Cartilage regeneration peptides and their applications

Abstract

The present application relates to a peptide having a cartilage regeneration effect and uses thereof, and provides a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S), a composition for cartilage regeneration containing the peptide, and a pharmaceutical composition for preventing or treating cartilage diseases containing the composition for cartilage regeneration as an active ingredient.

Description

Technical Field

[0001] This application relates to a peptide for cartilage regeneration and its uses.

Background Art

[0002] Due to the characteristics of cartilage tissue, when it is damaged extensively, it is difficult for tissue regeneration by natural healing. Therefore, treatments by surgical operations such as artificial joints, arthroscopic chondroplasty, and microperforation have been carried out. However, conventional methods have the problem that there remains a scar due to the incision, often resulting in the regeneration of fibrocartilage with reduced durability, and the treatment effect is lower compared to difficult surgical methods.

[0003] Therefore, injection solutions for intra-articular administration or compositions for cartilage tissue repair using hydrogels, collagen, etc., which show a treatment effect with a simple and rapid surgical process, have been developed (Korean Patent Publication No. 2013 - 0028012). However, although the above method can obtain a temporary reduction in pain, it is insufficient for inducing regeneration in cartilage tissue.

[0004] Also, in the case of treatment methods using cells, as a method of transplanting cells cultured in vitro again to the defect site to induce the regeneration of cartilage tissue, various treatment methods using autologous cartilage cells or stem cells, etc., have been developed (Korean Patent Publication No. 2013 - 0072983). However, in the case of autologous cartilage cell therapeutic agents, when the damaged site is large, there is a limit to treatment with only the cells collected and cultured from the patient. In the case of stem cell therapeutic agents, there are problems such as differences in cell number and differentiation ability depending on the collection site, changes in cell phenotypes due to dedifferentiation of cells during in vitro culture, low differentiation rate into cartilage cells after in vivo transplantation, and gene expression related to cell hypertrophy leading to cell death and calcification of cartilage cells due to the induction of vascular invasion.

[0005] Under such a technical background, although the development of effective factors that can enable more effective treatment of cartilage diseases by promoting the chondrogenic differentiation or chondrogenesis of stem cells or cartilage cells is required, the current situation is still incomplete.

Summary of the Invention

[0006] One embodiment is to provide a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0007] Another embodiment provides a cartilage regeneration composition containing as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0008] Another embodiment provides a pharmaceutical composition for the prevention or treatment of cartilage disease, comprising the cartilage regeneration composition as an active ingredient.

[0009] 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]

[0010] 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.

[0011] One embodiment provides a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0012] 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 synthesis techniques or liquid-phase synthesis techniques (US Patent No. 5,516,891). The inventors, through diligent efforts to develop a peptide with biologically effective activity, have identified a peptide consisting of the amino acid sequence represented by Sequence ID No. 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S). Here, the biologically effective activity is also one or more selected from the following characteristics: (a) induction of glycosaminoglycan production; (b) induction of COL2A1, COMP (Cartilage oligomeric matrix protein), COL11A, PCP (proteoglycan core protein), or aggrecan production; (c) induction of regulatory factors SOX5, SOX6, or SOX9 production; and (d) activation of the upstream activator of regulatory factor SOX9. Therefore, the peptide can be utilized for cartilage regeneration.

[0013] The peptides may also have a protecting group attached to the N-terminus 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 is bonded to one protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol (PEG); and / or, the C-terminus of the peptide may be bonded to one protecting group selected from the group consisting of amino, -NH2, tertiary alkyl, and azide (-NHNH2). The peptide may also selectively further include targeted sequences, tags, labeled residues, and amino acid sequences manufactured for specific purposes 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 cartilage regeneration composition containing as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

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

[0018] As used herein, the term "cartilage regeneration" also means repairing damaged cartilage tissue or improving cartilage tissue by inducing the generation of insufficient cartilage tissue. The term "improvement" may mean any action that alleviates or treats a condition, such as reducing the severity of symptoms.

[0019] The cartilage includes, but is not limited to, hyaline cartilage, fibrocartilage, or elastic cartilage. For example, the cartilage may also be one or more 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 vertebral cartilage.

