Cartilage regeneration peptides and their applications

Abstract

The present application relates to a peptide having a cartilage regeneration effect and its uses, and provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 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] The present invention relates to a peptide for cartilage regeneration and its uses. This application claims priority to Korean Patent Application No. 10-2022-0171936, filed with the Korean Intellectual Property Office on December 9, 2022, and the disclosure of the said patent application is incorporated herein by reference.

Background Art

[0002] Due to the characteristics of cartilage tissue, when it is damaged over a wide area, it is difficult to regenerate the tissue by natural healing, so treatments such as artificial joints, arthroscopic chondroplasty, and microperforation have been performed surgically. However, conventional methods often leave scars due to incisions, result in the regeneration of fibrocartilage with reduced durability, and have the problem that the treatment effect is lower compared to difficult surgical methods.

[0003] Therefore, injectable 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 said method can obtain a temporary reduction in pain, it is insufficient for inducing regeneration of cartilage tissue.

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

[0005] Against this technological backdrop, there is a need to develop effective factors that can promote chondrogenic differentiation or chondrogenesis of stem cells or chondrocytes, thereby enabling more effective treatment of cartilage-damage diseases, but the current situation is still inadequate. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] To solve the aforementioned problems, the inventors of the present invention developed a novel peptide and confirmed that this novel peptide can be usefully utilized to prevent or treat cartilage diseases and promote cartilage regeneration by significantly increasing various cartilage components such as glycosaminoglycans, collagen, COMP, and aggrecan, thereby completing the present invention.

[0007] One objective of the present invention is to provide a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] Another object of the present invention is to provide a cartilage regeneration composition containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising the cartilage regeneration composition described above.

[0010] However, the technical problems that this invention aims to solve are not limited to those described above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by experts skilled in the art to which this invention pertains. The nomenclature used herein is generally well known and commonly used in the art.

[0012] One embodiment provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0013] As used herein, the term "peptide" may mean a linear molecule formed by the bonding of amino acid residues to one another via peptide bonds. Such peptides can be produced by chemical synthesis methods known to those skilled in the art, particularly by solid-phase or liquid-phase synthesis techniques (US Patent No. 5,516,891). The inventors, through diligent efforts to develop a biologically effective peptide, have identified a peptide consisting of the amino acid sequence of Sequence ID No. 1 or Sequence ID No. 2. Here, the biologically effective activity is also one or more selected from the following properties: (a) induction of glycosaminoglycan production; (b) induction of COL2A1 (Collagen type II Alpha 1), COL11A1 (Collagen type XI Alpha 1), COMP (Cartilage oligomeric matrix protein), PCP (proteoglycan coreprotein), or aggrecan production; and (c) induction of regulatory factors 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). Therefore, the peptide can be utilized for cartilage regeneration.

[0014] 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.

[0015] 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.

[0016] 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).

[0017] Another embodiment provides a cartilage regeneration composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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 20 amino acids or less 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.

[0022] According to one embodiment, the peptide can significantly increase the expression of glycosaminoglycan, COL2A1, COMP, COL11A1, 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).

[0023] 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 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0024] Among the terms or elements mentioned in the description related to the peptide or composition, those same as those already mentioned are as described above.

[0025] As used herein, the term "prevention" means all acts of suppressing or delaying the onset of a disease by administration of the composition.

[0026] 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 the disease, delay of disease progression, delay of symptom occurrence, or attenuation of symptom progression. Therefore, the "treatment" and "prevention" are not intended to mean cure or complete removal of symptoms.

[0027] The "individual" means a subject that requires treatment for a disease, and more specifically, means mammals such as humans or non-human primates, mice, dogs, cats, horses, and cows.

[0028] 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.

[0029] 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.

[0030] The pharmaceutical composition may, but is not limited to, contain a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier.

[0031] As used herein, the term "pharmaceutical effective amount" may mean an amount sufficient to achieve the cartilage regeneration efficacy of the pharmaceutical composition.

[0032] 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.

[0033] 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).

[0034] 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.

[0035] 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.

[0036] The dosage of the aforementioned pharmaceutical composition may be approximately 0.0001 to 1000 μg (μg), approximately 0.001 to 1000 μg, approximately 0.01 to 1000 μg, approximately 0.1 to 1000 μg, or approximately 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.

[0037] 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.

