Agent for suppressing progression of osteoarthritis

Abstract

The purpose of the present invention is to provide a novel medicine for suppressing the progression of osteoarthritis, particularly a novel medicine for suppressing degeneration and / or disappearance of cartilage. Specifically, the inventors of the present invention have found that a value indicating the total amount of a cartilage matrix degradation product decreases when dabigatran etexilate methanesulfonic acid, which is a thrombin inhibitor having an action of inhibiting the production of fibrin from fibrinogen, is administered to a patient with osteoarthritis. Specifically, in the present invention, the means for solving the problem is: an agent for suppressing degeneration of cartilage in an osteoarthritic joint, the agent containing a fibrin formation inhibitor as an active ingredient; and provision of said agent.

Description

Inhibitor of the progression of osteoarthritis

[0001] The present invention relates to an agent for inhibiting the progression of osteoarthritis, for example, an agent for inhibiting the degeneration and / or loss of cartilage in osteoarthritis of the knee joint.

[0002] Osteoarthritis is a disease characterized by the degeneration and loss of articular cartilage, primarily due to aging. Once it develops, it causes pain, deformation, and impaired joint function. Osteoarthritis can occur in any synovial joint throughout the body, but knee osteoarthritis is the most clinically and socially problematic due to the large number of patients and the degree of functional impairment it causes. Osteoarthritis causes the degeneration and loss of cartilage in the knee joint, resulting in pain, swelling, joint deformation, and impaired knee function. There are 10 million patients in Japan alone, and including potential patients, it is estimated that 30 million people suffer from knee osteoarthritis. With the aging population, the number of patients with osteoarthritis, including knee osteoarthritis, is on the rise.

[0003] Osteoarthritis of the knee causes severe pain when performing activities that put stress on the knee, such as walking or climbing stairs, and also causes stiffness and swelling in the knee joint, limiting range of motion. Osteoarthritis of the knee therefore reduces the quality of life of elderly patients and can cause a loss of independence, making the establishment of appropriate treatments for this disease of great social importance. Although much research has been done on the mechanism by which cartilage degeneration and loss occur in osteoarthritis of the knee, the true mechanism remains unclear, and therefore no treatment has been established to prevent the progression of the disease.

[0004] Articular cartilage is primarily composed of type II collagen and aggrecan, a type of proteoglycan. Plasmin (EC 3.4.21.7), a type of serine protease, has the ability to directly denature aggrecan (Non-Patent Document 1). It has also been reported that in osteoarthritis, increased expression of urokinase (uPA, urokinase-type plasminogen activator, EC 3.4.21.73) and decreased expression of plasminogen activator inhibitor-1 (PAI-1), which inhibits uPA activity, result in increased plasmin activity (Non-Patent Document 2). However, the relationship between plasmin activity and the progression of osteoarthritis remains unclear.

[0005] Poe M et al. Arch Biochem Biophys 298:757-759 1992 Martel-Pelletier J et al. J Rheumatol 18:1863-1871 1991 Troeberg L et al. Biochim Biophys Acta 1824:133-145 2012 Yoshihara Y et al. Ann Rheum Dis 59:455-461 2000 Mitchell PG et al. J Clin Invest 1:761-768 1996 Weinberg JB et al. Arthritis Rheum 34:996-1005 1991 Fukui N et al. Arthritis Rheum 58:154-163, 2008 Mosesson MW. J Thromb Haemost 3:1894-1904 2005 Mosher DF. J Biol Chem 25:6614-6621 1975So AK et al. J Thromb Hoemost 1:2510-2515 2003Kummer JA et al. Arthritis Rheum 35:884-893 1992

[0006] In view of the above circumstances, an object of the present invention is to provide a novel drug for inhibiting the progression of osteoarthritis, particularly a novel drug for inhibiting the degeneration and / or loss of cartilage.

[0007] In osteoarthritis, cartilage degeneration and loss tend to occur locally in areas where a heavy load is applied. Specifically, in the knee joint, cartilage degeneration occurs on the inside of the joint where a heavy load is applied, while the cartilage on the outside of the knee joint often remains undegenerated. The present inventors focused on this fact and conducted research, suspecting that the activity of some kind of proteolytic enzyme may be increased in the degenerated areas of cartilage in osteoarthritis compared to non-degenerated areas.

