Cartilage regeneration peptides and their uses

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 use. This application claims priority to Korean Patent Application No. 10-2022-0171936, filed with the Korean Intellectual Property Office on December 9, 2022, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Due to the characteristics of cartilage tissue, when extensive damage occurs, natural tissue regeneration is difficult, so surgical treatments such as artificial joints, articular cartilage plastic surgery, and micro-treating have been used. However, these conventional methods often leave scars due to the incision and result in regeneration of fibrocartilage with reduced durability, which has the drawback of being less effective than more difficult surgical methods.

[0003] Therefore, intra-articular injection solutions or cartilage tissue repair compositions using hydrogels, collagen, etc., which have simple surgical procedures and rapid therapeutic effects, have been developed (Korean Patent Publication No. 2013-0028012). However, although these methods can temporarily reduce pain, they are insufficient to induce cartilage tissue regeneration.

[0004] Additionally, in the case of cell-based therapies, various treatment methods using autologous chondrocytes or stem cells have been developed as a method of inducing cartilage tissue regeneration by transplanting cells cultured in vitro into the defect site (Korea Patent Publication No. 2013-0072983). However, in the case of autologous chondrocyte treatments, when the damaged site is large, treatment is limited by the use of cells collected and cultured from the patient alone. In the case of stem cell treatments, there are problems such as differences in cell number and differentiation potential depending on the collection site, changes in cell phenotype due to cell dedifferentiation during in vitro culture, and cell death due to a low differentiation rate into chondrocytes and gene expression related to cell hypertrophy after implantation in vivo, as well as the induction of vascular infiltration, which can lead to chondrocyte calcification.

[0005] Under these technical backgrounds, there is a need to develop effective factors that can promote chondrogenesis or differentiation of stem cells or chondrocytes, thereby enabling more effective treatment of cartilage damage diseases, but the current situation is still insufficient. Summary of the Invention [Problem to be solved by the invention]

[0006] In order to solve the above-mentioned problems, the inventors of the present invention developed a novel peptide and confirmed that the novel peptide can be useful in preventing or treating cartilage diseases and promoting cartilage regeneration by significantly increasing various cartilage components such as glycosaminoglycans, collagen, COMP, and aggrecan, thereby completing the present invention.

[0007] One object 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 composition for cartilage regeneration, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0009] A further object of the present invention is to provide a pharmaceutical composition comprising the composition for cartilage regeneration.

[0010] However, the technical problems that the present invention aims to solve are not limited to the problems 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 one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is 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] The term "peptide" as used herein refers to a linear molecule formed by linking amino acid residues together via peptide bonds. The peptide can be prepared by chemical synthesis methods known to those skilled in the art, particularly solid-phase synthesis or liquid-phase synthesis (U.S. Patent No. 5,516,891). The present inventors have made extensive efforts to develop peptides with biologically effective activity, and as a result have identified peptides consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Here, the biologically effective activity may be any one or more of the following: (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 core protein), or aggrecan production; and (c) induction of the production 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 used for cartilage regeneration.

[0014] The peptides may also have protecting groups attached to their N- or C-termini to achieve chemical stability, enhanced pharmacological properties (half-life, absorbency, potency, efficacy, etc.), altered specificity (e.g., a broader spectrum of biological activity), or reduced antigenicity. In one embodiment, the N-terminus of the peptide may be conjugated with any 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); and / or the C-terminus of the peptide may be conjugated with any one protecting group selected from the group consisting of an amino group (-NH), a tertiary alkyl group, and an azide (-NHNH). Optionally, the peptide may further comprise a targeting sequence, a tag, a labeled residue, or an amino acid sequence specifically designed to increase half-life or peptide stability.

[0015] The peptides are artificially synthesized or non-naturally occurring or engineered, and the term "non-naturally occurring or engineered" refers to a state in which the peptides are produced by artificial modification, rather than in the state in which they exist in nature. Here, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking multiple amino acid structures, or engineering the peptides to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity, as described above.

[0016] The term "stability" as used herein may refer not only to in vivo stability, which protects the peptide from attack by in vivo proteolytic enzymes, but also to storage stability (eg, storage stability at room temperature).

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

[0018] Among the terms or elements mentioned in the description of the peptide, the same as those already mentioned are as described above.

[0019] The term "cartilage regeneration" as used herein refers to repairing damaged cartilage tissue or inducing the production of insufficient cartilage tissue to improve cartilage tissue. The term "improvement" may refer to any action that at least reduces a parameter related to the alleviation or treatment of a condition, for example, the severity of symptoms.

