Agent for inducing proliferation or differentiation of cartilage cells

Abstract

The present invention addresses the problem of providing a novel agent for inducing proliferation or differentiation of cartilage cells, which contains, as an active ingredient, a compound that has hitherto unknown ability to induce proliferation or differentiation of cartilage cells. The present invention provides an agent for inducing proliferation or differentiation of chondrocytes, which comprises compound (1) or a salt thereof.

Description

Technical Field

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority based on Japanese Patent Application No. 2022-061383 filed on March 31, 2022 (the entire disclosure of which is incorporated herein by reference). The present invention relates to a chondrocyte proliferation or differentiation inducing agent and a chondrocyte proliferation or differentiation inducing method. Background Art

[0002] Temporomandibular Disorder is a disease caused by problems in the jaw muscles or joints, or the fibrous tissues connecting them, and causes symptoms such as headaches, tenderness in the masticatory muscles, and clicking sounds in the jaw joints. It is said that when the number of patients with certain symptoms in the jaw joints is estimated, there are about 19 million people, so there is a desire to develop a treatment method for it.

[0003] Temporomandibular joint disorder syndrome may cause displacement or damage of cartilage in the joint. If cartilage can be regenerated, it can be effectively treated. In addition, cartilage is a connective tissue composed of chondrocytes and the matrix surrounding the chondrocytes, and is present in large quantities at the bone-to-bone connection site, such as the joint. In these joints, there are also many diseases caused by damage to the cartilage, and it is expected that if cartilage can be regenerated, it will be a useful option for treating these diseases. Prior art literature Patent Literature

[0004] Patent Document 1: Japanese Patent No. 5655179 Non-patent literature

[0005] Non-patent document 1: Mayo Clin Proc. 83, (2008) p1032-1045 Non-patent document 2: Journal of Pharmacological Sciences 131 (2016) p37-50. Non-patent literature 3: Nature review immunology, 2008, p.727 Summary of the invention Problems to be solved by the invention

[0006] An object of the present invention is to provide a novel agent for inducing proliferation or differentiation of chondrocytes, comprising as an active ingredient a compound whose ability to induce proliferation or differentiation of chondrocytes has been unknown so far. Solutions for solving problems

[0007] Under such circumstances, the present inventors have studied various compounds and unexpectedly found that the compound represented by the general formula (1) or a salt thereof described below has the ability to induce proliferation or differentiation of chondrocytes. The present invention has been accomplished based on this new finding.

[0008] Therefore, the present invention provides the following: Item 1. A chondrocyte proliferation or differentiation inducer comprising a compound represented by the general formula (1) or a salt thereof. [Chemistry 1] [In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group. R2 represents hydrogen or hydroxyl. n represents an integer from 0 to 3.]

[0009] Item 2. A preventive or therapeutic agent for a disease, which can treat the disease by inducing the proliferation or differentiation of chondrocytes, comprising a compound represented by the general formula (1) or a salt thereof. [Chemistry 2] [In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group. R2 represents hydrogen or hydroxyl. n represents an integer from 0 to 3.]

[0010] Item 3. The preventive or therapeutic agent according to Item 2, wherein the disease that can be treated by inducing the proliferation or differentiation of chondrocytes is osteoarthritis, inflammatory arthritis, cartilage damage, congenital cartilage defects, reduction of articular cartilage due to aging, or chondrodystrophy.

[0011] Item 4. The preventive or therapeutic agent according to Item 2 or 3, which is administered systemically or locally.

[0012] Item 5. The chondrocyte proliferation or differentiation inducer according to Item 1, or the preventive or therapeutic agent according to any one of Items 2 to 4, wherein the compound represented by the general formula (1) or a salt thereof is [4-(methylthio)phenylthio]-methanebisphosphonic acid or a salt thereof.

[0013] Item 6. A method for inducing proliferation or differentiation of chondrocytes, comprising the step of allowing a compound represented by the general formula (1) or a salt thereof to act on mesenchymal stem cells and / or chondrogenic progenitor cells in vitro. [Chemistry 3] [In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group. R2 represents hydrogen or hydroxyl. n represents an integer from 0 to 3.] Item 7. Use of a compound represented by the general formula (1) or a salt thereof for preparing a preventive or therapeutic agent for a disease that can be treated by inducing proliferation or differentiation of chondrocytes. [Chemistry 4] [In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group. R2 represents hydrogen or hydroxyl. n represents an integer from 0 to 3.]

[0014] Item 8. The use according to Item 7, wherein the disease that can be treated by inducing the proliferation or differentiation of chondrocytes is osteoarthritis, inflammatory arthritis, cartilage damage, congenital cartilage defects, reduction of articular cartilage due to aging, or chondrodystrophy.