[0008] The present inventors have confirmed that the activity of plasmin, a serine protease, is significantly elevated in degenerated cartilage in osteoarthritis compared with non-degenerated cartilage. Articular cartilage is primarily composed of type II collagen and aggrecan, a type of proteoglycan. Plasmin directly degrades aggrecan (Non-Patent Document 1). Plasmin also converts type II collagen-denaturing matrix metalloproteinase (MMP)-1 (EC 3.4.24.7) and MMP-13 from their latent forms to their active forms (Non-Patent Document 3). MMP-1 is known to be abundant in synovial fluid in patients with osteoarthritis (Non-Patent Document 4), and MMP-13 is known to be produced by chondrocytes in cartilage tissue in patients with osteoarthritis (Non-Patent Document 5). Therefore, increased plasmin activity in degenerated cartilage may be a direct cause of the degeneration and loss of cartilage matrix in those areas.

[0009] The present inventors also found that tissue plasminogen activator (tPA, EC 3.4.21.68) is present in large amounts in degenerated cartilage in patients with knee osteoarthritis. It is known that fibrin (a fibrous protein formed by the polymerization of fibrin monomers) precipitates within the joint in patients with knee osteoarthritis (Non-Patent Document 6). It is also known that chondrocytes in degenerated cartilage in patients with knee osteoarthritis express fibronectin (Non-Patent Document 7). Fibrin has the property of binding to fibronectin, and tPA's plasminogen activator activity is enhanced by binding to fibrin (Non-Patent Document 8). Based on these findings and those of the present inventors, it is possible that fibrin accumulates in degenerated cartilage by binding to the fibronectin produced in the degenerated cartilage, and that its binding enhances the activity of tPA, resulting in the local production of plasmin.

[0010] Based on the above reasoning, the inventors considered four potential methods for inhibiting the progression of osteoarthritis: (i) inhibition of fibrin formation, (ii) inhibition of tPA activity, (iii) inhibition of plasmin activity, and (iv) inhibition of MMP activity. Regarding (i) inhibition of fibrin formation, several drugs with this effect are currently in clinical use for the treatment of diseases other than osteoarthritis. Among these drugs, the inventors focused on dabigatran etexilate methanesulfonate (Pradaxa®; hereinafter referred to as "dabigatran"), which inhibits fibrin formation by inhibiting the activity of thrombin (EC 3.4.21.5). The effects of dabigatran administration on knee osteoarthritis were examined by synovial fluid analysis in patients receiving treatment for knee osteoarthritis who had been initiated during treatment.

[0011] In healthy knee joints, synovial fluid is rarely collected, but its volume often increases in osteoarthritis. Intra-articular injections of hyaluronic acid are commonly used as a treatment for knee osteoarthritis, and synovial fluid can be collected during this procedure in cases where synovial fluid is present. The present inventors selected patients who had received repeated intra-articular injections of hyaluronic acid for knee osteoarthritis and had continuous synovial fluid collection, but who had been administered dabigatran for the treatment of other diseases during treatment. They then investigated the changes in synovial fluid caused by dabigatran administration. As a result, dabigatran administration was confirmed to decrease intra-articular thrombin activity, decrease plasmin production, and fibrin degradation, as well as decrease MMP-1 (which acts to denature cartilage matrix) concentrations in synovial fluid, and inhibit aggrecan degeneration. The decreased thrombin activity and decreased fibrin degradation suggest a quantitative decrease in intra-articular fibrin. Therefore, the above results of synovial fluid analysis suggest that administration of dabigatran suppresses intra-articular fibrin formation and also inhibits cartilage degeneration and loss in knee osteoarthritis.

