Biocompatible cationic polymer-based injectable preparation and use thereof

Abstract

The present invention relates to: a biocompatible cationic polymer-based injectable preparation; and use for treating cartilage damage diseases, comprising same. It has been identified that the biocompatible cationic polymer-based injectable preparation of the present invention can maintain target cells in a captured state, and delivers the captured target cells to target tissues and engrafts same. Furthermore, an excellent regeneration effect of a target tissue is achieved in the engrafted tissue, and thus the biocompatible cationic polymer-based injectable preparation of the present invention can be variously utilized in the field of new cell therapeutic agents.

Description

Injectable formulation based on biocompatible cationic polymer and its use

[0001] The present invention relates to an injectable formulation based on a biocompatible cationic polymer; and a use thereof for treating cartilage damage diseases.

[0002] Conquering incurable diseases using stem cells is a crucial challenge for the life sciences of the present century, attracting attention across most medical fields, including cardiovascular, neurological, and hematological. However, treatments using stem cells alone have various clinical challenges. The most significant issues are those related to targetability and efficacy.

[0003] Research has revealed that conventional injectable treatments can fail to accurately reach the target area, and that injected cells can disperse, making it difficult for them to settle and differentiate. To address this issue of stem cell delivery efficiency, research is being conducted collaboratively across various fields to develop methods for delivering cells accurately and stably to the target area.

[0004] As part of these efforts, hybrid tissue-engineered products that combine stem cells with tissue engineering techniques are being actively researched. These tissue-engineered products involve introducing a delivery medium to stem cells. These materials are then cultured in a suitable biocompatible form, and then applied to the area requiring treatment. These tissue-engineered products can be manufactured in a variety of forms, depending on the specific disease site.

[0005] Furthermore, for stem cells to be used as a therapeutic agent to heal damaged tissue, they must be successfully delivered to the damaged tissue with minimal cell loss, and the stem cells must also maintain their differentiation potential. Furthermore, chronic diseases requiring stem cell therapy often involve not only necrosis of the damaged tissue but also damage to the surrounding blood vessels, resulting in limited blood flow. This creates an environment lacking the oxygen necessary for metabolism, making it difficult for cells to survive. Therefore, cell delivery systems for stem cell therapy must be biocompatible, easily deliver to the target damaged tissue, minimize cell loss, and maintain stem cell survival and differentiation potential. Consequently, the development of cell scaffolds to treat target diseases requires meeting the various conditions mentioned above, which has led to delays in their development.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] (Patent Document 0001) International Patent Publication No. WO2006 / 107183

[0009] (Patent Document 0002) International Patent Publication No. WO2005 / 092920

[0010] Accordingly, the present inventors prepared a cell scaffold by adding poloxamer at a concentration of 1% or less, for example, 0.7% (w / w), which is significantly lower than the previously known concentration, to coacervate (MAP-HA Coacervate) formed by mixing hyaluronic acid with mussel adhesive protein, and by this optimal composition, not only does the hydrogel not form but also maintains the properties of a liquid state, but also has shear viscosity characteristics suitable for injection, thereby providing a favorable environment for cell proliferation and spheroid formation, thereby demonstrating an excellent hyaline cartilage regeneration effect, thereby completing the present invention.

[0011] Accordingly, an object of the present invention is to provide a pre-formulation in powder form comprising (a) a first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; and (b) a second component comprising a non-ionic polymer.

[0012] Another object of the present invention is to provide an injectable formulation comprising (a) a first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; (b) a second component comprising a nonionic polymer; and (c) an aqueous solution.

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cartilage damage diseases, comprising a preparatory preparation or an injectable preparation according to the present invention.

[0014] Another object of the present invention is to provide a joint injection preparation comprising an injectable preparation according to the present invention.

[0015] Another object of the present invention is to provide a method for treating cartilage damage disease, comprising administering an injectable formulation according to the present invention to a subject in need thereof.

[0016] To achieve the above object, the present invention provides a powder-type preparatory preparation comprising (a) a first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; and (b) a second component comprising a non-ionic polymer.

[0017] The present invention also provides an injectable formulation comprising (a) a first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; (b) a second component comprising a nonionic polymer; and (c) an aqueous solution.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating cartilage damage disease comprising the above-described preparatory preparation.

[0019] The present invention also provides a pharmaceutical composition for preventing or treating cartilage damage disease comprising the injectable formulation.

[0020] The present invention also provides a joint injection preparation comprising the above injectable preparation.

[0021] The present invention also provides a method for treating a cartilage damage disease, comprising administering the injectable formulation to a subject in need thereof.

[0022] The biocompatible cationic polymer-based injectable formulation of the present invention can maintain target cells in a captured state and has been confirmed to deliver and engraft the captured target cells into target tissues. Furthermore, it achieves excellent regenerative effects in the engrafted tissues, suggesting that the biocompatible cationic polymer-based injectable formulation of the present invention can be utilized in various new cell therapy applications.

[0023] Figure 1 is a diagram showing the results of confirming whether coacervate is formed depending on the type of anionic polymer.

[0024] Figure 2 is a diagram showing the results of evaluating the apparent viscosity of mussel adhesive protein and anionic polymer coacervate according to the presence and type of nonionic polymer added.

[0025] Figure 3 is a diagram showing the results of evaluating the modulus of a mussel adhesive protein-based coacervate according to the poloxamer content.

[0026] Figure 4 is a diagram showing the results of evaluating the apparent viscosity of a mussel adhesive protein-based coacervate according to the content of poloxamer 188.

[0027] Figure 5 is a diagram showing the results of evaluating the underwater resistance of a mussel adhesive protein-based coacervate according to the content of poloxamer 188.

[0028] Figure 6 is a diagram showing the results of three-dimensional culture using a coacervate cell support based on mussel adhesive protein with or without poloxamer addition.

[0029] Figure 7 is a diagram showing the results of observing the appearance of cells captured within a mussel adhesive protein-based coacervate cell support.

[0030] Figures 8a and 8b are diagrams showing the results of confirming the presence or absence of cell death inside a mussel adhesive protein-based coacervate cell support depending on the addition of various nonionic polymers.

[0031] Figure 9 is a diagram showing the results of confirming the in vitro effectiveness of a mussel adhesive protein-based coacervate cell support through immunostaining.

[0032] Figure 10 is a diagram showing the results of confirming the in vivo effectiveness of a mussel adhesive protein-based coacervate cell support through Safranin O staining.

[0033] Figure 11 is a diagram showing the results of confirming the in vivo effectiveness of a mussel adhesive protein-based coacervate cell support through immunostaining.

[0034] Figure 12 is a diagram showing the results of measuring the moisture content of mussel adhesive protein and coacervate of anionic polymer.