[0015] Item 9. The use according to Item 7 or 8, which is for systemic administration or local administration.

[0016] Item 10. The chondrocyte proliferation or differentiation inducer according to Item 1, or the use according to any one of Items 7 to 9, wherein the compound represented by the general formula (1) or a salt thereof is [4-(methylthio)phenylthio]-methanebisphosphonic acid or a salt thereof.

[0017] Item 11. A compound represented by the general formula (1) or a salt thereof for use in preventing or treating a disease that can be treated by inducing proliferation or differentiation of chondrocytes. [Chemistry 5] [In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group. R2 represents hydrogen or hydroxyl. n represents an integer from 0 to 3.]

[0018] Item 12. The compound or salt thereof for use according to Item 11, wherein the disease that can be treated by inducing proliferation or differentiation of chondrocytes is osteoarthritis, inflammatory arthritis, cartilage damage, congenital cartilage defects, age-related decrease in articular cartilage, or chondrodystrophy.

[0019] Item 13. The compound or salt thereof for use according to Item 11 or 12, which is administered systemically or topically.

[0020] Item 14. A chondrocyte proliferation or differentiation inducer according to Item 1, or a compound or a salt thereof for use according to any one of Items 11 to 13, wherein the compound or a salt thereof represented by the general formula (1) is [4-(methylthio)phenylthio]-methanebisphosphonic acid or a salt thereof. Effects of the Invention

[0021] According to the present invention, by using a compound whose effect on the proliferation or differentiation of chondrocytes is unknown so far, i.e., a compound represented by the general formula (1) or a salt thereof as an active ingredient, a novel chondrocyte proliferation or differentiation inducer can be provided. In addition, according to the present invention, by using a compound represented by the general formula (1) or a salt thereof as an active ingredient, a novel preventive or therapeutic agent for a disease that can be treated by inducing the proliferation or differentiation of chondrocytes can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photograph of the hematoxylin-eosin-stained tissue of the mandibular cartilage of Example 1. The thickness of the cartilage layer (chondrocyte layer + hypertrophic chondrocyte layer) increased by the administration of MPMBP. F: fibrous layer, P: proliferating cell layer, C: chondrocyte layer, H: hypertrophic chondrocyte layer Figure 2 The results of histomorphometric measurement showing the effects of OVX and subsequent MPMBP administration on mandibular cartilage in Example 1. MPMBP administration increased the thickness of the cartilage layer (chondrocyte layer + hypertrophic chondrocyte layer). F: fibrous layer, P: proliferative cell layer, C: chondrocyte layer, H: hypertrophic chondrocyte layer Figure 3 The tissue image of the mandibular cartilage stained with toluidine blue in Example 1 is shown. In the cartilage layer (hypertrophic chondrocyte layer) with positive toluidine blue staining that was reduced by OVX, the intensity of toluidine blue staining increased in a dose-dependent manner by administering MPMBP, and the thickness of the stained cartilage layer increased. F: fibrous layer, P: proliferating cell layer, C: chondrocyte layer, H: hypertrophic chondrocyte layer Figure 4 The results of evaluating the effects of OVX and subsequent MPMBP administration in the mandibular cartilage matrix using toluidine blue stained sections in Example 1 are shown. The thickness of the toluidine blue-stained positive cartilage layer (hypertrophic chondrocyte layer) that was reduced by OVX was increased by MPMBP administration. F: fibrous layer, P: proliferating cell layer, C: chondrocyte layer, H: hypertrophic chondrocyte layer Figure 5 The results of immunostaining in Example 1 are shown. The chondrocyte layer of mandibular chondrocytes of OVX rats proliferated by administration of MPMBP. Figure 6 The results of the proliferation test of ATDC5 cells to which various compounds were added in Example 2 are shown. MPMBP significantly caused the cultured ATDC5 chondrocytes to proliferate. Figure 7 The results of the survival activity test of ATDC5 cells to which various compounds were added in Example 2 are shown. MPMBP increased the survival activity of cultured ATDC5 chondrocytes. Figure 8 The results of toluidine blue staining of the tibia in Example 3 are shown. The toluidine blue-positive cartilage layers of the articular cartilage and growth plate cartilage of the tibia increased by the MPMBP treatment. Fig. 9 The results of immunohistochemical staining in Example 3 are shown. PCNA-positive cells indicating proliferation were observed in the articular cartilage and growth plate cartilage of the tibia of OVX rats to which MPMBP was administered. DETAILED DESCRIPTION

[0023] In the present specification, singular forms (a, an, the, etc.) include the singular and the plural, unless otherwise explicitly stated in the present specification or there is a clear contradiction in the context. Disease preventive or therapeutic agent The present invention provides a preventive or therapeutic agent for a disease that can be treated by inducing proliferation or differentiation of chondrocytes, comprising or a salt thereof. [Chemistry 6] [In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group. R2 represents hydrogen or hydroxyl. n represents an integer from 0 to 3.] In the present invention, the compound represented by the above general formula (1) may be simply referred to as compound (1).