[0020] The cartilage may be, but is not limited to, hyaline cartilage, fibrocartilage, or elastic cartilage. For example, the cartilage may 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] Although conventional functional peptides have effective biological activities, they have the disadvantages of being unable to be effectively delivered to target tissues or cells due to their size, or of being rapidly eliminated in the body due to their short half-life. On the other hand, a composition for cartilage regeneration according to one embodiment contains a peptide consisting of approximately 20 or less amino acids as an active ingredient, which allows for excellent skin penetration of the active ingredient, and for example, when administered topically, provides effective cartilage regeneration effects.

[0022] According to one embodiment, the peptide can significantly increase the expression of cartilage-related substances such as glycosaminoglycans COL2A1, COMP, COL11A1, PCP, aggrecan, and regulatory factors SOX5, SOX6, or SOX9, and can be used as an active ingredient in 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] In yet another embodiment, there is provided a pharmaceutical composition for preventing or treating cartilage diseases, which comprises a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

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

[0025] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition.

[0026] As used herein, the term "treatment" refers to any form of care that provides a benefit to an individual suffering from or susceptible to a disease, including improvement of the individual's condition (e.g., one or more symptoms), delay in disease progression, delay in symptom onset, or slowing of symptom progression, etc. Thus, the terms "treatment" and "prevention" are not intended to mean a cure or complete elimination of symptoms.

[0027] The term "individual" refers to a subject in need of treatment for a disease, and more specifically refers to mammals such as human or non-human primates, mice, dogs, cats, horses, and cows.

[0028] As used herein, the term "cartilage disease" refers to any disease related to cartilage that requires cartilage differentiation or regeneration, and may be one or more selected from the group consisting of cartilage damage, cartilage defect, degenerative disc disease, disc prolapse, degenerative arthritis, bone fracture, muscle tissue damage, fracture nonunion or traumatic joint damage, osteomalacia, and chondromalacia.

[0029] The cartilage disorders may occur in the jaw 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 comprise, but is not limited to, a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier.

[0031] The term "pharmaceutical effective amount" as used herein may refer to 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. Examples of the weight ratio include, but are 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 are those commonly used in pharmaceutical preparations, 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0034] The pharmaceutical composition may further contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., but is not limited to these.

[0035] The pharmaceutical composition may be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, it may be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

[0036] The dosage of the pharmaceutical composition may be, but is not limited to, about 0.0001 to 1000 μg (μg), about 0.001 to 1000 μg, about 0.01 to 1000 μg, about 0.1 to 1000 μg, or about 1.0 to 1000 μg per day, and may vary depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity.

[0037] The pharmaceutical composition may be prepared in a unit dose form or in a multi-dose container by formulating it with pharmaceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person skilled 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 oily or aqueous medium, or in the form of an ointment, cream, gel, transdermal agent, cataplasm, patch, paste, extract, powder, granule, tablet or capsule, and may further comprise a dispersing agent and / or a stabilizing agent.

[0039] To further improve skin penetration or stability, the peptide can be incorporated into nanosomes or nanoparticles. For example, the nanosomes can be prepared using lecithin as a raw material using a microfluidizer, and then incorporated into lecithin particles. Any known method can be used to prepare the nanosomes. The nanosome particle size is preferably about 30 to 200 nm. If the nanosome particle size is less than about 30 nm, skin penetration is too rapid, resulting in skin side effects. If the nanosome particle size exceeds about 200 nm, skin penetration is slow, making it difficult to achieve the benefits of using the nanosome structure.

[0040] Yet another embodiment provides a method for preventing or treating cartilage diseases, comprising administering to an individual a pharmaceutical composition containing as an active ingredient a therapeutically effective amount of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0041] Among the terms or elements mentioned in the description of the peptides, compositions, etc., the same as those already mentioned are as described above.

[0042] As used herein, the terms "apply," "administer," and "apply" are used interchangeably and refer to at least partially localizing a composition according to an embodiment at a desired site or placing a composition according to an embodiment within an individual by a route of administration.

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

[0044] Among the terms or elements mentioned in the description of the peptides, compositions, etc., the same as those already mentioned are as described above.

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

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

[0047] Among the terms or elements mentioned in the description of the peptides, compositions, etc., the same as those already mentioned are as described above. [Effects of the Invention]

[0048] The peptide according to one embodiment significantly increases various cartilage components such as glycosaminoglycan, collagen, COMP, and aggrecan, thereby exhibiting excellent cartilage regeneration effects.

