Peptide for cartilage regeneration and use thereof

Abstract

The present application relates to a peptide having a cartilage regeneration effect and a use thereof, and provides a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 or Arg (R)-Leu (L)-Arg (R)-Ser (S), a composition for cartilage regeneration comprising the peptide, and a pharmaceutical composition for preventing or treating cartilage diseases, comprising the composition for cartilage regeneration as an active ingredient.

Description

Technical Field

[0001] The present application relates to a peptide for cartilage regeneration and its use. Background Art

[0002] Due to the characteristics of cartilage tissue, it is difficult to regenerate tissue through natural healing when damaged on a large scale. Therefore, surgical procedures such as artificial joints, arthroscopic chondroplasty, and microfracture are used for treatment. However, existing methods often leave scars due to incisions and result in low durability of fibrocartilage regeneration. Therefore, compared with the difficulty of the surgical method, there is a problem of low treatment effect.

[0003] Therefore, injections administered into joints or compositions for restoring cartilage tissue using hydrogels and collagen, etc. have been developed. Their surgical procedures are simple and the treatment effect is rapid (Korean Patent Publication No. 2013-0028012). However, although the above method can temporarily relieve pain, it is not sufficient to induce cartilage tissue regeneration.

[0004] In addition, in treatment methods using cells, various treatment methods using autologous chondrocytes or stem cells, etc. have been developed, that is, methods for inducing cartilage tissue regeneration by transplanting in vitro cultured cells to the defect site (Korean Patent Publication No. 2013-0072983). However, in autologous chondrocyte therapeutic agents, when the injury area is large, treatment using only cultured cells collected from the patient is limited. And in stem cell therapeutic agents, there are the following problems: due to different collection sites, there are differences in cell quantity and differentiation ability, cell dedifferentiation occurs during in vitro culture resulting in changes in cell phenotype, the ratio 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 calcification of chondrocytes.

[0005] In this technical background, there is a current need to develop effective factors that can more effectively treat cartilage diseases by promoting chondrogenic differentiation or chondrogenesis of stem cells or chondrocytes, but it is still not perfect. Summary of the Invention

[0006] Technical Problem

[0007] On the one hand, there is provided a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0008] On the other hand, there is provided a composition for cartilage regeneration, which comprises 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.

[0009] On the other hand, a pharmaceutical composition for preventing or treating cartilage diseases is provided, which comprises the composition for cartilage regeneration as an active ingredient.

[0010] Other objects and advantages of the present application will become more apparent in conjunction with the appended claims, the accompanying drawings, and the following detailed description. Regarding the content not described in this specification, as long as it can be fully recognized and analogized by those of ordinary skill in the art to which the present application pertains or those of ordinary skill in a similar technical field, the relevant description thereof is omitted.

[0011] Technical solution

[0012] The various descriptions and embodiments disclosed in the present application can also be applied to various other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. Moreover, it cannot be determined that the scope of the present application is limited to the specific descriptions recorded hereinafter.

[0013] On the one hand, a peptide is provided, which consists of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

[0014] The term "peptide" used in this specification may refer to a linear molecule formed by multiple amino acid residues bound to each other through peptide bonds. The peptide can be prepared by chemical synthesis methods well known in the art, especially solid-phase synthesis techniques or liquid-phase synthesis techniques (US Registered Patent No. 5,516,891). Through the efforts of the present inventors to develop peptides with biologically effective activities, a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S) has been successfully identified. Herein, the biologically effective activity may refer to at least any one selected from the following characteristics: (a) inducing the production of glycosaminoglycan; (b) inducing the production of COL2A1, cartilage oligomeric matrix protein (COMP), COL11A, proteoglycan core protein (PCP), or aggrecan; (c) inducing the production of regulatory factors SOX5, SOX6, or SOX9; and (d) activating the upstream activator of the regulatory factor SOX. Therefore, the peptide can be used for cartilage regeneration.