[0024] The compound (1) as the active ingredient of the present invention is known or can be produced using a known compound as a raw material. In the present invention, examples of hydrocarbon groups include alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, etc.), alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl, etc.), alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, etc.), cycloalkyl groups (e.g., cyclopropyl, cyclobutyl, cyclopentyl, etc.), and the like. The hydrocarbon group represented by R1 is C1 to C5, preferably C1 to C3, and more preferably C1. In the case where the hydrocarbon group is substituted, the substituent is not limited, and examples thereof include hydroxyl, halogen, alkoxy, acetyl, and the like. In the case where the hydrocarbon group represented by R1 has a substituent, the number thereof is not limited, and examples thereof include 1 to 3, and preferably 1. In the present invention, examples of the alkoxy group include alkoxy groups whose alkyl moieties are linear or branched alkyl groups (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, and the like), and the like, preferably C1 to C5 alkoxy groups, preferably C1 to C3 alkoxy groups, and more preferably methoxy groups. In the present invention, examples of the halogen include fluorine, chlorine, bromine, iodine, and the like. As the compound (1) or a salt thereof in the present invention, tetrahydro-4-methylthiophenylthiomethane bisphosphonate or a salt thereof is preferred. Tetrahydro-4-methylthiophenylthiomethane bisphosphonate is a known substance having the following structure.

[0025] [Chemistry 7]

[0026] As the salt of the compound (1) as the active ingredient of the present invention, for example, salts with bases can be mentioned. Specific examples of salts with bases include alkali metal or alkaline earth metal salts such as sodium salts, potassium salts or calcium salts. In the present invention, salts with bases such as alkali metal salts are preferred, and sodium salts are more preferred. In the present invention, disodium salts are preferred among the sodium salts, and disodium [4-(methylthio)phenylthio]-methanebisphosphonate (abbreviated as MPMBP) is particularly preferred. [Chemistry 8]

[0027] The compound (1) and a salt thereof which is an active ingredient of the present invention may exist in the form of a hydrate or a solvate. Therefore, these hydrates and solvates are also included in the compound which is an active ingredient of the present invention.

[0028] Examples of the solvent for forming a solvate include alcohols such as ethanol and propanol, organic acids such as acetic acid, esters such as ethyl acetate, ethers such as tetrahydrofuran and diethyl ether, ketones such as acetone, DMSO, etc. These solvents may be used alone or in combination of two or more thereof.

[0029] Diseases that can be treated by inducing the proliferation or differentiation of chondrocytes include, for example, arthritis such as osteoarthritis and inflammatory arthritis; cartilage defects that are congenital or caused by trauma or inflammation, etc. Examples of cartilage include cartilage of the nose, ear, jaw, knee, buttocks, elbows, shoulders, hands, feet, spine, etc. In addition, examples of arthritis include arthritis of the jaw, knee, buttocks, elbows, etc., typically temporomandibular joint disorder syndrome, knee arthritis, etc. In addition, in this specification, "the preventive or therapeutic agent for a disease that can be treated by inducing the proliferation or differentiation of chondrocytes of the present invention" is sometimes referred to as "the preventive or therapeutic agent of the present invention".

[0030] In the present invention, the compound (1) or its salt as an active ingredient can be used as the preventive or therapeutic agent of the present invention, or it can be used as a composition in combination with various carriers acceptable for pharmaceutical and food applications (e.g., isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, thickeners, etc.).

[0031] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol, polyols such as glycerol, polyethylene glycol, and propylene glycol, and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonic agents may be used alone or in combination of two or more.

[0032] Examples of the chelating agent include ethylenediaminetetraacetic acid salts such as disodium ethylenediaminetetraacetate, disodium calcium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, and calcium ethylenediaminetetraacetate, ethylenediaminetetraacetic acid salts, nitrilotriacetic acid or its salts, sodium hexametaphosphate, citric acid, etc. These chelating agents may be used alone or in combination of two or more.

[0033] As a stabilizer, sodium hydrogen sulfite etc. are mentioned, for example.

[0034] Examples of the pH adjuster include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, and further examples include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as tromethamine. These pH adjusters may be used alone or in combination of two or more.

[0035] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoates such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, quaternary ammonium salts such as chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, and cetylpyridinium chloride, alkyl polyaminoethyl glycine, chlorobutanol, polyquaternium salts, polyhexamethylene biguanide, chlorhexidine, etc. These preservatives may be used alone or in combination of two or more.