[0020] Conventional functional peptides, despite their effective biological activity, suffer from disadvantages such as not being effectively absorbed into target tissues or cells due to their size, or being eliminated from the body quickly due to their short half-life. On the other hand, the cartilage regeneration composition according to one example contains a peptide consisting of approximately 10 or fewer amino acids as an active ingredient, resulting in excellent skin penetration of the active ingredient. For example, when administered topically, an effective cartilage regeneration effect can be obtained.

[0021] According to one embodiment, the peptide can significantly increase the expression of glycosaminoglycan, COL2A1, COMP, COL11A, PCP, aggrecan, regulatory factors SOX5, SOX6 or SOX9, which are substances related to cartilage, and the peptide can be utilized as an active ingredient of a composition for cartilage regeneration (Orthop Res Rev. 2010 September 1; 2010(2): 85-94. doi:10.2147 / ORR.S7194, JOSPT Volume 28 Number 4 October 1998).

[0022] Still another aspect provides a pharmaceutical composition for preventing or treating cartilage diseases, comprising, as an active ingredient, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0023] Among the terms or elements mentioned in the description related to the peptide or composition, those same as the ones already mentioned are as described above.

[0024] As used herein, the term "prevention" means all acts of suppressing or delaying the onset of a disease by administration of the composition.

[0025] As used herein, the term "treatment" means any form of treatment that provides an effect including improvement of the state (e.g., one or more symptoms) of an individual suffering from a disease or having a potential for developing a disease, delay of disease progression, delay of symptom occurrence, or blunting of symptom progression. Thus, the "treatment" and "prevention" are not intended to mean cure or complete removal of symptoms.

[0026] The "individual" means a subject that requires treatment of a disease, and more specifically, means mammals such as humans or non-human primates, mice, dogs, cats, horses, and cows.

[0027] In this specification, the term "cartilage disease" means all diseases relating to cartilage that require cartilage differentiation or regeneration. The cartilage disease is also one or more selected from the group consisting of cartilage injury, cartilage defect, degenerative disc disease, disc herniation, degenerative arthritis, fracture, muscle tissue injury, fracture failure or traumatic joint injury, osteomalacia, and chondromalacia.

[0028] The aforementioned cartilage disorders can occur in the temporomandibular joint, shoulder joint, elbow joint, wrist joint, finger joint, spinal joint, hip joint, knee joint, ankle joint, or toe joint.

[0029] The pharmaceutical composition may, but is not limited to, contain a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier.

[0030] As used herein, the term "pharmaceutical effective amount" may mean an amount sufficient to achieve the cartilage regeneration efficacy of the pharmaceutical composition.

[0031] The weight ratio between the peptide and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500, and may also be, but is not limited to, 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2.

[0032] The pharmaceutically acceptable carriers mentioned above are those commonly used in the manufacture of pharmaceutical products and include, but are not limited to, lactose, dextrose, saccharose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0033] The aforementioned pharmaceutical composition may further contain, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspension agents, preservatives, and the like, in addition to the aforementioned components.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Another embodiment provides a method for preventing or treating cartilage disease, comprising the step of administering to an individual a pharmaceutical composition containing a therapeutically effective amount of a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S) as an active ingredient.

[0040] 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.

[0041] 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.

[0042] Another embodiment provides a cosmetic composition containing as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0043] 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.

[0044] The cosmetic composition may also contain, but is not limited to, a cosmetically effective amount of the peptide and / or a cosmetically acceptable carrier.

[0045] Another embodiment provides a method for regenerating cartilage, comprising the step of administering to an individual a composition containing as an active ingredient a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0046] 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. [Effects of the Invention]

[0047] According to one embodiment of the peptide, it significantly increases various cartilage components such as glycosaminoglycans, collagen, COMP, and aggrecan, thereby exhibiting excellent cartilage regeneration effects.

