Peptides for cartilage regeneration and uses thereof

Abstract

The present application relates to a peptide for cartilage regeneration and use thereof. Specifically disclosed is a peptide having a cartilage regeneration effect and use thereof, providing a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S), a composition for cartilage regeneration including the same, and a pharmaceutical composition for preventing or treating a cartilage disease including the composition for cartilage regeneration as an effective ingredient.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202280102442.X entitled “Peptides for Cartilage Regeneration and Use Thereof”, filed on December 16, 2022. Technical Field

[0002] This application relates to a peptide for cartilage regeneration and its uses. Background Technology

[0003] 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.

[0004] Therefore, injectable solutions or cartilage tissue recovery compositions using hydrogels and collagen, etc., have been developed for intra-articular application, with simple surgical 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.

[0005] 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 is low; and gene expression related to cell hypertrophy induces apoptosis and vascular permeability, leading to chondrocyte calcification.

[0006] 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

[0007] Technical issues

[0008] One aspect provides a peptide composed of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0009] On the other hand, a composition for cartilage regeneration is provided, comprising a peptide as an active ingredient consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0010] On the other hand, a pharmaceutical composition for the prevention or treatment of cartilage diseases is provided, comprising the composition for cartilage regeneration as an active ingredient.

[0011] 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.

[0012] Technical solution

[0013] 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.

[0014] One aspect provides a peptide composed of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0015] 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 No. 5,516,891). Through diligent research, the inventors have successfully identified peptides with biologically effective activity composed of amino acid sequences represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S). Here, the biologically effective activity can refer to at least one of the following properties: (a) induction of glycosaminoglycan formation; (b) induction of COL2A1, cartilage oligomeric matrix protein (COMP), COL11A, proteoglycan core protein (PCP), or aggrecan; (c) induction of regulatory factors SOX5, SOX6, or SOX9; and (d) activation of the upstream activator of the regulatory factor SOX. Therefore, the peptide can be used for cartilage regeneration.

[0016] 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 azide (-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.

[0017] 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.

[0018] 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).

[0019] On the other hand, a composition for cartilage regeneration is provided, comprising a peptide as an active ingredient consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0020] 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.

[0021] 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.

[0022] 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.

[0023] The drawback of existing functional peptides is that, even if they possess effective biological activity, their size limits their effective delivery to target tissues or cells, 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 comprises a peptide consisting of 10 or fewer amino acids as the active ingredient. Therefore, the skin penetration of the active ingredient is excellent, and for example, effective cartilage regeneration can be achieved when applied topically.

[0024] According to one embodiment, the peptide can significantly increase the expression of glycosaminoglycans, COL2A1, COMP, COL11A, PCP, aggregate proteoglycans, regulators SOX5, SOX6, or SOX9, which are cartilage-related substances, and therefore the peptide can be used as an active ingredient in compositions for cartilage regeneration (Orthop Res Rev. 2010 Sep 1; 2010(2): 85-94. doi: 10.2147 / ORR.S7194, JOSPT Vol. 28 No. 4 October 1998).

[0025] On the other hand, a pharmaceutical composition for the prevention or treatment of cartilage diseases is provided, comprising a peptide as an active ingredient consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0026] In the description of the peptide or composition, any terms or elements that are the same as those already mentioned are as described above.

[0027] In this specification, the term "prevention" refers to all actions that suppress or delay the occurrence of a disease by applying the composition.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The pharmaceutical composition may include, but is not limited to, a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier.

[0033] 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.

[0034] 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.

[0035] 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).

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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, penetration into the skin may be difficult, making it difficult to achieve the desired effects.

[0042] On the other hand, a method for preventing or treating cartilage diseases is provided, comprising the step of administering to an individual a therapeutically effective amount of a pharmaceutical composition consisting of a peptide composed of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S) as an active ingredient.

[0043] In the description of the peptides, compositions, etc., the terms or elements that are the same as those already mentioned are as described above.

[0044] 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.

[0045] On the other hand, a cosmetic composition is provided, comprising a peptide as an active ingredient, consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0046] In the description of the peptides, compositions, etc., the terms or elements that are the same as those already mentioned are as described above.

[0047] The cosmetic composition may include, but is not limited to, a cosmetically effective amount of the peptide and / or a cosmetically acceptable carrier.

[0048] On the other hand, a method for regenerating cartilage is provided, comprising the step of applying a peptide, consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S), as an active ingredient to an individual.

[0049] In the description of the peptides, compositions, etc., the terms or elements that are the same as those that have been mentioned are as described above.

[0050] Beneficial effects

[0051] According to one aspect, peptides exhibit excellent cartilage regeneration effects by significantly increasing various cartilage components such as glycosaminoglycans, collagen, COMP, and proteoglycans.