[0036] Examples of the antioxidant include sodium hydrogen sulfite, dried sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherols, etc. These antioxidants may be used alone or in combination of two or more.

[0037] Examples of the solubilizer include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropylmethylcellulose, polyvinyl pyrrolidone, polyethylene glycol, and D-mannitol. These solubilizers may be used alone or in combination of two or more.

[0038] Examples of the thickener include polyethylene glycol, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, xanthan gum, sodium chondroitin sulfate, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl pyrrolidone, polyvinyl alcohol, etc. These thickeners may be used alone or in combination of two or more.

[0039] Furthermore, in a preferred embodiment, the composition of the present invention can be used in combination with a method for preventing or treating a disease that can be treated by inducing proliferation or differentiation of chondrocytes, for example, the following method. (1) Anti-inflammatory analgesics and / or steroid preparations For example, in combination with diclofenac, naproxen, loxoprofen, etodolac, meloxicam, celecoxib, steroids, amitriptyline, pregabalin, etc. (2) Cartilage regeneration therapy and / or cartilage transplantation therapy Used in combination with cartilage regeneration therapy, transplantation, etc., such as filling of defect parts with chondrocytes and cartilage matrix, or a molded carrier containing them (3) Proliferation factors, growth factors, cytokines and / or matrix proteins acting on chondrocytes For example, combined use with TGFβ superfamily, BMP, IGF, VEGF, CNP, FGF, proteoglycan, type 2 collagen, PTHrP, etc. (4) Inhibitors of tissue destruction For example, it can be used in combination with anti-rheumatic drugs, immunosuppressants, biological agents (anti-TNFα monoclonal antibodies, fusion proteins of soluble TNFα receptor and human IgG), JAK inhibitor drugs, etc. used in severe inflammation, rheumatism, etc. (5) Drugs for the treatment of osteoporosis For example, it can be used in combination with denosumab, teriparatide, active vitamin D3 preparations, selective estrogen receptor agonists, romosuzumab, etc. Therefore, the composition of the present invention may further contain the above-mentioned components for combined use in addition to compound (1) or a salt thereof. When these components are used, they may be used alone or in combination of two or more.

[0040] In an embodiment of the composition, the content of compound (1) or a salt thereof in the composition is not particularly limited, and can be appropriately set, for example, from conditions such as 90% by mass or more, 70% by mass or more, 50% by mass or more, 30% by mass or more, 10% by mass or more, 5% by mass or more, or 1% by mass or more.

[0041] The preventive or therapeutic agent of the present invention can be used in the field of medicine. In addition, the preventive or therapeutic agent of the present invention can be used as a food additive; health food (functional food, nutritional supplement food (supplement, etc.), health supplement food, nutritionally fortified food, nutritionally formulated food, etc.); special purpose food, health functional food (specific health food, nutritional functional food), etc. In this specification, health food, special purpose food, health functional food, etc. are sometimes collectively referred to as health food, etc.

[0042] In the embodiment of medical use, the preparation form is not particularly limited, and examples thereof include oral administration agents such as tablets, pills, capsules, powders, granules, and syrups; injections (intravenous injection, local injection, intramuscular injection, etc.), drips, external preparations (gels, ointments, creams, patches, inhaled medicines), gargles, suppositories, and other non-oral administration agents. Among the above-mentioned preparation forms, as preferred forms, for example, oral administration agents such as tablets for systemic administration, injections for local administration, etc. can be listed.

[0043] In this embodiment, the dosage of compound (1) or its salt varies according to the route of administration, the age, weight, symptoms of the patient, etc., and cannot be uniformly specified. The daily dosage of an adult is generally about 5000 mg or less, preferably about 1000 mg or less. The lower limit of the dosage of compound (1) or its salt is not particularly limited. For example, as the dosage of compound (1), the daily dosage of an adult can be appropriately set within the range of 0.1 mg or more, preferably 0.5 mg or more. In the case of once daily administration, the amount can be contained in one preparation, and in the case of three daily administrations, one third of the amount can be contained in one preparation.

[0044] In medical use, the preventive or therapeutic agent of the present invention is administered to mammals, etc. Examples of mammals include humans, monkeys, mice, rats, rabbits, cats, dogs, pigs, cattle, horses, sheep, and the like.

