wherein, The N-terminus of the peptide is bound to any one protecting group 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 protecting group 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) Inducing the production of glycosaminoglycan; (b) Inducing the production of COL2A1, COMP, COL11A1, or aggrecan; and (c) Inducing the production of SOX5, SOX6, or SOX9 as a regulatory factor.

5. A composition for cartilage regeneration, comprising the peptide according to any one of claims 1 to 4 as an active ingredient.

6. A pharmaceutical composition for preventing or treating cartilage diseases, comprising the peptide according to any one of claims 1 to 4 as an active ingredient.

7. The pharmaceutical composition according to claim 6, which further comprises a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 6, wherein, The peptide is prepared in the form of a nanobody.

9. The pharmaceutical composition according to claim 6, wherein, The cartilage diseases are selected from at least one of the group consisting of cartilage injury, cartilage defect, degenerative disc disease, herniated disc, degenerative arthritis, fracture, muscle tissue injury, joint injury caused by non-union of fracture or trauma, osteomalacia, and chondromalacia.

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