[0038] 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.

[0039] 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 about 30 to 200 nm. If the size of the nanosome particles is less than about 30 nm, skin penetration proceeds very quickly, causing skin side effects, and if it exceeds about 200 nm, skin penetration is not easy, making it difficult to obtain the effect of using the nanosome structure.

[0040] 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 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0041] 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.

[0042] 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.

[0043] Another embodiment provides a cosmetic composition containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0044] 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.

[0045] The cosmetic composition may, but is not limited to, contain a cosmetically effective amount of the peptide and / or a cosmetically acceptable carrier.

[0046] Another embodiment provides a method for regenerating cartilage, comprising the step of administering to an individual a composition containing a peptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0047] 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]

[0048] 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.

[0049] 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]

[0050] [Figure 1] This shows the results of confirming the cytotoxicity of Peptide-1 against human adipose-derived mesenchymal stem cells (AD-MSCs) (a: microscopic image after SRB staining; b: graph of CCK-8 activity test results). [Figure 2]This shows the results of confirming the cytotoxicity of Peptide-2 against AD-MSCs (a: Microscopic image after SRB staining; b: Graph of CCK-8 activity test results). [Figure 3] This result shows an increase in glycosaminoglycan production after treating AD-MSCs with Peptide-1. [Figure 4] This result shows an increase in glycosaminoglycan production after treating AD-MSCs with Peptide-2. [Figure 5] The results show increased mRNA expression of extracellular matrix (ECM) components after treating AD-MSCs with Peptide-1, at 3 days (a), 7 days (b), and 14 days (c) of culture. [Figure 6] The results show increased mRNA expression of ECM components after treating AD-MSCs with Peptide-2, at 3 days (a), 7 days (b), and 14 days (c) of culture. [Figure 7] This result shows an increase in the expression of the ECM regulator SOX9 gene after treating AD-MSCs with Peptide-1. [Figure 8] The results show that after treating AD-MSCs with Peptide-2, increased expression of the ECM regulator SOX9 gene was confirmed 3 days after culture (a), 7 days after culture (b), and 14 days after culture (c). [Figure 9] The results show that after treating AD-MSCs with Peptide-1, increased expression of the ECM regulatory factors SOX5, SOX6, and SOX9 proteins was confirmed 3 days after culturing (a), 7 days after culturing (b), and 14 days after culturing (c). [Figure 10] The results show that after treating AD-MSCs with Peptide-2, increased expression of the ECM regulatory factors SOX5, SOX6, and SOX9 proteins was confirmed 3 days after culturing (a), 7 days after culturing (b), and 14 days after culturing (c). [Figure 11]The results show that after treating AD-MSCs with Peptide-1, increased expression of cartilage components aggrecan and COL2A1 was observed 3 days after culturing (a), 7 days after culturing (b), and 14 days after culturing (c). [Figure 12] The results show that after treating AD-MSCs with Peptide-2, increased expression of cartilage components aggrecan and COL2A1 was observed 3 days after culturing (a), 7 days after culturing (b), and 14 days after culturing (c). [Modes for carrying out the invention]

[0051] 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.

[0052] 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).

[0053] [Table 1]

[0054] Example 2. Confirmation of cytotoxicity We attempted to determine the cytotoxicity of human adipose-derived mesenchymal stem cells (AD-MSCs) by treating them with peptide-1 or peptide-2.

[0055] Specifically, the AD-MSC is approximately 1.5 x 103 After seeding cells / well in a 96-well plate, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for about 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and treated with Peptide-1 or Peptide-2 at varying concentrations (approximately 1, 10, 30, 50, 100, or 200 μg / ml). Subsequently, the medium was replaced with DMEM medium containing approximately 5% FBS every 3 days, and treated with Peptide-1 or Peptide-2 at varying concentrations (approximately 1, 10, 30, 50, 100, or 200 μg / ml). After 7 days of culture, approximately 1 / 10 the volume of the culture medium was added to the culture medium in a CCK-8 (Dojindo, CCK-8 kit) solution, and the cells were cultured for approximately 2 hours. After culturing, the culture medium was sampled, and the absorbance was measured at a wavelength of approximately 450 nm using a microplate reader. After measuring absorbance, the culture medium was removed from the culture medium in the 96-well plate, and approximately 60 μl of approximately 3.7% formalin was added to the 96-well plate to fix the cells for approximately 1 minute. After cell fixation, the formalin was removed, and approximately 70 μl of SRB staining solution (approximately 0.2 g of Sulforhodamine B sodium salt (sigma, S9012) dissolved in approximately 100 ml of secondary distilled water) was added to the fixed cells. The cells were then cultured overnight at room temperature under light-blocked conditions to stain them. After staining, the cells were washed with approximately 1% acetic acid and dried. The dried cells were observed under a microscope. An experimental group that was not treated with the peptide (CON) and an experimental group that was treated with a solution containing approximately 100 nM dexamethasone, approximately 50 μM ascorbic acid, approximately 40 μM proline, approximately 10 ng / ml TGFβ1, and 1X ITS instead of the peptide (CM) were used as control groups.