[0045] In the use of health food, for example, foods include dairy products such as yogurt and milk drinks; soups; jellies; fruit juices; coffee drinks; tea drinks; cookies; rice crackers; cakes, etc. The content of compound (1) or a salt thereof in food is not particularly limited as long as it is an acceptable amount. In addition, in the use of health food, the preventive or therapeutic agent of the present invention may be in the form of tablets, powders, granules, etc. In the case of adopting such a form, the content of compound (1) or a salt thereof in the tablets, powders, granules, etc. may be appropriately set. In the use of food additives, the preventive or therapeutic agent of the present invention can be added to food to give the food the ability to prevent or treat diseases that can be treated by chondrocyte proliferation or differentiation induction. As described above, compound (1) or its salt itself can be used as the preventive or therapeutic agent of the present invention, or it can be used as a composition combined with various carriers (such as isotonic agents, chelating agents, stabilizers, pH regulators, preservatives, antioxidants, cosolvents, thickeners, etc.). The proportion in the case of being used as a composition is as described above. In addition, as an amount added to food, for example, the content of compound (1) or its salt in food can be set within the range listed in the embodiments of health foods, etc.

[0046] Chondrocyte proliferation or differentiation inducer The compound (1) or its salt as the active ingredient of the present invention exerts the effect of promoting chondrocyte proliferation or differentiation induction. In addition, cartilage tissue can be increased by chondrocyte proliferation or differentiation induction. Therefore, the present invention also provides a chondrocyte proliferation or differentiation inducer, which comprises compound (1) or its salt. In addition, it is known that the compound (1) or its salt as the active ingredient of the present invention has the effect of inhibiting the function of osteoclasts that absorb bone (non-patent document 1). In addition, it is known that in compound (1) or its salt, MPMBP has a bone formation promoting effect in addition to the above-mentioned effect, but the proliferation or differentiation induction effect of chondrocytes is completely unknown. Here, cartilage and bone are tissues present in close parts, but are completely different in function and constituent elements. For example, in terms of function, the difference is that cartilage is a tissue with excellent resistance to mechanical pressure, while bone is a hard tissue with excellent bending resistance. Moreover, as constituent elements, the cell components of cartilage are chondroblasts and chondrocytes, and the cell components of bone are osteocytes, osteoblasts, and osteoclasts. In addition, regarding the matrix (extracellular matrix), the difference between the two is that the collagen fibers in cartilage are mainly type II collagen, while those in bones are mainly type I collagen, and bones contain a large amount of inorganic calcium salts (calcium phosphate, calcium carbonate, etc.).

[0047] In addition, the two differ structurally in that cartilage has basically undeveloped blood vessels and receives nutrients by passive diffusion from the cartilage surface, chondrocytes are dispersed in cavities (cartilage cavities) surrounded by the matrix they produce themselves to maintain the matrix, and the interior of the bone is occupied by bone marrow.

[0048] Mesenchymal stem cells are known to have the ability to differentiate into osteocytes, chondrocytes, adipocytes, etc. (non-patent document 3), and their differentiation is directed by different specific transcription factors. Moreover, as mentioned above, it is known that MPMBP also has a bone formation promoting effect (non-patent document 2). Therefore, by adding MPMBP, etc., differentiation from mesenchymal stem cells to osteoblasts and bone tissue is promoted, and it is expected to inhibit differentiation to cartilage. The inventors unexpectedly found that the proliferation or differentiation of chondrocytes was induced. The compound (1) or its salt in the present invention has an effect on the proliferation or differentiation of chondrocytes that is not expected by the prior art.

[0049] In addition, as the active ingredient, formulation form, usage amount, etc. of the chondrocyte proliferation or differentiation inducer, it can be selected in the same manner as the preventive or therapeutic agent of the present invention. Moreover, the chondrocyte proliferation or differentiation inducer of the present invention can also be added to mesenchymal stem cells and / or chondrogenic cells in vitro to induce chondrocyte proliferation or differentiation. Therefore, the present invention also provides a method for inducing chondrocyte proliferation or differentiation, comprising: a step of causing compound (1) or a salt thereof to act on mesenchymal stem cells and / or chondrogenic cells in vitro.

[0050] As mesenchymal stem cells, cells obtained from umbilical cord blood, bone marrow, etc. can be cited. As chondroprogenitor cells, cells induced by mesenchymal stem cell differentiation, etc. can be cited. As these cells, known cells or cells produced according to known methods, etc. can be cited. As these cells, commercially available cells, etc. can be used. In addition, as these cells, it can be isolated cells, or it can be in the form of cell blocks, tissue sheets, etc. In addition, as these cells, cells from humans, mice, rats, guinea pigs, monkeys, dogs, cats, African clawed frogs, etc. can be listed. The process of making compound (1) or its salt play a role can be performed by adding it to mesenchymal stem cells and / or chondroprogenitor cells. As the amount added, it is not particularly limited, for example, it can be listed in a cell culture medium, and it can be set in the range of 1 to 100 μM, preferably 5 to 15 μM, and more preferably about 10 μM.

