Peptides for cartilage regeneration and uses thereof

Abstract

The present application relates to a peptide for cartilage regeneration and use thereof, and provides a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D), a composition for cartilage regeneration including the same, and a pharmaceutical composition for preventing or treating cartilage disease, including the composition for cartilage regeneration as an effective ingredient.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202280102443.4 entitled “Peptides for Cartilage Regeneration and Use Thereof”, filed on December 26, 2022. Technical Field

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

[0003] Due to the nature of cartilage tissue, it is difficult for large-area damage to heal and regenerate naturally. Therefore, surgical treatments such as artificial joints, articular chondrotomy, and microfracture surgery are used. However, existing methods often leave scars due to incisions and result in low durability of fibrocartilage regeneration, thus leading to lower treatment outcomes compared to the difficulty of the procedures.

[0004] Therefore, injectable solutions or cartilage tissue recovery compositions using hydrogels and collagen, etc., have been developed for intra-articular application, with simple surgical procedures and rapid therapeutic effects (Korean Patent Publication No. 2013-0028012). However, while these methods can temporarily relieve pain, they are insufficient to induce cartilage tissue regeneration.

[0005] In addition, various treatment methods using autologous chondrocytes or stem cells have been developed in cell-based therapies, which involve inducing cartilage tissue regeneration by transplanting in vitro cultured cells to the defect site (Korean Patent Publication No. 2013-0072983). However, in autologous chondrocyte therapy, the use of cultured cells collected from the patient alone is limited when the damaged area is large. Furthermore, stem cell therapy has the following problems: cell numbers and differentiation capacity vary depending on the collection site; cell dedifferentiation during in vitro culture leads to changes in cell phenotype; the rate of differentiation into chondrocytes after transplantation is low; and gene expression related to cell hypertrophy induces apoptosis and vascular permeability, leading to chondrocyte calcification.

[0006] In this technological context, there is a need to develop effective factors that can more effectively treat cartilage diseases by promoting cartilage differentiation or cartilage formation in stem cells or chondrocytes, but these are still not perfect. Summary of the Invention

[0007] Technical issues

[0008] One aspect provides a peptide composed of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D).

[0009] On the other hand, a composition for cartilage regeneration is provided, comprising a peptide of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) as an active ingredient.

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

[0011] Other objects and advantages of this application will become clearer in conjunction with the appended claims and drawings, as well as the detailed description below. Regarding matters not described in this specification, which can be fully understood and deduced by those skilled in the art or similar fields, their descriptions are omitted.

[0012] Technical solution

[0013] The various descriptions and embodiments disclosed in this application can also be applied to various other descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, it cannot be determined that the scope of this application is limited to the specific descriptions set forth below.

[0014] One aspect provides a peptide composed of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D).

[0015] As used in this specification, the term "peptide" can refer to a linear molecule formed by multiple amino acid residues linked together by peptide bonds. The peptide can be prepared using chemical synthesis methods known in the art, particularly solid-phase or liquid-phase synthesis techniques (US Patent No. 5,516,891). Through diligent research, the inventors have successfully identified peptides with biologically effective activity, consisting of amino acid sequences represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D). Here, the biologically effective activity can refer to at least one of the following properties: (a) induction of glycosaminoglycan formation; (b) induction of COL2A1, cartilage oligomeric matrix protein (COMP), COL11A, proteoglycan core protein (PCP), or aggrecan; and (c) induction of regulatory factors SOX5, SOX6, or SOX9. Therefore, the peptide can be used for cartilage regeneration.

[0016] In the peptide, a protecting group may be bound to the N- or C-terminus to obtain chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad range of biological activities), and reduced antigenicity. In one embodiment, the N-terminus of the peptide may be bound to any protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, butoxycarbonyl, allyloxycarbonyl, and polyethylene glycol (PEG); and / or the C-terminus of the peptide may be bound to any protecting group selected from the group consisting of amino (-NH2), tertiary alkyl, and azide (-NHNH2). Furthermore, the peptide may optionally include a targeting sequence, a tag, labeled residues, or an amino acid sequence prepared for a specific purpose of increasing half-life or peptide stability.