[0056] As a result, as shown in Figures 1 and 2, when Peptide-1 and Peptide-2 were treated at concentrations of approximately 1 to 200 μg / ml, no changes in cell morphology occurred at any of the concentrations, confirming that no cytotoxicity was caused.

[0057] Example 3. Confirmation of glycosaminoglycan generation effect To confirm the chondrogenesis effects of Peptide-1 and Peptide-2, we treated adipose-derived mesenchymal stem cells (AD-MSCs) with Peptide-1 or Peptide-2 to check whether the production of glycosaminoglycans, which constitute the extracellular matrix (ECM) that is a component of cartilage, increased.

[0058] Specifically, the AD-MSC is approximately 1.5 x 10 3After seeding 96-well plates with cells / well concentration, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for about 24 hours. Subsequently, the medium was replaced with DMEM medium containing approximately 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (Peptide-1: approximately 30, 50, 100, or 200 μg / ml; Peptide-2: approximately 50, 100, or 200 μg / ml). Thereafter, the medium was replaced with DMEM medium containing approximately 5% FBS every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (Peptide-1: approximately 30, 50, 100, or 200 μg / ml; Peptide-2: approximately 50, 100, or 200 μg / ml). After 14 and 21 days of culture, the culture medium was removed from the 96-well plate, and approximately 60 μl of 3.7% formalin was added to the 96-well plate to fix the cells for approximately 1 minute. After cell fixation, the formalin was removed, and approximately 70 μl of Alcian blue staining solution (approximately 50 ml of 3% acetic acid + approximately 0.5 g of 1% Alcian blue 8GX; pH 2.5) was added to the fixed cells. The cells were then cultured at approximately 37°C for approximately 24 hours to stain them. After cell staining, the staining solution was removed, and the cells were washed with tertiary distilled water and dried. The dried cells were observed under a microscope. An experimental group that was not treated with the peptide (CON) and an experimental group that was treated with a solution containing approximately 100 nM dexamethasone, approximately 50 μM ascorbic acid, approximately 40 μM proline, approximately 10 ng / ml TGFβ1, and 1X ITS instead of the peptide (CM) were used as control groups.

[0059] As a result, as shown in Figures 3 and 4, when AD-MSCs were treated with Peptide-1 or Peptide-2, the production of glycosaminoglycans increased in a concentration-dependent manner of the treated peptide, which may promote the production of ECM, a component of cartilage. This confirmed that both Peptide-1 and Peptide-2 can promote cartilage formation or regeneration.

[0060] These results suggest that both Peptide-1 and Peptide-2 can either promote cartilage differentiation in stem cells or increase the ECM-producing capacity of stem cells, thereby promoting cartilage formation or regeneration.

[0061] Example 4. Confirmation of increased mRNA expression of ECM components To confirm the chondrogenesis effects of Peptide-1 and Peptide-2, we treated adipose-derived mesenchymal stem cells (AD-MSCs) with Peptide-1 or Peptide-2 and checked whether the expression of genes involved in the production of the extracellular matrix (ECM), a component of cartilage, such as COL2A1 (Collagen type II Alpha 1), COL11A1 (Collagen type XI Alpha 1), COMP (Cartilage oligomeric matrix protein), PCP (proteoglycan coreprotein), and ACAN (Aggrecan), increased.