[0049] According to one embodiment, the peptide can be used 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 drawings]

[0050] [Figure 1] The results show that Peptide-1 has a cytotoxic effect on human adipose-derived mesenchymal stem cells (AD-MSCs) (a: microscopic image after SRB staining; b: graph showing CCK-8 activity test results). [Figure 2]The results show that Peptide-2 has a cytotoxic effect on AD-MSCs (a: microscopic image after SRB staining; b: graph showing the results of the CCK-8 activity test). [Figure 3] This is the result of confirming increased glycosaminoglycan production after treating AD-MSCs with Peptide-1. [Figure 4] This is the result of confirming increased glycosaminoglycan production after treating AD-MSCs with Peptide-2. [Figure 5] After treating AD-MSCs with Peptide-1, increased mRNA expression of ECM (extracellular matrix) components was confirmed after 3 days of culture (a), 7 days of culture (b), and 14 days of culture (c). [Figure 6] After treating AD-MSCs with Peptide-2, increased mRNA expression of ECM components was confirmed after 3 days of culture (a), 7 days of culture (b), and 14 days of culture (c). [Figure 7] This is the result of confirming increased expression of the SOX9 gene, an ECM regulatory factor, after treating AD-MSCs with Peptide-1. [Figure 8] After treating AD-MSCs with Peptide-2, increased expression of the SOX9 gene, an ECM regulatory factor, was confirmed after 3 days of culture (a), 7 days of culture (b), and 14 days of culture (c). [Figure 9] After treating AD-MSCs with Peptide-1, increased expression of the ECM regulatory proteins SOX5, SOX6, and SOX9 was confirmed after 3 days of culture (a), 7 days of culture (b), and 14 days of culture (c). [Figure 10] After treating AD-MSCs with Peptide-2, increased expression of the ECM regulatory proteins SOX5, SOX6, and SOX9 was confirmed after 3 days of culture (a), 7 days of culture (b), and 14 days of culture (c). [Figure 11]After treating AD-MSCs with Peptide-1, increased expression of cartilage components aggrecan and COL2A1 was confirmed after 3 days of culture (a), 7 days of culture (b), and 14 days of culture (c). [Figure 12] After treating AD-MSCs with Peptide-2, increased expression of cartilage components aggrecan and COL2A1 was confirmed after 3 days of culture (a), 7 days of culture (b), and 14 days of culture (c). DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will be described in more detail below with reference to 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 listed in Table 1 below was synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified and separated using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA) on an ACQUITY UPLC BEH300 C18 column (2.1 mm x 100 mm, 1.7 μm, Waters Co., USA).

[0053] [Table 1]

[0054] Example 2. Confirmation of cytotoxicity Human adipose-derived mesenchymal stem cells (AD-MSCs) were treated with Peptide-1 or Peptide-2 to examine whether they were cytotoxic.

[0055] Specifically, approximately 1.5 x 10 AD-MSCs were cultured.3 After seeding at a concentration of 1000 cells / well into a 96-well plate, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for approximately 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS and treated with various concentrations of Peptide-1 or Peptide-2 (approximately 1, 10, 30, 50, 100, or 200 μg / ml). The medium was then replaced with DMEM medium containing approximately 5% FBS approximately every three days and treated with various concentrations of Peptide-1 or Peptide-2 (approximately 1, 10, 30, 50, 100, or 200 μg / ml). After 7 days of culture, the culture medium was treated with approximately 1 / 10 the volume of CCK-8 (Dojindo, CCK-8 kit) solution and cultured for approximately 2 hours. After incubation, 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 in the 96-well plate was removed, 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 double-distilled water) was added to the fixed cells. The cells were then stained by culturing overnight at room temperature in the dark. After staining, the cells were washed with approximately 1% acetic acid and dried. The dried cells were observed under a microscope. An experimental group (CON) not treated with the peptide and an experimental 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 served as controls.

[0056] As a result, as shown in Figures 1 and 2, when Peptide-1 and Peptide-2 were treated at concentrations of about 1 to 200 μg / ml, respectively, no changes in cell morphology occurred at any concentration, confirming that they did not cause cytotoxicity.

[0057] Example 3. Confirmation of glycosaminoglycan production effect To confirm the chondrogenic effects of Peptide-1 and Peptide-2, adipose-derived mesenchymal stem cells (Human Adipose-Derived Mesenchymal Stem Cells: AD-MSCs) were treated with Peptide-1 or Peptide-2 to examine whether the production of glycosaminoglycans, which make up the extracellular matrix (ECM), a component of cartilage, increased.

