Osteoarthritis progression inhibitor and progression inhibitor kit

Abstract

To provide an osteoarthritis progression inhibitor having improved efficacy and an osteoarthritis progression inhibitor kit that inhibit progression of OA.SOLUTION: An osteoarthritis progression inhibitor contains a G protein-coupled receptor kinase 5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof as active ingredients. The G-protein-coupled receptor kinase 5 inhibitor may be amlexanox. The osteoarthritis progression inhibitor may be administered by intra-articular injection. A kit for suppressing progression of osteoarthritis comprises a G protein-coupled receptor kinase 5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for inhibiting the progression of osteoarthritis and a kit for inhibiting the progression of osteoarthritis. [Background technology]

[0002] Osteoarthritis (OA) is a disease that causes cartilage to degenerate and wear down, and knee osteoarthritis (hereinafter sometimes abbreviated as "knee OA") is a typical disease that reduces the quality of life (QOL) of elderly people and threatens their healthy lifespan. As of 2010, the number of symptomatic knee OA patients in Japan was estimated to be 8 million (according to the ROAD (Research on Osteoarthritis Against Disability) project).

[0003] Current drug therapies for OA mainly focus on symptomatic treatment to relieve pain, and hyaluronic acid injections and steroid injections are commonly used clinically (see, for example, Non-Patent Document 1).

[0004] The inventors have previously demonstrated that G protein-coupled receptor kinase 5 (GRK5) is specifically highly expressed in OA cartilage and exacerbates cartilage degeneration due to catabolic reactions mediated by the NF-κB pathway, and further demonstrated that intra-articular administration of a GRK5 inhibitor could be a disease-modifying OA treatment drug that can structurally suppress cartilage degeneration in OA (see, for example, Non-Patent Document 2 and Patent Document 1, etc.). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2020-121957 [Non-patent literature]

[0006] [Non-Patent Document 1] Hiroshi Kawaguchi, "Domestic and International Guidelines for the Treatment of Osteoarthritis," Journal of the Japanese Society of Joint Diseases, Vol. 35, No. 1, pp. 1-9, 2016. [Non-patent document 2] Sueishi T et al., “GRK5 Inhibition Attenuates Cartilage Degradation via Decreased NF-κB Signaling.”, Arthritis and Rheumatology, Vol. 72, No. 4, pp. 620-631, 2020. Summary of the Invention [Problem to be solved by the invention]

[0007] Hyaluronic acid injections have been reported to have anti-inflammatory effects, but they are not a fundamental treatment. Steroid injections are highly effective at suppressing inflammation, but due to side effects, they are only suitable for a limited number of patients and cannot be used frequently. Furthermore, in the treatment methods using GRK5 inhibitors described in Patent Document 1 and Non-Patent Document 2, there is room for improvement in terms of reducing the frequency of use and improving efficacy in order to reduce the burden on patients caused by injections.

[0008] The present invention has been made in view of the above circumstances, and provides an osteoarthritis progression inhibitor and progression inhibition kit that can inhibit the progression of OA and have improved efficacy. [Means for solving the problem]

[0009] As a result of extensive research to achieve the above-mentioned objectives, the inventors discovered that simultaneous administration of hyaluronic acid and a GRK5 inhibitor can significantly suppress the expression of mRNA for interleukin 6 (IL6), a representative inflammatory cytokine involved in the pathogenesis of OA, matrix metalloproteinase 13 (MMP13), a substrate-degrading enzyme, and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), compared to administration of a GRK5 inhibitor alone, and thus completed the present invention.

[0010] That is, the present invention includes the following aspects. (1) An agent for inhibiting the progression of osteoarthritis, comprising as active ingredients a G protein-coupled receptor kinase 5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof. (2) The agent for suppressing the progression of osteoarthritis according to (1), wherein the G protein-coupled receptor kinase 5 inhibitor is amlexanox. (3) The agent for inhibiting the progression of osteoarthritis according to (1) or (2), which is administered by intra-articular injection. (4) a G protein-coupled receptor kinase 5 inhibitor; hyaluronic acid or a pharmaceutically acceptable salt thereof; A kit for inhibiting the progression of osteoarthritis, comprising: [Effects of the Invention]

[0011] The OA progression inhibitor and progression inhibitor kit of the above-mentioned aspects can inhibit the progression of OA, and have improved efficacy. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the relative expression levels of each gene involved in the pathogenesis of OA in human OA chondrocytes in Example 1. [Figure 2A] FIG. 1 shows the administration schedule for OA model mice in Example 2. [Figure 2B]1 shows images of Safranin O-Fast Green stained knee joint tissue sections from each group in Example 2. The scale bar indicates 100 μm. [Figure 2C] 1 is a graph quantifying the severity of OA in each group in Example 2. [Figure 3A] FIG. 1 shows the administration schedule for OA model mice in Example 3. [Figure 3B] 1 shows an image of Safranin O-Fast Green staining of a knee joint tissue section from the group administered amlexanox and hyaluronic acid every 10 days for 8 weeks in Example 3. The scale bar indicates 100 μm. [Figure 3C] 1 is a graph quantifying the severity of OA in each group in Example 3. [Figure 3D] 1 is a graph showing the results of measuring the pain threshold in response to pressure stimulation to the joints of each group in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] <OA progression inhibitor> The OA progression inhibitor of this embodiment contains a GRK5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof as active ingredients.