[0062] Specifically, the AD-MSC is approximately 1.5 x 10 3After seeding 96-well plates with cells / well concentration, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for about 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). Subsequently, the medium was replaced with DMEM medium containing approximately 5% FBS every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium was removed from the culture medium in the 96-well plates, and the cells were collected. RNA was then isolated from the collected cells. cDNA was synthesized from isolated RNA using a cDNA synthesis kit (Intron, Korea), and then PCR was performed using primers for hCOL2A1, hCOMP, hCOL11A1, hPCP, and hACAN and a PCR pre-mix (Intron, Korea). The primers used are shown in Table 2 below. A control group was used, consisting of an experimental group that was not treated with the peptide and an experimental group (CM) that was treated with a solution containing approximately 100 nM dexamethasone, approximately 50 μM ascorbic acid, approximately 40 μM proline, approximately 10 ng / ml TGFβ1, and 1X ITS instead of the peptide.

[0063] [Table 2]

[0064] As a result, as shown in Figure 5, when AD-MSCs were treated with Peptide-1, the expression of the COL2A1, COMP, and COL11A1 genes, which are involved in the production of ECM (endocartilage complex), a component of cartilage, increased in a concentration-dependent manner with the treated peptide. Furthermore, as shown in Figure 6, when AD-MSCs were treated with Peptide-2, the expression of the COL2A1, COMP, COL11A1, PCP, and ACAN genes, which are involved in the production of ECM (endocartilage complex), increased in a concentration-dependent manner with the treated peptide. This confirmed that both Peptide-1 and Peptide-2 promote the production of ECM (endocartilage complex), a component of cartilage, and as a result, can promote cartilage formation or regeneration.

[0065] These results suggest that both Peptide-1 and Peptide-2 can either promote cartilage differentiation in stem cells or increase the ECM-producing capacity of stem cells, thereby promoting cartilage formation or regeneration.

[0066] Example 5. Confirmation of the mRNA expression-inducing effect of the ECM regulatory factor SOX9. To confirm the chondrogenesis effects of Peptide-1 and Peptide-2, we treated adipose-derived mesenchymal stem cells (AD-MSCs) with Peptide-1 or Peptide-2 and checked whether the expression of the SOX9 (Sex determining region Y-Box Transcription Factor 9) gene, which regulates the production of the extracellular matrix (ECM), a component of cartilage, increased.

[0067] Specifically, the AD-MSC is approximately 1.5 x 10 3After seeding 96-well plates with cells / well concentration, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for about 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). Subsequently, the medium was replaced with DMEM medium containing approximately 5% FBS every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium was removed from the culture medium in the 96-well plates, and the cells were collected. RNA was isolated from the collected cells. cDNA was synthesized from the isolated RNA using a cDNA synthesis kit (Intron, Korea), and then PCR was performed using hSOX9 primers and a PCR pre-mix (Intron, Korea). The primers used are shown in Table 3 below. The experimental group that was not treated with the peptide and the experimental group (CM) that was treated with a solution containing approximately 100 nM dexamethasone, approximately 50 μM ascorbic acid, approximately 40 μM proline, approximately 10 ng / ml TGFβ1, and 1X ITS instead of the peptide were used as control groups.

[0068] [Table 3]

[0069] As a result, as shown in Figures 7 and 8, we confirmed that when AD-MSCs were treated with Peptide-1 or Peptide-2, the expression of the SOX9 gene, which regulates the production of ECM (endocartilage complex), a component of cartilage, increased in a concentration-dependent manner with the treated peptide. This confirmed that both Peptide-1 and Peptide-2 promote the production of ECM, a component of cartilage, and as a result, can promote cartilage formation or regeneration.

[0070] These results suggest that both Peptide-1 and Peptide-2 can either promote cartilage differentiation in stem cells or increase the ECM-producing capacity of stem cells, thereby promoting cartilage formation or regeneration.

[0071] Example 6. Confirmation of the expression-inducing effect of ECM regulatory factors SOX5, SOX6, and SOX9. To confirm the chondrogenesis effects of Peptide-1 and Peptide-2, we treated adipose-derived mesenchymal stem cells (AD-MSCs) with Peptide-1 or Peptide-2 to check whether the expression of SOX5 (Sex determining region Y-Box Transcription Factor 5), SOX6 (Sex determining region Y-Box Transcription Factor 6), and SOX9 (Sex determining region Y-Box Transcription Factor 9) proteins, which are involved in regulating the extracellular matrix (ECM) that is a component of cartilage, increased.

