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 uses thereof. [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 demand for the development of effective factors that can promote chondrogenesis or differentiation of stem cells or chondrocytes, thereby enabling more effective treatment of cartilage diseases. However, the reality is that such development is still lacking. Summary of the Invention [Problem to be solved by the invention]

[0006] One embodiment is to provide a peptide consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0007] Another aspect 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.

[0008] Yet another aspect is to provide a pharmaceutical composition for preventing or treating cartilage diseases, which comprises the above-mentioned composition for cartilage regeneration as an active ingredient.

[0009] Other objects and advantages of the present application will become more apparent from the following detailed description taken in conjunction with the claims and drawings. The contents not described in this specification are fully understood and can be inferred by those skilled in the art of the present application or a similar art, and therefore, the description thereof will be omitted. [Means for solving the problem]

[0010] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the specific descriptions described below are not intended to limit the scope of this application.

[0011] One embodiment provides a peptide consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0012] The term "peptide" as used herein refers to a linear molecule formed by amino acid residues linked to each other by 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 activities, and have identified peptides having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The biologically effective activities may include any one or more of the following: (a) induction of glycosaminoglycan synthesis; (b) induction of COL2A1, COMP (cartilage oligomeric matrix protein), COL11A, PCP (proteoglycan core protein), or aggrecan synthesis; and (c) induction of the synthesis of regulatory factors SOX5, SOX6, or SOX9. Therefore, the peptides can be used for cartilage regeneration.

[0013] 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.

[0014] The peptides are artificially synthesized or non-naturally occurring or engineered, where "non-naturally occurring or engineered" refers to a state in which the peptide is produced by artificial modification, rather than a state in which it exists in nature. Here, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking a plurality of amino acid structures, or engineering to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity, as described above.

[0015] 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).

[0016] 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.

[0017] Among the terms or elements mentioned in the description of the peptide, the same as those already mentioned are as described above.

[0018] 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.

[0019] 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.

[0020] 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 15 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.

[0021] According to one embodiment, the peptide can significantly increase the expression of cartilage-related substances such as glycosaminoglycans COL2A1, COMP, COL11A, 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).

[0022] 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.

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

[0024] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the composition.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

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

[0030] The term "pharmaceutical effective amount" as used herein may refer to 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. 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.

[0032] 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).

[0033] 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.

[0034] 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.

[0035] The dosage of the pharmaceutical composition may be, but is not limited to, 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, and may vary depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity of the patient. 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.

[0036] 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.

[0037] 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 30 to 200 nm. Nanosome particle sizes less than 30 nm can penetrate the skin too quickly, resulting in side effects. Nanosome particle sizes greater than 200 nm can penetrate the skin too quickly, making it difficult to achieve the benefits of using the nanosome structure.

[0038] 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.

[0039] Among the terms or elements mentioned in the description of the peptides, compositions, etc., the same as those already mentioned are as described above.

[0040] 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.

[0041] 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.

[0042] Among the terms or elements mentioned in the description of the peptides, compositions, etc., the same as those already mentioned are as described above.

[0043] The cosmetic composition may contain, but is not limited to, a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier.

[0044] 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.

[0045] 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]

[0046] The peptide according to one embodiment significantly increases various cartilage components such as glycosaminoglycan, collagen, COMP, PCP, and aggrecan, thereby exhibiting excellent cartilage regeneration effects.

[0047] 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, PCP, and aggrecan. [Brief explanation of the drawings]