[0015] In the peptide, in order to obtain chemical stability, enhanced pharmacological properties (half-life, absorbability, potency, efficacy, etc.), altered specificity (e.g., a wide range of biological activities), and reduced antigenicity, a protecting group may be bound to the N- or C-terminus at the end of the peptide. In one embodiment, the N-terminus of the peptide may be bound to any one of the protecting groups 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 a polyethylene glycol (PEG); and / or the C-terminus of the peptide may be bound to any one of the protecting groups selected from the group consisting of an amino group (-NH2), a tertiary alkyl group, and an azide (-NHNH2). Further, the peptide may optionally further include a targeting sequence, a tag, a labeled residue, and an amino acid sequence prepared for a specific purpose of increasing the half-life or peptide stability.

[0016] The peptide is artificially synthesized, or non-naturally occurring or engineered. The "non-naturally occurring or engineered" refers to a state generated by artificial modification, rather than the state of the existence itself produced in the natural state. Herein, the artificial modification may include artificially synthesizing an amino acid sequence by mimicking the structures of multiple amino acids, or may include being engineered as above to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

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

[0018] On the other hand, a composition for cartilage regeneration is provided, which includes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S) as an active ingredient.

[0019] Among the terms or elements mentioned in the description of the peptide, the same content as that already mentioned is as described above.

[0020] As used herein, the term "cartilage regeneration" may refer to improving cartilage tissue by repairing damaged cartilage tissue or inducing the generation of defective cartilage tissue. The "improvement" may refer to alleviation of the condition or any act of at least reducing treatment-related parameters such as the degree of symptoms.

[0021] The cartilage includes hyaline cartilage, fibrocartilage, or elastic cartilage, but is not limited thereto. 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 disadvantage of existing functional peptides is that even if they have effective biological activity, they cannot be effectively introduced into target tissues or cells due to the size of the peptide itself, or they disappear from the body within a short time because of their short half-life. On the other hand, a composition for cartilage regeneration according to an embodiment includes a peptide composed of 10 or fewer amino acids as an active ingredient. Therefore, the skin permeability of the active ingredient is very excellent. For example, when applied topically, an effective cartilage regeneration effect can be obtained.

[0023] According to an embodiment, the peptide can significantly increase the expression of glycosaminoglycan, COL2A1, COMP, COL11A, PCP, aggrecan, regulatory factors SOX5, SOX6, or SOX9, which are cartilage-related substances. Therefore, the peptide can be used as an active ingredient of a composition for cartilage regeneration (Orthop Res Rev. September 1, 2010; 2010(2): 85-94. doi: 10.2147 / ORR.S7194, JOSPT Vol. 28, No. 4, October 1998).

[0024] On the other hand, there is provided a pharmaceutical composition for preventing or treating cartilage diseases, which includes 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.

[0025] Among the terms or elements mentioned in the description of the peptide or composition, the same items as those already mentioned are as described above.

[0026] In this specification, the term "prevention" refers to all actions to inhibit or delay the onset of a disease by administering the composition.

[0027] In this specification, the term "treatment" refers to any form of treatment that provides an effect to an individual who already has a disease or is at risk of having a disease, and the effects include improvement of the individual's condition (e.g., one or more symptoms), delay in disease progression, delay in symptom onset, or deceleration of symptom progression, etc. Therefore, the "treatment" and "prevention" do not mean that the symptoms are cured or completely eliminated.

[0028] The "individual" refers to the target in need of treating a disease, and more specifically, refers to a human or non-human primate, and mammals such as a mouse, a dog, a cat, a horse, and a cow.

[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 group consisting of cartilage injury, cartilage defect, degenerative disc disease, herniated disc, degenerative arthritis, fracture, muscle tissue injury, joint injury caused by non-union of fracture or trauma, osteomalacia, and chondromalacia.

[0030] The cartilage disease may 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 a pharmaceutically effective amount of the peptide; and / or a pharmaceutically acceptable carrier, but is not limited thereto.

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

[0033] The weight ratio between the peptide and the pharmaceutically acceptable carrier may be, for example, 500:1 to 1:500. For example, the weight ratio may be 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] The pharmaceutically acceptable carrier is commonly used in the preparation of preparations and includes 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, etc., but is not limited thereto. Suitable pharmaceutically acceptable carriers and preparations are described in detail in Remington's Pharmaceutical Sciences (19th Edition, 1995).

[0035] In addition to the above-mentioned components, the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives, etc., but is not limited thereto.