[0014] In this specification, "containing as an active ingredient" means containing a therapeutically effective amount of each of a GRK5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof.

[0015] As shown in the examples described below, the inventors mixed an GRK5 inhibitor, anlequinox, and hyaluronic acid and allowed them to act on human chondrocytes, thereby finding an effect of synergistically suppressing the gene expression of IL6, an inflammatory cytokine, and MMP13 and ADAMTS4, proteolytic enzymes. Furthermore, by mixing anlequinox and hyaluronic acid and administering them intra-articularly to an animal model, a synergistic effect of structurally suppressing cartilage degeneration in OA was found. This is presumably because the simultaneous administration of anlequinox and hyaluronic acid can slow down the absorption of anlequinox from the joint after intra-articular administration.

[0016] Note that OA (Osteoarthritis) can occur in any joint of the body. In particular, the onset of OA leads to a decline in QOL and is likely to threaten the healthy life span. The joints that play a role in supporting the body and承受 a large body weight load, such as the knee joint, hip joint, spine, etc., are specifically mentioned. They are respectively called osteoarthritis of the knee (knee OA), osteoarthritis of the hip joint, and spondylosis deformans. Among them, the preventive or therapeutic agent for OA of the present embodiment is particularly preferably used for osteoarthritis of the knee. In addition, OA can be divided into primary osteoarthritis and secondary osteoarthritis according to its cause. Primary osteoarthritis is considered to be OA that has developed due to factors such as obesity and aging, although the cause has not been identified. Most cases of osteoarthritis of the knee are primary osteoarthritis. On the other hand, secondary osteoarthritis is OA that develops due to diseases, injuries, etc. Specific examples of the onset causes include, for example, rheumatoid arthritis, gout, fractures, ligament and meniscus injuries, congenital joint structure abnormalities, etc. Most cases of osteoarthritis of the hip joint are secondary osteoarthritis. Among them, the preventive or therapeutic agent for OA of the present embodiment is particularly preferably used for primary osteoarthritis.

[0017] <GRK5 inhibitor> Examples of GRK5 inhibitors include those that inhibit the phosphorylation of IκBα by GRK5. Specific examples of GRK5 inhibitors include, for example, low molecular weight compounds, GRK5 expression inhibitors, GRK5-specific binding substances, etc.

[0018] [Low molecular compound] Examples of low molecular weight compounds that are GRK5 inhibitors include compounds represented by the following general formula (I) (hereinafter, sometimes referred to as "compound (I)"). Compound (I) is an example of a GRK5 inhibitor, but GRK5 inhibitors are not limited to this.

[0019] [ka]

[0020] (In general formula (I), ring A may be substituted. R 11 is a hydrogen atom, an alkyl group, a phenyl group, an amino group, a carboxy group, or a hydroxyl group.

[0021] (Compound (I)) Compound (I) is a 1-azaxanthone-3-carboxylic acid derivative. Compound (I) will be described in detail below.

[0022] ·Ring A The benzene ring represented by ring A may be substituted. Examples of the substituent of ring A include a halogen atom, a nitro group, an amino group, a mono- or di-alkylamino group, an alkyl group, an alkoxy group, a carboxy group, and a hydroxyl group. Furthermore, the substituent may be a butadienylene group (-CH=CH-CH=CH-) in which two adjacent substituents consisting of carbon atoms form a benzene ring, among the substituents present at the 6th, 7th, 8th, or 9th positions of ring A. The alkyl group may be chain-like or cyclic. The chain-like alkyl group may be linear or branched. The chain-like alkyl group preferably has 1 to 6 carbon atoms, and specific examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a t-butyl group, a pentyl group, and a hexyl group. Among these, from a practical standpoint, a lower alkyl group having 1 to 3 carbon atoms is preferred. Examples of the cyclic alkyl group include a cyclopentyl group and a cyclohexyl group. Examples of halogen atoms include chlorine atoms, bromine atoms, iodine atoms, and fluorine atoms. Examples of mono- or dialkylamino groups include amino groups substituted with lower alkyl groups having from 1 to about 3 carbon atoms, such as methylamino, ethylamino, propylamino, dimethylamino, diethylamino, and dipropylamino groups. Examples of the alkoxy group include alkoxy groups in which the alkyl group moiety is an alkyl group having 1 to 4 carbon atoms, such as a methoxy group, an ethoxy group, a propoxy group, and a butoxy group. Ring A may have one or more of these substituents, but it is preferable that it has one. When it has two or more substituents, the substituents may be the same or different. Furthermore, these substituents may be substituted at any position on ring A, but it is preferable that they be substituted at the 7-position on ring A.

