Agents that induce the proliferation or differentiation of cartilage cells

Abstract

To provide a novel agent for inducing the proliferation or differentiation of chondrocytes, which contains, as an active ingredient, a compound that was previously unknown to have the capability of inducing the proliferation or differentiation of chondrocytes.SOLUTION: The present invention provides an agent for inducing the proliferation or differentiation of chondrocytes, which comprises a compound (1) or a salt thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a chondrocyte proliferation or differentiation inducing agent, and a method for inducing chondrocyte proliferation or differentiation.

Background Art

[0002] Temporomandibular disorder is a disease caused by problems in the jaw muscles or joints, or the fibrous tissues connecting them, and symptoms such as headache, tenderness of masticatory muscles, and clicking sound of the jaw joint appear. It is said that the estimated number of patients with some symptoms in the temporomandibular joint reaches as many as about 19 million, and development of a treatment method therefor is desired.

[0003] In temporomandibular disorder, displacement or damage of cartilage in the joint may occur, and regeneration of cartilage, if achievable, is effective for the treatment of temporomandibular disorder. Further, cartilage is a connective tissue composed of chondrocytes and a matrix surrounding the chondrocytes, and is abundantly present at sites where bones are connected to each other like joints. Many diseases caused by cartilage damage and the like also exist in these joints and the like, and it is expected that regeneration of cartilage, if achievable, can be a useful option for treatment of those diseases.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0006] The present invention aims to provide a novel chondrocyte proliferation or differentiation inducer containing a compound as an active ingredient whose ability to induce chondrocyte proliferation or differentiation has not been previously known. [Means for solving the problem]

[0007] Under these circumstances, the inventors investigated a wide variety of compounds and, as a result, unexpectedly discovered that a compound represented by general formula (1), described later, or a salt thereof, possesses the ability to induce the proliferation or differentiation of chondrocytes. The present invention is based on this new finding.

[0008] Accordingly, the present invention provides the following: Item 1. Chondrocyte proliferation or differentiation inducer containing a compound represented by general formula (1) or a salt thereof. [ka] [In the formula, R1 represents an optional C1-C5 hydrocarbon group. R2 represents a hydrogen or hydroxyl group. n represents an integer from 0 to 3.]

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

[0010] Item 3. The preventive or therapeutic agent according to Item 2, wherein the disease treatable by proliferation or induction of differentiation of chondrocytes is osteoarthritis, inflammatory arthritis, cartilage injury, congenital cartilage defect, age-related reduction in articular cartilage, or chondrodystrophy.

[0011] Item 4. The preventive or therapeutic agent according to Item 2 or 3, which is for systemic administration or local administration.

[0012] Item 5. The chondrocyte proliferation or differentiation-inducing agent according to Item 1, or the preventive or therapeutic agent according to any one of Items 2 to 4, wherein the compound represented by 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 the compound represented by general formula (1) or a salt thereof to act on mesenchymal stem cells and / or cartilage progenitor cells in vitro

Chemical Formula

Effects of the Invention

[0014] According to the present invention, a novel chondrocyte proliferation or differentiation-inducing agent can be provided by using, as an active ingredient, the compound represented by general formula (1) or a salt thereof, which has not been previously known to have an effect on chondrocyte proliferation or differentiation. Further, according to the present invention, a novel preventive or therapeutic agent for diseases treatable by proliferation or induction of differentiation of chondrocytes can be provided by using the compound represented by general formula (1) or a salt thereof as an active ingredient.