[0035] Hereinafter, the present invention will be described in detail.

[0036] According to an aspect of the present invention, the present invention provides a powder-formulated preparation comprising (a) a first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; and (b) a second component comprising a non-ionic polymer.

[0037] In a specific example of the present invention, the mass ratio of the first component: the second component may be 25 to 35:0.1 to 5, and the mass ratio of the biocompatible cationic polymer of the first component: the biocompatible anionic polymer may be 15 to 25:5 to 14. The mass ratio of the first component: the second component may be preferably 28 to 30:0.5 to 3, and most preferably 29:1. In addition, the mass ratio of the biocompatible cationic polymer of the first component: the biocompatible anionic polymer may be preferably 18 to 23:8 to 10, and most preferably 20:9.

[0038] In a preferred embodiment of the present invention, a first component was prepared by mixing 200 mg of a biocompatible cationic polymer and 90 mg of a biocompatible anionic polymer, and a second component comprising the first component and 10 mg of a nonionic polymer was mixed to prepare a powder-type preparatory formulation.

[0039] In a specific embodiment of the present invention, the biocompatible anionic polymer preferably has a molecular weight of 1 to 1500 kDa, and more preferably 10 to 1000 kDa. This is because if the molecular weight is exceeded or less than the above, coacervate may not be formed.

[0040] In the present invention, any polymer material capable of forming a coacervate by combining with the biocompatible cationic polymer may be used without limitation as a biocompatible anionic polymer.

[0041] In the present invention, “coacervate” is a type of colloidal material formed when anionic polymer electrolyte and cationic polymer electrolyte are mixed under specific conditions. When a coacervate is formed, the absorbance of the solution increases, and it exists in a spherical shape separated from the external solution in the solution phase. When a coacervate is formed, the participating electrolyte separates from the solution and condenses, still in a liquid phase, and at this time, the physical properties such as surface tension decreases and viscosity increases are changed. Coacervate can also be formed through mixing a protein with a polymer electrolyte that has opposite properties. Due to the low surface tension of the coacervate, it is used to immobilize functional materials such as drugs, enzymes, cells, and food additives in microcapsules.

[0042] In a specific embodiment of the present invention, the biocompatible anionic polymer may be a polysaccharide polymer.

[0043] In a specific embodiment of the present invention, the biocompatible anionic polymer is hyaluronic acid, nucleic acid, ferredoxin, poly styrene sulfonic acid, gum arabic, gelatin, albumin, sodium polyphosphate, carbopol, high or low methoxyl pectin, sodium carboxymethyl guar gum, xanthan gum, whey protein, faba bean legumin, carboxymethyl cellulose, alginate, carrageenan, sodium hexametaphosphate, sodium casinate, It may be at least one selected from the group consisting of hemoglobin, heparin, and exopolysaccharide B40, preferably hyaluronic acid, and more preferably hyaluronic acid having an extreme viscosity of 2.0 and a molecular weight of 1100 kDa.

[0044] In the present invention, nucleic acids are linear polymers of nucleotides, including DNA and RNA. Since the bond between a sugar (e.g., deoxyribose or ribose) and a phosphate group in nucleic acids carries a negative charge, this characteristic is utilized in the field of nucleic acid separation technology to separate nucleic acids. Therefore, nucleic acids can be applied as the biocompatible anionic polymer of the present invention.

[0045] In a most preferred embodiment of the present invention, the hyaluronic acid (ultimate viscosity 2.0, 1100 kDa) may be present in an amount of 0.1 to 10% (w / w), preferably 0.5 to 5% (w / w), and most preferably 1% (w / w).

[0046] In a specific embodiment of the present invention, the nonionic polymer may be a homopolymer or copolymer comprising polyethylene glycol.

[0047] In a preferred embodiment of the present invention, the single polymer may be polyethylene glycol.

[0048] In a preferred embodiment of the present invention, the copolymer may be a triblock polymer comprising polyethylene glycol, preferably poloxamer, and most preferably poloxamer 188.

[0049] In a more preferred embodiment of the present invention, the poloxamer 188 can hydrate the powdered coacervate in the form of a solution, and the final concentration of the poloxamer 188 after hydration can be 0.01 to 10% (w / w), preferably 0.1 to 5% (w / w), more preferably 0.35 to 2.8%, and most preferably 0.7% (w / w).

[0050] In a specific embodiment of the present invention, the biocompatible cationic polymer is preferably an amino acid polymer.

[0051] In a preferred embodiment of the present invention, the biocompatible cationic polymer is preferably an amino acid polymer, and the amino acid polymer is preferably biodegradable or adhesive.

[0052] An example of the biocompatible cationic polymer having the above-mentioned biodegradability and adhesive properties is a mussel adhesive protein. Specifically, the mussel adhesive protein is a protein having adhesive properties derived from a mussel or a byssus of a mussel, and may include at least one selected from the group consisting of mussel adhesive proteins derived from one or more species of mussels (e.g., byssus) selected from the group consisting of Mytilus edulis, Mytilus galloprovincialis, Mytilus coruscus, etc., and variants thereof. Preferably, all mussel adhesive proteins described in International Patent Publication No. WO2006 / 107183 or WO2005 / 092920 are included in the scope of the present invention.

[0053] Additionally, the mussel adhesive protein may be of natural origin or recombinantly synthesized, and if recombinantly synthesized, the naturally occurring protein may additionally include a methionine (Met) at the N-terminus due to an initiation codon. Accordingly, the mussel adhesive protein described herein may be interpreted as also including a protein that additionally includes a methionine (Met) at the N-terminus due to an initiation codon.

[0054] For example, the mussel adhesive protein is a protein comprising (1) one or more, two or more, or 2 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) decapeptides AKPSYPPTYK (SEQ ID NO: 1) that appear repeatedly in FP (foot protein)-1 (GenBank No. Q27409 or Genbank No. S23760 (Q25460)), or 2 to 10, e.g., 6 (e.g., 6x AKPSYPPTYK: SEQ ID NO: 2 or 11; hereinafter, FP-1) (2) FP-2 (SEQ ID NO: 3 or 12); (3) FP-3 (SEQ ID NO: 4 or 13); (4) FP-4 (SEQ ID NO: 5 or 14); (5) FP-5 (SEQ ID NO: 6, 7, 15, or 16); and (6) FP-6 (SEQ ID NO: 8 or 17), or (7) a fusion protein in which two or more selected from (1) to (6) are linked.