[0051] In the method of the present invention, mesenchymal stem cells and / or chondrogenic cells can be used in a state configured in a solution such as a culture medium. As a culture medium, a culture medium commonly used for culturing animal cells can be appropriately used. As the culture medium, for example, DMEM culture medium, αMEM culture medium, RPMI culture medium, BJG culture medium, etc. can be cited. In addition, serum, buffer, growth factor, differentiation factor, stabilizer, etc. can also be appropriately added to the culture medium.

[0052] In addition, in this process, mesenchymal stem cells and / or chondrocyte progenitor cells can be maintained in the presence of compound (1) or a salt thereof. In this embodiment, during the maintenance period, the cells can be left to stand, or they can be shaken or rotated. The holding time is not particularly limited, and for example, it can be set to 12 hours to 10 days, preferably between 36 and 72 hours. The temperature of this process is also not limited, and for example, it can be set to 30 to 40°C, preferably around 37°C. Through this process, chondrocytes differentiate and proliferate from mesenchymal stem cells and / or chondrocyte progenitor cells. The method of the present invention may include a process for recovering chondrocytes differentiated and proliferated through the above process. The recovery process can widely use methods used in the field of cell culture to which the present invention belongs. As methods used in the recovery process, centrifugation, filtration, adsorption, molding into sheets, molding using a 3D printer, etc. can be listed.

[0053] The specific embodiments of the present invention are described in detail below by way of examples, but the present invention is not limited to these examples. Example

[0054] Example 1 Fifty 12-week-old female Sprague Dawley rats underwent ovariectomy (OVX; n = 40) or sham OVX surgery (Sham; n = 10). Twelve weeks after surgery, the OVX rats were divided into four groups and 0 (0.9% NaCl), 1.2 mg / kg, 6.0 mg / kg, 30 mg / kg of MPMBP were administered by subcutaneous injection once a week for 12 weeks. As a control, rats in the Sham group were housed under the same conditions without drug administration, and their mandibular heads were removed at the end of the experiment. Figure 1 The effect of OVX and MPMBP administration on mandibular cartilage is shown. Using sagittal sections of mandibular cartilage stained with hematoxylin-eosin, four cartilage layers were distinguished from the thickest cartilage layer in the central area according to the method of Mizoguchi et al. (2013): fibrous layer (F), proliferative cell layer (P), chondrocyte layer (C), and hypertrophic cell layer (H). Representative histological images of mandibular cartilage hypertrophied in a dose-dependent manner by MPMBP administration are shown. A dose-dependent increase in the number of hypertrophic chondrocytes and cavities was observed. In detail, in the paper of Takizawa et al. (Takizawa A, Chiba M, Ota T, Yasuda M, Suzuki K, Kanemitsu T, Itoh T, Shinoda H, Igarashi K. 2016. The novel bisphosphonate disodium dihydrogen-4-[(methylthio)phenylthio]methanebisphosphonate increases bone mass in post-ovariectomy rats. J Pharmacol Sci. 131 (1): 37-50.), for example, in the 2. Materials and methods in the document, 2.2. Preparation of MPMBP for injection, 2.3 Animals and experimental schedule, etc. are described. The research and OVX schemes were approved by the Animal Research Committee of Tohoku University and the Animal Experiment Committee of SLC Inc., Japan, respectively. Animal husbandry and experiments were carried out in accordance with the guidelines of the Animal Experiment Facility of the Graduate School of Medicine, Tohoku University.

[0055] for Figure 1 The effects of OVX and subsequent MPMBP administration on mandibular cartilage were measured by tissue morphology. Figure 2 shown. There was no significant difference in the thickness of the mandibular cartilage between the sham group and the OVX+0 group. However, MPMBP significantly increased the combined thickness of the chondrocyte layer and the hypertrophic cell layer (OVX+6, OVX+30), resulting in significant hypertrophy of the mandibular cartilage. This indicates that MPMBP can dose-dependently cause hypertrophy of the cartilage layer, which helps repair and protect the mandibular cartilage in the ovariectomized rat model.

