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 the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 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 naturally after extensive damage. Therefore, surgical procedures such as artificial joints, arthroplasty, and microfracture are used for treatment. However, existing methods often leave scars due to incisions and result in a low durability of fibrocartilage regeneration. This leads to a problem of low therapeutic effectiveness, especially due to the difficulty of the surgical procedures.

[0003] Therefore, injections or cartilage tissue restoration compositions using hydrogels and collagen have been developed, which are simple to use and have rapid therapeutic effects (Korean Patent Publication No. 2013-0028012). However, although these methods can temporarily relieve pain, they are not sufficient to induce cartilage tissue regeneration.

[0004] In addition, various cell-based treatment methods have been developed using autologous chondrocytes or stem cells, specifically methods that induce cartilage tissue regeneration by transplanting cells cultured in vitro into the defect site (Korean Patent Publication No. 2013-0072983). However, autologous chondrocyte-based treatments are limited to treating large areas of injury using only cultured cells collected from the patient. Furthermore, stem cell-based treatments have the following problems: differences in cell number and differentiation capacity due to the site of collection; changes in cell phenotype caused by cell dedifferentiation during in vitro culture; a low rate of chondrocyte differentiation after in vivo transplantation; and chondrocyte calcification caused by the induction of apoptosis and vascular permeability due to the expression of genes associated with cell hypertrophy.

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

[0006] Technical issues

[0007] In one aspect, a peptide is provided, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] Another aspect provides a composition for cartilage regeneration, comprising a peptide having an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

[0009] On the other hand, there is provided a pharmaceutical composition for preventing or treating cartilage diseases, 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 similar technical fields, 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. In addition, it is not certain that the scope of the present application is limited to the specific descriptions recorded hereinafter.

[0013] On the one hand, there is provided a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[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 technology or liquid-phase synthesis technology (US Registered Patent No. 5,516,891). As a result of efforts to develop peptides with biologically effective activities, the present inventors have identified a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Herein, the biologically effective activity may refer to at least any one of the characteristics selected from (a) inducing the production of glycosaminoglycan; (b) inducing the production of COL2A1, COMP (Cartilage oligomeric matrix protein), COL11A, PCP (proteoglycan core protein) or aggrecan; and (c) inducing the production of SOX5, SOX6 or SOX9 as regulatory factors. 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 group (-NHNH2). Moreover, 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 term "non-naturally occurring or engineered" refers to a state generated by artificial modification, rather than the state of existence 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 having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 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 all actions that relieve the state or at least reduce 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 activities, 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 15 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, since the peptide can significantly increase the expression of glycosaminoglycan, COL2A1, COMP, COL11A, PCP, aggrecan, which are cartilage-related substances, and SOX5, SOX6, or SOX9, which are regulatory factors, 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 Volume 28 Number 4, October 1998).

[0024] On the other hand, a pharmaceutical composition for preventing or treating cartilage diseases is provided, which includes a peptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 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 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 occurrence, or deceleration of symptom progression, etc. Therefore, the "treatment" and "prevention" do not represent that the symptoms are cured or completely eliminated.

[0028] The "individual" refers to the target that needs to be treated for a disease, and more specifically, refers to a human or non-human primate, and mammals such as a mouse, dog, cat, horse, and 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, disc herniation, degenerative arthritis, fracture, muscle tissue injury, joint injury caused by nonunion 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 carriers are those commonly used in the preparation of pharmaceutical formulations, including 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 not limited thereto. Suitable pharmaceutically acceptable carriers and formulations 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 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 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 not limited thereto, and can be administered differently according to factors such as the formulation method, administration method, 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 pharmaceutically acceptable carriers and / or excipients according to methods easily implemented by those of ordinary skill in the art to which the present invention pertains, or can be prepared by filling into multi-volume containers.

[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 powders and / or stabilizers.

[0040] The peptide may be included in nanobodies or nanoparticles to further improve skin penetration problems or stability problems. For example, the nanobodies 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 nanobodies, any well-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, resulting in 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 effects of using the nanobodies.

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

[0042] Among the terms or elements mentioned in the description of the peptide, composition, etc., those identical to 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] In another aspect, a cosmetic composition is provided, which includes a peptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 as an active ingredient.

[0045] Among the terms or elements mentioned in the description of the peptide, composition, etc., those identical to 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] In another aspect, a method for cartilage regeneration is provided, which includes the step of administering a composition to an individual, wherein the composition includes a peptide consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 as an active ingredient.

