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 Glu (E)-Asp (D)-Asp (D), 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 represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D).

[0008] Another aspect provides a composition for cartilage regeneration, comprising a peptide having an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) 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 effective ingredient.

[0010] Other objects and advantages of the present application will become more apparent in conjunction with the attached claims and drawings as well as the following detailed description. Any content not described in this specification is omitted as long as it is fully understood and inferred by ordinary technicians in the field to which this application belongs or ordinary technicians in similar technical fields.

[0011] Technical Solution

[0012] The various descriptions and embodiments disclosed in this application are also applicable 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 is not intended that the scope of this application be limited to the specific descriptions described below.

[0013] In one aspect, a peptide is provided, which consists of the amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D).

[0014] As used herein, the term "peptide" may refer to a linear molecule formed by multiple amino acid residues bound together by peptide bonds. The peptide can be prepared by chemical synthesis methods known in the art, particularly solid-phase synthesis or liquid-phase synthesis (US Patent No. 5,516,891). The present inventors have diligently developed peptides with biologically effective activity and successfully identified a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D). The biologically effective activity may include at least one of the following properties: (a) induction of glycosaminoglycan production; (b) induction of production of COL2A1, cartilage oligomeric matrix protein (COMP), COL11A, proteoglycan core protein (PCP), or aggrecan; and (c) induction of production of regulatory factors SOX5, SOX6, or SOX9. Therefore, the peptide can be used for cartilage regeneration.

[0015] In the peptide, in order to obtain chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a wide range of biological activity), and reduced antigenicity, a protecting group may be bound to the N- or C-terminus 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 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). Furthermore, the peptide may optionally further include a targeting sequence, a tag, a labeled residue, or an amino acid sequence prepared for the specific purpose of increasing the half-life or stability of the peptide.

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

[0017] The term "stability" used in the present specification may refer not only to in vivo stability for protecting the peptide from attacks by proteolytic enzymes in the body but also to storage stability (eg, storage stability at room temperature).

[0018] Another aspect provides a composition for cartilage regeneration, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) as an effective ingredient.

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

[0020] The term "cartilage regeneration" used in this specification 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 alleviate the condition or at least reduce treatment-related parameters such as symptom severity.

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

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

[0023] According to one embodiment, the peptide can cause a significant increase in the expression of glycosaminoglycans, COL2A1, COMP, COL11A, PCP, aggrecan, regulatory factors SOX5, SOX6 or SOX9, which are cartilage-related substances, and thus the peptide can be used as an active ingredient in a composition for cartilage regeneration (Orthop Res Rev. 2010 Sep 01; 2010(2):85-94.doi:10.2147 / ORR.S7194, JOSPT Volume 28 Number 4 October 1998).

[0024] In another aspect, a pharmaceutical composition for preventing or treating cartilage diseases is provided, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) as an active ingredient.

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

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

[0027] In this specification, the term "treatment" refers to any form of treatment that provides an effect on an individual who already has a disease or is likely to develop a disease, including improvement in the individual's condition (e.g., one or more symptoms), delay in disease progression, delay in the onset of symptoms, or slowing down of symptom progression. Therefore, the terms "treatment" and "prevention" are not intended to mean that symptoms are cured or completely eliminated.

[0028] The "subject" refers to a target in need of disease treatment, and more specifically, refers to a human or non-human primate, a mammal 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 can be selected from at least one of the group consisting of cartilage damage, cartilage defects, degenerative disc disease, disc herniation, degenerative arthritis, bone fractures, muscle tissue damage, joint damage due to nonunion or trauma, osteomalacia, and chondromalacia.

[0030] The cartilage disease may occur in the jaw joint, shoulder joint, elbow joint, wrist joint, finger joint, spinal joint, hip joint, knee joint, ankle joint or toe joint.

[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 effect of the pharmaceutical composition.