[0048] [Figure 1] This shows the results of confirming CCK-8 activity after treating C28 / I2 cells with Peptide-1. [Figure 2] This shows the results of confirming CCK-8 activity after treating C28 / I2 cells with Peptide-2. [Figure 3] This is the result of confirming the increase in glycosaminoglycan production after treating C28 / I2 cells with Peptide-1. [Figure 4] This is the result of confirming the increase in glycosaminoglycan production after treating C28 / I2 cells with Peptide-2. [Figure 5] This shows the results of confirming increased mRNA expression of ECM components after treating C28 / I2 cells with Peptide-1. [Figure 6] This shows the results of confirming increased mRNA expression of ECM components after treating C28 / I2 cells with Peptide-2. [Figure 7] This is the result of confirming increased mRNA expression of SOX9, an ECM regulatory factor, after treating C28 / I2 cells with Peptide-1. [Figure 8] This is the result of confirming increased mRNA expression of SOX9, an ECM regulatory factor, after treating C28 / I2 cells with Peptide-2. [Figure 9] This is the result of confirming increased expression of ECM regulatory factors SOX5, SOX6, and SOX9 proteins after treating C28 / I2 cells with Peptide-1. [Figure 10] This is the result of confirming increased expression of SOX9 protein, an ECM regulator, after treating C28 / I2 cells with Peptide-2. [Figure 11] This shows the results of confirming the suppression of mRNA expression of hypertrophic markers COL10A1 and Runx2 after treating C28 / I2 cells with Peptide-2. [Figure 12] This is the result of confirming the increase in glycosaminoglycan production after treating 3D cultured C28 / I2 cells with Peptide-2. [Figure 13] This is the result of confirming increased mRNA expression of ECM components after treating 3D cultured C28 / I2 cells with Peptide-2. [Figure 14] This is the result of confirming the increased expression of SOX9 protein, an ECM regulator, after treating 3D cultured C28 / I2 cells with Peptide-2. [Figure 15]After treating AD-MSC cells with Peptide-1, cell morphological changes and CCK-8 activity were confirmed by SRB staining. [Figure 16] After treating AD-MSC cells with Peptide-2, cell morphological changes and CCK-8 activity were confirmed by SRB staining. [Figure 17] This is the result of confirming increased glycosaminoglycan production after treating AD-MSC cells with Peptide-1. [Figure 18] This is the result of confirming increased glycosaminoglycan production after treating AD-MSC cells with Peptide-2. [Figure 19] This shows the results of confirming increased mRNA expression of ECM components after treating AD-MSC cells with Peptide-1. [Figure 20] This shows the results of confirming increased mRNA expression of ECM components after treating AD-MSC cells with Peptide-2. [Figure 21] This result confirmed the increased production of SOX9, an ECM regulatory factor, after treating AD-MSC cells with Peptide-1. [Figure 22] This result confirmed the increased production of SOX9, an ECM regulatory factor, after treating AD-MSC cells with Peptide-2. [Figure 23] This shows the results of confirming increased expression of ECM regulatory factors SOX5, SOX6, and SOX9 after treating AD-MSC cells with Peptide-1. [Figure 24] This shows the results of confirming increased expression of ECM regulatory factors SOX5, SOX6, and SOX9 after treating AD-MSC cells with Peptide-2. [Figure 25] This is the result of confirming the increased expression of the cartilage component COL2A1 after treating AD-MSC cells with Peptide-1. [Figure 26] This is the result of confirming the increased expression of the cartilage component COL2A1 after treating AD-MSC cells with Peptide-2. DETAILED DESCRIPTION OF THE INVENTION

[0049] 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.

[0050] Example 1. Synthesis of peptides Peptides (Peptide-1 or Peptide-2) having the amino acid sequences of SEQ ID NO: 1 or 2 listed in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA). The synthesized peptides were purified and separated using C18 reverse-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was an ACQUITY UPLC BEH300 C18 (2.1 mm ∘ 100 mm, 1.7 μm, Waters Co., USA).

[0051] [Table 1]

[0052] Example 2. Confirmation of cytotoxicity The cytotoxicity of Peptide-1 or Peptide-2 in human chondrocytes (C28 / I2, human chondrocyte cell line) was analyzed using the CCK-8 assay.

[0053] Specifically, 3x10 human-derived chondrocytes 3After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium, and cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 3 days, CCK-8 (Dojindo, CCK-8 kit) solution was added at 1 / 10 the volume of the culture medium, followed by incubation for 2 hours. The culture medium was sampled and CCK-8 activity was measured using a microplate reader at 450 nm wavelength. A negative control group was used without the peptide.

[0054] As a result, as shown in Figures 1 and 2, it was found that Peptide-1 and Peptide-2 did not exhibit toxicity in human-derived chondrocytes.

[0055] Example 3. Confirmation of glycosaminoglycan production effect The effects of adding Peptide-1 or Peptide-2 to human-derived chondrocytes on increasing glycosaminoglycan production were confirmed, and the effects of the peptides on inducing chondrogenesis and promoting extracellular matrix (ECM) production were confirmed.