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

[0049] Beneficial effects

[0050] According to one aspect of the peptide, it can exhibit excellent cartilage regeneration effects by significantly increasing various cartilage components such as glycosaminoglycan, collagen, COMP, PCP, and aggrecan.

[0051] According to one aspect of the peptide, it can be applied to the prevention or treatment of cartilage diseases and promote cartilage regeneration by significantly increasing various cartilage components such as glycosaminoglycan, collagen, COMP, PCP, and aggrecan.

[0052] Brief Description of the Drawings

[0053] Figure 1 Shows the results of confirming the CCK-8 activity after treating C28 / I2 cells with peptide-1.

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

[0055] Figure 3 Shows the results of confirming an increase in the production of glycosaminoglycan after treating C28 / I2 cells with peptide-1.

[0056] Figure 4 Shows the results of confirming an increase in the production of glycosaminoglycan after treating C28 / I2 cells with peptide-2.

[0057] Figure 5 Shows the results of confirming an increase in the expression of mRNA of ECM (Extra Cellular Matrix) components after treating C28 / I2 cells with peptide-1.

[0058] Figure 6 Shows the results of confirming an increase in the expression of mRNA of ECM components after treating C28 / I2 cells with peptide-2.

[0059] Figure 7 Shows the results of confirming an increase in the mRNA expression of SOX9, which is an ECM regulator, after treating C28 / I2 cells with peptide-1.

[0060] Figure 8 Shows the results of confirming an increase in the mRNA expression of SOX9, which is an ECM regulator, after treating C28 / I2 cells with peptide-2.

[0061] Figure 9 Shows the results of confirming an increase in the expression of SOX5, SOX6, and SOX9 proteins, which are ECM regulators, after treating C28 / I2 cells with peptide-1.

[0062] Figure 10The results showing an increase in the expression of SOX9 protein, which is an ECM regulator, were confirmed after treating C28 / I2 cells with peptide-2.

[0063] Figure 11 The results showing that the mRNA expressions of COL10A1 and Runx2, which are hypertrophic markers, were inhibited were confirmed after treating C28 / I2 cells with peptide-2.

[0064] Figure 12 The results showing an increase in the production of glycosaminoglycan were confirmed after treating three-dimensionally cultured C28 / I2 cells with peptide-2.

[0065] Figure 13 The results showing an increase in the expression of mRNA of ECM components were confirmed after treating three-dimensionally cultured C28 / I2 cells with peptide-2.

[0066] Figure 14 The results showing an increase in the expression of SOX9 protein, which is an ECM regulator, were confirmed after treating three-dimensionally cultured C28 / I2 cells with peptide-2.

[0067] Figure 15 The results showing the cell morphological changes by SRB staining and CCK-8 activity were confirmed after treating AD-MSC cells with peptide-1.

[0068] Figure 16 The results showing the cell morphological changes by SRB staining and CCK-8 activity were confirmed after treating AD-MSC cells with peptide-2.

[0069] Figure 17 The results showing an increase in the production of glycosaminoglycan were confirmed after treating AD-MSC cells with peptide-1.

[0070] Figure 18 The results showing an increase in the production of glycosaminoglycan were confirmed after treating AD-MSC cells with peptide-2.

[0071] Figure 19 The results showing an increase in the expression of mRNA of ECM components were confirmed after treating AD-MSC cells with peptide-1.

[0072] Figure 20 The results showing an increase in the expression of mRNA of ECM components were confirmed after treating AD-MSC cells with peptide-2.

[0073] Figure 21 The results showing an increase in the production of SOX9, which is an ECM regulator, were confirmed after treating AD-MSC cells with peptide-1.

[0074] Figure 22The results showing an increase in the production of SOX9, a regulator of ECM, were confirmed after treating AD-MSC cells with peptide-2.

[0075] Figure 23 The results showing an increase in the expression of SOX5, SOX6, and SOX9, regulators of ECM, were confirmed after treating AD-MSC cells with peptide-1.

[0076] Figure 24 The results showing an increase in the expression of SOX5, SOX6, and SOX9, regulators of ECM, were confirmed after treating AD-MSC cells with peptide-2.

[0077] Figure 25 The results showing an increase in the expression of COL2A1, a cartilage component, were confirmed after treating AD-MSC cells with peptide-1.