[0055] The fusion protein (7) usable as the above mussel adhesive protein may be in a form in which two or more selected from (1) to (6) are each linked at least once in any order, and in one example, an FP-1 variant (SEQ ID NO: 2 or 11), an FP-5 (SEQ ID NO: 6, 7, 15, or 16), and an FP-1 variant (SEQ ID NO: 2 or 11) may be linked sequentially in the N-terminus to C-terminus direction or the C-terminus to N-terminus direction, more specifically, in the N-terminus to C-terminus direction. In the above fusion protein, a functional peptide may be additionally included (inserted) in the N-terminus to C-terminus direction, between the FP-1 variant and FP-5, and between FP-5 and the FP-1 variant, or both (i.e., either one or both of the N-terminus and the C-terminus of FP-5). The functional peptide may be selected from a group consisting of various enzyme cleavage sites, linkers, etc., and when two or more functional peptides are included (e.g., when included in both the N-terminus and the C-terminus of FP-5), the two or more functional peptides may be the same or different. For example, the functional peptide may be selected from the group consisting of PW (Pro-Trp), GS (Gly-Ser), etc. The fusion protein usable as the mussel adhesive protein may include the amino acid sequence of SEQ ID NO: 9, 10, 18, or 19.

[0056] The amino acid sequences of the mussel adhesive proteins described above are illustrated in Table 1 below:

[0057]

[0058] In the present invention, mutants of the mussel adhesive protein may preferably include an additional sequence at the carboxyl terminus or amino terminus of the mussel adhesive protein or may have some amino acids replaced with other amino acids, provided that the adhesiveness of the mussel adhesive protein is maintained.

[0059] The mussel adhesive protein of the present invention is not limited thereto, but can preferably be mass-produced by genetic engineering methods by inserting it into a conventional vector manufactured for the purpose of expressing an external gene so that it can be expressed. The vector can be appropriately selected or newly manufactured depending on the type and characteristics of the host cell for protein production. The method for transforming the host cell with the vector and the method for producing the recombinant protein from the transformant can be easily performed by conventional methods. Those skilled in the art can easily perform the methods of selecting, producing, transforming, and expressing the recombinant protein of the above-described vector, and some modifications of the conventional methods are also included in the scope of the present invention.

[0060] When the biocompatible cationic polymer of the present invention is the aforementioned mussel adhesive protein, its concentration may be 0.1 to 10% (w / w), preferably 1 to 5% (w / w), and most preferably 2.3% (w / w).

[0061] When the biocompatible cationic polymer of the present invention is the aforementioned mussel adhesive protein, it may be a polymer having a lower pI (isoelectric point) than the cationic mussel adhesive protein, more preferably a polymer having a pI of 2 to 6, and even more preferably a polymer having a pI of 2 to 4. When the pI is higher or lower than the above, it is difficult to form a coacervate, so it is preferable to use a biocompatible anionic polymer within the above pI range.

[0062] In a specific embodiment of the present invention, the powder diameter of the preliminary preparation may be 1 to 1000 μm, preferably 10 to 500 μm.

[0063] In a specific embodiment of the present invention, the preparatory preparation preferably has a moisture content of 450% or less, more preferably 450% or less, and most preferably 420% or less.

[0064] The preparative formulation according to the present invention can be rehydrated with an aqueous solution and used as an injectable formulation through the rehydration process.

[0065]

[0066] According to another aspect of the present invention, there is provided an injectable formulation comprising (a) a first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; (b) a second component comprising a non-ionic polymer; and (c) an aqueous solution.

[0067] In a specific embodiment of the present invention, the formulation may be a coacervate.

[0068] In a more preferred embodiment of the present invention, the poloxamer 188 can hydrate the powdered coacervate in the form of a solution, and the final concentration of the poloxamer 188 after hydration can be 0.01 to 10% (w / w), preferably 0.1 to 5% (w / w), more preferably 0.35 to 2.8%, and most preferably 0.7% (w / w).

[0069] By using a nonionic polymer at the above concentration, a cell support environment can be provided that maintains liquid-state properties without forming a hydrogel and provides a favorable environment for cell proliferation and spheroid formation.

[0070] In a specific embodiment of the present invention, the nonionic polymer may be a homopolymer or copolymer comprising polyethylene glycol.

[0071] In a preferred embodiment of the present invention, the nonionic polymer may be included at a concentration of 0.01 to 10% (w / w), preferably 0.1 to 5% (w / w), more preferably 0.35 to 2.8%, and most preferably 0.7% (w / w).

[0072] In a specific embodiment of the present invention, the biocompatible cationic polymer and the biocompatible anionic polymer are bonded by electrical attraction, and the first component and the second component are bonded through hydrophobic interaction or hydrogen bond.

[0073] In a specific example of the present invention, the complex viscosity of the formulation is preferably 20 to 2000 Pa·s when measured at 1 Hz.

[0074] In a specific example of the present invention, it is preferable that the modulus crossover point of the formulation is 6.2 Hz or less when measured in a frequency test under 1% strain conditions.

[0075] In a specific example of the present invention, the apparent viscosity of the formulation is preferably 200 to 755 Pa·s when measured at a shear rate of 1 1 / s.

[0076] The mussel adhesive protein-based coacervate manufactured according to an embodiment of the present invention was confirmed to have a complex viscosity of 20 to 2000 Pa·s when measured at 1 Hz, a modulus crossover point of 6.2 Hz or less when measured in a frequency test under 1% strain conditions, and an apparent viscosity of 200 to 755 Pa·s when measured at a shear rate of 1 1 / s.

[0077] In a specific embodiment of the present invention, the formulation may be capable of three-dimensional cell culture, and more preferably, of cell proliferation or differentiation. The cells capable of three-dimensional culture may be animal or plant-derived cells, and preferably stem cells, chondrocytes, or chondrocytes differentiated from stem cells.

[0078] In the present invention, “stem cell” refers to a cell that has the ability to self-replicate and differentiate into two or more cells, and can be classified into a totipotent stem cell, a pluripotent stem cell, and a multipotent stem cell.

[0079] The above stem cells may be appropriately selected without limitation according to the purpose, and may be derived from adult cells of mammals including humans, preferably all known tissues, cells, etc. derived from humans, and for example, may be derived from bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or fat tissue cells), etc.

[0080] For example, the stem cells may be stem cells obtained without limitation from bone marrow, adipose tissue, muscle tissue, ex vivo cultured autologous mesenchymal stem cells, allogeneic mesenchymal stem cells, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and adolescent skin, and blood, and may be stem cells derived from a fetus, postnatally, or adult.

[0081] Stem cells are not limited to the type of stem cells as long as they can achieve the desired effect, but preferably, the stem cells may be selected from the group consisting of adipose stem cells (ASC), mesenchymal stem cells (MSC), bone marrow stem cells, umbilical cord blood stem cells, neural stem cells, and induced pluripotent stem cells, and most preferably, adipose stem cells (ASC) or mesenchymal stem cells (MSC).