[0033] The weight ratio between the peptide and the pharmaceutically acceptable carrier can be, for example, 500:1 to 1:500, for example, the weight ratio can 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 when preparing a preparation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, hydroxybenzoate, 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 ingredients, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., but is not limited thereto.

[0036] The pharmaceutical composition can be administered parenterally 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), 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 can be administered differently depending on factors such as the formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, administration route, excretion rate and reaction sensitivity.

[0038] The pharmaceutical composition can be formulated using pharmaceutically acceptable carriers and / or excipients according to methods readily implemented by those skilled in the art to which the present invention pertains to, to prepare the pharmaceutical composition in a unit volume form, or can be injected into a multi-volume container for preparation.

[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, papule, patch, paste, extract, powder, granule, tablet or capsule, and can further include a powder and / or a stabilizer.

[0040] The peptide can be included in a nanobody or nanoparticle to further improve the skin penetration problem or stability problem. For example, the nanobody can be prepared using lecithin as a raw material using a microfluidizer, and can be included in lecithin particles. As the preparation method of the nanobody, any known method can be used. The size of the nanobody particles is preferably 30 to 200 nm. When the size of the nanobody particles is less than 30 nm, skin penetration may be very fast and cause skin side effects to occur. When the size of the nanobody particles is greater than 200 nm, it may not be easy to penetrate into the skin and it is difficult to obtain the effect of the use of the nanobody.

[0041] Another aspect provides a method for preventing or treating cartilage diseases, comprising administering to an individual a pharmaceutical composition comprising a therapeutically effective amount of a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) as an active ingredient.

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

[0043] The terms "apply," "apply," and "coating" used herein may be used interchangeably and may refer to localizing the composition according to an embodiment at least partially on a desired site, or placing the composition according to an embodiment within an individual via an administration route.

[0044] Another aspect provides a cosmetic composition comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) as an active ingredient.

[0045] Among the terms or elements mentioned in the description of the peptide, composition, etc., those that are the same as those 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] Another aspect provides a method for regenerating cartilage, comprising administering a composition comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) as an active ingredient to an individual.

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

[0049] Beneficial effects

[0050] The peptide according to one aspect exhibits an excellent cartilage regeneration effect by causing a significant increase in various cartilage components such as glycosaminoglycans, collagen, COMP, PCP, and aggrecan.

[0051] According to one aspect, the peptide causes a significant increase in various cartilage components such as glycosaminoglycans, collagen, COMP, PCP, aggrecan, and thus can be used to prevent or treat cartilage diseases and promote cartilage regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The results are shown for confirming CCK-8 activity after treating C28 / I2 cells with peptide-1.

[0053] Figure 2 The graph shows the results of confirming CCK-8 activity after treating C28 / I2 cells with peptide-2.

[0054] Figure 3 The figures show the results of confirming CCK-8 activity after treating human adipose-derived mesenchymal stem cells (AD-MSCs) with peptide-1.

[0055] Figure 4 The results show that treatment of C28 / I2 cells with peptide-1 confirmed an increase in glycosaminoglycan production.

[0056] Figure 5 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in glycosaminoglycan production.

[0057] Figure 6 Treatment of C28 / I2 cells with peptide-1 shows the results of confirming an increase in the mRNA expression of ECM components after peptide-1 treatment.

[0058] Figure 7 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in the mRNA expression of ECM components.

[0059] Figure 8 The results show that treatment of AD-MSC cells with peptide-1 confirmed an increase in the mRNA expression of ECM components.

[0060] Figure 9 The results show that treatment of C28 / I2 cells with peptide-1 confirmed increased production of the ECM regulatory factor SOX9.

[0061] Figure 10 The results show that treatment of C28 / I2 cells with peptide-2 confirmed increased production of the ECM regulatory factor SOX9.

[0062] Figure 11 Treatment of AD-MSC cells with peptide-1 shows the results of confirming that the production of the ECM regulatory factor SOX9 is increased after peptide-1 treatment.