[0023] ·R 11 R 11 is a hydrogen atom, an alkyl group, a phenyl group, an amino group, a carboxy group, or a hydroxyl group. The alkyl group is preferably a linear alkyl group having from 1 to 6 carbon atoms, and specific examples thereof include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-hexyl group. Among them, R 11 As the group, an amino group is preferred.

[0024] Specific examples of preferred compound (I) include compounds represented by the following general formula (I-1) (hereinafter, sometimes abbreviated as "compound (I-1)"). Note that compound (I-1) is an example of preferred compound (I), and preferred compound (I) is not limited thereto.

[0025] [ka]

[0026] (In general formula (I-1), R 111 is the above R11 is the same as R 112 is a halogen atom, a nitro group, an amino group, a mono- or di-alkylamino group, an alkyl group, an alkoxy group, a carboxy group, or a hydroxyl group.

[0027] ·R 111 R 111 is the above R 11 Among them, R 111 As the group, an amino group is preferred.

[0028] ·R 112 R 112 is a halogen atom, a nitro group, an amino group, a mono- or di-alkylamino group, an alkyl group, an alkoxy group, a carboxy group, or a hydroxyl group. Examples of the halogen atom, the mono- or di-alkylamino group, the alkyl group, and the alkoxy group include the same groups as those exemplified as the substituents of ring A above. Among these, R 112 As the alkyl group, an alkyl group is preferable, a chain alkyl group having 1 to 3 carbon atoms is more preferable, and an isopropyl group is even more preferable.

[0029] Preferable compounds (I-1) include, for example, R 111 is an amino group, and R 112 is an alkyl group. More preferred compounds (I-1) include, for example, R 111 is an amino group, and R 112 is a chain alkyl group having 1 to 3 carbon atoms.

[0030] Specific examples of preferred compound (I-1) include a compound represented by the following formula (I-1-1) (hereinafter, sometimes referred to as "compound (I-1-1)"). Note that compound (I-1-1) is an example of preferred compound (I-1), and preferred compound (I-1) is not limited thereto.

[0031] [ka]

[0032] Compound (I-1-1) is 2-amino-7-isopropyl-5-oxo-5H-[1]benzopyrano[2,3-b]pyridine-3-carboxylic acid, commonly known as amlexanox. Amlexanox exhibits potent inhibitory activity against IgE-mediated histamine release from mast cells, and is known to have potent antiallergic and anti-inflammatory effects through its inhibitory activity against leukotriene synthesis from macrophages (SRS-A production inhibitory activity) and antagonism against leukotrienes (SRS-A antagonism) (Reference 1: Japanese Patent Laid-Open Publication No. 111096 / 1978). Amlexanox is also clinically used as an antiallergic agent in the form of nasal sprays, eye drops, and tablets under the trade names Solfa (registered trademark) and Elix (registered trademark). In recent years, it has been reported that amlexanox selectively inhibits GRK5 (see Reference 2: Homan KT et al., "Identification and Characterization of Amlexanox as a GProtein-Coupled Receptor Kinase 5 Inhibitor," Molecules, Vol. 19, pp. 16937-16949, 2014). Since amlexanox has been used clinically up to now, it is considered to be highly safe and is suitably used as a GRK5 inhibitor in the preventive or therapeutic agent for OA of this embodiment.

[0033] Furthermore, salts of Compound (I) include salts of inorganic acids and salts of organic acids that are suitable for the separation or crystallization of Compound (I). Specific examples of salts of Compound (I) include organic amine salts, alkali metal salts, ammonium salts, pharmaceutically acceptable salts, etc. Pharmaceutically acceptable salts of Compound (I) include, for example, hydrochloride, bromate, sulfate, hydrogen sulfate, dihydrogen phosphate, methanesulfonate, methyl sulfate, maleate, fumarate, 2-naphthalenesulfonate, benzenesulfonate, glycolate, gluconate, citrate, isethionate, para-toluenesulfonate, and the like.

[0034] (Method for producing compound (I)) Compound (I) can be produced, for example, by the following method. That is, a compound represented by the following general formula (Ia) (hereinafter, sometimes referred to as "compound (Ia)") can be reacted with an active methylene compound and hydrolyzed to obtain compound (I) (see Reference 2 above).

[0035] [ka]

[0036] (In general formula (Ia), ring A may be substituted.)