[0082] In a preferred embodiment of the present invention, when chondrocytes are cultured three-dimensionally on the above formulation, it is preferable that the cultured chondrocytes highly express SOX9 compared to chondrocytes not cultured on the above formulation.

[0083]

[0084] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cartilage damage disease comprising the above-described preparatory preparation or injectable preparation.

[0085] In a specific embodiment of the present invention, the composition may further comprise stem cells.

[0086] In the present invention, the cartilage damage disease may be at least one disease selected from the group consisting of degenerative arthritis, rheumatoid arthritis, fracture, muscle tissue damage, plantar fasciitis, lateral epicondylitis, calcific myositis, nonunion of fracture, and cartilage damage due to trauma, but the scope of the present invention is not limited thereto.

[0087] When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention can be formulated and used in various forms according to conventional methods. For example, it can be formulated in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, and can be formulated and used in the form of topical preparations, suppositories, and sterile injectable solutions.

[0088] The composition of the present invention may contain at least one known effective ingredient having a preventive or therapeutic effect on cartilage damage disease together with the preparative or injectable preparation of the present invention.

[0089] The composition of the present invention may further include a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, etc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0090] The composition of the present invention can be administered in various oral or parenteral dosage forms during actual clinical administration. When formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. It is preferable to use suitable formulations known in the art as disclosed in the literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA).

[0091] The above solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, the above liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

[0092] The above-mentioned parenteral administration formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0093] The dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the method of administration, the time of administration, and / or the route of administration, and may vary depending on various factors including the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, and similar factors well known in the medical field, and a person having ordinary knowledge in the relevant technical field can easily determine and prescribe an effective dosage for the desired treatment.

[0094] The dosage of the pharmaceutical composition of the present invention is preferably administered at a concentration of, for example, 0.05 to 5 mg / kg, more preferably 0.1 to 0.4 mg / kg, even more preferably 0.2 to 0.35 mg / kg, and even more preferably 0.25 mg / kg, but the dosage does not limit the scope of the present invention in any way.

[0095] The route and method of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in their method, and any route and method of administration may be followed as long as the pharmaceutical composition can reach the target area.

[0096] The above pharmaceutical composition may be administered orally or parenterally, with parenteral administration being preferred. Examples of parenteral administration include intravenous, intraperitoneal, intramuscular, transdermal, intra-articular, or subcutaneous administration.

[0097] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of cartilage damage diseases.

[0098]

[0099] According to another aspect of the present invention, the present invention provides a joint injection preparation comprising the injectable preparation.

[0100] The above joint injection preparation is intended for intra-articular injection for the purpose of treating cartilage damage disease, and may further include a preservative, an isotonic agent, or a pH adjusting agent.

[0101] The above preservative may include, but is not limited to, benzyl alcohol, lidocaine, procaine, and chlorobutanol. The concentration of the preservative included in the joint injection preparation of the present invention may be 0.1% (w / v) to 2% (w / v).

[0102] The above isotonic agent plays a role in appropriately maintaining (regulating) the osmotic pressure when the joint injection preparation of the present invention is administered into the body, and also exhibits the secondary effect of further stabilizing the joint injection preparation of the present invention in the solution phase. The isotonic agent may be a pharmaceutically acceptable sugar, salt, or any combination or mixture thereof, and examples thereof include glucose as a sugar, or sodium chloride, calcium chloride, sodium sulfate, glycerin, and propylene glycol as a water-soluble inorganic salt, polyethylene glycol having a molecular weight of 1000 or less, etc. These may be used in the form of one or a combination of two or more. The concentration of the isotonic agent may be 0.1% (w / v) to 5% (w / v), and depending on the types, amounts, etc. of the components included in the injection composition of the present invention, the content may be adjusted to an appropriate amount so that the solution formulation containing all of the respective mixtures becomes an isotonic solution.

[0103] The pH regulator of the present invention serves to regulate the pH of a joint injection preparation, and includes both acidic and basic substances.

[0104] The acidic substances include, but are not limited to, hydrochloric acid, acetic acid, adipic acid, ascorbic acid, sodium ascorbate, sodium etherate, malic acid, succinic acid, tartaric acid, fumaric acid, citric acid, etc. The basic substances include, but are not limited to, inorganic bases (e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, magnesium carbonate, calcium carbonate, magnesium oxide, ammonia, synthetic hydrotalcite), organic salts (e.g., basic amino acids such as lysine, arginine, etc., meglumine, etc.), etc. In the present invention, the pH adjusting agent may include an acidic substance and a basic substance alone in the composition, or two or more of each substance may be combined and used. The amount of the pH regulator added to the joint injection preparation of the present invention may vary depending on the types and amounts of the components constituting the joint injection preparation of the present invention, and may be 0.01% (w / v) to 1% (w / v).

[0105] The joint injection preparation of the present invention may be provided in a range of pH 7.5 to pH 8.5, and the type and amount of pH adjusting agent may be changed by a person skilled in the art according to the specific composition of the solution.

[0106]

[0107] According to another aspect of the present invention, the present invention provides a method for treating a cartilage damage disease, comprising the step of administering the injectable formulation to a subject in need thereof.

[0108] In a specific example of the present invention, the subject may be, but is not limited to, a subject expected to develop a cartilage damage disease; a subject that has developed the disease; or a subject that has been judged to be cured.

[0109]

[0110] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0111] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0112]

[0113] Example 1. Production of recombinant mussel adhesive protein

[0114] Among the naturally occurring mussel adhesive protein fp-1, a decapeptide consisting of 10 amino acids repeated approximately 80 times was synthesized to enable expression in E. coli. An fp-1 mutant consisting of 6 decapeptides was synthesized, and the Mgfp-5 gene (Genbank No. AAS00463 or AY521220) was inserted between two fp-1 mutants, and then produced in E. coli to produce the mussel adhesive protein fp-151 (SEQ ID NO: 1).

[0115] Specifically, in the amino acid sequence of fp-1 (Genbank No. Q27409 or S23760), a fp-1 variant (hereinafter referred to as 6xAKPSYPPTYK) in which a peptide consisting of AKPSYPPTYK is linked 6 times was prepared, and the 6xAKPSYPPTYK was combined with the N-terminus of Mgfp-5 and also combined with the C-terminus of Mgfp-5 to prepare fp-151. The specific preparation of the mussel adhesive protein is the same as that disclosed in International Patent Publication No. WO2006 / 107183 or WO2005 / 092920, and the above patent documents are incorporated herein by reference in their entirety.