[0056] Figure 3 This is a tissue image of mandibular cartilage stained with toluidine blue. Figure 3 Starting from the top, magnifications are x4, x10, and x20. For OVX rats, MPMBP was administered at 0 mg / kg / week (OVX+0), 1.2 mg / kg / week (OVX+1.2), 6 mg / kg / week (OVX+6), or 30 mg / kg / week (OVX+30). Tissue image of the central sagittal section of the thickest part of the mandibular head: fibrous layer (F), proliferative cell layer (P), chondrocyte layer (C) and hypertrophic chondrocyte layer (H), subchondral bone (SC). Toluidine blue staining positive cartilage matrix is ​​shown. By administering MPMBP, an increase in the intensity of toluidine blue staining was observed in the cartilage layer (chondrocyte layer + hypertrophic chondrocyte layer) reduced by OVX, and an increase in the thickness of the stained cartilage layer. Scale bar = 100um. The above shows that by administering MPMBP, the proteoglycan-rich mandibular cartilage matrix is ​​increased. In the mandibular cartilage matrix of OVX rats, a lower staining intensity of toluidine blue was observed, and a decrease in proteoglycan expression was observed (OVX+0). Then, MPMBP increased the proteoglycan expression in the mandibular cartilage matrix, and its staining intensity was greater than that observed in sham-operated rats (Sham). (OVX+1.2, OVX+6, OVX+30). In particular, the toluidine blue-positive matrix was confined to the hypertrophic chondrocyte layer of the mandibular cartilage.

[0057] Next, the sections were stained with toluidine blue to evaluate the effects of OVX and subsequent MPMBP administration in the mandibular cartilage matrix. Figure 4 As shown. The area of ​​toluidine blue-positive cartilage matrix (circled by dotted circle) was measured with a width of 500um. MPMBP significantly increased the thickness of toluidine blue-positive proteoglycan-rich mandibular cartilage matrix in a dose-dependent manner (OVX+6, OVX+30). These findings indicate that MPMBP significantly enhances the proteoglycan expression of mandibular cartilage matrix that is reduced after OVX, and the thickness of the proteoglycan-rich mandibular cartilage matrix is ​​proportional to the dose-dependent increase of MPMBP.

[0058] Proliferating Cell Nuclear Antigen (PCNA) is a factor that promotes the activity of DNA polymerase ▲f▼Β. Immunostaining to detect this protein can prove that cells are proliferating. Anti-PCNA antibody was used as the primary antibody for immunohistochemistry, and red fluorescence was detected using a secondary antibody fluorescently labeled with Alexa 555. All samples were stained under the same conditions, and photos were taken using a fluorescence microscope under the same conditions. The results are shown in the figure. Figure 5 shown. PCNA is present in the nucleus of the cell of propagation, so the nucleus is stained. In the bone marrow (Bone marrow), proliferating cells are more, and a large number of positive cells that emit red fluorescence in PCNA immunostaining are observed in the bone marrow. By MPMBP administration, PCNA positive proliferating cells are observed in the chondrocyte layer. On the other hand, in the fibrous layer, proliferative layer, hypertrophic cartilage layer, do not observe. In the chondrocyte layer of the mandibular head chondrocyte of the rat administered with MPMBP, PCNA positive cells representing propagation are observed.

[0059] Example 2 5x10 5 ATDC5 cells were cultured at a cell density of 10 cells / 35 mm culture dish. As the culture medium, DMEM-Ham's F12 medium containing 5% FBS and 1% penicillin-streptomycin mixed solution (Pen / Strep) was used. The final concentrations were adjusted to 10 μM (M10) and 100 μM (M100) for MPMBP, 10 μM (C10) and 100 μM (C100) for clodronate, 10 μM (Z10) and 100 μM (Z100) for zoledronate, and control (Vehicle). The cells were cultured under the conditions of 5% CO2 and 37°C, and the culture medium was replaced every three days. The results are shown in Figure 6 shown. On the third day of culture, the cell numbers of BP-treated cells were all less than those of the control, decreased in Z10 and Z100, but increased in C10, C100, M10, and M100 treatments. On the sixth day of culture, Z10 and Z100 decreased significantly compared with the control, C10, C100, M10, and M100, and especially increased in the M10 and M100 treatments.

[0060] Next, 5x10 5 ATDC5 cells were cultured at a cell density of 10 cells / 35 mm culture dish. As the culture medium, DMEM-Ham's F12 medium containing 5% FBS and 1% penicillin-streptomycin mixed solution (Pen / Strep) was used. The final concentrations were adjusted to 0.1 μM (M0.1), 1 μM (M1), 10 μM (M10) and 100 μM (M100) of MPMBP, and the control (Vehicle) was cultured under the conditions of 5% CO2 and 37°C, and the culture medium was replaced every three days. After three days of culture or six days of culture, the Alamar Blue assay was performed. After incubation for three hours at 37°C and 5% CO2, the assay was performed with an excitation wavelength of 544 nm and a measurement wavelength of 590 nm. The results are as shown in Figure 7 shown. On the third day of culture, the survival activity of cells was increased by treatment with M10 and M100. On the sixth day of culture, the survival activity of cells was increased by treatment with M0.1, M1, M10, and M100.

