Peptides for Cartilage Regeneration and Their Uses

Abstract

This application relates to a peptide with cartilage regeneration effect and its use, providing a peptide composed of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a composition for cartilage regeneration including said peptide, and a pharmaceutical composition for the prevention or treatment of cartilage diseases including said composition for cartilage regeneration as an active ingredient.

Description

[Technical Field]

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

[0002] Due to the nature of cartilage tissue, it is difficult for large-area damage to heal and regenerate naturally. Therefore, surgical treatments such as artificial joints, articular chondrotomy, and microfracture surgery are used. However, existing methods often leave scars due to incisions and result in low durability of fibrocartilage regeneration, thus leading to lower treatment outcomes compared to the difficulty of the procedures.

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

[0004] In addition, various treatment methods using autologous chondrocytes or stem cells have been developed in cell-based therapies, which involve inducing cartilage tissue regeneration by transplanting in vitro cultured cells to the defect site (Korean Patent Publication No. 2013-0072983). However, in autologous chondrocyte therapy, the use of cultured cells collected from the patient alone is limited when the damaged area is large. Furthermore, stem cell therapy has the following problems: cell numbers and differentiation capacity vary depending on the collection site; cell dedifferentiation during in vitro culture leads to changes in cell phenotype; the rate of differentiation into chondrocytes after transplantation into the body is low; and gene expression related to cell hypertrophy induces apoptosis and vascular permeability, leading to chondrocyte calcification.

[0005] In this technological context, there is a need to develop effective factors that can more effectively treat cartilage diseases by promoting cartilage differentiation or cartilage formation in stem cells or chondrocytes, but these are still not perfect. [Summary of the Invention]

[0006] [Technical Issues]

[0007] One aspect provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] On the other hand, a composition for cartilage regeneration is provided, comprising a peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0009] Another aspect provides a pharmaceutical composition for the prevention or treatment of cartilage diseases, comprising the composition for cartilage regeneration as an active ingredient.

[0010] Other objects and advantages of this application will become clearer in conjunction with the appended claims and drawings, as well as the detailed description below. Regarding matters not described in this specification, which can be fully understood and deduced by those skilled in the art or similar fields, their descriptions are omitted.

[0011] [Technical Solution]

[0012] The various descriptions and embodiments disclosed in this application can also be applied to various other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, it cannot be determined that the scope of this application is limited to the specific descriptions set forth below.

[0013] One aspect provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0014] As used in this specification, the term "peptide" can refer to a linear molecule formed by multiple amino acid residues linked together by peptide bonds. The peptide can be prepared using chemical synthesis methods known in the art, particularly solid-phase or liquid-phase synthesis techniques (US Patent Registration No. 5,516,891). As a result of efforts to develop peptides with biologically effective activity, the inventors have identified a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Here, biologically effective activity can refer to at least one of the following properties: (a) inducing the formation of glycosaminoglycans; (b) inducing the formation of COL2A1, COMP (Cartilage oligomeric matrix protein), COL11A, or aggrecan; and (c) inducing the formation of SOX5, SOX6, or SOX9 as regulatory factors. Therefore, the peptide can be used for cartilage regeneration.

[0015] In the peptide, a protecting group may be bound to the N- or C-terminus to obtain chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad range of biological activities), and reduced antigenicity. In one embodiment, the N-terminus of the peptide may be bound to any 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 bound to any protecting group selected from the group consisting of amino (-NH2), tertiary alkyl, and hydrazino (-NHNH2). Furthermore, the peptide may optionally include a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose of increasing half-life or peptide stability.

[0016] The peptide is artificially synthesized or non-naturally occurring or engineered. "Non-naturally occurring or engineered" refers to a state generated through artificial modification, rather than its inherent state as a naturally occurring entity. Here, artificial modification may include the artificial synthesis of an amino acid sequence by mimicking the structure of multiple amino acids, or may include the above-mentioned engineering to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0017] The term "stability" as used in this specification may refer not only to the in vivo stability of the peptide protected from attack by proteolytic enzymes in vivo, but also to storage stability (e.g., room temperature storage stability).

[0018] On the other hand, a composition for cartilage regeneration is provided, comprising a peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0019] In the description of the peptide, the terms or elements that are the same as those that have been mentioned before are as described above.

[0020] As used in this specification, the term "cartilage regeneration" can refer to improving cartilage tissue by repairing damaged cartilage tissue or inducing the generation of defective cartilage tissue. "Improvement" can refer to the alleviation of the condition or at least a reduction in all behaviors related to treatment-related parameters such as symptom severity.