[0078] Figure 26 The results showing an increase in the expression of COL2A1, a cartilage component, were confirmed after treating AD-MSC cells with peptide-2. Detailed Description of the Invention

[0079] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only for illustrative description of the present invention, and the scope of the present invention is not limited to these examples.

[0080] Example 1, Synthesis of Peptides

[0081] Peptides (peptide-1 or peptide-2) having the amino acid sequences of SEQ ID NO: 1 or 2 described in Table 1 below were synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), 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.

[0082] [Table 1]

[0083] Name Amino Acid Sequence (N-terminal -> C-terminal) SEQ ID NO. Peptide-1 KVSYAAEKNRKNY 1 Peptide-2 SSFYF 2

[0084] Example 2, Confirmation of Cytotoxicity

[0085] The aim was to analyze the cytotoxicity caused by adding peptide-1 or peptide-2 in human chondrocytes (C28 / I2, a human chondrocyte cell line) using the CCK-8 assay.

[0086] Specifically, human chondrocytes were cultured at a rate of 3 × 10 3 After the density of cells / well was seeded in a 96-well plate, it was cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, it was replaced with a new culture medium and peptide-1 or peptide-2 was treated at different concentrations. Then, after 3 days, CCK-8 (Dojindo, CCK-8 kit) solution was added to one tenth of the volume of the culture medium and incubated for 2 hours. The culture medium was sampled and CCK-8 activity was confirmed at a wavelength of 450nm using a microplate reader. On the other hand, a test group to which the peptide was not added was used as a negative control group.

[0087] The results, such as Figure 1 and Figure 2 As shown, it was found that both peptide-1 and peptide-2 showed no toxicity in human chondrocytes.

[0088] Example 3: Confirmation of the production effect of glycosaminoglycans

[0089] The purpose was to confirm the effect of increasing the production of glycosaminoglycans by adding peptide-1 or peptide-2 to human chondrocytes, thereby confirming the effect of the peptides on inducing chondrogenesis and promoting extracellular matrix (ECM) production.

[0090] Specifically, human chondrocytes were cultured at a rate of 3 × 10 3 After the cells were seeded in a 96-well plate at a density of 100 cells / well, they were cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, new culture medium was replaced, and peptide-1 or peptide-2 was treated at different concentrations. Next, the culture medium was replaced every 3 days, and peptide-1 or peptide-2 was treated at different concentrations. After 7 days, after suctioning the culture medium, 60 μL of 3.7% formaldehyde was added to the 96-well plate for staining and fixed for 1 minute. After suctioning 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 added. After incubation at 37°C for 24 hours, the staining solution was sucked, washed and dried with three distilled water, and observed under a microscope. On the other hand, a test group to which the peptide was not added was used as a negative control group, and a test group to which TGF-β was added was used as a positive control group.

[0091] The results, such as Figure 3 and Figure 4 As shown, it was found that both peptide-1 and peptide-2 could promote the production of glycosaminoglycans.

[0092] Example 4: Confirmation of increased mRNA expression of ECM components

[0093] The aim is to confirm whether the treatment with peptide-1 or peptide-2 increases the mRNA expression of ECM components in human chondrocytes.

[0094] Specifically, after inoculating human chondrocytes into a 6-well plate at a density of 8.9×10 4 cells / well, they were cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, the medium was replaced with fresh medium, and 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 1 day, 3 days, and 7 days, the medium was aspirated, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit and a PCR premix (Intron, Korea), PCR was performed using the hCOL2A1, COMP, hCOL11A, ACAN, and GAPDH primers shown in Table 2 below. On the other hand, an experimental group supplemented with TGF-β was used as a positive control group. In Table 2 below, hCOL2A1 encodes α1 of type II collagen, hCOMP encodes cartilage oligomeric matrix protein, hCOL11A encodes the α-chain of type XI collagen, hPCP encodes proteoglycan core protein, hACAN encodes aggrecan, and hGAPDH encodes glyceraldehyde 3-phosphate dehydrogenase.

[0095] [Table 2]

[0096]

[0097] As a result, as Figure 5 and Figure 6 shown, it was found that both peptide-1 and peptide-2 could induce the production of ACAN, COL2A1, COMP, and COL11A, which are ECM components.

[0098] Example 5. Confirm the effect of inducing mRNA expression of SOX9, an ECM regulator

[0099] The aim is to confirm whether the treatment with peptide-1 or peptide-2 increases the mRNA expression of SOX9, an ECM regulator, in human chondrocytes.