[0036] The pharmaceutical composition can be administered enterally or parenterally, preferably parenterally. For parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc., but is not limited thereto.

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

[0038] The pharmaceutical composition can be formulated into a unit volume form by using a pharmaceutically acceptable carrier and / or excipient according to a method easily implemented by those of ordinary skill in the technical field to which the present invention pertains, or can be prepared by injecting it into a multi-volume container.

[0039] The dosage form can be in the form of a solution, suspension, or emulsion in an oily or aqueous solvent, and can also be in the form of an ointment, cream, gel, transdermal absorbent, cataplasm, patch, paste, extract, powder, granule, lozenge, or capsule, and can further include a powder and / or a stabilizer.

[0040] The peptide may be included in a nanobody or nanoparticle to further improve skin penetration or stability issues. For example, the nanobody can be prepared using a microfluidizer with lecithin as a raw material and can be included in lecithin particles. As a method for preparing the nanobody, any known method can be used. The size of the nanobody particles is preferably 30 to 200 nm. In the case where the size of the nanobody particles is less than 30 nm, skin penetration may be very fast, leading to skin side effects. In the case where the size of the nanobody particles is greater than 200 nm, it may not be easy to penetrate the skin, making it difficult to obtain the usage effect of the nanobody.

[0041] On the other hand, a method for preventing or treating cartilage diseases is provided, which includes the step of administering to an individual a pharmaceutical composition comprising a therapeutically effective amount of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S) as an active ingredient.

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

[0043] The terms "apply", "administer", and "coat" used in this specification can be used interchangeably and can refer to localizing at least part of the composition according to an embodiment at a desired site or disposing the composition according to an embodiment within an individual through an administration route.

[0044] On the other hand, a cosmetic composition is provided, which includes a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S) as an active ingredient.

[0045] Among the terms or elements mentioned in the description of the peptide, composition, etc., those same as the items already mentioned are as described above.

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

[0047] On the other hand, a method for regenerating cartilage is provided, which includes the step of administering to an individual a peptide comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S) as an active ingredient.

[0048] Among the terms or elements mentioned in the description of the peptide, composition, etc., those same as the content already mentioned are as described above.

[0049] Beneficial effects

[0050] The peptide according to one aspect exhibits excellent cartilage regeneration effects by causing a significant increase in various cartilage components such as glycosaminoglycan, collagen, COMP, aggrecan, etc.

[0051] The peptide according to one aspect causes a significant increase in various cartilage components such as glycosaminoglycan, collagen, COMP, aggrecan, etc., and thus can be applied to the prevention or treatment of cartilage diseases and the promotion of cartilage regeneration. Brief description of the drawings

[0052] Figure 1 Shows the results of confirming the morphological changes of AD-MSC cells after treatment with peptide-1 by sulforhodamine B (SRB) staining.

[0053] Figure 2 Shows the results of confirming the morphological changes of AD-MSC cells after treatment with peptide-2 by SRB staining.

[0054] Figure 3 Shows the results of confirming the CCK-8 activity after treatment of AD-MSC cells with peptide-1.

[0055] Figure 4 Shows the results of confirming the CCK-8 activity after treatment of AD-MSC cells with peptide-2.

[0056] Figure 5 Shows the results of confirming the increased production of glycosaminoglycan after treatment of AD-MSC cells with peptide-1.

[0057] Figure 6 Shows the results of confirming the increased production of glycosaminoglycan after treatment of AD-MSC cells with peptide-2.

[0058] Figure 7 Shows the results of confirming the increased mRNA expression of ECM components after treatment of AD-MSC cells with peptide-1.

[0059] Figure 8 Shows the results of confirming the increased mRNA expression of ECM components after treatment of AD-MSC cells with peptide-2.

[0060] Figure 9 Shows the results of confirming the increased production of ECM regulator SOX9 after treatment of AD-MSC cells with peptide-1.

[0061] Figure 10 Shows the results of confirming the increased production of ECM regulator SOX9 after treatment of AD-MSC cells with peptide-2.

[0062] Figure 11 It shows the result of confirming the increased expression of SOX5, SOX6, and SOX9, which are ECM regulators, after treating AD-MSC cells with Peptide-1.