[0058] Specifically, approximately 1.5 x 10 AD-MSCs were cultured. 3After seeding at a concentration of 1000 cells / well into a 96-well plate, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for approximately 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (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 approximately every 3 days, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (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 in the 96-well plate was removed, 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 Alcian blue staining solution (approximately 50 mL of approximately 3% acetic acid + approximately 0.5 g of approximately 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 the cells. After cell staining, the staining solution was removed, and the cells were washed with triple-distilled water and dried. The dried cells were observed under a microscope. An experimental group (CON) not treated with the peptide and an experimental 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 served as controls.

[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 peptide concentration-dependent manner, which may promote the production of ECM, a component of cartilage. This confirms 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 promote chondrogenic differentiation of stem cells or increase the ECM-producing ability of stem cells, thereby promoting cartilage formation or regeneration.

[0061] Example 4. Confirmation of increased mRNA expression of ECM components To confirm the chondrogenic effects of Peptide-1 and Peptide-2, adipose-derived mesenchymal stem cells (AD-MSCs) were treated with Peptide-1 or Peptide-2 to examine whether the expression of COL2A1 (Collagen type II Alpha 1), COL11A1 (Collagen type XI Alpha 1), COMP (Cartilage oligomeric matrix protein), PCP (Proteoglycan core protein), and ACAN (Aggrecan) genes, which are involved in the production of extracellular matrix (ECM), a component of cartilage, was increased.

[0062] Specifically, approximately 1.5 x 10 AD-MSCs were cultured. 3After seeding at a concentration of 1000 cells / well into a 96-well plate, the cells were cultured in DMEM medium (approximately 10% FBS + DMEM medium) for approximately 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (approximately 30, 50, or 100 μg / ml). Thereafter, the medium was replaced with DMEM medium containing approximately 5% FBS approximately every three days, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium in the 96-well plate was removed, and cells were harvested. RNA was isolated from the harvested cells. cDNA was synthesized from the isolated RNA using a cDNA synthesis kit (Intron, Korea), and PCR was performed using primers for hCOL2A1, hCOMP, hCOL11A1, hPCP, and hACAN and PCR premix (Intron, Korea). The primers used are listed in Table 2. Control groups included an experimental group not treated with the peptide and an experimental 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.

[0063] [Table 2]

[0064] As a result, as shown in Figure 5, when AD-MSCs were treated with Peptide-1, the expression of COL2A1, COMP, and COL11A1 genes, which are genes involved in the production of ECM, a component of cartilage, increased in a peptide concentration-dependent manner. Furthermore, as shown in Figure 6, when AD-MSCs were treated with Peptide-2, the expression of COL2A1, COMP, COL11A1, PCP, and ACAN genes, which are genes involved in the production of ECM, a component of cartilage, increased in a peptide concentration-dependent manner. These results demonstrate 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.

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

[0066] Example 5. Confirmation of the mRNA expression induction effect of the ECM regulatory factor SOX9 To confirm the chondrogenic effects of Peptide-1 and Peptide-2, adipose-derived mesenchymal stem cells (Human Adipose-Derived Mesenchymal Stem Cells: AD-MSCs) were treated with Peptide-1 or Peptide-2 to examine whether the expression of the SOX9 (Sex Determining Region Y-Box Transcription Factor 9) gene, which regulates the production of extracellular matrix (ECM), a component of cartilage, was increased.

[0067] Specifically, approximately 1.5 x 10 AD-MSCs were cultured. 3Cells were seeded into 96-well plates at a concentration of 1000 cells / well and cultured in DMEM medium (approximately 10% FBS + DMEM medium) for approximately 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS and treated with Peptide-1 or Peptide-2 at various concentrations (approximately 30, 50, or 100 μg / ml). The medium was then replaced with DMEM medium containing approximately 5% FBS approximately every three days and treated with Peptide-1 or Peptide-2 at various concentrations (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium was removed from the 96-well plates, the cells were harvested, and RNA was isolated from the harvested cells. cDNA was synthesized from the isolated RNA using a cDNA synthesis kit (Intron, Korea), followed by PCR using hSOX9 primers and PCR premix (Intron, Korea). The primers used are listed in Table 3 below. An experimental group not treated with the peptide and an experimental 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 as controls.

[0068] [Table 3]

[0069] As a result, as shown in Figures 7 and 8, when AD-MSCs were treated with Peptide-1 or Peptide-2, the expression of SOX9 gene, which regulates the production of ECM, a component of cartilage, increased in a peptide concentration-dependent manner. This indicates 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 promote chondrogenic differentiation of stem cells or increase the ECM-producing ability 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 chondrogenic effects of Peptide-1 and Peptide-2, adipose-derived mesenchymal stem cells (Human Adipose-Derived Mesenchymal Stem Cells: AD-MSCs) were treated with Peptide-1 or Peptide-2 to examine 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), which are involved in regulating the extracellular matrix (ECM), a component of cartilage, was increased.