[0056] Specifically, 3x10 human-derived chondrocytes 3After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium and treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 7 days, the medium was removed by suction, and 60 μL of 3.7% formalin was added to the 96-well plate to be stained and fixed for 1 minute. After suctioning the 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 added. After 24 hours of incubation at 37°C, the staining solution was removed by suction, the cells were washed with triple-distilled water, dried, and observed under a microscope. On the other hand, an experimental group to which the peptide was not added was used as a negative control group, and an experimental group to which TGF-β was added was used as a positive control group.

[0057] As a result, as shown in Figures 3 and 4, it was found that Peptide-1 and Peptide-2 each promoted the production of glycosaminoglycans.

[0058] Example 4. Confirmation of increased mRNA expression of ECM components It was confirmed whether the mRNA expression of ECM components was increased by treatment with Peptide-1 or Peptide-2 in human-derived chondrocytes.

[0059] Specifically, 8.9 x 10 human-derived chondrocytes 4The cells were seeded into 6-well plates at a density of 100 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium and treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three days and treated with Peptide-1 or Peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was suctioned, the cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and PCR was performed using the hCOL2A1, COMP, hCOL11A, ACAN, and GAPDH primers listed in Table 2 below. The experimental group, supplemented with TGF-β, served as a positive control. In Table 2 below, hCOL2A1 encodes collagen type II α1, hCOMP encodes cartilage oligomeric matrix protein, hCOL11A encodes the α chain of collagen type XI, hPCP encodes proteoglycan core protein, hACAN encodes aggrecan, and hGAPDH encodes glyceraldehyde-3-phosphate dehydrogenase.

[0060] [Table 2]

[0061] As a result, as shown in Figures 5 and 6, it was found that Peptide-1 and Peptide-2 each induced the production of ECM components ACAN, COL2A1, COMP, and COL11A.

[0062] Example 5. Confirmation of the mRNA expression induction effect of the ECM regulatory factor SOX9 We examined whether the mRNA expression of the ECM regulator SOX9 was increased by treatment with Peptide-1 or Peptide-2 in human-derived chondrocytes.

[0063] Specifically, 8.9 x 10 human-derived chondrocytes 4 The cells were seeded into 6-well plates at a density of 1000 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium and treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was suctioned, the cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and PCR was performed using the primers listed in Table 3 below. The negative control group was an experimental group without the peptide, and the positive control group was an experimental group with TGFβ1 (20 ng / ml). In Table 3 below, SOX9 refers to (sex determining region Y)-box 9.

[0064] [Table 3]

[0065] As a result, as shown in Figures 7 and 8, it was found that Peptide-1 and Peptide-2 each promoted the production of SOX9, an ECM regulatory factor.

[0066] Example 6: Confirmation of the expression-inducing effect of ECM regulatory factors SOX5, SOX6, and SOX9 It was confirmed whether the expression of ECM regulatory factors SOX5, SOX6, and SOX9 proteins was increased by treatment with Peptide-1 or Peptide-2 in human-derived chondrocytes.

[0067] Specifically, 8.9 x 10 human-derived chondrocytes 4Cells were seeded into 6-well plates at a density of 100 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium and treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every 3 days, and cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was suctioned, and cells were harvested to prepare lysates, followed by Western blotting. A negative control group lacking the peptide served as a negative control, while a positive control group receiving TGFβ1 (20 ng / ml) served as a positive control. 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.

[0068] As a result, as shown in FIGS. 9 and 10, it was found that Peptide-1 and Peptide-2 increased the expression of ECM regulatory factors SOX5, SOX6, and SOX9, respectively.

[0069] Example 7. Confirmation of the effect of suppressing the expression of hypertrophic markers COL10A1 and Runx2 We examined whether the mRNA expression of hypertrophic markers, COL10A1 and Runx2, increased with Peptide-2 treatment in human-derived chondrocytes.

[0070] Specifically, 8.9 x 10 human-derived chondrocytes 4The cells were seeded into 6-well plates at a density of 100 cells / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium and treated with Peptide-2 at various concentrations. After 3 days, the medium was suctioned, the cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR pre-mix (Intron, Korea), and PCR was performed using the primers listed in Table 4 below. The negative control group was an experimental group without the peptide, and the positive control group was an experimental group with TGFβ1 (20 ng / ml). In Table 4 below, COL10A1 encodes collagen type Xα1, and Runx2 encodes runt-related transcription factor 2.

[0071] [Table 4]

[0072] As a result, as shown in FIG. 11, it was found that Peptide-2 suppressed the expression of the hypertrophic markers COL10A1 and Runx2.