[0100] Specifically, after inoculating human chondrocytes into a 6-well plate at a density of 8.9×10 4The cells were seeded at a density of cells / well in a 6-well plate and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was replaced with fresh medium, and the cells were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and the cells were treated with peptide-1 or peptide-2 at different concentrations. After 1 day, 3 days, and 7 days, the medium was aspirated, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit and a PCR premix (Intron, Korea), PCR was performed using the primers shown in Table 3 below. On the other hand, a test group without the addition of the peptide was used as a negative control group, and a test group with the addition of TGF-β1 (20 ng / ml) was used as a positive control group. In Table 3 below, SOX9 refers to (sex determining region Y)-box 9.

[0101] [Table 3]

[0102]

[0103] As a result, as shown in Figure 7 and Figure 8 , it was found that both peptide-1 and peptide-2 could promote the production of SOX9, an ECM regulator.

[0104] Example 6. Confirmation of the effect of inducing the expression of SOX5, SOX6, and SOX9 as ECM regulators

[0105] The aim was to confirm whether the treatment with peptide-1 or peptide-2 would increase the expression of SOX5, SOX6, and SOX9 proteins, which are ECM regulators, in human chondrocytes.

[0106] Specifically, human chondrocytes were seeded at 8.9×10 4The cells were seeded at a density of [number] cells / well in a 6-well plate and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was changed to fresh medium, and the cells were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and the cells were treated with peptide-1 or peptide-2 at different concentrations. After 1 day, 3 days, and 7 days, the medium was aspirated, the cells were harvested, lysates were prepared, and western blotting was performed. On the other hand, an experimental group without the addition of the peptide was used as a negative control group, and an experimental group with the addition of TGF-β1 (20 ng / ml) was used as a positive control group. As detection antibodies, 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.

[0107] As a result, as Figure 9 and Figure 10 shown, it was found that both peptide-1 and peptide-2 could increase the expression of SOX5, SOX6, and SOX9, which are ECM regulators.

[0108] Example 7. Confirmation of the effect of inhibiting the expression of COL10A1 and Runx2 as hypertrophic markers

[0109] The aim was to confirm whether the treatment with peptide-2 could increase the mRNA expression of COL10A1 and Runx2, which are hypertrophic markers, in human chondrocytes.

[0110] Specifically, human chondrocytes were seeded at a density of 8.9×10 4 cells / well in a 6-well plate and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, the medium was changed to fresh medium, and the cells were treated with peptide-2 at different concentrations. After 3 days, the medium was aspirated, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit and a PCR premix (Intron, Korea), PCR was performed using the primers shown in Table 4 below. On the other hand, an experimental group without the addition of the peptide was used as a negative control group, and an experimental group with the addition of TGF-β1 (20 ng / ml) was used as a positive control group. In Table 4 below, α1 encodes type X collagen, and Runx2 encodes runt-related transcription factor-2.

[0111] [Table 4]

[0112]

[0113]

[0114] As a result, as Figure 11 shown, it was found that Peptide-2 inhibited the expression of COL10A1 and Runx2, which are hypertrophic markers, respectively.

[0115] Example 8. Verification of Efficacy in Three-Dimensionally Cultured Human Chondrocytes

[0116] To verify the biological efficacy of Peptide-2 again, three-dimensionally cultured human chondrocytes using the micromass culture method were used as the subject.

[0117] Specifically, human chondrocytes were inoculated into a 48-well plate at a density of 2×10 5 cells / 25 μl / well and in the form of droplets, and then cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, as described above, for the three-dimensionally cultured human chondrocytes, the production of glycosaminoglycan was confirmed in the same manner as in Example 3, the production of mRNA of ACAN, COL2A1, COMP, and COL11A was confirmed in the same manner as in Example 4, and the expression of SOX9 protein was confirmed in the same manner as in Example 6.

[0118] As a result, as Figures 12 to 14 shown, the same as the test results, it was found that Peptide-2 could also promote the production of glycosaminoglycan in three-dimensionally cultured human chondrocytes, induce the production of ACAN, COL2A1, COMP, and COL11A as ECM components, and increase the expression of SOX9 protein as an ECM regulator.

[0119] Example 9. Confirmation of Cytotoxicity

[0120] The aim was 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) using SRB staining and CCK-8 assays, respectively.