[0052] Based on the fact that one type of peptide leads to a significant increase in various cartilage components such as glycosaminoglycans, collagen, COMP, and agglutinin, it can be applied to the prevention or treatment of cartilage diseases and the promotion of cartilage regeneration. Attached Figure Description

[0053] Figure 1 The results show the changes in cell morphology confirmed by sulfonylrhodamine B (SRB) staining after treatment of AD-MSC cells with peptide-1.

[0054] Figure 2 The results of SRB staining confirm the changes in cell morphology after treatment of AD-MSC cells with peptide-2 are shown.

[0055] Figure 3 The results show the confirmation of CCK-8 activity after treatment of AD-MSC cells with peptide-1.

[0056] Figure 4 The results show the confirmation of CCK-8 activity after treatment of AD-MSC cells with peptide-2.

[0057] Figure 5 The results show that treatment of AD-MSC cells with peptide-1 confirmed an increase in glycosaminoglycan production.

[0058] Figure 6 The results show that treatment of AD-MSC cells with peptide-2 confirmed an increase in glycosaminoglycan production.

[0059] Figure 7 The results show that treatment of AD-MSC cells with peptide-1 confirmed an increase in the mRNA expression of ECM components.

[0060] Figure 8 The results show that treatment of AD-MSC cells with peptide-2 confirmed an increase in the mRNA expression of ECM components.

[0061] Figure 9 The results show that treatment of AD-MSC cells with peptide-1 confirmed an increase in the production of the ECM regulator SOX9.

[0062] Figure 10 The results show that treatment of AD-MSC cells with peptide-2 confirmed an increase in the production of the ECM regulator SOX9.

[0063] Figure 11 The results show that treatment of AD-MSC cells with peptide-1 confirmed the increased expression of SOX5, SOX6, and SOX9, which are ECM regulators.

[0064] Figure 12 The results show that treatment of AD-MSC cells with peptide-2 confirmed the increased expression of ECM regulatory factors SOX5, SOX6, and SOX9.

[0065] Figure 13 The results show that treatment of AD-MSC cells with peptide-1 confirmed increased expression of cartilage component aggregates, proteoglycans, and COL2A1.

[0066] Figure 14 The results show that treatment of AD-MSC cells with peptide-2 confirmed increased expression of cartilage component aggregates, proteoglycans, and COL2A1.

[0067] Figure 15 The results show that treatment of AD-MSC cells with peptide-2 confirmed the activation of the upstream activator of SOX9, an ECM regulator of chondrocytes. Detailed Implementation

[0068] 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.

[0069] Example 1: Peptide Synthesis

[0070] Peptide-1 or peptide-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 C18 reversed-phase high-performance liquid chromatography (HPLC) (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.

[0071] [Table 1]

[0072] Example 2: Confirmation of the presence of cytotoxicity

[0073] The aim was to analyze the changes in cell morphology and cytotoxicity caused by the addition of peptide-1 or peptide-2 in human adipose-derived mesenchymal stem cells (AD-MSCs) by SRB staining and CCK-8 assay, respectively.

[0074] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3 Cells were seeded at a density of 10% FBS in 96-well plates and cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium). Then, the medium was replaced with DMEM supplemented with 5% FBS, and peptide-1 or peptide-2 was applied at different concentrations. The medium was then changed every 3 days, and peptide-1 or peptide-2 was applied at different concentrations. To confirm CCK-8 activity, CCK-8 (Dojindo, CCK-8 kit) solution was added to 1 / 10 volume of the culture medium after 7 days and incubated for 2 hours. The culture medium was sampled and CCK-8 activity at 450 nm was confirmed using a microplate reader.

[0075] Furthermore, for sulfonylrhodamine B (SRB) staining, after suctioning the culture medium from the plate, 60 μL of 3.7% formaldehyde solution was added to the 96-well plate for staining and fixed for 1 minute. After suctioning the 3.7% formaldehyde solution, 70 μL of SRB staining solution (sulfonylrhodamine B sodium salt (sigma, S9012): 0.2 g in 100 mL DDW) was added for staining. The plate was covered with silver foil and incubated overnight at room temperature to block light. After washing with 100 μL of 1% acetic acid using multiple pipettes, the plate was dried and observed under a microscope. In this example, a group supplemented with dexamethasone (100 nM), acetic acid (50 μM), proline (40 μM), TGFβ1 (10 ng / ml), and 1X ITS was used as the positive control group (CM).