[0116]

[0117] Example 2. Preparation of coacervate based on mussel adhesive protein

[0118] We aimed to develop a novel formulation of a cell scaffold for the treatment of osteoarthritis with biological efficacy utilizing a mussel adhesive protein-based coacervate.

[0119] Here, the mussel adhesive protein fp-151 manufactured in Example 1 was mixed with an anionic polymer, and then a nonionic polymer was added to manufacture a coacervate. Specifically, the mussel adhesive protein fp-151 was dissolved in distilled water at a concentration of 2.3% (w / w). The anionic polymer was dissolved in distilled water at a concentration of 1% (w / w). Thereafter, the two solutions were mixed in a volume ratio of 1:1 and centrifuged to recover the coacervate. Thereafter, the recovered coacervate was freeze-dried and freeze-pulverized to be processed into a powder state. The coacervate obtained through the above process was named 'coacervate of mussel adhesive protein and anionic polymer (MAP-HA Coacervate)'.

[0120] Nonionic polymer solutions were then prepared by dissolving various types and concentrations of nonionic polymers in PBS. The dry powders of mussel adhesive proteins and anionic polymer coacervates were rehydrated with each nonionic polymer solution to prepare 'mussel adhesive protein-based coacervates' [1 ml of solvent was used per 290 mg of mussel adhesive proteins and anionic polymer coacervate (MAP 200 mg and HA 90 mg) powder].

[0121]

[0122] Example 3. Evaluation of physical properties of coacervate based on mussel adhesive protein

[0123] 3-1. Coacervate formation between mussel adhesive protein and anionic polymer coacervate (MAP-HA Coacervate) according to the type of anionic polymer

[0124] The formation of mussel adhesive proteins and anionic polymer coacervates (MAP-HA Coacervate) according to the type of anionic polymer was investigated. Specifically, mussel adhesive protein fp-151 was dissolved in distilled water at a concentration of 2.3% (w / w) to prepare a mussel adhesive protein solution. Anionic polymer solutions were prepared by dissolving them in distilled water at a concentration of 1% (w / w). The anionic polymers used in this experiment were sodium hyaluronate, carboxymethyl cellulose, sodium caseinate, sodium polyphosphate, K-carrageenan, and alginic acid. Nonionic polymers polyethylene glycol and (Hydroxypropyl)methyl cellulose were used as controls. 3 ml of each of the two prepared solutions were mixed and centrifuged. After centrifugation, coacervate formation was visually confirmed. The results of confirming whether coacervate was formed depending on the type of anionic polymer are shown in Figure 1.

[0125] As shown in Figure 1, it was confirmed that anionic polymers, Sodium Hyaluronate, Carboxymethyl cellulose, Sodium caseinate, Sodium polyphosphate, K-carrageenan, and Alginic acid, formed coacervates. In contrast, it was confirmed that mussel adhesive proteins, nonionic polymers, Polyethylene glycol and (Hydroxypropyl)methyl cellulose did not form coacervates.

[0126] Among anionic polymers, hyaluronic acid (extreme viscosity 2.0, 1100 kDa), which forms coacervate best, was selected as an anionic polymer at 1% (w / w).

[0127]

[0128] In the experiments described below, a mussel adhesive protein-based coacervate prepared by the method of Example 2 using hyaluronic acid, an anionic polymer, was used.

[0129]

[0130] 3-2. Apparent viscosity according to the type and addition of nonionic polymer

[0131] The apparent viscosity of mussel adhesive protein-based coacervates was evaluated according to the presence and type of nonionic polymer added. Specifically, the mussel adhesive protein-based coacervates used in this experiment were prepared using hyaluronic acid and various nonionic polymers (Poloxamer 188, Poloxamer 407, PEG 8000, PVP, or Tween 80) according to the method of Example 2. The apparent viscosity was measured according to the presence and type of various nonionic polymers added. The apparent viscosity measurement information is as follows.

[0132]

[0133] - Instrument: Discovery HR20(TAInstruments)

[0134] - Geometry: 20 mm parallel plate

[0135] - GAP: 1 mm

[0136] - Temperature: 25 ℃

[0137] - Shear rate: 0.1-100 1 / s

[0138]

[0139] The results of measuring the apparent viscosity are shown in Fig. 2.

[0140] As shown in Fig. 2, compared to the non-polymer-added group (PBS group), the low-shear (1 1 / s) viscosity was confirmed to have increased in the polymer-added group. Compared to the non-polymer-added group (PBS group in the above figure), the high-shear (100 1 / s) viscosity was decreased in the polymer-added group excluding PVP.

[0141] Additionally, when poloxamer 188 was added to the mussel adhesive protein-based coacervate, the complex viscosity of the coacervate was confirmed to be 20 to 2000 Pa·s.

[0142] Therefore, the above results mean that the mussel adhesive protein-based coacervate with added Poloxamer 188 or PEG (polyethylene glycol) improves physical properties by increasing viscosity in a static state and reduces viscosity during injection, making it advantageous for injection.

[0143] In the experiments described below, a mussel adhesive protein-based coacervate prepared by the method of Example 2 using Poloxamer 188 as a nonionic polymer was used.

[0144]

[0145] 3-3. Modulus measurement according to the content of poloxamer 188

[0146] The modulus of mussel adhesive protein-based coacervates was evaluated according to the poloxamer content. Specifically, the mussel adhesive protein-based coacervates used in this experiment were prepared using hyaluronic acid and poloxamer 188 according to the method of Example 2. The powdered coacervates were hydrated by adding 0.5, 1, 2, and 4% poloxamer solutions, respectively, resulting in final concentrations of 0.35, 0.7, 1.4, and 2.8% (w / w), respectively. The modulus according to the poloxamer 188 content was measured under the following conditions.

[0147]

[0148] - Instrument: Discovery HR20 (TA Instruments)

[0149] - Geometry: 20 mm parallel plate

[0150] - GAP: 1 mm

[0151] - Temperature: 25 ℃

[0152] - Strain: 1%

[0153] - Frequency: 0.01-100 Hz

[0154]

[0155] The results of modulus measurements of mussel adhesive protein-based coacervates according to poloxamer content are shown in Fig. 3.

[0156] As shown in Fig. 3, when poloxamer 188 was added to a mussel adhesive protein-based coacervate, the viscosity was improved as the concentration of poloxamer 188 in the coacervate increased, while hydrogel formation was not observed as with the conventional high-concentration (20% or more) addition of poloxamer 188. In addition, the modulus crossover point was confirmed to be 6.2 Hz or less when measured in a frequency test under 1% strain conditions.

[0157]

[0158] Based on the modulus measurement results above, the complex viscosity of mussel adhesive protein-based coacervate was calculated according to the poloxamer content. The results of the complex viscosity calculation are shown in Table 2.