[0072] Specifically, the AD-MSC is approximately 1.5 x 10 3After seeding 96-well plates with cells / well concentration, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for about 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). Subsequently, the medium was replaced with DMEM medium containing approximately 5% FBS every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium was removed from the culture medium in the 96-well plates, and the cells were collected. The collected cells were lysed, and the resulting cell lysates were analyzed by Western blotting. For analysis, SOX5 antibody (Santa Cruz, sc-293215, USA), SOX6 antibody (Santa Cruz, sc-393314, USA), and SOX9 antibody (Cell Signaling, 82630S, USA) were used. A control group (untreated with the peptide) and a control group (CM) treated with a solution containing approximately 100 nM dexamethasone, approximately 50 μM ascorbic acid, approximately 40 μM proline, approximately 10 ng / ml TGFβ1, and 1X ITS instead of the peptide were used.

[0073] As a result, as shown in Figures 9 and 10, when AD-MSCs were treated with Peptide-1 or Peptide-2, the expression of SOX5, SOX6, and SOX9 proteins, which are involved in regulating the extracellular matrix (ECM), a component of cartilage, increased in a concentration-dependent manner with respect to the treated peptide. This confirmed that both Peptide-1 and Peptide-2 promote the production of ECM, a component of cartilage, and consequently, can promote cartilage formation or regeneration.

[0074] These results suggest that both Peptide-1 and Peptide-2 can either promote cartilage differentiation in stem cells or increase the ECM-producing capacity of stem cells, thereby promoting cartilage formation or regeneration.

[0075] Example 7. Confirmation of the expression-promoting effect of aggrecan and COL2A1. To confirm the chondrogenesis effects of Peptide-1 and Peptide-2, we treated adipose-derived mesenchymal stem cells (AD-MSCs) with Peptide-1 or Peptide-2 to check whether the expression of COL2A1 (Collagen type II Alpha 1) and Aggrecan proteins, which constitute the extracellular matrix (ECM) that is a component of cartilage, increased.

[0076] Specifically, the AD-MSC is approximately 1.5 x 10 3 After seeding 96-well plates with cells / well concentration, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for about 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). Subsequently, the medium was replaced with DMEM medium containing approximately 5% FBS every 3 days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium was removed from the culture medium in the 96-well plates, and the cells were collected. The collected cells were lysed, and the resulting cell lysates were analyzed by Western blotting. COL2A1 antibody (Santa Cruz, sc-518017, USA) and Aggrecan antibody (Santa Cruz, sc-33695, USA) were used for analysis. A control group was used in which no peptide was treated, and a control group (CM) was used in which a solution containing approximately 100 nM dexamethasone, approximately 50 μM ascorbic acid, approximately 40 μM proline, approximately 10 ng / ml TGFβ1, and 1X ITS was used instead of the peptide.

[0077] As a result, as shown in Figures 11 and 12, when AD-MSCs were treated with Peptide-1 or Peptide-2, the expression of COL2A1 and Aggrecan proteins, which constitute the cartilage's ECM, increased in a concentration-dependent manner with respect to the treated peptide. This confirmed that both Peptide-1 and Peptide-2 promote the production of the ECM, a component of cartilage, and consequently, can promote cartilage formation or regeneration.

[0078] These results suggest that both Peptide-1 and Peptide-2 can either promote cartilage differentiation in stem cells or increase the ECM-producing capacity of stem cells, thereby promoting cartilage formation or regeneration.

[0079] In summary, the experimental results showed that both Peptide-1 and Peptide-2, as used in one example, have the effect of inducing cartilage regeneration.

[0080] 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.

[0081] 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

[0082] 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

[0083] 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

[0084] 2-4. Manufacturing of injectable drugs The following ingredients are manufactured 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

[0085] 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)

[0086] Having described in detail certain aspects of the present invention, it will be clear to those skilled in the art that such specific technologies are merely desirable embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the claims and their equivalents.

Claims

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

2.

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

3. The C-terminus of the peptide is an amino group (-NH₂). 2 ), tertiary alkyl group, and 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) induction of COL2A1, COMP, COL11A1, PCP, or aggrecan production; and (c) Induction of SOX5, SOX6, or SOX9 production.

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, fracture, muscle tissue injury, fracture failure or joint injury due to trauma, osteomalacia, and chondromalacia.

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