[0063] Figure 12 The results show that treatment of C28 / I2 cells with peptide-1 confirmed increased expression of ECM regulatory factors SOX5, SOX6, and SOX9.

[0064] Figure 13 The results show that treatment of C28 / I2 cells with peptide-2 confirmed increased expression of SOX5, SOX6, and SOX9, which are ECM regulatory factors.

[0065] Figure 14 The results show that treatment of AD-MSC cells with peptide-1 confirmed increased expression of ECM regulatory factors SOX5, SOX6, and SOX9.

[0066] Figure 15 The results show that treatment of C28 / I2 cells with peptide-1 confirmed increased expression of aggrecan and COL2A1, which are cartilage components.

[0067] Figure 16 The results show that treatment of C28 / I2 cells with peptide-2 confirmed increased expression of aggrecan and COL2A1, both cartilage components.

[0068] Figure 17 The results show that treatment of AD-MSC cells with peptide-1 confirmed increased expression of the cartilage component COL2A1.

[0069] Figure 18 The results are shown for confirming changes in cell morphology by sulforhodamine B (SRB) staining after AD-MSC cells were treated with peptide-1. DETAILED DESCRIPTION

[0070] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are only used to exemplify the present invention, and the scope of the present invention is not limited to these examples.

[0071] Example 1. Synthesis of peptide

[0072] Peptide-1 or peptide-2 described in Table 1 below were synthesized using an automatic peptide synthesizer (Milligen 9050, Millipore, USA), and purified using C18 reverse-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 a column.

[0073] [Table 1]

[0074] name Amino acid sequence (N-terminus → C-terminus) SEQ ID NO Peptide-1 KRRRKRK 1 Peptide-2 EDD -

[0075] Example 2: Confirming whether there is cytotoxicity

[0076] The purpose was to analyze the cytotoxicity caused by the addition of peptide-1 or peptide-2 to human chondrocytes (C28 / I2, Human chondrocyte cellline) and human adipose-derived mesenchymal stem cells (AD-MSC) using CCK-8 assay.

[0077] Specifically, human chondrocytes were cultured at a rate of 3 × 10 3 The density of cells / well was seeded in a 96-well plate and cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, after the culture medium was replaced with a new culture medium, peptide-1 or peptide-2 was treated at different concentrations. Then, after 3 days, CCK-8 (Dojindo, CCK-8 kit) 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.

[0078] In order to further confirm the toxicity of peptide-1, AD-MSC cells were cultured at 1.5×10 3 The cells were seeded in a 96-well plate at a density of 100 μg / well and cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS for 24 hours. Then, the culture medium was replaced with DMEM supplemented with 5% FBS and treated with peptide-1 at different concentrations. Next, the culture medium was replaced every 3 days and treated with peptide-1 at different concentrations. After 7 days, CCK-8 (Dojindo, CCK-8 kit) 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.

[0079] As a result, we can see that Figures 1 to 2 As shown in Figure 2, peptide-1 and peptide-2 each showed no toxicity in human chondrocytes. Figure 3 As shown, peptide-1 also showed no toxicity in human adipose-derived mesenchymal stem cells.

[0080] Example 3: Confirmation of glycosaminoglycan production effect

[0081] The purpose was to confirm the effect of increasing glycosaminoglycan production in human chondrocytes by adding peptide-1 or peptide-2, respectively, and to confirm the effects of the peptides in inducing chondrogenesis and promoting extracellular matrix (ECM) production.

[0082] Specifically, human chondrocytes were cultured at a rate of 3 × 10 3 The density of cells / well was seeded in 96-well plates and cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, after replacing with new culture medium, peptide-1 or peptide-2 was treated at different concentrations. Then, the culture medium was replaced every 3 days, and peptide-1 or peptide-2 was treated according to the concentration. After 7 days, 60 μL of 3.7% formaldehyde solution was added to the 96-well plate for staining and fixed for 1 minute after suctioning the culture medium. After suctioning the 3.7% formaldehyde solution, 70 μL of new blue staining solution (3% acetic acid 50 mL + 1% new blue 8GX 0.5 g, pH 2.5) was added. After incubation at 37°C for 24 hours, the staining solution was sucked and washed with three distilled water, dried and observed under a microscope. The group to which 20 ng / ml of TGFβ1 was added was used as the positive control group.