[0048] According to one embodiment of the peptide, it can be applied to prevent or treat cartilage diseases and promote cartilage regeneration by significantly increasing various cartilage components such as glycosaminoglycans, collagen, COMP, and aggrecan. [Brief explanation of the drawing]

[0049] [Figure 1]This shows the results of treating AD-MSC cells with Peptide-1 and then observing changes in cell morphology by SRB staining. [Figure 2] This shows the results of treating AD-MSC cells with Peptide-2 and then confirming changes in cell morphology by SRB staining. [Figure 3] This is the result of checking CCK-8 activity after treating AD-MSC cells with Peptide-1. [Figure 4] This is the result of checking CCK-8 activity after treating AD-MSC cells with Peptide-2. [Figure 5] This result shows an increase in glycosaminoglycan production after treating AD-MSC cells with Peptide-1. [Figure 6] This result shows an increase in glycosaminoglycan production after treating AD-MSC cells with Peptide-2. [Figure 7] This is the result of confirming increased mRNA expression of ECM components after treating AD-MSC cells with Peptide-1. [Figure 8] This is the result of confirming increased mRNA expression of ECM components after treating AD-MSC cells with Peptide-2. [Figure 9] This result shows an increase in the production of SOX9, an ECM regulator, after treating AD-MSC cells with Peptide-1. [Figure 10] This result shows an increase in the production of SOX9, an ECM regulator, after treating AD-MSC cells with Peptide-2. [Figure 11] This result shows that after treating AD-MSC cells with Peptide-1, the expression of ECM regulators SOX5, SOX6, and SOX9 increased. [Figure 12] This result shows that after treating AD-MSC cells with Peptide-2, the expression of ECM regulators SOX5, SOX6, and SOX9 increased. [Figure 13]This result shows that after treating AD-MSC cells with Peptide-1, the expression of cartilage components aggrecan and COL2A1 was increased. [Figure 14] This result shows that after treating AD-MSC cells with Peptide-2, the expression of cartilage components aggrecan and COL2A1 was increased. [Figure 15] This study confirmed that treating AD-MSC cells with Peptide-2 activated the superior activators of the ECM regulator SOX9 in chondrocytes. [Modes for carrying out the invention]

[0050] 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.

[0051] Example 1. Synthesis of peptides Peptide-1 or Peptide-2, as listed in Table 1 below, were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the 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).

[0052] [Table 1]

[0053] Example 2. Confirmation of cytotoxicity We attempted to analyze the changes in cell morphology and cytotoxicity in human adipose-derived mesenchymal stem cells (AD-MSCs) induced by the addition of peptide-1 or peptide-2, using SRB staining and the CCK-8 assay, respectively.

[0054] Specifically, AD-MSC cells 1.5 x 10 3 After seeding cells in a 96-well plate at a cell / well density, the cells were cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with 10% FBS. The medium was then replaced with DMEM medium supplemented with 5% FBS, followed by treatment with Peptide-1 or Peptide-2 at different concentrations. The medium was then changed every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. To confirm CCK-8 activity, after 7 days, a 1 / 10 volume CCK-8 (Dojindo, CCK-8 kit) solution was added to the culture medium, followed by incubation for 2 hours. The culture medium was sampled and CCK-8 activity was confirmed at a 450 nm wavelength using a microplate reader.

[0055] Furthermore, for SRB staining, after suctioning the culture medium from the plate, 60 μL of 3.7% formalin was added to a 96-well plate to be stained and fixed for 1 minute. After suctioning with 3.7% formalin, 70 μL of SRB staining solution (sulfolodamine B sodium salt (Sigma-A, S9012): 0.2 g of 100 mL DDW) was added and stained. The plate was covered with silver foil, light was blocked, and it was incubated at room temperature overnight. After washing with 100 μL of 1% acetic acid using a multipipette, it was dried and observed under a microscope. On the other hand, in this example, the positive control group (CM) used was a plate to which dexamethasone (100 nM), acetic acid (50 μM), proline (40 μM), TGFβ1 (10 ng / ml), and 1X ITS had been added.

[0056] As a result, as shown in Figures 1 and 2, it was found that Peptide-1 or Peptide-2 did not cause any changes in cell morphology when applied to adipose-derived mesenchymal stem cells, as measured by SRB staining. Furthermore, as shown in Figures 3 and 4, it was found that Peptide-1 or Peptide-2 did not exhibit toxicity to adipose-derived mesenchymal stem cells.

[0057] Example 3. Confirmation of glycosaminoglycan generation effect We aimed to confirm the cartilage-inducing and extracellular matrix (ECM)-promoting effects of the peptides by examining the effect of adding Peptide-1 or Peptide-2 on increasing glycosaminoglycan production in adipose-derived mesenchymal stem cells.