[0159] Concentration of poloxamer solution (%) Complex viscosity (Pa·s) at 1 Hz0148.40.5236.71376.92430.44547.5

[0160] As shown in Table 2, when poloxamer 188 was added to the mussel adhesive protein-based coacervate, the complex viscosity of the coacervate was confirmed to be 20 to 2000 Pa·s.

[0161]

[0162] That is, at low shear, it is liquid (Loss modulus > Storage modulus), and the crossover point where the Loss modulus and Storage modulus values ​​are reversed was brought forward as the Poloxamer concentration increased. The overall physical properties were also brought forward as the concentration of Poloxamer 188 increased. In addition, unlike the hydrogel made with the existing high content of Poloxamer 188 (more than 20%), the cell scaffold with Poloxamer 188 added to the coacervate is liquid and has different physical properties from Poloxamer 188.

[0163]

[0164] 3-4. Apparent viscosity according to the content of poloxamer 188

[0165] The apparent viscosity of mussel adhesive protein-based coacervates according to the content of poloxamer 188 was evaluated. Specifically, the mussel adhesive protein-based coacervates used in this experiment were prepared using hyaluronic acid and various concentrations of poloxamer 188 according to the method of Example 2. The apparent viscosity according to the content of poloxamer 188 was measured. The powdered coacervates were hydrated by adding 0.5, 1, 2, and 4% poloxamer solutions, respectively, so the final concentrations were 0.35, 0.7, 1.4, and 2.8% (w / w), respectively. The apparent viscosity measurement was performed in the same manner as in Example 3-2. The results of the apparent viscosity measurement are shown in Fig. 4 and Table 3.

[0166] Concentration of poloxamer solution (%) Apparent viscosity (Pa·s) at 1 1 / s0200.20.5206.11349.32475.04627.5

[0167] As shown in Fig. 4 and Table 3, it was confirmed that the viscosity significantly improved at low shear (0.01 to 1 Hz) and decreased at high shear (10 to 100 Hz) as the concentration of poloxamer 188 increased. In addition, it was confirmed that the apparent viscosity according to the poloxamer 188 content was 200 to 755 Pa·s when measured at a shear rate of 1 1 / s.

[0168] This allows for the prevention of detachment from the lesion site while maintaining the injectable liquid properties. In other words, by adding poloxamer 188, it is possible to produce a liquid that maintains its shape (increases low-shear viscosity) at the lesion site while also being injectable (reduces high-shear viscosity).

[0169]

[0170] 3-5. Evaluation of water resistance according to the content of poloxamer 188

[0171] The underwater resistance of mussel adhesive protein-based coacervates according to the content of poloxamer 188 was evaluated. The mussel adhesive protein-based coacervates were prepared using hyaluronic acid, an anionic polymer, and poloxamer 188, a nonionic polymer, at various concentrations (0, 20, 40, 50, 60 mg) according to the method of Example 2. 1 ml of PBS was added and mixed to the mussel adhesive protein-based coacervates containing various concentrations of poloxamer 188, and observed with the naked eye. The results of evaluating the underwater resistance of the mussel adhesive protein-based coacervates are shown in Fig. 5.

[0172] As shown in Fig. 5, it was confirmed that re-liquefaction occurred when poloxamer 188 was less than 50 mg. In contrast, it was confirmed that re-liquefaction did not occur when poloxamer 188 was more than 50 mg, and that precipitation occurred and the particles were obtained in the form of a dispersed paste. In other words, the above results indicate that, as the paste was obtained, it lost its underwater resistance and its surface easily dispersed in water, making it unusable.

[0173]

[0174] 3-6. Analysis of Freeze-grinding Time and Average Particle Size of Mussel Adhesive Protein and Anionic Polymer Coacervate (MAP-HA Coacervate)

[0175] A mussel adhesive protein-based coacervate (MAP-HA Coacervate) is prepared by adding non-ionic polymer poloxamer 188 to the freeze-dried powder of mussel adhesive protein and anionic polymer. The mussel adhesive protein and anionic polymer coacervate were freeze-pulverized at 10 CPS using a freezer mill (SPEX 6875) under the following conditions.

[0176]

[0177] - 1 cycle: 2 minutes

[0178] - 2 cycles: 2 minutes x 2 (4 minutes total)

[0179] - 3 cycles: 2 minutes x 3 (6 minutes total)

[0180]

[0181] The results of analyzing the average particle size of mussel adhesive protein and anionic polymer coacervate according to freeze-grinding time are shown in Table 4 below.

[0182] Sample name Freezing grinding time Average particle size (distribution) FM_1cycle2 minutes 130.0 (13.95~400.0) μm FM_2cycle2 minutes x 2 (4 minutes) 35.32 (5.519~72.19) μm FM_3cycle2 minutes x 3 (6 minutes) 38.02 (6.725~77.08) μm

[0183] As shown in Table 4, the average particle size of the mussel adhesive protein and the coacervate of the anionic polymer was confirmed to be 10 to 500 μm.

[0184]

[0185] 3-7. Optimal Manufacturing Conditions for the Coacervate Cell Support Based on the Derived Mussel Adhesive Protein

[0186] - Manufacture of coacervate dry powder of mussel adhesive protein and anionic polymer

[0187] Mussel adhesive protein fp-151 was dissolved in distilled water at a concentration of 2.3% (w / w) to prepare a cationic polymer solution. Hyaluronic acid (intrinsic viscosity 2.0, approximately 1100 kDa) was dissolved in distilled water at a concentration of 1% (w / w) to prepare an anionic polymer solution.

[0188] Afterwards, the above two solutions were mixed in a volume ratio of 1:1 and centrifuged to recover the coacervate. The recovered coacervate was then freeze-dried and freeze-pulverized to process it into a powder form (i.e., obtaining a powdered coacervate of mussel adhesive protein and anionic polymer).

[0189]

[0190] -Manufacture of coacervate cell scaffolds based on mussel adhesive proteins

[0191] A mixture (total 300 mg) was prepared by mixing 10 mg of nonionic polymer poloxamer 188 and 290 mg of coacervate powder of mussel adhesive protein and anionic polymer (200 mg of MAP and 90 mg of HA), and the mixture was named 'CartFix'.

[0192] 1 ml of PBS was added to 300 mg of the above mixture and mixed, and the mixture was re-liquefied to obtain a mussel adhesive protein-based coacervate cell support.

[0193]

[0194] Example 4. 3D culture using a coacervate cell support based on mussel adhesive proteins.