[0017] The peptide is artificially synthesized or non-naturally occurring or engineered. "Non-naturally occurring or engineered" refers to a state generated through artificial modification, rather than its inherent state as a naturally occurring entity. Here, artificial modification may include the artificial synthesis of an amino acid sequence by mimicking the structure of multiple amino acids, or may include the above-mentioned engineering to obtain chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0018] The term "stability" as used in this specification may refer not only to the in vivo stability of the peptide protected from attack by proteolytic enzymes in vivo, but also to storage stability (e.g., room temperature storage stability).

[0019] Another aspect provides a composition for cartilage regeneration comprising a peptide as an active ingredient, consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D).

[0020] In the description of the peptide, the terms or elements that are the same as those that have been mentioned before are as described above.

[0021] As used in this specification, the term "cartilage regeneration" can refer to improving cartilage tissue by repairing damaged cartilage tissue or inducing the generation of defective cartilage tissue. "Improvement" can refer to the alleviation of the condition or at least a reduction in all behaviors related to treatment-related parameters such as symptom severity.

[0022] The cartilage includes, but is not limited to, hyaline cartilage, fibrocartilage, or elastic cartilage. For example, the cartilage may be at least one selected from the group consisting of articular cartilage, ear cartilage, nasal cartilage, elbow cartilage, meniscus cartilage, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage, and spinal cartilage.

[0023] The drawback of existing functional peptides is that, even if they possess effective biological activity, they cannot be effectively delivered to target tissues or cells due to their size, or they disappear from the body quickly due to their short half-life. On the other hand, a composition for cartilage regeneration according to one embodiment includes a peptide composed of 10 or fewer amino acids as the active ingredient. Therefore, the skin penetration of the active ingredient is excellent, and for example, when applied topically, an effective cartilage regeneration effect can be obtained.

[0024] According to one embodiment, the peptide can lead to a significant increase in the expression of glycosaminoglycans, COL2A1, COMP, COL11A, PCP, aggregate proteoglycans, regulators SOX5, SOX6 or SOX9, which are cartilage-related substances, and therefore the peptide can be used as an active ingredient in a cartilage regeneration composition (Orthop Res Rev. 2010 Sep 1; 2010(2): 85-94. doi: 10.2147 / ORR.S7194, JOSPT Volume 28 Number 4 October 1998).

[0025] On the other hand, a pharmaceutical composition for the prevention or treatment of 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.

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

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

[0028] In this specification, the term "treatment" refers to any form of treatment that provides an effect to an individual who has a disease or is at risk of developing a disease, including improvement of the individual's condition (e.g., one or more symptoms), delay of disease progression, delay of symptom onset, or slowing of symptom progression. Therefore, "treatment" and "prevention" are not intended to represent a cure or complete elimination of symptoms.

[0029] The term "individual" refers to a target for treatment of a disease, and more specifically, to humans or non-human primates, mammals such as mice, dogs, cats, horses, and cattle.

[0030] In this specification, the term "cartilage disease" refers to all cartilage-related diseases that require cartilage differentiation or regeneration. The cartilage disease may be selected from at least one of the following groups: cartilage lesions, cartilage defects, degenerative disc diseases, herniated discs, degenerative arthritis, fractures, muscle tissue injuries, joint injuries due to nonunion of fractures or trauma, osteomalacia, and chondromalacia.

[0031] The cartilage disease can occur in the temporomandibular joint, shoulder joint, elbow joint, wrist joint, finger joint, spinal joint, hip joint, knee joint, ankle joint, or toe joint.

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

[0033] The term "pharmaceuticalally effective amount" as used in this specification refers to an amount sufficient to achieve the cartilage regeneration effect of the pharmaceutical composition.

[0034] The weight ratio between the peptide and the pharmaceutically acceptable carrier can be, for example, 500:1 to 1:500, such as 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2, but is not limited thereto.