[0037] Examples of the active methylene compound used in the reaction include methyl acetoacetate, ethyl acetoacetate, methyl cyanate, cyanoacetamide, malononitrile, acetic acid oxaloacetate, diethyl malonate, dimethyl malonate, ethyl benzoylacetate, methyl-3-oxo-n-caproate, etc. The amount of these active methylene compounds used is usually about 1 mole to 10 moles per mole of compound (Ia).

[0038] The above reaction is generally desirably carried out in the presence of a base, and examples of the base used include organic amines. Specific examples of organic amines include primary amines, secondary amines, tertiary amines, heterocyclic bases, etc. Primary amines include n-butylamine, benzylamine, aniline, etc. Secondary amines include diethylamine, dipropylamine, dibutylamine, piperidine, pyrrolidine, morpholine, etc. Tertiary amines include 1,8-diazabicyclo[5,4,0]-7-undecene, triethylamine, etc. Heterocyclic bases include imidazole, 2-methylimidazole, etc. The amount of base used is usually from a catalytic amount to 5 moles per mole of compound (Ia).

[0039] The reaction is preferably carried out in an organic solvent, and examples of the solvent include alcohols, aromatic hydrocarbons, dimethylformamide, etc. Examples of the alcohols include methanol, ethanol, propanol, butanol, etc. Examples of the aromatic hydrocarbons include benzene, toluene, etc.

[0040] There are no particular limitations on the reaction temperature, reaction time, and other reaction conditions, but the reaction is generally carried out at a temperature above room temperature and below the boiling point of the solvent used, for about 1 hour to 24 hours.

[0041] The compound obtained as described above is hydrolyzed to obtain Compound (I). Conventional acidic hydrolysis methods are used as the hydrolysis conditions. For example, an excess of sulfuric acid, hydrochloric acid, phosphoric acid, or the like is used, and the hydrolysis is carried out by heating the acid alone or together with an organic solvent, usually at a temperature of 50°C to 150°C. Examples of organic solvents used for hydrolysis include organic acids and alcohols. Examples of organic acids include formic acid and acetic acid. Examples of alcohols include methanol, ethanol, and propanol. The reaction time varies depending on the type of target Compound (I), but is usually from one hour to several days.

[0042] Compound (I) can also be produced, for example, by the method shown below. That is, compound (I) can be obtained by reacting compound (Ia) with an acetylene carboxylic acid and hydrolyzing the reaction product.

[0043] Examples of acetylene carboxylic acids used in the reaction include dimethyl acetylene dicarboxylic acid ester, diethyl acetylene dicarboxylic acid ester, and ethyl propiolate. When ethyl propiolate is used, the aminoacrylate derivative generated as an intermediate can be isolated, but the ring-closing reaction can also be carried out directly without isolation. The amount of these acetylene carboxylic acids used is usually about 1 to 10 moles per mole of compound (Ia).

[0044] The above reaction is generally desirably carried out in the presence of a base. Examples of the base used include organic amines. Examples of organic amines include secondary amines, tertiary amines, and heterocyclic bases. Examples of secondary amines include piperidine, pyrrolidine, morpholine, diethylamine, dipropylamine, and dibutylamine. Examples of tertiary amines include triethylamine, tripropylamine, and tributylamine. Examples of heterocyclic bases include pyridine, quinoline, imidazole, and 2-methylimidazole. The amount of these bases used is usually from a catalytic amount to about 10 moles per mole of compound (Ia).

[0045] The reaction is preferably carried out in an organic solvent, and examples of the solvent include alcohols, aromatic hydrocarbons, dimethylformamide, etc. Examples of the alcohols include methanol, ethanol, propanol, butanol, etc. Examples of the aromatic hydrocarbons include benzene, toluene, xylene, etc.

[0046] There are no particular limitations on the reaction temperature, reaction time, and other reaction conditions, but the reaction is generally carried out at a temperature above room temperature and below the boiling point of the solvent used, for about 1 hour to 24 hours.

[0047] The compound obtained as above is hydrolyzed to obtain Compound (I). The hydrolysis conditions can be the same as those described above.

[0048] Also, R 11 Compound (I) in which R is a carboxy group is heated without solvent at a temperature slightly higher than the temperature at which decarboxylation occurs. 11 is a hydrogen atom, the compound (I) is obtained.

[0049] Furthermore, an organic amine salt, alkali metal salt or ammonium salt of compound (I) can be obtained by reacting compound (I) with an organic amine, an alkali metal hydroxide, ammonia or the like in an appropriate solvent using a known method, such as mixing and heating.

[0050] [GRK5 expression inhibitor] Examples of GRK5 expression inhibitors include siRNA, shRNA, miRNA, ribozymes, antisense nucleic acids, and small molecular weight compounds. Administration of these GRK5 expression inhibitors can reduce the expression level of GRK5 and suppress NF-κB signaling. As a result, the expression of inflammatory cytokines and substrate enzymes is suppressed, thereby inhibiting the progression of OA. In other words, administration of these GRK5 expression inhibitors can prevent or treat OA.