[0083] As a result, we can see that Figure 4 and Figure 5 As shown, peptide-1 and peptide-2 each promoted the production of glycosaminoglycans in human chondrocytes.

[0084] Example 4: Confirmation of increased mRNA expression of ECM components

[0085] The purpose was to determine whether treatment of human chondrocytes and human adipose-derived mesenchymal stem cells with peptide-1 or peptide-2, respectively, would increase the mRNA expression of ECM components.

[0086] Specifically, human chondrocytes were cultured at a rate of 8.9×10 4After the density of cells / well was seeded in a 6-well plate, it was cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, after the culture medium was replaced with a new culture medium, peptide-1 or peptide-2 was treated at different concentrations. Then, the culture medium was replaced every 3 days, and peptide-1 or peptide-2 was treated at different concentrations. After aspirating the culture medium after 1 day, 3 days, and 7 days, the cells were recovered and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, South Korea), the hCOL2A1, hCOMP, hCOL11A, hACAN and GAPDH primers shown in Table 2 below were used to perform polymerase chain reaction (PCR).

[0087] In order to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5×10 3 After the cells were seeded in a 96-well plate at a density of 100 μg / well, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the culture medium was replaced with DMEM medium supplemented with 5% FBS and peptide-1 was treated at different concentrations. Next, the culture medium was changed every 3 days and peptide-1 was treated at different concentrations. After 3 days and 7 days, the culture medium was aspirated, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, South Korea), PCR was performed using the hCOL2A1, COMP, hCOL11A, 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 proteoglycan core protein, hACAN is used to encode aggrecan, and hGAPDH is used to encode glyceraldehyde-3-phosphate dehydrogenase.

[0088] [Table 2]

[0089]

[0090]

[0091] As a result, we can see that Figure 6 and Figure 7 As shown in Figure 2, peptide-1 and peptide-2 each increased the mRNA production of COL2A1, COMP, COL11A, and ACAN, which are ECMs, in human chondrocytes. Figure 8As shown, peptide-1 also led to increased mRNA production of COL2A1, COMP1, COL11A, PCP, and ACAN in human adipose-derived mesenchymal stem cells.

[0092] Example 5: Confirmation of the mRNA expression induction effect of the ECM regulatory factor SOX9

[0093] The aim was to determine whether treatment with peptide-1 or peptide-2 in human chondrocytes and human adipose-derived mesenchymal stem cells leads to increased mRNA expression of the ECM regulatory factor SOX.

[0094] Specifically, human chondrocytes were cultured at a rate of 8.9×10 4 After the density of cells / well was seeded in a 6-well plate, it was cultured in DMEM medium (cat.11995-065, Gibco) for 24 hours. Then, after replacing with a new culture medium, peptide-1 or peptide-2 was treated at different concentrations. Then, the culture medium was replaced every 3 days, and peptide-1 or peptide-2 was treated at different concentrations. After 1 day, 3 days, and 7 days, the culture medium was aspirated, the cells were recovered, and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, South Korea), PCR was performed using the primers shown in Table 3 below.

[0095] In order to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5×10 3 After the cells / well were seeded in a 96-well plate at a density of 100 μg / well, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, after replacing the DMEM medium supplemented with 5% FBS, peptide-1 was treated at different concentrations. Next, the culture medium was replaced every 3 days, and peptide-1 was treated at different concentrations. After aspirating the culture medium after 3 days and 7 days, the cells were harvested and RNA was isolated. After synthesizing cDNA using a cDNA synthesis kit & PCR pre-mix (Intron, South 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.