[0035] Pharmaceutically acceptable carriers are those commonly used in the preparation of formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are detailed in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0036] In addition to the ingredients mentioned above, the pharmaceutical composition may further include, but is not limited to, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc.

[0037] The pharmaceutical composition can be administered via the gastrointestinal tract or parenterally, preferably via parenteral administration. For parenteral administration, it can be administered via intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local application, percutaneous application, etc., but is not limited thereto.

[0038] The dosage of the pharmaceutical composition may be 0.0001 to 1000 μg (micrograms), 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg per day, but is not limited thereto, and may vary depending on factors such as formulation method, administration method, patient's age, weight, gender, symptoms, diet, administration time, route of administration, excretion rate, and reaction sensitivity.

[0039] The pharmaceutical composition can be formulated in a unit volume form using pharmaceutically acceptable carriers and / or excipients according to methods readily practiced by those skilled in the art to which this invention pertains, or can be prepared by injection into multi-volume containers.

[0040] The dosage form may be a solution, suspension or emulsion in an oily or aqueous solvent, or may be an ointment, cream, gel, transdermal absorbent, poultice, patch, paste, extractant, powder, granule, lozenge or capsule, and may further include powder and / or stabilizer.

[0041] The peptides may be incorporated into nanobodies or nanoparticles to further improve skin penetration or stability. For example, the nanobodies can be prepared using lecithin as a raw material via a microfluidic apparatus and may be incorporated into lecithin particles. Any known method may be used to prepare the nanobodies. The size of the nanobodies is preferably 30 to 200 nm. If the size of the nanobodies is less than 30 nm, skin penetration may be very rapid, leading to skin side effects; if the size of the nanobodies is greater than 200 nm, penetration into the skin may be difficult, making it difficult to achieve the desired effects.

[0042] On the other hand, a method for preventing or treating cartilage diseases is provided, comprising the step of administering to an individual a 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.

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

[0044] The terms “application,” “apply,” and “coat” used in this specification are used interchangeably and can refer to at least partially localizing a composition according to an embodiment onto a desired site or distributing a composition according to an embodiment into an individual by means of application.

[0045] On the other hand, a cosmetic composition is provided, comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1 or Glu(E)-Asp(D)-Asp(D) as an active ingredient.

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

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

[0048] On the other hand, a method for regenerating cartilage is provided, comprising the step of applying a 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.

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

[0050] Beneficial effects

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

[0052] Based on the fact that one type of peptide leads to a significant increase in various cartilage components such as glycosaminoglycans, collagen, COMP, PCP, and aggregated proteoglycans, it can be used to prevent or treat cartilage diseases and promote cartilage regeneration. Attached Figure Description

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

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

[0055] Figure 3 The results show the confirmation of CCK-8 activity after treatment of human adipose-derived mesenchymal stem cells (AD-MSCs) with peptide-1.

[0056] Figure 4 The results show that treatment of C28 / I2 cells with peptide-1 confirmed an increase in glycosaminoglycan production.

[0057] Figure 5 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in glycosaminoglycan production.

[0058] Figure 6 The results show that treatment of C28 / I2 cells with peptide-1 resulted in increased mRNA expression of ECM components.

[0059] Figure 7 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in the mRNA expression of ECM components.

[0060] Figure 8 The results show that treatment of AD-MSC cells with peptide-1 confirmed an increase in the mRNA expression of ECM components.

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

[0062] Figure 10 The results show that treatment of C28 / I2 cells with peptide-2 confirmed an increase in the production of the ECM regulator SOX9.

[0063] Figure 11 The results show that treatment of AD-MSC cells with peptide-1 confirmed an increase in the production of the ECM regulator SOX9.

[0064] Figure 12 The results show that treatment of C28 / I2 cells with peptide-1 confirmed the increased expression of SOX5, SOX6 and SOX9, which are ECM regulators.