[0051] siRNA (small interfering RNA) is a small double-stranded RNA of 21 to 23 base pairs that is used for gene silencing by RNA interference. When siRNA is introduced into cells, it binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA with a sequence complementary to the siRNA, thereby suppressing gene expression in a sequence-specific manner.

[0052] siRNA can be prepared by synthesizing sense and antisense strand oligonucleotides using an automatic DNA / RNA synthesizer, denaturing them in an appropriate annealing buffer at approximately 90°C to 95°C for about 1 minute, and then annealing them at approximately 30°C to 70°C for about 1 hour to 8 hours.

[0053] siRNA, shRNA, miRNA, ribozymes, and antisense nucleic acids may contain various chemical modifications to improve stability and activity. For example, to prevent degradation by hydrolases such as nucleases, phosphate residues may be substituted with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. Furthermore, at least a portion of the nucleic acid may be composed of a nucleic acid analog such as peptide nucleic acid (PNA).

[0054] [GRK5 specific binding substance] Examples of GRK5-specific binding substances include those that specifically bind to GRK5 and inhibit the function of GRK5, such as antibodies, antibody fragments, and aptamers. Antibodies can be produced, for example, by immunizing animals such as mice with the GRK5 protein or a fragment thereof as an antigen. Alternatively, they can be produced, for example, by screening a phage library. Examples of antibody fragments include Fv, Fab, and scFv. The above-mentioned antibodies are preferably monoclonal antibodies. Alternatively, commercially available antibodies may be used.

[0055] An aptamer is a substance that has the ability to specifically bind to a target substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers that have the ability to specifically bind to a target peptide can be selected, for example, by the systematic evolution of ligand by exponential enrichment (SELEX) method. Peptide aptamers that have the ability to specifically bind to a target peptide can be selected, for example, by the two-hybrid method using yeast.

[0056] <Hyaluronic acid or a pharmaceutically acceptable salt thereof> Hyaluronic acid (hyaluronan) is basically a disaccharide or more containing at least one disaccharide unit formed by bonding the 1-position of β-D-glucuronic acid to the 3-position of β-DN-acetylglucosamine, and may be a sugar formed by bonding one or more disaccharide units to these elements, as long as it is basically composed of β-D-glucuronic acid and β-DN-acetylglucosamine, or may be a derivative thereof, for example, one having a hydrolyzable protecting group such as an acyl group. The sugar may be an unsaturated sugar, and examples of the unsaturated sugar include non-reducing terminal sugars, typically those unsaturated between the 4- and 5-carbon positions of glucuronic acid.

[0057] As hyaluronic acid, specifically, any of the hyaluronic acid extracted from natural products such as animals, obtained by cultivating microorganisms, or chemically or enzymatically synthesized can be used.For example, it can be obtained from living tissues such as cockscomb, crocus, skin, synovial fluid, etc. by known extraction and purification methods.It can also be produced by fermentation using bacteria such as Streptococcus.

[0058] Hyaluronic acid also includes hyaluronan oligosaccharide, and can be used from low molecular weight hyaluronic acid such as the disaccharide consisting of one disaccharide unit and its derivative, to high molecular weight hyaluronic acid with a weight-average molecular weight of about 4 million.Because it has excellent tissue permeability, etc., hyaluronic acid with a weight-average molecular weight of about 380 to 4,000,000 is preferred, and hyaluronic acid with a weight-average molecular weight of about 2 to 20 sugars is more preferred.

[0059] Specifically, low-molecular-weight hyaluronic acid can be produced by known methods such as enzymatic degradation, alkaline degradation, heat treatment, and ultrasonic treatment to reduce the molecular weight of hyaluronic acid, or by chemical or enzymatic synthesis. For example, enzymatic degradation methods include a method in which hyaluronan is treated with a hyaluronic acid-degrading enzyme (e.g., hyaluronidase (testis-derived), hyaluronidase (Streptomyces-derived), hyaluronidase SD, etc.), chondroitinase AC, chondroitinase ACII, chondroitinase ACIII, chondroitinase ABC, or another enzyme that degrades hyaluronic acid, to produce hyaluronan oligosaccharides.

[0060] In addition, examples of alkaline decomposition methods include adding a base such as about 1N sodium hydroxide to a solution of hyaluronic acid, heating it for several hours to lower the molecular weight, and then adding an acid such as hydrochloric acid to neutralize it, thereby obtaining low-molecular-weight hyaluronic acid.

[0061] Hyaluronic acid includes salt form, and can use its pharmaceutically acceptable salt according to the need of preparation.For example, can include alkali metal salt such as sodium salt, potassium salt, alkaline earth metal salt such as calcium salt, magnesium salt, amine salt such as tri(n-butyl)amine salt, triethylamine salt, pyridine salt, amino acid salt, etc.