Brief Description of the Drawings

[0015] [Figure 1]This shows a histological photograph of the mandibular condyle cartilage stained with hematoxylin-eosin in Example 1. The administration of MPMBP increased the thickness of the cartilage layer (chondrocyte layer + hypertrophic chondrocyte layer). F: Fibrous layer, P: Proliferating cell layer, C: Chondrocyte layer, H: Hypertrophic chondrocyte layer [Figure 2] This shows the histomorphometric measurement results regarding the effects of OVX and subsequent MPMBP administration on the mandibular condyle cartilage in Example 1. The administration of MPMBP increased the thickness of the cartilage layer (chondrocyte layer + hypertrophic chondrocyte layer). F: Fibrous layer, P: Proliferating cell layer, C: Chondrocyte layer, H: Hypertrophic chondrocyte layer [Figure 3] This shows the histological image of the mandibular condyle cartilage stained with toluidine blue in Example 1. The toluidine blue-stain positive cartilage layer (hypertrophic chondrocyte layer), which was reduced by OVX, showed a dose-dependent increase in toluidine blue staining intensity and an increase in the thickness of the stained cartilage layer after MPMBP administration. F: Fibrous layer, P: Proliferating cell layer, C: Chondrocyte layer, H: Hypertrophic chondrocyte layer [Figure 4] This shows the results of evaluating the effects of OVX and subsequent MPMBP administration on the mandibular condyle cartilage matrix using toluidine blue-stained sections in Example 1. The administration of MPMBP increased the thickness of the toluidine blue-stain positive cartilage layer (hypertrophic chondrocyte layer) that had been reduced by OVX. F: Fibrous layer, P: Proliferating cell layer, C: Chondrocyte layer, H: Hypertrophic chondrocyte layer [Figure 5] This shows the results of immunostaining in Example 1. The administration of MPMBP promoted the proliferation of the chondrocyte layer of mandibular condyle chondrocytes in OVX rats. [Figure 6] This shows the results of a proliferation test of ATDC5 cells supplemented with various compounds in Example 2. MPMBP significantly proliferated the cultured chondrocyte ATDC5 cells. [Figure 7] This shows the results of a viability test of ATDC5 cells supplemented with various compounds in Example 2. MPMBP increased the viability of the cultured chondrocyte ATDC5 cells. [Figure 8] This shows the results of toluidine blue staining of the tibia in Example 3. MPMBP treatment increased the toluidine blue-positive cartilage layers of the articular cartilage and growth plate cartilage of the tibia. [Figure 9] The results of immunohistochemical staining in Example 3 are shown. PCNA-positive cells showing proliferation were observed in the articular cartilage and growth plate cartilage of OVX rats administered MPMBP. [Modes for carrying out the invention]

[0016] A preventive or therapeutic agent for disease The present invention provides a preventive or therapeutic agent for diseases that can be treated by inducing the proliferation or differentiation of chondrocytes, or a salt thereof. [ka] [In the formula, R1 represents an optional C1-C5 hydrocarbon group. R2 represents a hydrogen or hydroxyl group. n represents an integer from 0 to 3.] In the present invention, the compound represented by the above general formula (1) may also be simply referred to as compound (1).

[0017] Compound (1), which is the active ingredient of the present invention, is either known or can be produced using known compounds as raw materials. In the present invention, examples of hydrocarbon groups include alkyl groups (e.g., methyl group, ethyl group, n-propyl group, i-propyl group, n-butyl group, i-butyl group, t-butyl group, n-pentyl group, etc.), alkenyl groups (e.g., vinyl group, propenipyr group, butenyl group, pentenyl group, etc.), alkynyl groups (e.g., ethynyl group, propinipyr group, butynyl group, pentynyl group, etc.), and cycloalkyl groups (e.g., cyclopropyl group, cyclobutyl group, cyclopentyl group, etc.). The hydrocarbon group represented by R1 is C1 to C5, preferably C1 to C3, and more preferably C1. When the hydrocarbon group is substituted, the substituent is not limited, but examples include hydroxyl groups, halogens, alkoxy groups, acetyl groups, etc. When the hydrocarbon group represented by R1 has substituents, the number is not limited, but for example, it is 1 to 3, preferably 1. In the present invention, examples of alkoxy groups include alkoxy groups in which the alkyl portion is a linear or branched alkyl group (for example, a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a t-butyl group, an n-pentyl group, etc.), preferably a C1-C5 alkoxy group, preferably a C1-C3 alkoxy group, and more preferably a methoxy group. Examples of halogens in the present invention include fluorine, chlorine, bromine, iodine, etc. In the present invention, compound (1) or a salt thereof is preferably tetrahydrogen-4-methylthiophenylthiomethanebisphosphonate or a salt thereof. Tetrahydrogen-4-methylthiophenylthiomethanebisphosphonate is a known substance having the following structure:

[0018] [ka]

[0019] Examples of salts of compound (1), which is the active ingredient of the present invention, include salts with a base. Specific examples of salts with a base include alkali metal or alkaline earth metal salts such as sodium salt, potassium salt, or calcium salt. In the present invention, salts with a base such as an alkali metal salt are preferred, and sodium salts are even more preferred. In the present invention, among sodium salts, disodium salts are preferred, and disodium dihydrogen[4(methylthio)pheylthio]methanebisphosphonate (abbreviated as MPMBP) is particularly preferred. [ka]

[0020] Since compound (1), which is the active ingredient of the present invention, and its salts may also exist in the form of hydrates or solvates, these hydrates and solvates are also included in the compound that is the active ingredient of the present invention.

[0021] Examples of solvents that form solvates 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, and DMSO. These solvents can be used individually or as a mixture of two or more solvents.

[0022] Diseases that can be treated by the proliferation or differentiation induction of chondrocytes include, for example, osteoarthritis such as osteoarthritis and inflammatory arthritis; and congenital or cartilage defects due to trauma or inflammation. Examples of cartilage include that of the nose, ears, jaw, knees, hips, elbows, shoulders, hands, feet, and spine. Examples of osteoarthritis include osteoarthritis of the jaw, knees, hips, and elbows, and typically include temporomandibular joint disorder and knee osteoarthritis. In this specification, "the preventive or therapeutic agent for diseases that can be treated by the proliferation or differentiation induction of chondrocytes of the present invention" may be simply referred to as "the preventive or therapeutic agent of the present invention."

[0023] In the present invention, the active ingredient compound (1) or its salt itself may be used as the preventive or therapeutic agent of the present invention, or it may be used as a composition in combination with various carriers that are acceptable in pharmaceutical and food applications (for example, isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity modifiers, etc.).

[0024] Examples of isotonic agents include sugars such as glucose, trehalose, lactose, fructose, mannitol, xylitol, and sorbitol; polyhydric alcohols such as glycerin, polyethylene glycol, and propylene glycol; and inorganic salts such as sodium chloride, potassium chloride, and calcium chloride. These isotonic agents can be used individually or in combination of two or more.

[0025] Examples of chelating agents include edetates such as disodium edetate, disodium calcium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate, and citric acid. These chelating agents can be used individually or in combination of two or more.

[0026] Examples of stabilizers include sodium bisulfite.

[0027] Examples of pH adjusting agents include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid, as well as alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, and bases such as trometamol. These pH adjusting agents can be used individually or in combination of two or more.

[0028] Examples of preservatives include sorbic acid, potassium sorbate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, and butyl parahydroxybenzoate, chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, quaternary ammonium salts such as cetylpyridinium chloride, alkyl polyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, and chlorhexidine. These preservatives can be used individually or in combination of two or more.

[0029] Examples of antioxidants include sodium bisulfite, anhydrous sodium sulfite, sodium pyrosulfite, and concentrated mixed tocopherols. These antioxidants can be used individually or in combination of two or more.

[0030] Examples of solubilizers include sodium benzoate, glycerin, D-sorbitol, glucose, propylene glycol, hydroxypropyl methylcellulose, polyvinylpyrrolidone, macrogol, and D-mannitol. These solubilizers can be used individually or in combination of two or more.

[0031] Examples of viscosity-reducing agents include polyethylene glycol, methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, chondroitin sulfate sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. These viscosity-reducing agents can be used individually or in combination of two or more.