[0021] The cartilage includes, but is not limited to, hyaline cartilage, fibrocartilage, or elastic cartilage. For example, the cartilage may be at least one selected from the group consisting of articular cartilage, ear cartilage, nasal cartilage, elbow cartilage, meniscus cartilage, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage, and spinal cartilage.

[0022] The drawback of existing functional peptides is that, even if they possess effective biological activity, they cannot be effectively delivered to target tissues or cells due to their size, or they disappear from the body quickly due to their short half-life. On the other hand, a composition for cartilage regeneration according to one embodiment includes a peptide composed of 10 or fewer amino acids as the active ingredient. Therefore, the skin penetration of the active ingredient is excellent, and for example, when applied topically, an effective cartilage regeneration effect can be obtained.

[0023] According to one embodiment, since the peptide can significantly increase the expression of glycosaminoglycans, COL2A1, COMP, COL11A, aggregate proteoglycans as cartilage-related substances, and SOX5, SOX6 or SOX9 as regulatory factors, the peptide can be used as an active ingredient in a composition for cartilage regeneration (Orthop Res Rev., September 1, 2010; 2010(2):85-94. doi:10.2147 / ORR.S7194, JOSPT Volume 28 Number 4, October 1998).

[0024] Another aspect provides a pharmaceutical composition for the prevention or treatment of cartilage diseases, comprising a peptide of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0025] In the description of the peptide or composition, any terms or elements that are the same as those already mentioned are as described above.

[0026] In this specification, the term "prevention" refers to all actions that suppress or delay the occurrence of a disease by applying the composition.

[0027] In this specification, the term "treatment" refers to any form of treatment that provides an effect to an individual who has a disease or is at risk of developing a disease, including improvement of the individual's condition (e.g., one or more symptoms), delay of disease progression, delay of symptom onset, or slowing of symptom progression. Therefore, "treatment" and "prevention" are not intended to represent a cure or complete elimination of symptoms.

[0028] The term "individual" refers to a target for treatment of a disease, and more specifically, to humans or non-human primates, mammals such as mice, dogs, cats, horses, and cattle.

[0029] In this specification, the term "cartilage disease" refers to all cartilage-related diseases that require cartilage differentiation or regeneration. The cartilage disease may be selected from at least one of the following groups: cartilage lesions, cartilage defects, degenerative disc diseases, herniated discs, degenerative arthritis, fractures, muscle tissue injuries, joint injuries due to nonunion of fractures or trauma, osteomalacia, and chondromalacia.

[0030] The cartilage disease 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.

[0031] The pharmaceutical composition may include, but is not limited to, a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier.

[0032] The term "pharmaceuticalally effective amount" as used in this specification refers to an amount sufficient to achieve the cartilage regeneration effect of the pharmaceutical composition.

[0033] The weight ratio between the peptide and the pharmaceutically acceptable carrier can be, for example, 500:1 to 1:500, such as 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2, but is not limited thereto.

[0034] Pharmaceutically acceptable carriers are those commonly used in the preparation of formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0035] In addition to the ingredients mentioned above, the pharmaceutical composition may further include, but is not limited to, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc.

[0036] The pharmaceutical composition can be administered via the gastrointestinal tract or parenterally, preferably via parenteral administration. For parenteral administration, it can be administered via intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local application, percutaneous application, etc., but is not limited thereto.

[0037] The dosage of the pharmaceutical composition may be 0.0001 to 1000 μg (micrograms), 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg per day, but is not limited thereto, and may vary depending on factors such as formulation method, administration method, patient's age, weight, gender, symptoms, diet, administration time, route of administration, excretion rate, and reaction sensitivity.

[0038] The pharmaceutical composition can be formulated in a unit volume form using pharmaceutically acceptable carriers and / or excipients according to methods readily practiced by those skilled in the art to which this invention pertains, or can be prepared by injection into multi-volume containers.

[0039] The dosage form may be a solution, suspension or emulsion in an oily or aqueous solvent, or may be an ointment, cream, gel, transdermal absorbent, poultice, patch, paste, extractant, powder, granule, lozenge or capsule, and may further include powder and / or stabilizer.

[0040] The peptides may be incorporated into nanobodies or nanoparticles to further improve skin penetration or stability. For example, the nanobodies can be prepared using lecithin as a raw material via a microfluidic apparatus and may be incorporated into lecithin particles. Any known method may be used to prepare the nanobodies. The size of the nanobodies is preferably 30 to 200 nm. If the size of the nanobodies is less than 30 nm, skin penetration may be very rapid, leading to skin side effects; if the size of the nanobodies is greater than 200 nm, they may not easily penetrate the skin, making it difficult to achieve the desired effects.