[0065] Figure 13 The results show that treatment of C28 / I2 cells with peptide-2 confirmed the increased expression of SOX5, SOX6 and SOX9, which are ECM regulators.

[0066] Figure 14 The results show that treatment of AD-MSC cells with peptide-1 confirmed the increased expression of ECM regulatory factors SOX5, SOX6, and SOX9.

[0067] Figure 15 The results show that treatment of C28 / I2 cells with peptide-1 confirmed increased expression of cartilage components, namely aggregates of proteoglycans and COL2A1.

[0068] Figure 16 The results show that treatment of C28 / I2 cells with peptide-2 confirmed increased expression of cartilage component aggregates, proteoglycans, and COL2A1.

[0069] Figure 17 The results show that treatment of AD-MSC cells with peptide-1 confirmed an increase in the expression of the cartilage component COL2A1.

[0070] Figure 18 The results show the results of morphological changes in AD-MSC cells after treatment with peptide-1, confirmed by sulfonylrhodamine B (SRB) staining. Detailed Implementation

[0071] The present invention will now be described in more detail through embodiments. However, these embodiments are merely illustrative of the invention, and the scope of the invention is not limited to these embodiments.

[0072] Example 1: Peptide Synthesis

[0073] Peptide-1 or peptide-2 as described in Table 1 below were synthesized using an automated peptide synthesizer (Milligen 9050, Millipore, USA), and the synthesized peptides were purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). An ACQUITY UPLC BEH300 C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Co., USA) was used as the column.

[0074] [Table 1]

[0075] Example 2: Confirmation of cytotoxicity

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

[0077] Specifically, human chondrocytes were used at a concentration of 3 × 10⁻⁶. 3 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, after replacing the medium with fresh medium, cells were treated with either peptide-1 or peptide-2 at different concentrations. Following this, CCK-8 (Dojindo, CCK-8 kit) was added at 1 / 10 volume of the culture medium after 3 days and incubated for 2 hours. The culture medium was sampled and CCK-8 activity at 450 nm was confirmed using a microplate reader.

[0078] Furthermore, to further confirm the toxicity of peptide-1, AD-MSC cells were cultured at 1.5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS. Then, the medium was replaced with DMEM supplemented with 5% FBS, and peptide-1 was treated at different concentrations. The medium was then changed every 3 days, and peptide-1 was treated at different concentrations each time. After 7 days, CCK-8 (Dojindo, CCK-8 kit) was added to 1 / 10 volume of the culture medium and incubated for 2 hours. The culture medium was sampled, and CCK-8 activity at 450 nm was confirmed using a microplate reader.

[0079] As a result, it can be seen that... Figures 1 to 2 As shown, peptide-1 and peptide-2 did not exhibit toxicity in human chondrocytes. Furthermore, it is known that... Figure 3 As shown, peptide-1 did not exhibit toxicity in human adipose-derived mesenchymal stem cells.

[0080] Example 3: Confirmation of glycosaminoglycan formation effect

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

[0082] Specifically, human chondrocytes were used at a concentration of 3 × 10⁻⁶. 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, after changing to fresh medium, cells were treated with either peptide-1 or peptide-2 at different concentrations. The medium was then changed every 3 days, and peptide-1 or peptide-2 treatment was performed at the appropriate concentrations. After 7 days, 60 μL of 3.7% formaldehyde solution was added to the 96-well plates for staining after suturing with the 3.7% formaldehyde solution, followed by 70 μL of New Blue staining solution (50 mL of 3% acetic acid + 8 g x 0.5 g of 1% New Blue, pH 2.5). After incubation at 37°C for 24 hours, the staining solution was aspirated, washed with triple-distilled water, dried, and observed under a microscope. A group supplemented with 20 ng / mL TGFβ1 was used as a positive control.

[0083] As a result, it can be seen that... Figure 4 and Figure 5 As shown, peptide-1 and peptide-2 each promote the production of glycosaminoglycans in human chondrocytes.