[0058] Specifically, AD-MSC cells 1.5 x 10 3 After seeding 96-well plates at a cell / well density, the cells were cultured for 24 hours in DMEM medium supplemented with 10% FBS. The medium was then replaced with DMEM medium supplemented with 5% FBS, followed by treatment with Peptide-1 or Peptide-2 at different concentrations. The medium was then changed every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 14 days, the medium was added to a 96-well plate by suction and stained with 60 μL of 3.7% formalin, and fixed for 1 minute. After suctioning with 3.7% formalin, 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 dispensed. After incubation at 37°C for 24 hours, the staining solution was suctioned, washed with tertiary distilled water, dried, and observed under a microscope.

[0059] As a result, as shown in Figures 5 and 6, it was found that Peptide-1 and Peptide-2, respectively, increase glycosaminoglycan production, induce cartilage formation, and promote extracellular matrix production.

[0060] Example 4. Confirmation of increased mRNA expression in the ECM. We aimed to determine whether treatment with Peptide-1 or Peptide-2 increased mRNA expression of ECM components in adipose-derived mesenchymal stem cells.

[0061] Specifically, AD-MSC cells 1.5 x 10 3After seeding 96-well plates at a cell / well density, cells were cultured for 24 hours in DMEM medium supplemented with 10% FBS. The medium was then replaced with DMEM medium supplemented with 5% FBS, followed by treatment with Peptide-1 or Peptide-2 at different concentrations. The medium was then changed every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After suctioning the medium at 3, 7, and 14 days, cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), and PCR was performed using the hCOL2A1, COMP, hCOL11A, PCP, and ACAN primers shown in Table 2 below. In Table 2 below, hCOL2A1 codes for collagen type IIα1, hCOMP codes for cartilage oligomeric matrix protein, hCOL11A codes for the α chain of collagen type XI, hPCP codes for proteoglycan core protein, hACAN codes for aggrecan, and hGAPDH codes for glyceraldehyde-3-phosphate dehydrogenase.

[0062] [Table 2]

[0063] As a result, as shown in Figures 7 and 8, it was found that Peptide-1 and Peptide-2, respectively, induce the production of ECM components COL2A1, COMP, COL11A, PCP, and ACAN.

[0064] Example 5. Confirmation of the mRNA expression induction effect of the ECM regulatory factor SOX9. We aimed to determine whether treatment with Peptide-1 or Peptide-2 increased mRNA expression of the ECM regulator SOX9 in adipose-derived mesenchymal stem cells.

[0065] Specifically, AD-MSC cells 1.5 x 103 After seeding 96-well plates at a cell / well density, cells were cultured for 24 hours in DMEM medium supplemented with 10% FBS. The medium was then replaced with DMEM medium supplemented with 5% FBS, followed by treatment with Peptide-1 or Peptide-2 at different concentrations. The medium was then changed every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After suctioning the medium at 3, 7, and 14 days, cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), and PCR was performed using the primers shown in Table 3 below. In Table 3, SOX9 refers to (sex determining region Y)-box 9.

[0066] [Table 3]

[0067] As a result, as shown in Figures 9 and 10, it was found that Peptide-1 and Peptide-2, respectively, promote the production of SOX9, an ECM regulator.

[0068] Example 6. Confirmation of the expression-inducing effect of ECM regulatory factors SOX5, SOX6, and SOX9. We aimed to determine whether treatment with Peptide-1 or Peptide-2 increased the expression of ECM regulators SOX5, SOX6, and SOX9 in adipose-derived mesenchymal stem cells.

[0069] Specifically, AD-MSC cells 1.5 x 10 3After seeding 96-well plates at a cell / well density, cells were cultured for 24 hours in DMEM medium supplemented with 10% FBS. The medium was then replaced with DMEM medium supplemented with 5% FBS, followed by treatment with Peptide-1 or Peptide-2 at different concentrations. The medium was then changed every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 3, 7, and 14 days, the medium was suctioned, cells were harvested to prepare lysates, and Western blotting was performed. Antibodies used for detection were sc-293215 (Santa Cruz, USA) for SOX5, sc-393314 (Santa Cruz, USA) for SOX6, and 82630S (Cell Signaling, USA) for SOX9.