[0073] Example 8: Efficacy verification using 3D cultured human-derived chondrocytes We attempted to re-examine the biological efficacy of Peptide-2 using human-derived chondrocytes cultured in three dimensions using the micromass culture method.

[0074] Specifically, 2x10 human-derived chondrocytes 5The cells were seeded in droplet form into a 48-well plate at a density of 25 μl / well and cultured for 24 hours in DMEM medium (cat. 11995-065, Gibco). The human chondrocytes cultured in micromass were then examined for glycosaminoglycan production in the same manner as in Example 3, ACAN, COL2A1, COMP, and COL11A mRNA production in the same manner as in Example 4, and SOX9 protein expression in the same manner as in Example 6.

[0075] As a result, as shown in Figures 12 to 14, Peptide-2, as in the above-mentioned experimental results, promoted the production of glycosaminoglycans in 3D-cultured human chondrocytes, induced the production of ECM components ACAN, COL2A1, COMP, and COL11A, and increased the expression of SOX9 protein, an ECM regulatory factor.

[0076] Example 9. Confirmation of cytotoxicity The cell morphological changes and cytotoxicity caused by the addition of Peptide-1 or Peptide-2 in human adipose-derived mesenchymal stem cells (AD-MSCs) were analyzed by SRB staining and CCK-8 assay, respectively.

[0077] Specifically, 1.5x10 AD-MSC cells 3After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS. The medium was then replaced with DMEM supplemented with 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. To confirm CCK-8 activity, after seven days, a CCK-8 (Dojindo, CCK-8 kit) solution was added at 1 / 10 the volume of the culture medium and incubated for two hours. The culture medium was sampled and CCK-8 activity was measured using a microplate reader at 450 nm wavelength. For SRB staining, the medium was removed from the plate by suction, and 60 μL of 3.7% formalin was added to the 96-well plate for fixation and fixation for one minute. After suctioning off the 3.7% formalin, 70 μL of SRB staining solution (sulforhodamine B sodium salt (Sigma, S9012): 0.2 g per 100 mL of DDW) was added for staining. The plate was covered with silver foil and incubated overnight at room temperature in the dark. After washing with 100 μL of 1% acetic acid using a multi-pipette, the plate was dried and observed under a microscope.

[0078] As a result, as shown in Figures 15 and 16, when adipose-derived mesenchymal stem cells were treated with Peptide-1 or Peptide-2, no changes in cell morphology were observed by SRB staining, indicating that Peptide-1 or Peptide-2 does not exhibit toxicity to adipose-derived mesenchymal stem cells.

[0079] Example 10. Confirmation of glycosaminoglycan production effect By confirming the effect of increasing glycosaminoglycan production by adding Peptide-1 or Peptide-2 to adipose-derived mesenchymal stem cells, the effects of the peptides on inducing chondrogenesis and promoting extracellular matrix (ECM) production were confirmed.

[0080] Specifically, 1.5x10 AD-MSC cells 3After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured in DMEM medium supplemented with 10% FBS for 24 hours. The medium was then replaced with DMEM medium supplemented with 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 14 days, the medium was removed by suction, and 60 μL of 3.7% formalin was added to the 96-well plate to be stained and fixed for 1 minute. After suctioning the 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 added. After 24 hours of incubation at 37°C, the staining solution was removed by suction, the cells were washed with triple-distilled water, dried, and observed under a microscope. On the other hand, the positive control group (CM) was an experimental group containing 100 nM dexamethasone, 50 μM ascorbic acid, 40 μM proline, 10 ng / ml TGFβ1, and 1 XITS.

[0081] As a result, as shown in Figures 17 and 18, it was found that Peptide-1 and Peptide-2 each increased the production of glycosaminoglycans, induced cartilage production, and promoted extracellular matrix production.

[0082] Example 11. Confirmation of increased mRNA expression of ECM components It was confirmed whether the mRNA expression of ECM components increased upon treatment with Peptide-1 or Peptide-2 in adipose-derived mesenchymal stem cells.

[0083] Specifically, 1.5x10 AD-MSC cells 3After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured in DMEM medium supplemented with 10% FBS for 24 hours. The medium was then replaced with DMEM medium supplemented with 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three 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, the cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and PCR was performed using the hCOL2A1, hCOMP, hCOL11A, ACAN, and hPCP primers listed in Table 5 below.