[0195] The formation of cell spheroids inside the mussel adhesive protein-based coacervate cell support was examined depending on the addition of nonionic polymers. Specifically, 3 mg of mussel adhesive protein-based coacervate dry powder was weighed and added to each well of a 24-well plate, and 10 μL of PBS solution containing 1% (w / w) of nonionic polymer was dropped, mixed, and applied to the bottom of the well. The mixture was then left in a 37°C incubator for 30 minutes to ensure that the liquid cell support was evenly spread. 1 mL (1x10) of L929 cell suspension was added to each well coated with the cell support. 5 cells / mL) were seeded. The cells were cultured for 24 hours at 37°C under 5% CO2 conditions, and then spheroid formation was visually observed using an optical microscope. The test group used non-ionic polymers Poloxamer 188 and Poloxamer 407. The control group used the group without polymer and Tween 80. The results of the 3D culture are shown in Figure 6.

[0196] As shown in Figure 6, spheroids were not formed when the mussel adhesive protein-based coacervate alone was used, but spheroids were formed when 1% poloxamer was added. On the other hand, it was confirmed that cell viability decreased when other surfactants were added. In other words, cell proliferation and 3D culture were difficult when the mussel adhesive protein-based coacervate alone was used, but cell proliferation and 3D culture were confirmed when mixed with poloxamer. Other surfactants actually caused cell death.

[0197] Therefore, when a mussel adhesive protein-based coacervate is produced under the optimal conditions of the present invention, a hydrogel is not formed, the properties of a liquid state are maintained, and an environment favorable for cell proliferation and spheroid formation is provided.

[0198]

[0199] Example 5. Cell capture using a coacervate cell support based on mussel adhesive proteins.

[0200] ADSc cells (rat) were seeded and cultured on a mussel adhesive protein-based coacervate cell support. Paraffin sections were then stained with H&E, and the morphology of the cells trapped within the coacervate was observed. The results of observing the morphology of cells trapped within the mussel adhesive protein-based coacervate cell support are shown in Figure 7.

[0201] As shown in Fig. 7, unlike conventional coacervates, it was confirmed that cells were well attached in the form of clusters within the formed pores in the coacervate containing 0.7% (w / w) of Poloxamer.

[0202] This is different from existing coacervate-based cell therapy in that it is a liquid-type scaffold capable of 3D cell culture.

[0203] Therefore, it is demonstrated that cell clusters are well formed within the pores when coacervate and poloxamer 188 are used together.

[0204]

[0205] Example 6. Confirmation of cell death within a coacervate cell support based on mussel adhesive proteins.

[0206] The presence or absence of cell death inside the mussel adhesive protein-based coacervate cell support was confirmed according to the addition of nonionic polymers. Specifically, coverslips were adhered to the bottom of a 24-well plate one by one. 15 μL of coacervate containing nonionic polymers (MAP 2.3 mg, HA 1 mg, and 150 μg of each nonionic polymer) and 1 mL (1x10) of L929 cell suspension were added. 5After evenly mixing (cells / mL), the cells were seeded into each well of a 24-well plate. The seeded cells were then cultured for 16 hours at 37°C under 5% CO2 conditions. Afterwards, Calcein AM (Live) and 7-AAD (Dead) were added to each well for fluorescent labeling, and the cells were mounted on slide glass with DAPI mounting medium and observed under a fluorescence microscope. The experimental group used non-ionic polymers Poloxamer 188, Poloxamer 407, and PEG 8000. The control group used the group without polymer addition, PVP, and Tween 80. The results of the fluorescence microscope observation are shown in Figures 8a and b.

[0207] As shown in Figures 8a and b, cells survived in coacervate containing Poloxamer 188, Poloxamer 407, and PEG 8000, and a clear live signal was observed. In the groups without polymer addition, PVP, and Tween 8000, cells died, and only the red fluorescence of 7-AAD was observed. The above results imply that only Poloxamer 188, 407, and PEG can create an environment in which cells can survive in a high-viscosity liquid.

[0208]

[0209] Example 7. In vitro validation of a coacervate cell support based on mussel adhesive proteins.

[0210] The in vitro effectiveness of the mussel adhesive protein-based coacervate cell scaffold of the present invention was confirmed. Specifically, adipose stem cell suspension medium containing 1% (w / w) poloxamer was added dropwise to the coacervate dry powder, mixed, and cultured for 7 and 14 days at 37°C in a 5% CO2 environment to obtain a mussel adhesive protein-based coacervate cell scaffold. The chondrogenic differentiation medium was changed every 2 days. The coacervate was then fixed in formalin, paraffin-embedded, and analyzed by immunostaining (Ki67, Alcian Blue, Collagen Type 2). The immunostaining results are shown in Fig. 9.

[0211] As shown in Fig. 9, when Ki-67 staining, a cell proliferation marker, was performed, red staining was confirmed around blue-stained cells, indicating that the cells were actively proliferating until the 7th day. In addition, after the 7th day, when Alcian Blue staining was performed to confirm cartilage differentiation, blue hyaluronic acid staining was confirmed around red-marked cells, indicating that chondrocyte differentiation had occurred. In addition, when staining Collagen type 2, a representative indicator of hyaline cartilage, the expression of red-stained Collagen type 2 was confirmed around blue cells, indicating that cartilage was well formed.

[0212]

[0213] Example 8. In vivo validation of a coacervate cell scaffold based on mussel adhesive proteins.

[0214] The in vivo efficacy of a mussel adhesive protein-based coacervate-cell scaffold (CartiFix) was confirmed. Specifically, to demonstrate the effects of cartilage defect and microfracture, a 2 mm diameter and 2 mm deep defect was created in the femoral trochlear groove of rats, and bleeding was observed. A total of four groups were treated: a group in which the cartilage defect was left untreated (control group 1), a group in which chondrogenic stem cells were transplanted and then fixed with fibrin sealant (control group 2), a group in which chondrogenic stem cells were transplanted and then fixed with CartiFix (experimental group 1), and a group in which CartiFix was applied (experimental group 2).

[0215] Information on the specific test and control groups is shown in Table 5 below.

[0216] Total number of animals tested per section Number of animals tested per section Test substance by sex Number of application sites per animal 100 Weeks 1 / 2 / 4 / 8 / 12 after surgery (total 5 sections) 20 Male Cartifix (test group 1, CF group) 1 Cartifix + stem cell (test group 2, CFC group) 1 Fibrin glue + stem cell (positive control group, FGC group) 1 Negative control group (NC group) 1

[0217] Afterwards, the rats were sacrificed and autopsied, and tissue analysis and immunoassay were performed.

[0218]

[0219] - Organizational analysis

[0220] Tissue samples embedded in Paraffin were sectioned at 4 μm thickness. The sectioned tissues were rehydrated, stained with Safranin O / Fast Green, and observed under an optical microscope to determine the degree of regeneration of damaged cartilage.