[0041] Another aspect provides a method for preventing or treating cartilage diseases, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition to an individual, wherein the pharmaceutical composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

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

[0043] The terms “application,” “apply,” and “coat” used in this specification are used interchangeably and can refer to at least partially localizing a composition according to an embodiment onto a desired site or distributing a composition according to an embodiment into an individual by means of application.

[0044] Another aspect 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.

[0045] In the description of the peptides, compositions, etc., the terms or elements that are the same as those already mentioned are as described above.

[0046] The cosmetic composition may include, but is not limited to, a cosmetically effective amount of the peptide; and / or a cosmetically acceptable carrier.

[0047] Another aspect provides a method for cartilage regeneration, comprising the step of applying a composition to an individual, wherein the composition comprises a peptide consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0048] In the description of the peptides, compositions, etc., the terms or elements that are the same as those already mentioned are as described above.

[0049] [Beneficial Effects]

[0050] According to one type of peptide, excellent cartilage regeneration effects can be achieved by significantly increasing various cartilage components such as glycosaminoglycans, collagen, COMP, and agglutinin.

[0051] According to one type of peptide, it 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 agglutinin.

[0052] [Brief explanation of the attached image]

[0053] Figure 1 The results show the confirmation of CCK-8 activity after treatment of C28 / I2 cells with peptide-1.

[0054] Figure 2 The results show the confirmation of CCK-8 activity after treating C28 / I2 cells with peptide-2.

[0055] Figure 3 The results show that treatment of C28 / I2 cells with peptide-1 confirmed an increase in glycosaminoglycan production.

[0056] Figure 4 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in glycosaminoglycan production.

[0057] Figure 5 The results show that treatment of C28 / I2 cells with peptide-1 confirmed an increase in the expression of mRNAs of ECM (Extra Cellular Matrix) components.

[0058] Figure 6 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in the expression of ECM component mRNA.

[0059] Figure 7 The results show that treatment of C28 / I2 cells with peptide-1 confirmed an increase in the mRNA expression of SOX9, an ECM regulator.

[0060] Figure 8 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in the mRNA expression of SOX9, an ECM regulator.

[0061] Figure 9 The results show that treatment of C28 / I2 cells with peptide-1 confirmed an increase in the expression of SOX9 protein, a regulator of ECM.

[0062] Figure 10The results show that treatment of C28 / I2 cells with peptide-2 resulted in increased expression of SOX5, SOX6, and SOX9 proteins, which are ECM regulators.

Detailed Implementation Methods

[0063] The present invention will now be described in more detail through embodiments. However, these embodiments are merely illustrative of the invention, and the scope of the invention is not limited to these embodiments.

[0064] [Example 1. Peptide Synthesis]

[0065] Peptides (peptide-1 or peptide-2) with the amino acid sequence SEQ ID NO: 1 or 2 as described in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified using a C18 reversed-phase high-performance liquid chromatography (HPLC) system (Waters Associates, USA). An ACQUITY UPLC BEH300 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Co., USA) was used as the column.

[0066] [Table 1]

[0067] Peptide-1 PYYRI 1 Peptide-2 KFLIK 2

[0068] [Example 2. Confirmation of Cytotoxicity]

[0069] The aim was to analyze the cytotoxicity induced by the addition of peptide-1 or peptide-2 in human chondrocytes (C28 / I2, human chondrocyte cell line) using the CCK-8 assay.

[0070] Specifically, human chondrocytes were used at a rate of 3 × 10⁻⁶. 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured for 24 hours in DMEM (Dulbecco's Modified Essential Medium) (cat. 11995-065, Gibco). Then, the medium was replaced with fresh medium, and peptide-1 or peptide-2 were treated at different concentrations. After 3 days, CCK-8 (Dojindo, CCK-8 kit) solution was added to one-tenth of the culture volume, and the plates were incubated for 2 hours. The culture medium was sampled, and CCK-8 activity was confirmed at 450 nm using a microplate reader. A negative control group was used, without the added peptides.

[0071] The result, such as Figure 1 and Figure 2As shown, neither peptide-1 nor peptide-2 exhibited toxicity in human chondrocytes.