[0063] Figure 12 It shows the result of confirming the increased expression of ECM regulators SOX5, SOX6, and SOX9 after treating AD-MSC cells with Peptide-2.

[0064] Figure 13 It shows the result of confirming the increased expression of the cartilage components aggrecan and COL2A1 after treating AD-MSC cells with Peptide-1.

[0065] Figure 14 It shows the result of confirming the increased expression of the cartilage components aggrecan and COL2A1 after treating AD-MSC cells with Peptide-2.

[0066] Figure 15 It shows the result of confirming the activation of the upstream activator of the ECM regulator SOX9 in chondrocytes after treating AD-MSC cells with Peptide-2. Detailed Description of the Invention

[0067] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only for exemplarily describing the present invention, and the scope of the present invention is not limited to these examples.

[0068] Example 1, Synthesis of Peptides

[0069] An automatic peptide synthesizer (Milligen 9050, Millipore, USA) was used to synthesize Peptide-1 or Peptide-2 described in Table 1 below, and the synthesized peptides were purified by C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co. USA) was used as the column.

[0070] [Table 1]

[0071]

[0072]

[0073] Example 2, Confirmation of Cytotoxicity

[0074] The aim is to analyze the cell morphological changes and cytotoxicity caused by the addition of peptide-1 or peptide-2 in human adipose-derived mesenchymal stem cells (AD-MSC) through SRB staining and CCK-8 assay respectively.

[0075] Specifically, AD-MSC cells were seeded in a 96-well plate at a density of 1.5×10 3 cells / well and cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with 10% FBS for 24 hours. Then, after changing to DMEM medium supplemented with 5% FBS, peptide-1 or peptide-2 was treated at different concentrations. Subsequently, the medium was changed every 3 days, and peptide-1 or peptide-2 was treated at different concentrations. To confirm the CCK-8 activity, after 7 days, a CCK-8 (Dojindo, CCK-8 kit) solution was added at 1 / 10 volume of the culture medium and incubated for 2 hours. The culture medium was sampled and the CCK-8 activity at a wavelength of 450 nm was confirmed using a microplate reader.

[0076] Moreover, for sulfanilamide rhodamine B (SRB) staining, after sucking the 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 sucking the 3.7% formaldehyde solution, 70 μL of SRB staining solution (sulfanilamide rhodamine 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 with light blocked. After washing with 100 μL of 1% acetic acid using a multi-pipette, it 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).

[0077] As a result, as Figure 1 and Figure 2 shown, even when peptide-1 or peptide-2 was treated on human adipose-derived mesenchymal stem cells, there were no cell morphological changes during SRB staining. Moreover, as Figure 3 and Figure 4 shown, peptide-1 or peptide-2 did not show toxicity to human adipose-derived mesenchymal stem cells.

[0078] Example 3. Confirm the glycosaminoglycan production effect

[0079] The purpose is to confirm the increased effect of glycosaminoglycan production caused by the addition of Peptide-1 or Peptide-2 in human adipose-derived mesenchymal stem cells, and to confirm the effect of the peptide in inducing chondrogenesis and promoting the production of extracellular matrix (ECM).

[0080] Specifically, after inoculating AD-MSC cells at a density of 1.5×10 3 cells / well in a 96-well plate, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, after changing to DMEM medium with 5% FBS, Peptide-1 or Peptide-2 was treated at different concentrations. Subsequently, the medium was changed once every 3 days, and Peptide-1 or Peptide-2 was treated at different concentrations. After 14 days, the medium was aspirated, and 60 μL of 3.7% formaldehyde solution was added to the 96-well plate for staining and fixed for 1 minute. After aspirating the 3.7% formaldehyde solution, 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 incubating at 37 °C for 24 hours, the staining solution was aspirated, washed three times with distilled water, dried, and observed under a microscope.

[0081] As a result, as Figure 5 and Figure 6 shown, Peptide-1 and Peptide-2 respectively caused an increase in glycosaminoglycan production to induce chondrogenesis and promote the production of extracellular matrix.