[0032] In a preferred embodiment, the composition of the present invention may also be used in combination with methods used for the prevention or treatment of diseases that can be treated by the proliferation or differentiation induction of chondrocytes, such as the following methods. (1) Anti-inflammatory analgesics and steroid preparations For example, in combination with diclofenac, naproxen, loxoprofen, etodocla, meloxicam, celecoquidib, steroids, aminotriptyline, pregabalin, etc. (2) Cartilage regeneration therapy / Cartilage transplantation therapy Combined use with cartilage regeneration therapies such as cartilage regeneration therapy or transplantation, including the replacement of defective areas with cartilage cells and cartilage matrix, or molded carriers containing them. (3) Growth factors, cytokines, and substrate proteins that act on chondrocytes For example, in combination with TGFβ superfamily, BMP, IGF, VEGF, CNP, FGF, proteoglycans, type 2 collagen, PTHrP, etc. (4) Factors that suppress tissue destruction For example, in combination with anti-rheumatic drugs used in cases of severe inflammation or rheumatism, immunosuppressants, biological agents (anti-TNFα monoclonal antibodies, fusion proteins of soluble TNFα receptors and human IgG), and JAK inhibitors. (5) Osteoporosis treatment drugs For example, in combination with denosumab, teriparatide, activated vitamin D3 preparations, selective estrogen receptor agonists, romosozumab, etc. Accordingly, the above-described composition of the present invention may further contain components used in combination with compound (1) or its salt. When these components are used, they can be used individually or in combination of two or more.

[0033] In embodiments of the composition, the content of compound (1) or its salt in the composition is not particularly limited and can be appropriately set 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, 1% by mass or more, etc.

[0034] The preventive or therapeutic agent of the present invention can be used in the field of medicine. Furthermore, the preventive or therapeutic agent of the present invention can be used as a food additive; health foods (functional foods, nutritional supplements, health foods, fortified foods, nutritionally prepared foods, etc.); foods for special dietary uses; foods with health claims (foods for specified health uses, foods with nutritional function claims), etc. In this specification, health foods, foods for special dietary uses, foods with health claims, etc., may be collectively referred to as "health foods, etc."

[0035] In embodiments of pharmaceutical use, the formulation form is not particularly limited and can include various formulation forms such as oral preparations like tablets, pills, capsules, powders, granules, and syrups; and parenteral preparations such as injections (intravenous injection, local injection, intramuscular injection, etc.), drips, topical preparations (gels, ointments, creams, patches, inhalants), gargles, and suppositories. Among the above formulation forms, preferred ones include, for example, oral preparations such as tablets for systemic administration, and injections for local administration.

[0036] In this embodiment, the dosage of compound (1) or its salt varies depending on the route of administration, the patient's age, weight, symptoms, etc., and cannot be specified in general terms. However, the daily dose for adults should be approximately 5000 mg or less, preferably approximately 1000 mg or less. The lower limit of the dosage of compound (1) or its salt is not particularly limited. For example, the dosage of compound (1) for adults can be appropriately set within the range of 0.1 mg or more, preferably 0.5 mg or more, for a daily dose. If administered once a day, this amount should be contained in one formulation. If administered three times a day, one-third of this amount should be contained in one formulation.

[0037] In pharmaceutical applications, 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.

[0038] In applications such as health foods, examples of foods include dairy products such as yogurt and milk beverages; soups; jellies; juices; coffee beverages; tea beverages; cookies; rice crackers; cakes, etc. The content of compound (1) or its salt in the food is not particularly limited as long as it is within an acceptable amount. Furthermore, in applications such as health foods, the preventive or therapeutic agent of the present invention may be in the form of tablets, powders, granules, etc. When taking such a form, the content of compound (1) or its salt in the tablets, powders, granules, etc. can be set as appropriate. In food additive applications, the preventive or therapeutic agent of the present invention can be added to food to impart to the food the ability to prevent or treat diseases that can be treated by inducing the proliferation or differentiation of chondrocytes. As described above, compound (1) or its salt itself may be used as the preventive or therapeutic agent of the present invention, or it may be used as a composition in combination with various carriers (e.g., isotonic agents, chelating agents, stabilizers, pH adjusters, preservatives, antioxidants, solubilizers, viscosity modifiers, etc.). The mixing ratio when used as a composition is as described above. Furthermore, the amount added to food can be set, for example, so that the content of compound (1) or its salt in the food is within the range given in the embodiments of health foods, etc.