[0070] As a result, as shown in Figures 11 and 12, it was found that Peptide-1 and Peptide-2, respectively, increase the expression of the ECM regulators SOX5, SOX6, and SOX9.

[0071] Example 7. Confirmation of the expression-inducing effect of aggrecan and COL2A1. We aimed to determine whether treatment with Peptide-1 or Peptide-2 increased the expression of cartilage components aggrecan and COL2A1 in adipose-derived mesenchymal stem cells.

[0072] Specifically, AD-MSC cells 1.5 x 10 3After seeding 96-well plates at a cell / well density, cells were cultured for 24 hours in DMEM medium supplemented with 10% FBS. The medium was then replaced with DMEM medium supplemented with 5% FBS, followed by treatment with either Peptide-1 or Peptide-2 at different concentrations. The medium was then changed every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After suction of the medium at 3, 7, and 14 days, cells were harvested to prepare lysates, and Western blotting was performed. For detection, sc-33695 (Santa Cruz, USA) was used for aggrecan and sc-393314 (Santa Cruz, USA) for COL2A1.

[0073] As a result, as shown in Figures 13 and 14, Peptide-1 and Peptide-2 each increased the expression of COL2A1, and Peptide-2 also increased the expression of aggrecan.

[0074] Example 8. Confirmation of the human activity induction effect of the ECM regulator SOX9, which is a higher activity level. We aimed to confirm the effect of peptide-2 treatment on inducing the activity of upstream activators of the ECM regulatory factor SOX9 in adipose-derived mesenchymal stem cells.

[0075] Specifically, AD-MSC cells 1.5 x 10 3 After seeding 96-well plates at a cell / well density, cells were cultured for 24 hours in DMEM medium supplemented with 10% FBS. The medium was then replaced with DMEM medium supplemented with 5% FBS, followed by treatment with Peptide-2 at varying concentrations. After 15 minutes, the medium was suctioned, cells were harvested, lysates were prepared, and Western blotting was performed. Antibodies used for detection were #8685S (Cell Signaling, USA) for Smad 2 / 3 and #8828S (Cell Signaling, USA) for p-Smad 2 / 3.

[0076] As a result, as shown in Figure 15, it was found that Peptide-2 induces the activity of the top activators of SOX9, a regulatory factor of the ECM.

[0077] In summary, the experimental results described above show that Peptide-1 and Peptide-2, as used in one example, each have the effect of inducing cartilage regeneration.

[0078] 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.

[0079] Dosage Form Examples 2. Pharmaceutical Preparations 2-1. Manufacturing of powdered medicines The following ingredients are mixed and filled into an airtight cloth to produce the powder. 20 mg of the peptide of the present invention Lactose 100mg Talc 10mg

[0080] 2-2. Manufacturing of Tablets After mixing the following ingredients, tablets are manufactured by compressing them using a standard tablet manufacturing method. 10 mg of the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0081] 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. 10 mg of the peptide of the present invention Crystalline cellulose 3 mg Lactose 14.8mg Magnesium stearate 0.2 mg

[0082] 2-4. Manufacturing of injectable drugs The following ingredients are produced per ampoule (2 ml) using a standard method for manufacturing injectable drugs. 10 mg of the peptide of the present invention Mannitol 180mg Sterile distilled water for injection 2974 mg Na2HPO4·2H2O 26mg

[0083] 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. 10 mg of the peptide of the present invention Isomerized sugar 10g Mannitol 5g Purified water (appropriate amount)

[0084] 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 an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

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 hydrazino (-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) Induction of glycosaminoglycan production; (b) COL2A1, COMP, COL11A, PCP, or aggrecan production induction; (c) Induction of the production of regulatory factors SOX5, SOX6, or SOX9; and (d) Activation of upstream activators of the regulatory factor SOX9.

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

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

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

8. The pharmaceutical composition according to claim 6, wherein the peptide is formulated into nanosomes.

9. The pharmaceutically active composition according to claim 6, wherein the cartilage disease is one or more selected from the group consisting of cartilage injury, cartilage defect, degenerative disc disease, disc herniation, degenerative arthritis, articular cartilage injury due to fracture, articular cartilage injury due to muscle tissue injury, articular cartilage injury due to non-union of fracture or articular cartilage injury due to trauma, and chondromalacia.

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