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

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

[0086] Specifically, human chondrocytes were used at a concentration of 8.9 × 10⁻⁶. 4Cells were seeded at a density of 6-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, after replacing the medium with fresh medium, cells were treated with either peptide-1 or peptide-2 at different concentrations. The medium was then changed every 3 days, and cells were treated with either peptide-1 or peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was aspirated, cells were recovered, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit & PCR premix (Intron, Korea), and polymerase chain reaction (PCR) was performed using the hCOL2A1, hCOMP, hCOL11A, hACAN, and GAPDH primers shown in Table 2 below.

[0087] Furthermore, to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5 × 10⁻⁶ m². 3 Cells were seeded at a density of 96-well plates and cultured in DMEM medium supplemented with 10% FBS for 24 hours. Then, the medium was replaced with DMEM medium supplemented with 5% FBS, and peptide-1 was treated at different concentrations. The medium was then changed every 3 days, and peptide-1 was treated at different concentrations each time. After 3 and 7 days, the medium was aspirated, cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit and PCR premix (Intron, Korea), and PCR was performed using the primers hCOL2A1, COMP, hCOL11A, PCP, and ACAN shown in Table 2 below. In Table 2, hCOL2A1 encodes type II collagen α1, hCOMP encodes Cartilage oligomeric matrix protein, hCOL11A encodes the α chain of type XI collagen, hPCP encodes the proteoglycan core protein, hACAN encodes agglutinin, and hGAPDH encodes glyceraldehyde-3-phosphate dehydrogenase.

[0088] [Table 2]

[0089] As a result, it can be seen that... Figure 6 and Figure 7 As shown, peptide-1 and peptide-2 each led to increased mRNA production of COL2A1, COMP, COL11A, and ACAN, which are ECM molecules, in human chondrocytes. Furthermore, it is known that... Figure 8 As shown, peptide-1 also leads to increased mRNA production of COL2A1, COMP1, COL11A, PCP, and ACAN in human adipose-derived mesenchymal stem cells.

[0090] Example 5: Confirmation of the mRNA expression induction effect of ECM regulator SOX9

[0091] The aim was to determine whether treatment with peptide-1 or peptide-2 in human chondrocytes and human adipose-derived mesenchymal stem cells resulted in increased mRNA expression of the ECM regulator SOX.

[0092] Specifically, human chondrocytes were used at a concentration of 8.9 × 10⁻⁶. 4 Cells were seeded at a density of 6-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, after replacing the medium, cells were treated with either peptide-1 or peptide-2 at different concentrations. The medium was then changed every 3 days, and cells were treated with either peptide-1 or peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was aspirated, cells were recovered, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit & PCR premix (Intron, Korea), and PCR was performed using the primers shown in Table 3 below.

[0093] Furthermore, to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5 × 10⁻⁶ m². 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours in DMEM medium supplemented with 10% FBS. Then, the medium was replaced with DMEM medium supplemented with 5% FBS, followed by peptide-1 treatment at different concentrations. The medium was then changed every 3 days, and peptide-1 treatment at different concentrations was performed again. After 3 and 7 days, the medium was aspirated, cells were harvested, and RNA was isolated. cDNA was synthesized using a cDNA synthesis kit & PCRpre-mix (Intron, Korea), and PCR was performed using the primers shown in Table 3 below. In Table 3, SOX9 refers to the sex-determining region Y-box protein 9.

[0094] [Table 3]

[0095] As a result, it can be seen that... Figure 9 and Figure 10 As shown, peptide-1 and peptide-2 each induce the production of the ECM regulator SOX9 in human chondrocytes. Furthermore, it is known that... Figure 11 As shown, peptide-1 promotes the production of the regulatory factor SOX9 in human adipose-derived mesenchymal stem cells.

[0096] Example 6: Confirmation of the expression induction effects of ECM regulators SOX5, SOX6, and SOX9.

[0097] The aim was to determine whether treatment with peptide-1 or peptide-2 in human chondrocytes and human adipose-derived mesenchymal stem cells, respectively, led to an increase in the expression of ECM regulatory factors SOX5, SOX6, and SOX9.