[0039] Agents that induce the proliferation or differentiation of cartilage cells Compound (1) or a salt thereof, which is the active ingredient of the present invention, has the effect of promoting the proliferation or inducing the differentiation of chondrocytes. Furthermore, by inducing the proliferation or differentiation of chondrocytes, the amount of cartilage tissue can be increased. Accordingly, the present invention also provides a chondrocyte proliferation or differentiation inducer containing compound (1) or a salt thereof. Furthermore, compound (1) or its salt, which is the active ingredient of the present invention, is known to have the effect of suppressing the function of osteoclasts that resorb bone (Non-Patent Literature 1). In addition, MPMBP, one of compound (1) or its salt, is known to have a bone formation promoting effect in addition to the above effect, but no effect on the proliferation or differentiation induction of chondrocytes is known at all. Here, cartilage and bone are tissues that are located in close proximity, but they are completely different in terms of function and constituent elements. For example, functionally, cartilage is a tissue with excellent resistance to mechanical pressure, while bone is a hard tissue with excellent resistance to flexion. In terms of constituent elements, the cellular components of cartilage are chondroblasts and chondrocytes, while the cellular components of bone are osteocytes, osteoblasts, and osteoclasts. Furthermore, regarding the extracellular matrix, cartilage consists mainly of Type II collagen fibers, while bone consists mainly of Type I collagen, and bone also differs in that it contains a large amount of inorganic calcium salts (calcium phosphate, calcium carbonate, etc.).

[0040] Furthermore, structurally, cartilage differs from bone in several ways: cartilage generally lacks well-developed blood vessels and receives nutrients through passive diffusion from its surface; chondrocytes are scattered within small lumens (cartilage lumens) surrounded by a matrix they themselves produce, maintaining the matrix; and bone is predominantly composed of bone marrow.

[0041] Mesenchymal stem cells are known to have the ability to differentiate into osteocytes, chondrocytes, adipocytes, etc. (Non-Patent Literature 3), and differentiation is directed by different specific transcription factors. Furthermore, as mentioned above, MPMBP is known to have a bone formation promoting effect (Non-Patent Literature 2). Therefore, it was expected that adding MPMBP, etc., would promote differentiation from mesenchymal stem cells to osteoblasts and bone tissue, and suppress differentiation to cartilage. However, the inventors unexpectedly found that it induced the proliferation or differentiation of chondrocytes. The effect of compound (1) or its salt in the present invention on the proliferation or differentiation of chondrocytes is unpredictable from the prior art.

[0042] Furthermore, the active ingredient, formulation form, and amount used of the chondrocyte proliferation or differentiation inducer can be selected in the same way as for the preventive or therapeutic agent of the present invention. In addition, the chondrocyte proliferation or differentiation inducer of the present invention can also induce the proliferation or differentiation of chondrocytes by adding it to mesenchymal stem cells and / or chondrocytes in vitro. Accordingly, the present invention also provides a method for inducing the proliferation or differentiation of chondrocytes, comprising the step of reacting mesenchymal stem cells and / or chondrocytes with compound (1) or a salt thereof in vitro.

[0043] Examples of mesenchymal stem cells include cells obtained from umbilical cord blood, bone marrow, etc. Examples of chondrogenic progenitor cells include those differentiated from mesenchymal stem cells. These cells may be known or prepared according to known methods. Commercially available cells may also be used. Furthermore, these cells may be isolated cells, cell aggregates, tissue fragments, or other forms. Examples of these cells include those derived from humans, mice, rats, guinea pigs, monkeys, dogs, cats, African clawed frogs, etc. The step of treating with compound (1) or its salt can be carried out, for example, by adding it to mesenchymal stem cells and / or chondrogenic cells. The amount added is not particularly limited, but for example, it can be set in the range of 1 to 100 μM, preferably 5 to 15 μM, and more preferably about 10 μM in the cell culture medium.