[0076] As a result, it can be seen that... Figure 1 and Figure 2 As shown, even after treatment with peptide-1 or peptide-2, human adipose-derived mesenchymal stem cells did not exhibit any changes in cell morphology during SRB staining. Furthermore, it can be seen that... Figure 3 and Figure 4 As shown, peptide-1 or peptide-2 did not exhibit toxicity to human adipose-derived mesenchymal stem cells.

[0077] Example 3: Confirmation of glycosaminoglycan formation effect

[0078] The aim is to confirm the effect of the peptides in inducing chondrogenesis and promoting extracellular matrix (ECM) generation by confirming the increased glycosaminoglycan production induced by the addition of peptide-1 or peptide-2 in human adipose-derived mesenchymal stem cells.

[0079] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3 Cells were seeded at a density of 10% FBS in 96-well plates and cultured for 24 hours. Then, the medium was replaced with 5% FBS in DMEM, and cells were treated with either peptide-1 or peptide-2 at different concentrations. The medium was changed every 3 days, and peptide-1 or peptide-2 treatments were performed at different concentrations. After 14 days, the medium was drained, and 60 μL of 3.7% formaldehyde solution was added to the 96-well plates for staining and fixed for 1 minute. Following the 3.7% formaldehyde solution, 70 μL of New Blue staining solution (50 mL of 3% acetic acid + 8 g x 0.5 g of 1% New Blue, pH 2.5) was added. After incubation at 37°C for 24 hours, the staining solution was drained, washed with triple-distilled water, dried, and observed under a microscope.

[0080] As a result, it can be seen that... Figure 5 and Figure 6 As shown, peptide-1 and peptide-2 lead to increased glycosaminoglycan production to induce chondrogenesis and promote extracellular matrix production, respectively.

[0081] Example 4: Confirmation of increased ECM mRNA expression

[0082] The aim was to determine whether treatment with peptide-1 or peptide-2 in human adipose-derived mesenchymal stem cells resulted in increased mRNA expression of ECM components.

[0083] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3Cells were seeded at a density of 96-well plates and cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was replaced with DMEM medium supplemented with 5% FBS, and cells were treated with either peptide-1 or peptide-2 at different concentrations. The medium was then changed every 3 days, and cells were treated with either peptide-1 or peptide-2 at different concentrations. Cells were harvested and RNA isolated after 3, 7, and 14 days. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and PCR was performed using the hCOL2A1, chondrocyte oligomeric matrix protein (COMP), hCOL11A, proteoglycan core protein (PCP), and ACAN primers shown in Table 2 below. In Table 2 below, hCOL2A1 is used to encode type II collagen α1, hCOMP is used to encode Cartilage oligomeric matrix protein, hCOL11A is used to encode the α chain of type XI collagen, hPCP is used to encode the proteoglycan core protein, hACAN is used to encode aggregated proteoglycan, and hGAPDH is used to encode glyceraldehyde-3-phosphate dehydrogenase.

[0084] [Table 2]

[0085] As a result, it can be seen that... Figure 7 and Figure 8 As shown, peptide-1 and peptide-2 induce the generation of ECM components, namely COL2A1, COMP, COL11A, PCP, and ACAN, respectively.

[0086] Example 5: Confirmation of the mRNA expression induction effect of ECM regulator SOX9

[0087] The aim was to determine whether treatment with peptide-1 or peptide-2 in human adipose-derived mesenchymal stem cells led to an increase in the mRNA expression of the ECM regulator SOX9.

[0088] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3Cells were seeded at a density of 96-well plates and cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was replaced with DMEM medium supplemented with 5% FBS, and cells were treated with either peptide-1 or peptide-2 at different concentrations. The medium was then changed every 3 days, and cells were treated with either peptide-1 or peptide-2 at different concentrations. Cells were harvested and RNA isolated after 3, 7, and 14 days. cDNA was synthesized using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), and PCR was performed using the primers shown in Table 3 below. In Table 3, SOX9 refers to the sex-determining region Y-box protein 9.

[0089] [Table 3]

[0090] As a result, it can be seen that... Figure 9 and Figure 10 As shown, peptide-1 and peptide-2 promote the generation of the ECM regulator SOX9, respectively.

[0091] Example 6: Confirmation of the expression induction effects of ECM regulators SOX5, SOX6, and SOX9.

[0092] The aim was to determine whether peptide-1 or peptide-2 treatment in human adipose-derived mesenchymal stem cells led to an increase in the expression of ECM regulatory factors SOX5, SOX6, and SOX9.

[0093] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours in DMEM medium supplemented with 10% FBS. Then, the medium was replaced with 5% FBS DMEM medium, and cells were treated with different concentrations of peptide-1 or peptide-2. The medium was then changed every 3 days, and cells were treated with different concentrations of peptide-1 or peptide-2. After 3, 7, and 14 days, the medium was aspirated, cells were harvested, and lysates were prepared for Western blotting. The 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.