[0062] Examples of commercially available hyaluronic acid products in clinical use include Artzdispo (purified sodium hyaluronate, weight-average molecular weight of 500,000 to 1,200,000) and Suvenyl (purified sodium hyaluronate, weight-average molecular weight of 1,500,000 to 3,900,000).

[0063] The above-mentioned GRK5 inhibitor and the above-mentioned hyaluronic acid or its pharmaceutically acceptable salt may be in the form of a mixture thereof, or the above-mentioned GRK5 inhibitor and the above-mentioned hyaluronic acid or its pharmaceutically acceptable salt may be sealed in separate containers and be in the form of a kit that can be prepared before use.That is, in one embodiment, the present invention provides a kit for inhibiting the progression of osteoarthritis, comprising a GRK5 inhibitor and hyaluronic acid or its pharmaceutically acceptable salt.

[0064] <Pharmaceutical Composition> The OA progression inhibitor of this embodiment can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition for inhibiting the progression of OA.

[0065] The pharmaceutical composition of this embodiment can suppress the progression of OA, and has improved efficacy compared to administration of a GRK5 inhibitor alone.

[0066] The pharmaceutical composition of this embodiment may be in a dosage form for oral administration or a dosage form for parenteral administration, but is preferably in a dosage form for parenteral administration. Examples of dosage forms for oral administration include tablets, capsules, elixirs, microcapsules, etc. Examples of dosage forms for parenteral administration include injections, ointments, patches, etc.

[0067] As the pharmaceutically acceptable carrier, those usually used in the preparation of pharmaceutical compositions can be used without any particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum, gum arabic, etc.; excipients such as starch, crystalline cellulose, etc.; swelling agents such as alginic acid, solvents for injections such as water, ethanol, glycerin, etc.; adhesives such as rubber-based adhesives, silicone-based adhesives, etc.

[0068] The pharmaceutical composition may contain additives, such as lubricants such as calcium stearate and magnesium stearate, sweeteners such as sucrose, lactose, saccharin, and maltitol, flavorings such as peppermint and rhizome oil, stabilizers such as benzyl alcohol and phenol, buffers such as phosphates and sodium acetate, solubilizers such as benzyl benzoate and benzyl alcohol, antioxidants, and preservatives.

[0069] The pharmaceutical composition can be formulated by appropriately combining the above-mentioned OA progression inhibitor with the above-mentioned pharmaceutically acceptable carriers and additives, and mixing them in a unit dosage form required for generally accepted pharmaceutical practice.

[0070] The pharmaceutical composition may be used in combination with at least one agent selected from the group consisting of therapeutic agents having anti-inflammatory activity other than the OA progression inhibitor and therapeutic agents for other diseases. The OA progression inhibitor and the other agent may be formulated in the same formulation or in separate formulations. Furthermore, each formulation may be administered via the same administration route or via separate administration routes. Furthermore, each formulation may be administered simultaneously, sequentially, or separately at a certain time or interval. In one embodiment, the OA progression inhibitor and the other agent may be formulated as a kit containing them.

[0071] <Administration method> Subjects to be administered include, but are not limited to, humans, monkeys, dogs, cattle, horses, sheep, pigs, rabbits, mice, rats, guinea pigs, hamsters, and cells thereof. Among these, mammals or mammalian cells are preferred, and humans or human cells are particularly preferred.

[0072] The administration route is preferably a parenteral route such as subcutaneous administration, transdermal administration, intramuscular administration or intraarticular administration, with intraarticular administration being particularly preferred.

[0073] The dosage of OA progression inhibitor varies depending on the type of GRK5 inhibitor and hyaluronic acid or its pharmaceutically acceptable salt, the symptoms of the subject, the administration site, the administration method, etc. Those skilled in the art can appropriately select the appropriate dosage, for example, when administering locally, for GRK5 inhibitor, generally, for an adult (body weight 60 kg), per day, it is usually about 0.001 mg to 10 mg, preferably about 0.01 mg to 5 mg, more preferably about 0.02 mg to 2 mg. For hyaluronic acid or its pharmaceutically acceptable salt, generally, for an adult (body weight 60 kg), per day, it is usually about 0.08 mg to 800 mg, preferably about 0.8 mg to 400 mg, more preferably about 1.6 mg to 100 mg. Alternatively, in clinical practice, one syringe (2.5 mL) (containing approximately 25 mg of purified sodium hyaluronate) is usually administered per dose, so hyaluronic acid or a pharmaceutically acceptable salt thereof can be administered in a range of 25 mg or more or 25 mg or less per dose. The administration of the OA progression inhibitor can be a single administration or multiple administrations.When multiple administrations are used, for example, it can be administered every 2 hours or more and 12 hours or less, every day, or every 2 days, 5 days, 1 week, 1.5 weeks, several weeks, 1 month or several months etc. frequency.Usually, in clinical practice, for the treatment of OA, one syringe (25 mg of purified sodium hyaluronate per administration) is administered to an adult in the knee joint cavity every week for 5 consecutive times, and then, for the purpose of maintaining symptoms, it is administered every 2 weeks or more and 4 weeks or less.However, as shown in the examples below, the OA progression inhibitor of this embodiment can be used in combination with GRK5 inhibitor and hyaluronic acid or its pharmaceutically acceptable salt, so that sufficient effect can be obtained even if the administration interval is increased, and therefore it can also be administered less frequently than before.