[0072] [Example 3. Confirmation of the glycosaminoglycan formation effect]

[0073] The aim was to confirm the effect of adding peptide-1 or peptide-2 to human chondrocytes on increasing glycosaminoglycan production, thereby confirming the effect of the peptides on inducing chondrogenesis and promoting extracellular matrix (ECM) production.

[0074] Specifically, human chondrocytes were used at a rate of 3 × 10⁻⁶. 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium, and peptide-1 or peptide-2 were treated at different concentrations, respectively. The medium was then replaced every 3 days, and peptide-1 or peptide-2 were treated at different concentrations, respectively. After 7 days, the medium was drained, and 60 μL of 3.7% formaldehyde was added to the 96-well plates used for staining, and the plates were fixed for 1 minute. After draining the 3.7% formaldehyde, 70 μL of Alcian blue staining solution (50 mL of 3% acetic acid + 0.5 g of 1% Alcian blue 8GX, pH 2.5) was extracted. After incubation at 37°C for 24 hours, the staining solution was drained, washed with triple-distilled water, and dried for microscopic observation. A group supplemented with TGFβ1 (20 ng / mL) served as a positive control. On the other hand, the experimental group without the added peptide was used as a negative control group, and the experimental group with added TGF-β was used as a positive control group.

[0075] The result, such as Figure 3 and Figure 4 As shown, both peptide-1 and peptide-2 can promote the formation of glycosaminoglycans.

[0076] [Example 4. Confirmation of increased mRNA expression of ECM components]

[0077] The aim was to determine whether treatment with peptide-1 or peptide-2 in human chondrocytes would increase the mRNA expression of ECM components.

[0078] Specifically, human chondrocytes were seeded at a density of 8.9 × 10⁴ cells / well in 6-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was replaced, and peptide-1 or peptide-2 was treated at different concentrations. The medium was then replaced every 3 days, and peptide-1 or peptide-2 was treated at different concentrations. After 1, 3, and 7 days, the medium was aspirated, cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and a PCR premix (Intron, Korea), and PCR was performed using the hCOL2A1, COMP, hCOL11A, ACAN, and GAPDH primers shown in Table 2. On the other hand, an experimental group without the added peptides was used as a negative control, and an experimental group with added TGF-β was used as a positive control. In Table 2 below, hCOL2A1 encodes the α1 chain of type II collagen, hCOMP encodes cartilage oligomeric matrix protein, hCOL11A encodes the α chain of type XI collagen, hACAN encodes agglutinin, and hGAPDH encodes glyceraldehyde-3-phosphate dehydrogenase.

[0079] [Table 2]

[0080]

[0081] The result, such as Figure 5 and Figure 6 As shown, both peptide-1 and peptide-2 were found to increase the production of COL2A1, COMP, COL11A and ACAN mRNA as ECM.

[0082] [Example 5. Confirmation of the effect of SOX9 as an ECM regulator on inducing mRNA expression]

[0083] The aim was to determine whether treatment with peptide-1 or peptide-2 in human chondrocytes would increase the mRNA expression of SOX9, an ECM regulator.

[0084] Specifically, human chondrocytes were used at a concentration of 8.9 × 10⁻⁶. 4Cells were seeded at a density of 6-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was replaced, and peptide-1 or peptide-2 was treated at different concentrations. The medium was then replaced every 3 days, treating peptide-1 or peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was aspirated, cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and a PCR premix (Intron, Korea), and PCR was performed using the primers shown in Table 3. A negative control group was used without the peptides, and a positive control group was used with TGF-β. In Table 3, SOX9 refers to (sex determining region Y)-box 9.

[0085] [Table 3]

[0086]

[0087] The result, such as Figure 7 and Figure 8 As shown, both peptide-1 and peptide-2 can induce the generation of SOX9, an ECM regulator.

[0088] [Example 6. Confirmation of the effects of SOX5, SOX6, and SOX9 as ECM regulators on induced expression]

[0089] The aim was to determine whether treatment with peptide-1 or peptide-2 in human chondrocytes would increase the expression of SOX5, SOX6, and SOX9, which are ECM regulators.

[0090] Specifically, human chondrocytes were used at a concentration of 8.9 × 10⁻⁶. 4 Cells were seeded at a density of 6-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The medium was then replaced with fresh medium, and peptide-1 or peptide-2 were treated at different concentrations. The medium was then replaced every 3 days, and peptide-1 or peptide-2 were treated at different concentrations. After 1, 3, and 7 days, the medium was aspirated, cells were harvested, lysates were prepared, and Western blotting was performed. As detection antibodies, sc-293215 (Santa Cruz, USA) was used for SOX5, sc-393314 (Santa Cruz, USA) for SOX6, and 82630S (Cell Signaling, USA) for SOX9. On the other hand, a negative control group was used without the stated peptides, and a positive control group was used with TGF-β.