[0221]

[0222] - Immunostaining

[0223] Tissue samples embedded in Paraffin were sectioned at 4 μm thickness. The sectioned tissues were rehydrated, then subjected to endogenous peroxidase blocking and endogenous serum blocking. Anti-Type II Collagen, anti-Type I Collagen, and anti-Type X Collagen solutions were spotted as primary antibodies on each slide and incubated, and then Bionylated secondary antibody solutions corresponding to the host of each primary antibody were spotted and incubated. Streptavid-HRP was spotted and incubated, and color development was performed using AEC (red). After color development, the slides were counterstained with hematoxylin.

[0224]

[0225] The results of tissue analysis and immunoassay are shown in Figures 10 and 11, respectively.

[0226] As shown in Fig. 10, when microfracture was performed and treated in the rat model, fibrotic cartilage was observed macroscopically in the untreated group (control group 1), whereas cartilage regeneration was well observed macroscopically in the treatment group (control group 2) and the CartiFix treatment group (experimental groups 1 and 2) where Fibrin and Cell were applied in the same way as the existing cell therapy agent. In addition, as a result of performing Safranin O staining for tissue analysis, the regenerated cartilage in control group 1 was completely fibrosed at 12 weeks, and most of the cartilage in control group 2 was also fibrosed. In the CartiFix + cell group, the regenerated cartilage was well maintained, and fibrosis of the cartilage was hardly observed in the group where CartiFix was applied alone.

[0227] In addition, as shown in Fig. 11, when the collagen of the regenerated cartilage was analyzed using an immunological method, the expression of Collagen Type 2 was low in the control groups 1 and 2 and Type I was highly expressed, whereas Collagen Type 2 was well expressed in the experimental groups CartiFix + cell and CartiFix groups. This means that the regenerated cartilage is close to normal cartilage.

[0228]

[0229] Example 9. Measurement of the moisture content of mussel adhesive protein and coacervate of anionic polymers.

[0230] Approximately 0.1 g of mussel adhesive protein and anionic polymer coacervate (MAP-HA Coacervate) powder were prepared, and 2 mL of distilled water was added to each powder and mixed thoroughly. After mixing, the powder was left to stand in a sealed container at 25°C for 24 hours to completely hydrate. After removing excess water, the surface moisture of the coacervate was carefully removed, and the weight was measured.

[0231] The functional ratio was calculated as follows, and the initial and final functional ratios are shown in Figure 12.

[0232]

[0233] Absorption rate (%) = (weight after absorption - initial powder weight) / powder weight * 100

[0234]

[0235] As shown in Figure 12, the maximum absorption rate based on 1100 kDa was confirmed to be 425.5%.

[0236]

[0237] While specific aspects of the present invention have been described in detail, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (a) a first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; and (b) a second component comprising a non-ionic polymer; Pre-formulation in powder form.

2. In the first paragraph, the mass ratio of the first component: the second component is 25 to 35:0.1 to 5, A preparatory formulation, wherein the mass ratio of the biocompatible cationic polymer of the first component: the biocompatible anionic polymer is 15 to 25:5 to 14.

3. A preparatory preparation according to claim 1, wherein the biocompatible anionic polymer has a molecular weight of 1 to 1500 kDa.

4. A preparatory preparation according to claim 1, wherein the biocompatible anionic polymer is a polysaccharide polymer.

5. In the first paragraph, the biocompatible anionic polymer is hyaluronic acid, nucleic acid, ferredoxin, poly styrene sulfonic acid, gum arabic, gelatin, albumin, sodium polyphosphate, carbopol, high or low methoxyl pectin, sodium carboxymethyl guar gum, xanthan gum, whey protein, faba bean legumin, carboxymethyl cellulose, alginate, carrageenan, sodium hexametaphosphate, sodium casinate, A preparative preparation comprising at least one selected from the group consisting of hemoglobin, heparin and exopolysaccharide B40.

6. A preparatory preparation according to claim 1, wherein the non-ionic polymer is a single polymer or copolymer containing polyethylene glycol.

7. A preparatory preparation according to claim 6, wherein the single polymer is polyethylene glycol.

8. A preparatory formulation according to claim 6, wherein the copolymer is a triblock polymer containing polyethylene glycol.

9. A preparatory preparation according to claim 8, wherein the copolymer is a poloxamer.

10. A preparatory preparation according to claim 1, wherein the biocompatible cationic polymer is an amino acid polymer.

11. A preparatory preparation according to claim 10, wherein the amino acid polymer has biodegradability or adhesiveness.

12. A preparatory preparation according to claim 1, wherein the powder diameter of the preparatory preparation is 1 to 1000 μm.

13. In the first paragraph, the preliminary preparation has a water content of 450% or less. 14.(a) A first component comprising a biocompatible cationic polymer and a biocompatible anionic polymer; (b) a second component comprising a nonionic polymer; and (c) An injectable preparation comprising an aqueous solution.

15. A formulation according to claim 14, wherein the formulation is a coacervate.

16. A formulation according to claim 14, wherein the non-ionic polymer is a single polymer or copolymer containing polyethylene glycol.

17. A formulation according to claim 16, wherein the non-ionic polymer is included at a concentration of 0.01 to 10% (w / w).

18. In paragraph 14, the biocompatible cationic polymer and the biocompatible anionic polymer are combined by electrical attraction, A formulation wherein the first component and the second component are combined through hydrophobic interaction or hydrogen bond.

19. A formulation according to claim 14, wherein the complex viscosity of the formulation is 20 to 2000 Pa·s when measured at 1 Hz.

20. In claim 14, the formulation has a modulus crossover point of 6.2 Hz or less when measured in a frequency test under 1% strain conditions.

21. A formulation according to claim 14, wherein the apparent viscosity of the formulation is 200 to 755 Pa·s when measured at a shear rate of 1 1 / s.

22. In the 14th paragraph, the preparation is capable of three-dimensional cell culture.

23. In claim 22, the three-dimensional cell culture is a preparation capable of cell proliferation or differentiation.

24. A pharmaceutical composition for preventing or treating cartilage damage disease, comprising a preparatory preparation according to any one of claims 1 to 13.

25. A pharmaceutical composition for preventing or treating cartilage damage disease, wherein the composition further comprises stem cells in claim 24.

26. A pharmaceutical composition for preventing or treating cartilage damage disease, comprising an injectable formulation of any one of claims 14 to 23.

27. A pharmaceutical composition for preventing or treating cartilage damage disease, wherein the composition further comprises stem cells in claim 26.

28. A joint injection preparation comprising an injectable preparation according to any one of claims 14 to 23.

29. A method for treating a cartilage damage disease, comprising administering to a subject in need thereof an injectable formulation of any one of claims 14 to 23.

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