[0084] [Table 5]

[0085] As a result, as shown in Figures 19 and 20, it was found that Peptide-1 and Peptide-2 each induced the production of ECM components COL2A1, COMP, COL11A, ACAN, and hPCP.

[0086] Example 12: Confirmation of the mRNA expression induction effect of the ECM regulatory factor SOX9 We examined whether the mRNA expression of the ECM regulator SOX9 was increased by treatment with Peptide-1 or Peptide-2 in adipose-derived mesenchymal stem cells.

[0087] Specifically, 1.5x10 AD-MSC cells 3After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured in DMEM medium supplemented with 10% FBS for 24 hours. The medium was then replaced with DMEM medium supplemented with 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three 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, and the cells were harvested and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and PCR was performed using the primers listed in Table 3.

[0088] As a result, as shown in Figures 21 and 22, it was found that Peptide-1 and Peptide-2 each promoted the production of SOX9, an ECM regulatory factor.

[0089] Example 13: Confirmation of the expression-inducing effect of ECM regulatory factors SOX5, SOX6, and SOX9 It was examined whether the expression of ECM regulatory factors SOX5, SOX6, and SOX9 was increased by treatment with Peptide-1 or Peptide-2 in adipose-derived mesenchymal stem cells.

[0090] Specifically, 1.5x10 AD-MSC cells 3After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured in DMEM medium supplemented with 10% FBS for 24 hours. The medium was then replaced with DMEM medium supplemented with 5% FBS and treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three days and treated with Peptide-1 or Peptide-2 at different concentrations. After 3, 7, and 14 days, the medium was removed by suction, and cells were harvested to prepare lysates, followed by Western blotting. 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.

[0091] As a result, as shown in Figures 23 and 24, it was found that Peptide-1 and Peptide-2 increased the expression of ECM regulatory factors SOX5, SOX6, and SOX9, respectively.

[0092] Example 14: Confirmation of COL2A1 expression induction effect It was confirmed whether the expression of the cartilage component COL2A1 was increased by treatment with Peptide-1 or Peptide-2 in adipose-derived mesenchymal stem cells.

[0093] Specifically, 1.5x10 AD-MSC cells 3 After seeding at a density of 100 cells / well into a 96-well plate, the cells were cultured in DMEM medium supplemented with 10% FBS for 24 hours. The medium was then replaced with DMEM medium supplemented with 5% FBS, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. The medium was then replaced every three days, and the cells were treated with Peptide-1 or Peptide-2 at different concentrations. After 3, 7, and 14 days, the medium was removed by suction, and the cells were harvested to prepare lysates, followed by Western blotting. The antibody used for detection was sc-393314 (Santa Cruz, USA) against COL2A1.

[0094] As a result, as shown in Figures 25 and 26, it was found that Peptide-1 and Peptide-2 each increased the expression of COL2A1.

[0095] To summarize the above experimental results, it was found that each of Peptide-1 and Peptide-2 according to one embodiment has the effect of inducing cartilage regeneration.

[0096] 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 1 L with distilled water. The mixture was then emulsified under high pressure in a microfluidizer to produce peptide nanosomes with a size of approximately 100 nm.

[0097] Dosage form example 2. Pharmaceutical preparations 2-1. Powder manufacturing The following ingredients are mixed and packed into an airtight cloth to produce a powder. The present invention Peptide 20mg Lactose 100mg Talc 10mg

[0098] 2-2. Tablet manufacturing The following ingredients are mixed and compressed into tablets by a conventional tablet manufacturing method. The present invention Peptide 10mg Corn starch 100mg Lactose 100mg Magnesium stearate 2mg

[0099] 2-3. Capsule manufacturing The following ingredients are mixed and filled into gelatin capsules according to a conventional capsule manufacturing method to produce capsules. The present invention Peptide 10mg Crystalline cellulose 3mg Lactose 14.8mg Magnesium stearate 0.2mg

[0100] 2-4. Manufacturing of injections The following ingredients are prepared per ampoule (2 ml) using the usual injection manufacturing method. The present invention Peptide 10mg Mannitol 180mg Sterile distilled water for injection 2974mg Na2HPO4·2H2O 26mg

[0101] 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. The present invention Peptide 10mg Isomerized sugar 10g Mannitol 5g Purified water (appropriate amount)

[0102] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

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 group and azide (-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, COL11A, PCP, or aggrecan production; and (c) Induction of the production of regulatory factors SOX5, SOX6, or SOX9.

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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