[0074] Other Embodiments In one embodiment, the present invention provides a method for preventing, treating, or inhibiting the progression of OA, comprising administering an effective amount of a GRK5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof to a patient or animal in need of treatment. Here, the GRK5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof include those similar to those described above. In addition, the OA includes those similar to those described above.

[0075] In one embodiment, the present invention provides a GRK5 inhibitor and hyaluronic acid or its pharmaceutically acceptable salt for preventing, treating, or inhibiting the progression of OA.Here, the GRK5 inhibitor and hyaluronic acid or its pharmaceutically acceptable salt can be the same as those described above.In addition, the OA can be the same as those described above.

[0076] In one embodiment, the present invention provides the use of GRK5 inhibitor and hyaluronic acid or its pharmaceutically acceptable salt for producing an agent for suppressing the progression of OA.Here, GRK5 inhibitor and hyaluronic acid or its pharmaceutically acceptable salt can be the same as those described above.In addition, OA can be the same as those described above. [Example]

[0077] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0078] [material] 1. Collection of human chondrocytes Human chondrocytes were isolated and cultured from cartilage fragments excised after total knee arthroplasty, which are usually discarded. All donors consented to participate in a clinical study approved by the ethical review board. All donors' personal information was handled with care. The donor knee joints were classified as osteoarthritis grade III to IV (Kellgren-Lawrence) on radiographs.

[0079] 2. Mouse C57BL / 6J mice (hereinafter sometimes referred to as "wild-type mice") were purchased from Charles River Japan. GRK5 knockout (KO) mice (GRK5 - / -) were obtained from Dr. Kurose (Kyushu University, Faculty of Pharmaceutical Sciences). There were no significant differences in the thickness of the cartilage in the growth plate and articular surface, body weight, or appearance between wild-type and GRK5KO mice, and no differences in development were observed. Immature chondrocytes were collected from the knees of 5-day-old mice.

[0080] [Example 1] Synergistic inhibitory effect of amlexanox and hyaluronic acid on IL6, MMP13, and ADAMTS4 expression in human chondrocytes Human OA chondrocytes were incubated for 48 hours after adding amlexanox (100 μM in culture medium) and hyaluronic acid (1 mg / mL in culture medium, weight-average molecular weight: 500,000 to 1,490,000). As controls, cells were incubated for 48 hours without any treatment, and cells were incubated for 48 hours after adding amlexanox (100 μM in culture medium) or hyaluronic acid (1 mg / mL in culture medium, weight-average molecular weight: 500,000 to 1,490,000).

[0081] Human OA chondrocytes supplemented with amlexanox and hyaluronic acid were stimulated with lipopolysaccharide (LPS) (10 μg / mL in the culture medium) and incubated for 6 hours. mRNA was then isolated from the cells. After reverse transcription, quantitative real-time PCR was performed using the LightCycler 2.0 system (Roche). GAPDH was used as an internal standard. The sequences of the primers used are shown in Table 1 below. The results are shown in Figure 1.

[0082] [Table 1]

[0083] As shown in Figure 1, administration of hyaluronic acid alone did not significantly reduce the expression of IL6, MMP13, and ADAMTS4 (relative to control: 96.6%, 90.13%, and 76.84%). Administration of amlexanox alone significantly reduced the expression of IL6, MMP13, and ADAMTS4 (relative to control: 32.99%, 43.83%, and 42.04%). Co-administration of amlexanox and hyaluronic acid significantly suppressed the expression of IL6, MMP13, and ADAMTS4 by 10.58%, 26.25%, and 18.95% compared to control, and by 32.07%, 59.89%, and 45.08% compared to amlexanox alone, demonstrating a synergistic effect.