[0094] As a result, it can be seen that... Figure 11 and Figure 12 As shown, peptide-1 and peptide-2 led to increased expression of ECM regulators SOX5, SOX6 and SOX9, respectively.

[0095] Example 7: Confirmation of the expression-inducing effect of agglutinin and COL2A1.

[0096] The aim was to determine whether treatment with peptide-1 or peptide-2 in human adipose-derived mesenchymal stem cells resulted in increased expression of cartilage component aggregates, proteoglycans, and COL2A1.

[0097] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours in DMEM medium supplemented with 10% FBS. Then, the medium was replaced with 5% FBS DMEM medium, and cells were treated with either peptide-1 or peptide-2 at different concentrations. The medium was then changed every 3 days, and cells were treated with either peptide-1 or peptide-2 at different concentrations. After 3, 7, and 14 days, the medium was aspirated, cells were harvested, and lysates were prepared for Western blotting. SC-33695 (Santa Cruz, USA) was used as the antibody for detection against proteoglycans, and SC-518017 (Santa Cruz, USA) was used against COL2A1.

[0098] As a result, it can be seen that... Figure 13 and Figure 14 As shown, peptide-1 and peptide-2 led to increased expression of COL2A1, and peptide-2 also led to increased expression of aggregate proteoglycans.

[0099] Example 8: Confirmation of the induction effect of upstream activator activity of ECM regulator SOX9

[0100] The aim was to confirm the effect of peptide-2 treatment on the induction of the activity of the upstream activator of the ECM regulator SOX9 in human adipose-derived mesenchymal stem cells.

[0101] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3 Cells were seeded at a density of 10% FBS in 96-well plates and cultured for 24 hours. Then, the medium was replaced with DMEM supplemented with 5% FBS, and cells were treated with different concentrations of peptide-2. After 15 minutes, the medium was aspirated, cells were harvested, and lysates were prepared for Western blotting. #8685S (Cell Signaling, USA) was used as the detection antibody for Smad2 / 3, and #8828S (Cell Signaling, USA) was used for p-Smad2 / 3.

[0102] As a result, it can be seen that... Figure 15 As shown, peptide-2 induces the activity of the upstream activator of the ECM regulator SOX.

[0103] Based on the experimental results, peptide-1 and peptide-2, according to one embodiment, each have the effect of inducing cartilage regeneration.

[0104] Dosage Form Example 1: Preparation of Peptide Nanobody

[0105] 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 peptide nanobody with a size of approximately 100 nm was then prepared by emulsification under high pressure using a high-pressure microfluidic homogenizer.

[0106] Dosage form example 2, pharmaceutical preparation

[0107] 2-1. Preparation of Powders

[0108] Mix the following ingredients and fill them into a sealed bag to prepare a powder.

[0109] 20 mg of the peptide of the present invention

[0110] 100 mg lactose

[0111] 10 mg talc

[0112] 2-2. Preparation of Tablets

[0113] Mix the following ingredients and then compress them into tablets according to the usual tablet preparation method.

[0114] 10 mg of the peptide of the present invention

[0115] 100 mg corn starch

[0116] 100 mg lactose

[0117] 2 mg magnesium stearate

[0118] 2-3. Preparation of Capsules

[0119] Following standard capsule preparation methods, the following ingredients are mixed and filled into gelatin capsules to prepare capsules.

[0120] 10 mg of the peptide of the present invention

[0121] 3 mg crystalline cellulose

[0122] 14.8 mg lactose

[0123] 0.2 mg magnesium stearate

[0124] 2-4. Preparation of Injectables

[0125] Following standard injectable preparation methods, each ampoule (2 ml) is prepared according to the following ingredient content.

[0126] 10 mg of the peptide of the present invention

[0127] 180 mg mannitol

[0128] 2974 mg sterile distilled water for injection

[0129] 26 mg Na2HPO4·2H2O

[0130] 2-5. Preparation of Liquid Formulation

[0131] 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 100 ml, and then it is filled into a brown bottle, sterilized, and prepared as a liquid.

[0132] 10 mg of the peptide of the present invention

[0133] 10 g isomerized sugar

[0134] 5 g mannitol

[0135] An appropriate amount of purified water

[0136] 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 represented by 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 the 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 as an active ingredient according to any one of claims 1 to 3.

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 a peptide comprising the amino acid sequence represented by 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 one or more of the group consisting of cartilage injury, cartilage defect, degenerative intervertebral disc disease, intervertebral disc herniation, degenerative arthritis, fracture, injury to muscle tissue, joint injury due to nonunion of fracture or trauma, osteomalacia, and chondromal malformation.

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