[0082] Example 4. Confirm the increased mRNA expression of ECM

[0083] The purpose is to confirm whether the mRNA expression of ECM components is increased by the treatment with Peptide-1 or Peptide-2 in human adipose-derived mesenchymal stem cells.

[0084] Specifically, after inoculating AD-MSC cells at a density of 1.5×10 3The cells were seeded at a density of

[0085] [Table 2]

[0086]

[0087] As a result, as shown in 7 and Figure 8 as shown, Peptide-1 and Peptide-2 respectively induced the production of ECM components, namely COL2A1, COMP, COL11A, PCP, and ACAN.

[0088] Example 5. Confirmation of the induction effect of mRNA expression of the ECM regulator SOX9

[0089] The aim was to confirm whether treatment with Peptide-1 or Peptide-2 in human adipose-derived mesenchymal stem cells resulted in an increase in the mRNA expression of the ECM regulator SOX9.

[0090] Specifically, AD-MSC cells were seeded at a density of 1.5×10 3 cells / well in a 96-well plate and cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, after changing to DMEM medium with 5% FBS, Peptide-1 or Peptide-2 was treated at different concentrations. Subsequently, the medium was changed once every 3 days, and Peptide-1 or Peptide-2 was treated at different concentrations. After aspirating the medium at 3 days, 7 days, and 14 days, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, Korea), PCR was performed using the primers shown in Table 3 below. In Table 3 below, SOX9 refers to sex-determining region Y-box protein 9.

[0091] [Table 3]

[0092]

[0093] As a result, it can be seen that as Figure 9 and Figure 10 shown, Peptide-1 and Peptide-2 respectively promote the production of the ECM regulator SOX9.

[0094] Example 6. Confirmation of the expression induction effect of ECM regulators SOX5, SOX6 and SOX9

[0095] The purpose is to confirm whether the treatment with Peptide-1 or Peptide-2 in human adipose-derived mesenchymal stem cells results in an increase in the expression of ECM regulators SOX5, SOX6 and SOX9.

[0096] Specifically, after inoculating AD-MSC cells into a 96-well plate at a density of 1.5×10 3 cells / well, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, after changing to DMEM medium with 5% FBS, Peptide-1 or Peptide-2 was treated at different concentrations. Subsequently, the medium was changed every 3 days, and Peptide-1 or Peptide-2 was treated at different concentrations. After aspirating the medium at 3 days, 7 days and 14 days, the cells were harvested and lysates were prepared, followed by immunoblotting. As the antibodies to be detected, sc-293215 (Santa Cruz, USA) was used for SOX5, sc-393314 (Santa Cruz, USA) was used for SOX6, and 82630S (Cell Signaling, USA) was used for SOX9.

[0097] As a result, it can be seen that as Figure 11 and Figure 12 shown, Peptide-1 and Peptide-2 respectively result in an increase in the expression of ECM regulators SOX5, SOX6 and SOX9.

[0098] Example 7. Confirmation of the expression induction effect of aggrecan and COL2A1

[0099] The purpose is to confirm whether the treatment with Peptide-1 or Peptide-2 in human adipose-derived mesenchymal stem cells results in an increase in the expression of the cartilage components aggrecan and COL2A1.

[0100] Specifically, the AD-MSC cells were seeded at 1.5×10 3The cells were seeded at a density of cells per well in a 96-well plate and cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, after changing to DMEM medium with 5% FBS, they were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and they were treated with peptide-1 or peptide-2 at different concentrations. After aspirating the medium at 3 days, 7 days, and 14 days, the cells were harvested and lysates were prepared, followed by immunoblotting. As the antibodies to be detected, sc-33695 (Santa Cruz, USA) was used for aggrecan and sc-518017 (Santa Cruz, USA) was used for COL2A1.

[0101] As a result, as Figure 13 and Figure 14 shown, peptide-1 and peptide-2 respectively led to an increase in the expression of COL2A1, and peptide-2 also led to an increase in the expression of aggrecan.

[0102] Example 8. Confirmation of the effect of inducing the activity of the upstream activator of the ECM regulator SOX9

[0103] The aim was to confirm the effect of inducing the activity of the upstream activator of the ECM regulator SOX9 by treatment with peptide-2 in human adipose-derived mesenchymal stem cells.