[0044] In the method of the present invention, mesenchymal stem cells and / or chondrogenic cells may be used in a state where they are placed in a solution such as a culture medium. As the culture medium, any medium commonly used for culturing animal cells can be used as appropriate. Examples of such media include DMEM medium, αMEM medium, RPMI medium, and BJG medium. In addition, serum, buffers, growth factors, differentiation factors, stabilizers, etc. may be added to the culture medium as appropriate.

[0045] Furthermore, in this step, mesenchymal stem cells and / or chondrogenic cells may be held in the presence of compound (1) or a salt thereof. In such embodiments, the holding period may be static, shaken, or rotated. The holding time is not particularly limited, but can be set, for example, between 12 hours and 10 days, preferably between 36 and 72 hours. The temperature in this step is also not limited, but can be set, for example, between 30 and 40°C, preferably around 37°C. Through this step, chondrocytes are differentiated and proliferated from mesenchymal stem cells and / or chondrogenic cells. The method of the present invention may include a step of recovering the chondrocytes differentiated and proliferated by the above step. The recovery step can use a wide range of methods used in the field of cell culture to which the present invention belongs. Methods used in the recovery step include centrifugation, filtration, adsorption, molding into a sheet, molding with a 3D printer, etc.

[0046] The following examples illustrate specific embodiments of the present invention, but the present invention is not limited to these embodiments. [Examples]

[0047] Example 1 Fifty 12-week-old female Sprague-Dawley rats underwent either ovariectomy (OVX; n = 40) or simulating OVX surgery (Sham; n = 10). For 12 weeks post-surgery, the OVX rats were divided into four groups and administered MPMBP at doses of 0 (0.9% NaCl), 1.2, 6.0, and 30 mg / kg once weekly via subcutaneous injection for 12 weeks. As a control, the rats in the Sham group were not administered the drug, were raised under the same conditions, and their mandibular condyles were removed at the end of the experiment. Figure 1 shows the effects of OVX and MPMBP administration to the mandibular condylar cartilage. Using hematoxylin-eosin stained tissue sections of the sagittal section of the mandibular condyle, the thickest cartilage layer in the central region was divided into four cartilage layers: fibrous layer (F), proliferating cell layer (P), chondrocyte layer (C), and hypertrophic cell layer (H), based on the method of Mizoguchi et al (2013). This image shows a typical histological feature of mandibular condylar cartilage thickened in a dose-dependent manner by MPMBP administration. A dose-dependent increase in the number of hypertrophied chondrocytes and empty foveals is observed. Further details are provided in the paper by 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 sections 2. Materials and methods, specifically 2.2. Preparation of MPMBP for injection and 2.3 Animals and experimental schedule. The study and OVX protocol were approved by the Animal Research Committee of Tohoku University and the Animal Experiment Committee of SLC Inc. Japan, respectively. Animal rearing and experiments were conducted in accordance with the guidelines of the Animal Experiment Facility of the Graduate School of Medicine, Tohoku University.

[0048] Histological morphological measurements were performed on the mandibular condylar cartilage shown in Figure 1, and the subsequent effects of MPMBP administration. The results are shown in Figure 2. There was no significant difference in the thickness of the mandibular condyle cartilage between the Sham group and the OVX + 0 group. However, MPMBP significantly increased the total thickness of the chondrocyte layer and hypertrophic cell layer (OVX + 6, OVX + 30), resulting in a substantial thickening of the mandibular condyle cartilage. This suggests that MPMBP induces thickening of the cartilage layer in a dose-dependent manner and may contribute to the repair and protection of the mandibular condyle cartilage in an ovariectomized rat model.