[0121] Specifically, AD-MSC cells were seeded at 1.5×10 3After seeding cells at a density of [number of cells] per well in a 96-well plate, they were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS for 24 hours. Then, the medium was changed to DMEM supplemented with 5% FBS, and 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 CCK-8 activity, after 7 days, CCK-8 (Dojindo, CCK-8 kit) solution was added at one-tenth of the culture medium volume and incubated for 2 hours. The culture medium was sampled, and the CCK-8 activity was confirmed using a microplate reader at a wavelength of 450 nm. Additionally, for SRB staining, after sucking the medium from the plate, 60 μL of 3.7% formaldehyde was added to the 96-well plate for staining and fixed for 1 minute. After sucking the 3.7% formaldehyde, 70 μL of SRB staining solution (sulforhodamine B sodium salt (sigma, S9012): 0.2 g in 100 mL of DDW) was added for staining. The plate was covered with aluminum foil to block light and incubated overnight at room temperature. After washing with 100 μL of 1% acetic acid using a multi-channel pipette and drying, it was observed under a microscope.

[0122] As a result, as Figure 15 and Figure 16 shown, it was found that even when treated in human adipose mesenchymal stem cells, neither peptide-1 nor peptide-2 showed any change in cell morphology in SRB staining and did not show any toxicity to human adipose mesenchymal stem cells.

[0123] Example 10. Confirmation of the production effect of glycosaminoglycan

[0124] The purpose is to confirm the effect of increasing the production of glycosaminoglycan by adding peptide-1 or peptide-2 in human adipose mesenchymal stem cells, so as to confirm the effect of the peptide on inducing chondrogenesis and promoting the production of extracellular matrix (ECM).

[0125] Specifically, AD-MSC cells were seeded at a density of 1.5×10 3After inoculating the cells at a density of [cells / well] into a 96-well plate, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was changed to DMEM medium supplemented with 5% FBS, and 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 14 days, after sucking out the medium, 60 μL of 3.7% formaldehyde was added to the 96-well plate for staining and fixed for 1 minute. After sucking out 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 added in aliquots. After incubating at 37 °C for 24 hours, the staining solution was sucked out, washed three times with distilled water and dried, and observed under a microscope. On the other hand, an experimental group supplemented with 100 nM dexamethasone, 50 μM ascorbic acid, 40 μM proline, 10 ng / ml TGFβ1 and 1X ITS was used as a positive control group (CM).

[0126] As a result, as Figure 17 and Figure 18 shown, it was found that both peptide-1 and peptide-2 could increase the production of glycosaminoglycans, thereby inducing cartilage formation and promoting the production of extracellular matrix.

[0127] Example 11. Confirming an increase in mRNA expression of ECM components

[0128] The aim was to confirm whether the treatment with peptide-1 or peptide-2 would increase the mRNA expression of ECM components in human adipose mesenchymal stem cells.

[0129] Specifically, after inoculating AD-MSC cells at a density of 1.5×10 3 cells / well into a 96-well plate, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was changed to DMEM medium supplemented with 5% FBS, and 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 3 days, 7 days and 14 days, the medium was sucked out, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit and a PCR premix (Intron, Korea), PCR was performed using the hCOL2A1, hCOMP, hCOL 11A, ACAN and hPCP primers shown in Table 5 below.

[0130] [Table 5]

[0131]

[0132]

[0133] As a result, as Figure 19 and Figure 20 shown, it was found that both peptide-1 and peptide-2 could induce the production of COL2A1, COMP, COL 11A, ACAN, and hPCP, which are components of the ECM.

[0134] Example 12. Confirmation of the effect of inducing mRNA expression of SOX9 as an ECM regulator

[0135] The aim was to confirm whether the treatment with peptide-1 or peptide-2 would increase the mRNA expression of SOX9, which is an ECM regulator, in human adipose mesenchymal stem cells.

[0136] 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, the medium was changed to DMEM medium supplemented with 5% FBS, and 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 3 days, 7 days, and 14 days, the medium was aspirated, the cells were harvested, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit (synthesis kit) and a PCR premix (pre-mix) (Intron, Korea), PCR was performed using the primers shown in Table 3 above.

[0137] As a result, as Figure 21 and Figure 22 shown, it was found that both peptide-1 and peptide-2 could promote the production of SOX9, which is an ECM regulator.