[0084] [Example 2] Synergistic inhibitory effect of intra-articular administration of amlexanox and hyaluronic acid on the progression of cartilage degeneration in osteoarthritis 1 OA was induced in 12-week-old male mice (wild-type and GRK5KO mice) by transection of the ligament connecting the medial meniscus to the tibia, resulting in knee joint instability (DMM model) (hereafter referred to as the "DMM group"). Control mice were also treated with a sham operation without ligament transection (hereafter referred to as the "sham operation group"). Immediately after OA induction, mice were divided into four groups: saline (10 μL), hyaluronic acid (10 mg / mL, 10 μL), amlexanox (100 μM, 10 μL), or a mixture of amlexanox (100 μM) and hyaluronic acid (10 mg / mL) (10 μL) every 5 days for 8 weeks (see Figure 2A). Sections of knee joint tissue from each group 8 weeks after OA induction surgery were stained with Safranin O. The results are shown in Figure 2B (stained image).

[0085] We also quantified the severity of OA by evaluating stained sections according to the histopathological grading recommended by OARSI. The results are shown in Figure 2C.

[0086] As shown in Figures 2B and 2C, there was no significant improvement in OARSI scores in the group administered hyaluronic acid alone (92.57% compared to the control group). A significant decrease in OARSI scores was observed in the group administered amlexanox alone (56.95% compared to the control group). The group administered amlexanox and hyaluronic acid simultaneously showed a significant decrease in OARSI scores of 32.58% compared to the control group and 57.21% compared to the amlexanox alone group, demonstrating a synergistic inhibitory effect on cartilage degeneration. It was speculated that simultaneous administration of amlexanox and hyaluronic acid slowed the absorption of amlexanox from the joint after intra-articular administration. The half-life of hyaluronic acid in synovial fluid is approximately 20 hours, and it is detectable in synovial fluid for up to approximately three days after administration. Amlexanox is a low-molecular-weight compound with a molecular weight of 300, and its half-life within the joint is several hours. It was speculated that simultaneous administration of amlexanox and hyaluronic acid resulted in a sustained pharmacological effect, resulting in greater efficacy in the mouse model than administration of amlexanox alone.

[0087] [Example 3] (Synergistic inhibitory effect of simultaneous intra-articular administration of amlexanox and hyaluronic acid on the progression of cartilage degeneration in osteoarthritis 2) Using the same method as in Example 2, OA-induced mice were administered a mixed solution of amlexanox (100 μM) and hyaluronic acid (10 mg / mL) every 10 days for 8 weeks (see Figure 3A). Eight weeks after OA-induction surgery, sections of knee joint tissue from each group were stained with Safranin O. The results are shown in Figure 3B (stained image).

[0088] The severity of OA was also quantified by evaluating stained sections based on the histopathological grading recommended by OARSI. The results are shown in Figure 3C. Figure 3C also shows the results for the four groups examined in Example 2.

[0089] As shown in Figures 3B and 3C, the group receiving simultaneous administration of amlexanox and hyaluronic acid had a significantly higher inhibitory effect on cartilage degeneration than the group receiving amlexanox alone, even when the administration interval was extended to 10 days.

[0090] In addition, OA-induced mice were treated with a mixture of amlexanox (100 μM) and hyaluronic acid (10 mg / mL) every 10 days for 8 weeks, and with amlexanox solution (100 μM, 10 μL) every 5 days for 8 weeks. At 8 weeks after OA-induction surgery, pain thresholds to pressure stimuli on the joints were measured using a SMALGO small animal algometer (Bioseb, model number BIO-SMALGO+). The results are shown in Figure 3D.

[0091] As shown in FIG. 3D, it was revealed that the group administered amlexanox and hyaluronic acid simultaneously was able to reduce pain more than the group administered amlexanox alone.

[0092] These results suggest that the simultaneous administration of the GRK5 inhibitor amlexanox and hyaluronic acid has a synergistic effect in inhibiting OA progression, making it possible for the drug to become a disease-modifying OA treatment with a yet-to-be-existent inhibitory effect on cartilage degeneration. Because hyaluronic acid alone does not have the inhibitory effect on OA progression, the results above can be said to be a novel effect of hyaluronic acid that can enhance the effect of the GRK5 inhibitor (amlexanox). [Industrial Applicability]

[0093] According to the osteoarthritis progression inhibitor and progression inhibition kit of this embodiment, the progression of OA can be inhibited, and the effectiveness is improved compared to the administration of a GRK5 inhibitor alone.

Claims

1. The composition contains a G protein-coupled receptor kinase 5 inhibitor and hyaluronic acid or a pharmaceutically acceptable salt thereof as active ingredients, An agent for suppressing the progression of osteoarthritis, wherein the G protein-coupled receptor kinase 5 inhibitor is amlexanox.

2. The agent for inhibiting the progression of osteoarthritis according to claim 1, which is administered by intra-articular injection.

3. a G protein-coupled receptor kinase 5 inhibitor; hyaluronic acid or a pharmaceutically acceptable salt thereof; Equipped with A kit for inhibiting the progression of osteoarthritis, wherein the G protein-coupled receptor kinase 5 inhibitor is amlexanox.

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