[0049] Figure 3 shows histological images of mandibular condylar cartilage stained with toluidine blue. From top to bottom in Figure 3, the magnifications are x4, x10, and x20. OVX rats were administered MPMBP 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). Histological view of the thickest part of the mandibular condyle in the central sagittal section: fibrous layer (F), proliferating cell layer (P), chondrocyte layer (C), and hypertrophic chondrocyte layer (H), subchondral bone (SC). Toluidine blue-positive cartilage matrix was shown. MPMBP administration increased the intensity of toluidine blue staining and the thickness of the stained cartilage layer in the cartilage layer (chondrocyte layer + hypertrophic chondrocyte layer) that was reduced in OVX. Scale bar = 100 μm. These findings suggest that MPMBP administration increases the proteoglycan-rich cartilage matrix of the mandibular condyle. In OVX rats, low toluidine blue staining intensity was observed in the mandibular condylar matrix, indicating decreased proteoglycan expression (OVX + 0). MPMBP increased proteoglycan expression in the mandibular condylar matrix, staining more strongly than that observed in sham rats (OVX + 1.2, OVX + 6, OVX + 30). In particular, the toluidine blue-positive matrix was localized to the hypertrophic chondrocyte layer of the mandibular condylar cartilage.

[0050] Next, the effects of OVX and subsequent MPMBP administration on the mandibular condylar matrix were evaluated using toluidine blue-stained sections. The results are shown in Figure 4. The area of ​​toluidine blue-positive cartilage matrix (outlined by dashed lines) was measured in 500 μm widths. MPMBP significantly increased the thickness of toluidine blue-positive proteoglycan-rich mandibular condylar matrix in a dose-dependent manner (OVX + 6, OVX + 30). These findings suggest that MPMBP significantly enhances proteoglycan expression in the mandibular condylar matrix, which decreased after OVX, and that the thickness of proteoglycan-rich mandibular condylar matrix is ​​directly proportional to the dose-dependent increase in MPMBP.

[0051] Proliferating cell nuclear antigen (PCNA) is a factor that promotes the activity of DNA polymerase β, and detecting this protein by immunohistochemistry can prove that cells are proliferating. Immunohistochemistry was performed using an anti-PCNA antibody as the primary antibody, and detection was performed using a secondary antibody fluorescently labeled with Alexa555, which induced red fluorescence. All samples were stained under the same conditions and photographed using a fluorescence microscope under the same conditions. The results are shown in Figure 5. PCNA is present in the nucleus of proliferating cells, and therefore stains the nucleus. Bone marrow contains many proliferating cells, and PCNA-positive cells that emit red fluorescence in PCNA immunostaining are frequently observed in the bone marrow. PCNA-positive proliferating cells are observed in the chondrocyte layer after MPMBP administration. On the other hand, they are not observed in the fibrous layer, proliferative layer, or hypertrophic cartilage layer. PCNA-positive cells showing proliferation were observed in the chondrocyte layer of mandibular condylar chondrocytes of rats administered MPMBP.

[0052] Example 2 ATDC5 cells, 5 x 10 5Cells were cultured at a cell density of 35 mm dishes. DMEM-Ham's F12 medium containing 5% FBS and 1% Pen / Strep was used as the culture medium. The final concentrations were adjusted to 10 μM (M10) and 100 μM (M100) of MPMBP, 10 μM (C10) and 100 μM (C100) of clodronate, 10 μM (Z10) and 100 μM (Z100) of zoledronate, and a control (Vehicle). The cells were cultured under conditions of 5% CO2 and 37°C, and the medium was changed every 3 days. The results are shown in Figure 6. On day 3 of culture, the cell count of BP-treated cells was lower than that of the control in all cases. The count decreased in Z10 and Z100, but increased in C10, C100, M10, and M100 treatments. On day 6 of culture, Z10 and Z100 levels were significantly reduced compared to the control, C10, C100, M10, and M100 groups, and were particularly increased in the M10 and M100 treatments.

[0053] Next, ATDC5 cells were divided into 5x10 5 Cells were cultured at a cell density of 35 mm dishes. DMEM-Ham's F12 medium containing 5% FBS and 1% Pen / Strep was used as the culture medium. The final concentrations of MPMBP were adjusted to 0.1 μM (M0.1), 1 μM (M1), 10 μM (M10), and 100 μM (M100), and a control (Vehicle). The cells were cultured under conditions of 5% CO2 and 37°C, and the medium was changed every 3 days. After 3 or 6 days of culture, an Allamer Blue assay was performed. After incubation for 3 hours under conditions of 37°C and 5% CO2, measurements were taken at an excitation wavelength of 544 nm and a measurement wavelength of 590 nm. The results are shown in Figure 7. On day 3 of culture, cell viability increased with M10 and M100 treatments. On day 6 of culture, cell viability increased with M0.1, M1, M10, and M100 treatments.