[0138] Example 13. Confirmation of the effect of inducing the expression of SOX5, SOX6, and SOX9 as ECM regulators

[0139] The aim was to confirm whether the treatment with peptide-1 or peptide-2 would increase the expression of SOX5, SOX6, and SOX9, which are ECM regulators, in human adipose mesenchymal stem cells.

[0140] Specifically, after inoculating AD-MSC cells into a 96-well plate at a density of 1.5×10 3After seeding cells at a density of cells / well in a 96-well plate, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was changed to DMEM medium supplemented with 5% FBS, and the cells were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and the cells were treated with peptide-1 or peptide-2 at different concentrations. After 3 days, 7 days, and 14 days, the medium was aspirated, the cells were harvested, lysates were prepared, and Western blotting was performed. As the detection antibodies, 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.

[0141] As a result, as Figure 23 and Figure 24 shown, it was found that both peptide-1 and peptide-2 could increase the expression of SOX5, SOX6, and SOX9, which are ECM regulators.

[0142] Example 14. Confirmation of the effect of COL2A1 induction expression

[0143] The aim was to confirm whether the treatment with peptide-1 or peptide-2 would increase the expression of COL2A1, which is a cartilage component, in human adipose mesenchymal stem cells.

[0144] Specifically, after seeding 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, the medium was changed to DMEM medium supplemented with 5% FBS, and the cells were treated with peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and the cells were treated with peptide-1 or peptide-2 at different concentrations. After 3 days, 7 days, and 14 days, the medium was aspirated, the cells were harvested, lysates were prepared, and Western blotting was performed. As the detection antibody, sc-518017 (Santa Cruz, USA) was used for COL2A1.

[0145] As a result, as Figure 25 and Figure 26 shown, it was found that both peptide-1 and peptide-2 could increase the expression of COL2A1.

[0146] Based on the above test results, it was found that both peptide-1 and peptide-2 according to one embodiment have the effect of inducing cartilage regeneration.

[0147] Formulation Example 1. Preparation of peptide nanobodies

[0148] Dissolve 50 mg of the peptide of Example 1 in 500 ml of distilled water with sufficient stirring. 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, adjust the volume with distilled water until the total volume is 1 L, and then use a high-pressure microfluidizer to emulsify at high pressure to prepare peptide nanoparticles with a size of about 100 nm.

[0149] Formulation Example 2, Pharmaceutical Preparation

[0150] 2-1. Preparation of Powder

[0151] Mix the following components and fill them into a sealed bag to prepare a powder.

[0152] 20 mg of the peptide of the present invention

[0153] 100 mg of lactose

[0154] 10 mg of talc

[0155] 2-2. Preparation of Lozenge

[0156] Mix the following components and then press them into lozenges according to the usual method for preparing lozenges.

[0157] 10 mg of the peptide of the present invention

[0158] 100 mg of corn starch

[0159] 100 mg of lactose

[0160] 2 mg of magnesium stearate

[0161] 2-3. Preparation of Capsule

[0162] According to the usual method for preparing capsules, mix the following components and fill them into gelatin capsules to prepare capsules.

[0163] 10 mg of the peptide of the present invention

[0164] 3 mg of crystalline cellulose

[0165] 14.8 mg of lactose

[0166] 0.2 mg of magnesium stearate

[0167] 2-4. Preparation of Injection

[0168] According to the usual method for preparing injections, each ampoule (2 ml) is prepared with the following component contents.

[0169] 10 mg of the peptide of the present invention

[0170] 180 mg of mannitol

[0171] 2974 mg of distilled water for injection, sterilized

[0172] 26 mg of Na2HPO4·2H2O

[0173] 2 - 5. Preparation of the liquid preparation

[0174] According to the usual method for preparing a liquid preparation, each component is added and dissolved in pure water, and the following components are mixed. Pure water is added to adjust the total volume to 100 ml, and then it is filled into a brown bottle and sterilized to prepare the liquid preparation.

[0175] 10 mg of the peptide of the present invention

[0176] 10 g of isomerized sugar

[0177] 5 g of mannitol

[0178] An appropriate amount of pure water

[0179] 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 and not restrictive.

Claims

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

2.

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 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 characteristics: (a) Inducing the production of glycosaminoglycan; (b) Inducing the production of COL2A1, COMP, COL11A, PCP, or aggrecan; and (c) Inducing the production of SOX5, SOX6, or SOX9 as a regulatory factor.

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 pharmaceutical 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 diseases are selected from at least one of 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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