[0096] [Table 3]

[0097]

[0098] As a result, we can see that Figure 9 and Figure 10 As shown in Figure 2, peptide-1 and peptide-2 each induce the production of the ECM regulatory factor SOX9 in human chondrocytes. Figure 11 As shown, peptide-1 promotes the production of the regulatory factor SOX9 in human adipose-derived mesenchymal stem cells.

[0099] Example 6: Confirmation of the Expression Induction Effect of ECM Regulatory Factors SOX5, SOX6, and SOX9

[0100] The purpose was to confirm whether treatment with peptide-1 or peptide-2 in human chondrocytes and human adipose-derived mesenchymal stem cells, respectively, leads to increased expression of the ECM regulatory factors SOX5, SOX6, and SOX9.

[0101] Specifically, human chondrocytes were cultured at a rate of 8.9×10 4 Cells were seeded in 6-well plates at a density of 100 μg / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The culture medium was then replaced with fresh medium and treated with peptide-1 or peptide-2 at varying concentrations. The culture medium was then replaced every three days, and peptide-1 or peptide-2 was treated at varying concentrations. After 1, 3, and 7 days, the culture medium was aspirated, the cells were harvested, and lysates were prepared and immunoblotted.

[0102] In order to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5×10 3 After the cells were seeded in a 96-well plate at a density of 100 μg / well, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was replaced with DMEM medium supplemented with 5% FBS and peptide-1 was treated at different concentrations. Subsequently, the medium was replaced every 3 days and peptide-1 was treated at different concentrations. After aspirating the medium after 3 days and 7 days, the cells were harvested and lysates were prepared and immunoblotting was performed. 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.

[0103] As a result, we can see that Figure 12 and Figure 13 As shown in Figure 2, peptide-1 and peptide-2 each increased the expression of ECM regulatory factors SOX5, SOX6, and SOX9 in human chondrocytes. Figure 14 As shown, peptide-1 also led to increased expression of SOX6 and SOX9 in human adipose-derived mesenchymal stem cells.

[0104] Example 7: Confirmation of the effect of promoting aggrecan and COL2A1 expression

[0105] The aim was to determine whether peptide-1 or peptide-2 treatment leads to increased expression of the cartilage components aggrecan and COL2A1 in human chondrocytes and human adipose-derived mesenchymal stem cells.

[0106] Specifically, human chondrocytes were cultured at a rate of 8.9×10 4 Cells were seeded in 6-well plates at a density of 100 μg / well and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. The culture medium was then replaced with fresh medium and treated with peptide-1 or peptide-2 at varying concentrations. The culture medium was then replaced every three days, and peptide-1 or peptide-2 was treated at varying concentrations. After 1, 3, and 7 days, the culture medium was aspirated, the cells were harvested, and lysates were prepared and immunoblotted.

[0107] In order to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5×10 3 After the cells were seeded in a 96-well plate at a density of 100 μg / well, they were cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was replaced with DMEM medium supplemented with 5% FBS and peptide-1 was treated at different concentrations. Subsequently, the medium was replaced every 3 days and peptide-1 was treated at different concentrations. After aspirating the medium after 3 days, 7 days, and 14 days, the cells were harvested and lysates were prepared and immunoblotting was performed. As detection antibodies, sc-33695 (Santa Cruz, USA) was used for aggrecan and sc-518017 (Santa Cruz, USA) was used for COL2A1.

[0108] As a result, we can see that Figure 15 and Figure 16 As shown in Figure 2, peptide-1 or peptide-2 increased the expression of aggrecan and COL2A1 in human chondrocytes. Figure 17 As shown, peptide-1 also led to increased expression of COL2A1 in human adipose-derived mesenchymal stem cells.

[0109] Example 8: Confirming whether cell deformation occurs

[0110] The purpose was to analyze whether the addition of peptide-1 to human adipose-derived mesenchymal stem cells (AD-MSCs) leads to cell morphological changes by SRB staining.