[0054] Example 3 Female SD rats were subjected to ovariectomy (OVX) or simulated surgery (Sham) at 12 weeks of age. They were fed MPMBP once a week for 12 months from 24 to 36 weeks of age (1.2, 6, 30 mg / kg / week, or 0.9% NaCl (OVX) as a control group). At 36 weeks of age, the rats were sacrificed, and the excised tibias were stained with toluidine blue according to the instructions. The results are shown in Figure 8. The significant increase in toluidine blue-positive cartilage layer seen in the mandibular condyle cartilage is not observed in the tibia. However, an increase in cartilage layer is observed in both the articular cartilage and growth plate cartilage of the tibia. This difference is thought to be due to the fact that the mandibular condylar cartilage is a non-weight-bearing bone and does not experience pressure at rest, resulting in a different mechanical environment compared to the tibia, which bears weight. Furthermore, during development, the mandibular condylar cartilage is cartilage that develops in the articular portion of the mandible, which is formed through the process of membranous ossification, while the tibia is formed through the process of chondrodic ossification, which may explain the difference in responsiveness. It was suggested that both cartilage tissues increased in volume. PCNA (Proliferating Cell Nuclear Antigen) was detected by immunohistochemistry using an anti-PCNA antibody as the primary antibody, followed by red fluorescence staining with a secondary antibody fluorescently labeled with Alexa555. Nuclei: The nuclei were stained with blue fluorescence using DAPI. Merge: PCNA staining and DAPI staining are superimposed. All samples were stained under the same conditions and photographed using a fluorescence microscope under the same conditions. The results are shown in Figure 9. PCNA is present in the nucleus of proliferating cells, causing the nucleus to stain. Bone marrow contains many proliferating cells, and PCNA-positive cells, which emit red fluorescence in PCNA immunostaining, are frequently observed in the bone marrow. MPMBP administration (1.2 mg / kg / week) resulted in the observation of PCNA-positive proliferating cells in the superficial chondrocyte layer of the tibial articular cartilage and in the growth plate cartilage. However, this was not observed in the cartilage layer of OVX rats. This suggests that MPMBP administration induces proliferation of chondrocytes in the tibial articular cartilage and growth plate cartilage. [Industrial applicability]

[0055] According to the present invention, compound (1) can be used to promote the proliferation or induce the differentiation of chondrocytes by using its salt. Therefore, the present invention is useful for the prevention or treatment of diseases that can be treated by the proliferation or differentiation of chondrocytes. Furthermore, the present invention is also useful in the testing and research of chondrocytes and the like.

Claims

1. A chondrocyte proliferation inducer comprising a compound represented by general formula (1) or a salt thereof. 【Chemistry 1】 [In the formula, R １ R represents a C1-C5 hydrocarbon group that may be substituted. ２ [where n represents a hydrogen or hydroxyl group, and n represents an integer from 0 to 3.]

2. A compound represented by general formula (1) or a salt thereof, comprising an agent for the prevention or treatment of osteoarthritis, cartilage damage, or age-related reduction of articular cartilage. 【Chemistry 2】 [In the formula, R １ R represents a C1-C5 hydrocarbon group that may be substituted. ２ [where n represents a hydrogen or hydroxyl group, and n represents an integer from 0 to 3.]

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

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

5. A method for inducing the proliferation of chondrocytes, comprising the step of reacting mesenchymal stem cells and / or chondrocytes with a compound represented by general formula (1) or a salt thereof in vitro. 【Transformation 3】 [In the formula, R １ R represents a C1-C5 hydrocarbon group that may be substituted. ２ [where n represents a hydrogen or hydroxyl group, and n represents an integer from 0 to 3.]

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