[0091] The result, such as Figure 9 and Figure 10 As shown, both peptide-1 and peptide-2 can increase the expression of SOX5, SOX6 and SOX9, which are ECM regulators.

[0092] Based on the above experimental results, it was found that peptide-1 and peptide-2 according to one embodiment both have the effect of inducing cartilage regeneration.

[0093] [Dosage Form Example 1. Preparation of Peptide Nanobody]

[0094] 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water by thorough stirring. The complex solution was then mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil phase. The mixture was adjusted with distilled water until the total volume was 1 L. The mixture was then emulsified under high pressure using a high-pressure microfluidic homogenizer to prepare peptide nanobody particles with a size of approximately 100 nm.

[0095] [Dosage Form Example 2. Pharmaceutical Preparation]

[0096] 【2-1. Preparation of Powders】

[0097] Mix the following ingredients and fill them into a sealed bag to prepare a powder.

[0098] 20 mg of the peptide of the present invention

[0099] 100 mg lactose

[0100] 10 mg talc

[0101] 【2-2. Preparation of Tablets】

[0102] Mix the following ingredients and then compress them into tablets according to the usual tablet preparation method.

[0103] 10 mg of the peptide of the present invention

[0104] 100 mg corn starch

[0105] 100 mg lactose

[0106] 2 mg magnesium stearate

[0107] [2-3. Preparation of Capsules]

[0108] Following standard capsule preparation methods, the following ingredients are mixed and filled into gelatin capsules to prepare capsules.

[0109] 10 mg of the peptide of the present invention

[0110] 3 mg crystalline cellulose

[0111] 14.8mg lactose

[0112] 0.2mg magnesium stearate

[0113] [2-4. Preparation of Injectable Formulations]

[0114] Following standard injection preparation methods, each ampoule (2 ml) is prepared according to the following ingredient content.

[0115] 10 mg of the peptide of the present invention

[0116] 180 mg mannitol

[0117] 2974 mg sterile distilled water for injection

[0118] 26mg Na2HPO4·2H2O

[0119] 【2-5. Preparation of Liquid Formulation】

[0120] Following the usual liquid preparation method, each ingredient is added and dissolved in purified water, and the following ingredients are mixed. Purified water is added to adjust the total volume to 100ml, and then it is filled into a brown bottle, sterilized, and prepared as a liquid.

[0121] 10 mg of the peptide of the present invention

[0122] 10 g isomerized sugar

[0123] 5 g mannitol

[0124] An appropriate amount of purified water

[0125] The description of this invention is for illustrative purposes only, and those skilled in the art should understand that the invention can be readily modified into other specific forms without changing the technical concept or essential features of the invention. Therefore, the embodiments described above should be understood as exemplary in all respects, and not as limiting.

Claims

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

1.

2. The peptide according to claim 1, wherein, The N-terminus of the peptide is bound to any one of the protecting groups selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristoyl, stearoyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol.

3. The peptide according to claim 1, wherein, The C-terminus of the peptide is bound to a protecting group selected from the group consisting of amino, tertiary alkyl, and hydrazine groups.

4. A composition for cartilage regeneration, comprising a peptide according to any one of claims 1 to 3 as an active ingredient.

5. A pharmaceutical composition for the prevention or treatment of cartilage diseases, comprising a peptide according to any one of claims 1 to 3 as an active ingredient.

6. The pharmaceutical composition according to claim 5, further comprising a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 5, wherein, The peptide was prepared in the form of nanobody.

8. Use of the peptide comprising the amino acid sequence of SEQ ID NO: 1 in the preparation of a pharmaceutical composition for the prevention or treatment of cartilage diseases, wherein the cartilage disease is selected from at least one of the group consisting of: cartilage defects, degenerative arthritis, and chondromalacia.

9. The use according to claim 8, wherein, The cartilage defect is an injury to the articular cartilage caused by trauma.

10. The use according to claim 8 or 9, wherein, The N-terminus of the peptide is bound to any one of the protecting groups selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristoyl, stearoyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol.

11. The use according to claim 8 or 9, wherein, The C-terminus of the peptide is bound to a protecting group selected from the group consisting of amino, tertiary alkyl, and hydrazine groups.

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