[0061] Example 3 Female SD rats aged 12 weeks were subjected to ovariectomy (OVX) or sham surgery (Sham), and were administered MPMBP once a week (1.2, 6, 30 mg / kg / week, or 0.9% NaCl (OVX) as a control group) during 12 months from 24 weeks to 36 weeks of age. They were killed at 36 weeks of age, and the removed tibia was stained with toluidine blue as reported. The results are shown in Figure 8 shown. In the tibia, no significant increase in the toluidine blue-positive cartilage layer was found, as in the mandibular cartilage. However, an increase in the cartilage layer was found in both the articular cartilage and the growth plate cartilage of the tibia. This difference is believed to be due to the fact that the mandibular cartilage is a non-load-bearing bone and is not subjected to pressure at rest, so the mechanical environment is different from the tibia that bears weight. In addition, during the production process, the mandibular cartilage is a cartilage produced in the joint part of the mandible, and is formed through the process of membranous ossification, while the tibia is a bone formed through the process of cartilaginous ossification, so it is believed that their reactivity may be different. This shows that both cartilage tissues have increased. PCNA: Proliferating Cell Nuclear Antigen (PCNA) Immunohistochemical staining was performed using anti-PCNA antibody as the primary antibody, and red fluorescent staining was performed using a secondary antibody fluorescently labeled with Alexa 555. Nuclei: Nuclei were stained with blue fluorescence using DAPI. Merge: Overlay of PCNA staining and DAPI staining. All samples were stained under the same conditions and photographed using a fluorescence microscope under the same conditions. Fig. 9 shown. PCNA is present in the nucleus of the proliferated cells, so the nucleus is stained. Proliferating cells are more in the bone marrow, and a large number of positive cells emitting red fluorescence in PCNA immunostaining are observed in the bone marrow. By MPMBP administration (1.2mg / kg / week), PCNA-positive proliferating cells are observed in the chondrocyte layer of the surface layer of tibial articular cartilage and the growth plate cartilage. On the other hand, it is not observed in the cartilage layer of OVX rats. It shows that by MPMBP administration, the chondrocytes of the articular cartilage and the growth plate cartilage of the tibia proliferate. Industrial Applicability

[0062] According to the present invention, compound (1) can promote the proliferation or differentiation induction of chondrocytes by using its salt. Therefore, the present invention can be used to prevent or treat diseases that can be treated by the proliferation or differentiation induction of chondrocytes. In addition, the present invention can also be used for experimental research on chondrocytes, etc.

Claims

1. A chondrocyte proliferation or differentiation inducing agent, comprising a compound represented by general formula (1) or a salt thereof, [Chemistry 1] In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group, R2 represents hydrogen or hydroxyl, and n represents an integer from 0 to 3.

2. A preventive or therapeutic agent for a disease, which can treat the disease by inducing the proliferation or differentiation of chondrocytes, comprising a compound represented by the general formula (1) or a salt thereof, [Chemistry 2] In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group, R2 represents hydrogen or hydroxyl, and n represents an integer from 0 to 3.

3. The preventive or therapeutic agent according to claim 2, wherein Diseases that can be treated by inducing proliferation or differentiation of chondrocytes are osteoarthritis, inflammatory arthritis, cartilage damage, congenital cartilage defects, reduction of articular cartilage due to aging, or chondrodystrophy.

4. The preventive or therapeutic agent according to claim 2 or 3, which is administered systemically or locally.

5. The chondrocyte proliferation or differentiation inducing agent according to claim 1, or the preventive or therapeutic agent according to claim 2 or 3, wherein The compound represented by the general formula (1) or a salt thereof is [4-(methylthio)phenylthio]-methanebisphosphonic acid or a salt thereof.

6. A method for inducing proliferation or differentiation of chondrocytes, comprising: a step of allowing the compound represented by the general formula (1) or a salt thereof to act on mesenchymal stem cells and / or chondroprogenitor cells in vitro, [Chemistry 3] In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group, R2 represents hydrogen or hydroxyl, and n represents an integer from 0 to 3.

7. Use of a compound represented by general formula (1) or a salt thereof for preparing a preventive or therapeutic agent for a disease that can be treated by inducing proliferation or differentiation of chondrocytes, [Chemistry 4] In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group, R2 represents hydrogen or hydroxyl, and n represents an integer from 0 to 3.

8. A compound represented by the general formula (1) or a salt thereof for use in preventing or treating a disease that can be treated by inducing proliferation or differentiation of chondrocytes, [Chemistry 5] In the formula, R1 represents an optionally substituted C1-C5 hydrocarbon group, R2 represents hydrogen or hydroxyl, and n represents an integer from 0 to 3.

Images