[0104] Specifically, AD-MSC cells were seeded at a density of 1.5×10 3 cells per well in a 96-well plate and cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, after changing to DMEM medium supplemented with 5% FBS, they were treated with peptide-2 at different concentrations. After 15 minutes, the medium was aspirated, the cells were harvested and lysates were prepared, followed by immunoblotting. As the detection antibodies, #8685S (Cell Signaling, USA) was used for Smad2 / 3 and #8828S (Cell Signaling, USA) was used for p-Smad2 / 3.

[0105] As a result, as Figure 15 shown, peptide-2 induced the activity of the upstream activator of the ECM regulator SOX.

[0106] Based on the above experimental results, it can be seen that peptide-1 and peptide-2 according to one embodiment each have the effect of inducing cartilage regeneration.

[0107] Formulation Example 1. Preparation of peptide nanobodies

[0108] 50 mg of the peptide of Example 1 was dissolved by stirring well with 500 ml of distilled water. After mixing the complex solution with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol and a small amount of oil phase, the volume was adjusted to 1 L with distilled water, and then emulsified under high pressure using a high-pressure microfluidizer to prepare peptide nanobodies with a size of about 100 nm.

[0109] Formulation Example 2, Pharmaceutical Preparation

[0110] 2-1. Preparation of Powder

[0111] The following components were mixed and filled into a sealed bag to prepare a powder.

[0112] 20 mg of the peptide of the present invention

[0113] 100 mg of lactose

[0114] 10 mg of talc

[0115] 2-2. Preparation of Lozenge

[0116] The following components were mixed and then pressed into lozenges according to the usual method for preparing lozenges.

[0117] 10 mg of the peptide of the present invention

[0118] 100 mg of corn starch

[0119] 100 mg of lactose

[0120] 2 mg of magnesium stearate

[0121] 2-3. Preparation of Capsule

[0122] According to the usual method for preparing capsules, the following components were mixed and filled into gelatin capsules to prepare capsules.

[0123] 10 mg of the peptide of the present invention

[0124] 3 mg of crystalline cellulose

[0125] 14.8 mg of lactose

[0126] 0.2 mg of magnesium stearate

[0127] 2-4. Preparation of Injection

[0128] According to the usual method for preparing injections, each ampoule (2 ml) was prepared with the following component contents.

[0129] 10 mg of the peptide of the present invention

[0130] 180 mg of mannitol

[0131] 2974 mg Sterile Distilled Water for Injection

[0132] 26mg Na2HPO4·2H2O

[0133] 2-5. Preparation of Liquid Agent

[0134] According to the usual method for preparing a liquid agent, each component is added and dissolved in pure water, and the following components are mixed. The volume is adjusted to 100 ml with pure water, and then it is filled into a brown bottle and sterilized to prepare a liquid agent.

[0135] 10 mg of the peptide of the present invention

[0136] 10 g of isomerized sugar

[0137] 5 g of mannitol

[0138] Appropriate amount of pure water

[0139] The description of the present invention is only for providing examples. Those of ordinary skill in the art should be able to understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood as exemplary in all aspects, rather than restrictive.

Claims

1. A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Arg(R)-Leu(L)-Arg(R)-Ser(S).

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, fluorenylmethyloxycarbonyl, 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 any one of the protecting groups selected from the group consisting of amino, tertiary alkyl, and azide.

4. The peptide according to claim 1, wherein, The peptide exhibits at least any one of the following properties: (a) Inducing glycosaminoglycan production; (b) Inducing the production of COL2A1, COMP, COL11A, PCP, or aggrecan; (c) Inducing the production of regulatory factors SOX5, SOX6, or SOX9; and (d) Activating the upstream activator of regulatory factor 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. The composition according to claim 6, further comprising a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 6, wherein, The peptide is prepared in the form of a nanobody.

9. The pharmaceutical composition according to claim 6, wherein, The cartilage disease is at least one selected from the group consisting of cartilage injury, cartilage defect, degenerative disc disease, disc herniation, degenerative arthritis, fracture, muscle tissue injury, joint injury caused by nonunion or trauma of fracture, osteomalacia, and chondromalacia.

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