[0072] Specifically, approximately 1.5 x 10 AD-MSCs were cultured. 3Cells were seeded into 96-well plates at a concentration of 1000 cells / well and cultured in DMEM medium (approximately 10% FBS + DMEM medium) for approximately 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (approximately 30, 50, or 100 μg / ml). The medium was then replaced with DMEM medium containing approximately 5% FBS approximately every three days, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium in the 96-well plates was removed and the cells were harvested. The harvested 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. Control groups included an experimental group not treated with the peptide and an experimental 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.

[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 the regulation of ECM, a component of cartilage, increased in a peptide concentration-dependent manner. This indicates 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.

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

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

[0076] Specifically, approximately 1.5 x 10 AD-MSCs were cultured. 3 Cells were seeded into 96-well plates at a concentration of 1000 cells / well and cultured in DMEM medium (approximately 10% FBS + DMEM medium) for approximately 24 hours. The medium was then replaced with DMEM medium containing approximately 5% FBS, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (approximately 30, 50, or 100 μg / ml). The medium was then replaced with DMEM medium containing approximately 5% FBS approximately every three days, and cells were treated with various concentrations of Peptide-1 or Peptide-2 (approximately 30, 50, or 100 μg / ml). After 3, 7, and 14 days of culture, the medium in the 96-well plates was removed and the cells were harvested. The harvested cells were lysed, and the resulting cell lysates were analyzed by Western blotting. For analysis, COL2A1 antibody (Santa Cruz, sc-518017, USA) and Aggrecan antibody (Santa Cruz, sc-33695, USA) were used. Control groups included an experimental group not treated with the peptide and an experimental 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.

[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 are components of ECM, a component of cartilage, increased in a peptide concentration-dependent manner. This indicates 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.

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

[0079] To summarize the above experimental results, it was found that both Peptide-1 and Peptide-2 according to one embodiment 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 with thorough stirring. The resulting solution was mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil, and then the mixture was adjusted to a total volume of 1 L with distilled water. The mixture was then emulsified using high pressure in a microfluidizer to produce peptide nanosomes with a size of approximately 100 nm.

[0081] Dosage form example 2. Pharmaceutical preparations 2-1. Manufacturing of powders The following ingredients are mixed and packed into an airtight cloth to produce a powder. 20 mg of the peptide of the present invention Lactose 100mg Talc 10mg

[0082] 2-2. Tablet manufacturing The following ingredients are mixed and compressed into tablets by a conventional tablet manufacturing method. 10 mg of the peptide of the present invention Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0083] 2-3. Capsule manufacturing The following ingredients are mixed and filled into gelatin capsules according to a conventional capsule manufacturing method to produce capsules. 10 mg of the peptide of the present invention Crystalline cellulose 3mg Lactose 14.8mg Magnesium stearate 0.2mg

[0084] 2-4. Manufacturing of injections The following ingredients are prepared per ampoule (2 ml) using the usual injection manufacturing method. 10 mg of the peptide of the present invention Mannitol 180mg Sterile distilled water for injection 2974mg Na2HPO4·2H2O 26mg

[0085] 2-5. Liquid drug manufacturing Using the usual method for producing liquid preparations, add each ingredient to purified water and dissolve it, then mix the ingredients listed below, add purified water to bring the total volume to 100 ml, and fill into a brown bottle and sterilize to prepare the liquid. 10 mg of the peptide of the present invention Isomerized sugar 10g Mannitol 5g Purified water (appropriate amount)

[0086] While certain aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific techniques are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the following 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. 2. The peptide of claim 1, wherein the N-terminus of the peptide is bound to any 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 (-NH 2 ), tertiary alkyl groups, and azides (-NHNH 2 2. The peptide of claim 1, wherein the peptide is bound to any one of the protecting groups selected from the group consisting of:

4. The peptide of claim 1, wherein the peptide exhibits one or more of the following properties: (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 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. 7. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable carrier.

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

9. The pharmaceutical composition of claim 6, wherein the cartilage disease is one or more selected from the group consisting of cartilage damage, cartilage defect, degenerative disc disease, disc prolapse, degenerative arthritis, bone fracture, muscle tissue damage, fracture non-union or traumatic joint damage, osteomalacia, and chondromalacia.

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