[0098] Specifically, human chondrocytes were used at a concentration of 8.9 × 10⁻⁶. 4 Cells were seeded at a density of 1 / well in 6-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, after replacing the medium, cells were treated with either peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and cells were treated with either peptide-1 or peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was aspirated, cells were harvested, and lysates were prepared for Western blotting.

[0099] Furthermore, to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5 × 10⁻⁶ m². 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours in DMEM medium supplemented with 10% FBS. Then, the medium was replaced with DMEM medium supplemented with 5% FBS, and cells were treated with different concentrations of peptide-1. Subsequently, the medium was changed every 3 days, and different concentrations of peptide-1 were applied. After 3 and 7 days, the medium was aspirated, cells were harvested, and lysates were prepared for Western blotting. As detection antibodies, sc-293215 (Santa Cruz, USA) was used for SOX5, sc-393314 (Santa Cruz, USA) for SOX6, and 82630S (Cell Signaling, USA) for SOX9.

[0100] As a result, it can be seen that... Figure 12 and Figure 13 As shown, peptide-1 and peptide-2 each led to increased expression of ECM regulators SOX5, SOX6, and SOX9 in human chondrocytes. Furthermore, it can be seen that... Figure 14 As shown, peptide-1 also leads to increased expression of SOX6 and SOX9 in human adipose-derived mesenchymal stem cells.

[0101] Example 7: Confirmation of the promoting effect of agglutinin and COL2A1 expression.

[0102] The aim was to determine whether peptide-1 or peptide-2 treatment in human chondrocytes and human adipose-derived mesenchymal stem cells led to increased expression of cartilage component aggregates, proteoglycans, and COL2A1.

[0103] Specifically, human chondrocytes were used at a concentration of 8.9 × 10⁻⁶. 4Cells were seeded at a density of 1 / well in 6-well plates and cultured in DMEM medium (cat. 11995-065, Gibco) for 24 hours. Then, after replacing the medium, cells were treated with either peptide-1 or peptide-2 at different concentrations. Subsequently, the medium was changed every 3 days, and cells were treated with either peptide-1 or peptide-2 at different concentrations. After 1, 3, and 7 days, the medium was aspirated, cells were harvested, and lysates were prepared for Western blotting.

[0104] Furthermore, to further confirm the effect of peptide-1, AD-MSC cells were cultured at 1.5 × 10⁻⁶ m². 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours in DMEM medium supplemented with 10% FBS. Then, the medium was replaced with DMEM medium supplemented with 5% FBS, and cells were treated with different concentrations of peptide-1. Subsequently, the medium was changed every 3 days, and different concentrations of peptide-1 were applied. After 3, 7, and 14 days, the medium was aspirated, cells were harvested, and lysates were prepared for Western blotting. As detection antibodies, sc-33695 (Santa Cruz, USA) was used for proteoglycans, and sc-518017 (Santa Cruz, USA) was used for COL2A1.

[0105] As a result, it can be seen that... Figure 15 and Figure 16 As shown, peptide-1 or peptide-2 leads to increased expression of aggregated proteoglycans and COL2A1 in human chondrocytes. Furthermore, it is known that... Figure 17 As shown, peptide-1 also leads to increased expression of COL2A1 in human adipose-derived mesenchymal stem cells.

[0106] Example 8: Confirm whether cell deformation has occurred.

[0107] The aim was to analyze, by means of SRB staining, whether the addition of peptide-1 to human adipose-derived mesenchymal stem cells (AD-MSCs) resulted in changes in cell morphology.