[0111] Specifically, AD-MSC cells were cultured at a density of 1.5×10 3The cells / well were seeded in a 96-well plate at a density of 100 μg / well and cultured in a DMEM (Dulbecco's Modified Eagle's Medium) medium supplemented with 10% FBS for 24 hours. Then, after replacing the DMEM medium supplemented with 5% FBS, peptide-1 was treated at different concentrations. Next, the culture medium was replaced every 3 days, and peptide-1 was treated at different concentrations. 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 absorbing the 3.7% formaldehyde solution, 70 μL of SRB staining solution (sulforhodamine Bsodium salt (sigma, S9012): 0.2 g in 100 mL DDW) was added for staining. Cover the plate with silver foil and block light to incubate overnight at room temperature. Wash with 100 μL of 1% acetic acid with a multi-pipette, dry, and observe under a microscope.

[0112] As a result, we can see that Figure 18 As shown, even when human adipose-derived mesenchymal stem cells were treated with peptide-1, no cell morphology changes were observed when stained with SRB.

[0113] Based on the experimental results, it can be seen that peptide-1 and peptide-2 according to one embodiment each have the effect of inducing cartilage regeneration.

[0114] Dosage Form Example 1: Preparation of Peptide Nanobodies

[0115] 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water by thorough stirring. The complex solution was mixed 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 approximately 100 nm.

[0116] Dosage Form Example 2: Pharmaceutical Preparations

[0117] 2-1. Preparation of powder

[0118] The following ingredients were mixed and filled into a sealed bag to prepare a powder.

[0119] 20 mg of the peptide of the present invention

[0120] 100 mg lactose

[0121] 10 mg talc

[0122] 2-2. Preparation of tablets

[0123] The following ingredients are mixed and compressed into tablets according to the usual tablet preparation method.

[0124] 10 mg of the peptide of the present invention

[0125] 100 mg corn starch

[0126] 100 mg lactose

[0127] 2 mg magnesium stearate

[0128] 2-3. Preparation of capsules

[0129] The following ingredients were mixed according to a conventional capsule preparation method and filled into gelatin capsules to prepare capsules.

[0130] 10 mg of the peptide of the present invention

[0131] 3 mg crystalline cellulose

[0132] 14.8mg lactose

[0133] 0.2mg magnesium stearate

[0134] 2-4. Preparation of injection

[0135] According to the usual method for preparing injections, each ampoule (2 ml) is prepared according to the following ingredient contents.

[0136] 10 mg of the peptide of the present invention

[0137] 180 mg mannitol

[0138] 2974 mg sterile distilled for injection

[0139] 26mgNa2HPO4·2H2O

[0140] 2-5. Preparation of liquid

[0141] According to the usual liquid preparation method, each component was added and dissolved in purified water, and the following components were mixed, and purified water was added to adjust the total volume to 100 ml, which was then filled in a brown bottle and sterilized to prepare a liquid.

[0142] 10 mg of the peptide of the present invention

[0143] 10 g isomerized sugar

[0144] 5 g mannitol

[0145] Appropriate amount of purified water

[0146] The description of the present invention is intended to provide examples only. It should be understood by those skilled in the art that the present invention can be easily modified into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all aspects and not restrictive.

Claims

A peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D).

2. The peptide according to claim 1, wherein The N-terminus of the peptide is bound to any one protecting group 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 protecting group selected from the group consisting of an amino group, a tertiary alkyl group, and an azide group.

4. The peptide according to claim 1, wherein The peptide exhibits at least one property selected from the following properties: (a) Inducing glycosaminoglycan production; (b) inducing COL2A1, COMP, COL11A, PCP, or aggrecan production; and (c) Induction of SOX5, SOX6, or SOX9 production. 5 . A composition for cartilage regeneration, comprising the peptide according to claim 1 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. The composition according to claim 6 , further comprising a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 6, wherein The peptides are prepared in the form of nanobodies.

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 due to nonunion of fracture or trauma, osteomalacia and chondromalacia.

Images