[0108] Specifically, AD-MSC cells were stored at a concentration of 1.5 × 10⁻⁶. 3Cells were seeded at a density of 10% FBS in 96-well plates and cultured for 24 hours in DMEM (Dulbecco's Modified Eagle's Medium). Then, the medium was replaced with DMEM supplemented with 5% FBS, and peptide-1 was treated at different concentrations. The medium was then changed every 3 days, and peptide-1 was treated at different concentrations each time. After suction of the medium, 60 μL of 3.7% formaldehyde solution was added to the 96-well plates for staining and fixed for 1 minute. After suction of 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. The plates were covered with silver foil and incubated overnight at room temperature to block light. After washing with 100 μL of 1% acetic acid using multiple pipettes, the plates were dried and observed under a microscope.

[0109] As a result, it can be seen that... Figure 18 As shown, even after human adipose-derived mesenchymal stem cells were treated with peptide-1, no changes in cell morphology were observed during SRB staining.

[0110] Based on the experimental results, peptide-1 and peptide-2, according to one embodiment, each have the effect of inducing cartilage regeneration.

[0111] Dosage Form Example 1: Preparation of Peptide Nanobody

[0112] 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water by thorough stirring. The complex solution was then mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil phase. The mixture was adjusted with distilled water until the total volume was 1 L. The peptide nanobody with a size of approximately 100 nm was then prepared by emulsification under high pressure using a high-pressure microfluidic homogenizer.

[0113] Dosage form example 2, pharmaceutical preparation

[0114] 2-1. Preparation of Powders

[0115] Mix the following ingredients and fill them into a sealed bag to prepare a powder.

[0116] 20 mg of the peptide of the present invention

[0117] 100 mg lactose

[0118] 10 mg talc

[0119] 2-2. Preparation of Tablets

[0120] Mix the following ingredients and then compress them into tablets according to the usual tablet preparation method.

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

[0122] 100 mg corn starch

[0123] 100 mg lactose

[0124] 2 mg magnesium stearate

[0125] 2-3. Preparation of Capsules

[0126] Following standard capsule preparation methods, the following ingredients are mixed and filled into gelatin capsules to prepare capsules.

[0127] 10 mg of the peptide of the present invention

[0128] 3 mg crystalline cellulose

[0129] 14.8 mg lactose

[0130] 0.2 mg magnesium stearate

[0131] 2-4. Preparation of Injectables

[0132] Following standard injectable preparation methods, each ampoule (2 ml) is prepared according to the following ingredient content.

[0133] 10 mg of the peptide of the present invention

[0134] 180 mg mannitol

[0135] 2974 mg Sterile Distilled Injection

[0136] 26 mg Na2HPO4·2H2O

[0137] 2-5. Preparation of Liquid Formulation

[0138] Following the usual liquid preparation method, each ingredient is added and dissolved in purified water, and the following ingredients are mixed. Purified water is added to adjust the total volume to 100 ml, and then it is filled into a brown bottle, sterilized, and prepared as a liquid.

[0139] 10 mg of the peptide of the present invention

[0140] 10 g isomerized sugar

[0141] 5 g mannitol

[0142] An appropriate amount of purified water

[0143] The description of this invention is for illustrative purposes only, and those skilled in the art should understand that the invention can be readily modified into other specific forms without changing the technical concept or essential features of the invention. Therefore, the embodiments described above should be understood as exemplary in all respects, and not as limiting.

Claims

1. Use of a peptide comprising an amino acid sequence represented by Glu(E)-Asp(D)-Asp(D) in the preparation of a pharmaceutical composition for the prevention or treatment of cartilage diseases, wherein, The cartilage disease is selected from at least one of the following: cartilage defects, degenerative intervertebral disc disease, fracture, joint damage caused by trauma, and chondromalacia.

2. Use of a peptide comprising an amino acid sequence represented by Glu(E)-Asp(D)-Asp(D) in the preparation of a pharmaceutical composition for the prevention or treatment of cartilage diseases, wherein, The cartilage disease is selected from at least one of the following groups: cartilage injury, intervertebral disc herniation, and nonunion of fracture.

3. The peptide according to claim 1 or 2, 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.

4. The peptide according to claim 1 or 2, wherein, The C-terminus of the peptide is bound to a protecting group selected from the group consisting of amino, tertiary alkyl, and hydrazine groups.

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