[0012] The present invention was completed based on the above findings. Specifically, the present invention comprises the following (1) to (15): (1) An agent for inhibiting cartilage degeneration in osteoarthritic joints, the agent comprising a fibrin formation inhibitor as an active ingredient. (2) The agent for inhibiting cartilage degeneration according to (1) above, wherein the fibrin formation inhibitor is a thrombin inhibitor. (3) The agent for inhibiting cartilage degeneration according to (2) above, wherein the thrombin inhibitor is one or more selected from the group consisting of dabigatran etexilate methanesulfonate, argatroban hydrate, and hirudin. (4) The agent for inhibiting cartilage degeneration according to (1) above, wherein the fibrin formation inhibitor is warfarin potassium. (5) The agent for inhibiting cartilage degeneration according to (1) above, wherein the fibrin formation inhibitor is a coagulation factor Xa inhibitor. (6) The cartilage degeneration inhibitor according to (5) above, wherein the coagulation factor Xa inhibitor is one or more selected from the group consisting of rivaroxaban, apixaban, edoxaban tosilate hydrate, and fondaparinux sodium. (7) The cartilage degeneration inhibitor according to (1) above, wherein the fibrin formation inhibitor is heparin. (8) The cartilage degeneration inhibitor according to (7) above, wherein the heparin is one or more selected from the group consisting of heparin sodium and heparin calcium. (9) The cartilage degeneration inhibitor according to (1) above, wherein the fibrin formation inhibitor is a low molecular weight heparin. (10) The cartilage degeneration inhibitor according to (9) above, wherein the low molecular weight heparin is enoxaparin sodium, parnaparin sodium, dalteparin sodium, or danaparoid sodium. (11) The cartilage degeneration inhibitor according to (1) above, wherein the fibrin formation inhibitor is dried concentrated human antithrombin III. (12) The cartilage degeneration inhibitor according to (1) above, wherein the fibrin formation inhibitor is antithrombin gamma. (13) The cartilage degeneration inhibitor according to (1) above, wherein the fibrin formation inhibitor is dried concentrated human activated protein C. (14) The cartilage degeneration inhibitor according to (1) above, wherein the fibrin formation inhibitor is thrombomodulin alpha. (15) The cartilage degeneration inhibitor according to (1) above, wherein the fibrin formation inhibitor is batroxobin.In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of it.

[0013] According to the present invention, it is possible to inhibit the degeneration and loss of cartilage in osteoarthritis and to suppress the progression of various symptoms associated with osteoarthritis.

[0014] Figure 1 shows the results of measuring plasmin activity in cartilage tissue samples taken from macroscopically non-degenerated and degenerated areas of cartilage in seven cases of knee osteoarthritis. Panel A shows the measured values ​​and the mean ± standard deviation of the measured values ​​for each individual sample. Panel B shows the same data, plotted as a line segment for the measured values ​​in the non-degenerated and degenerated areas of each individual case. In each graph, the vertical axis represents the amount of plasmin extracted per gram of wet cartilage tissue. Statistical tests were performed using a paired t-test. Figure 2 shows the results of measuring the amount of uPA (A), tPA (B), and PAI-1 (C) extracted from cartilage tissue samples taken from macroscopically non-degenerated and degenerated areas of cartilage in 17 cases of knee osteoarthritis. The graphs show the measured values ​​and the mean ± standard deviation of the measured values ​​for each individual sample. For tPA, a line segment is also shown for the amount of tPA extracted from the non-degenerated and degenerated areas of each individual case (B, right graph). In each graph, the vertical axis represents the amount of each protein extracted per gram of wet cartilage tissue. Statistical testing was performed using a paired t-test. Figure 3 shows the results of measuring the enzyme activity of uPA (A) and tPA (B) in cartilage tissue samples taken from macroscopically non-denatured and degenerated cartilage tissue in 11 cases of knee osteoarthritis. Proteins were extracted from each sample and measured. The graphs show the measured values ​​for each sample and the mean ± standard deviation of the measured values. For tPA, a line segment representing the activity of proteins extracted from the non-denatured and degenerated sections of each individual case is also shown (B, right graph). In each graph, the vertical axis represents the activity of each enzyme extracted per gram of wet cartilage tissue. Statistical testing was performed using a paired t-test, with a significance level of p = 0.05. In Figure 3A, ns indicates no statistically significant difference. Figure 4 shows the results of measuring the amount of fibronectin (A) and the total amount of FDP (fibrin degeneration products) and D-dimer extracted from cartilage tissue collected from macroscopically non-degenerated and degenerated areas of cartilage in 17 cases of knee osteoarthritis (OA). The graphs show the measured values ​​for each sample and the mean ± standard deviation of the measured values. In each graph, the vertical axis represents the amount of each protein extracted per gram of wet weight of cartilage tissue.Statistical tests were performed using paired t-tests. Figure 5 shows the changes in the concentrations of various factors in the synovial fluid (TAT (A), D-dimer (B), PAP (C), MMP-1 (D)) and the product (E) of the aggrecan (PG) concentration and synovial fluid volume after the initiation of dabigatran administration in a case (male, 52 years old at the time of initiation of dabigatran administration) in which synovial fluid was collected from osteoarthritis of the knee through continuous intra-articular injections and dabigatran administration was initiated for the treatment of another disease. Figure 6 shows the changes in the concentrations of various factors in the synovial fluid (TAT (A), D-dimer (B), PAP (C), MMP-1 (D)) and the product (E) of the concentration of aggrecan (PG) and the synovial fluid volume in a case (female, 80 years old at the time of initiation of dabigatran administration) in a patient who had been receiving continuous intra-articular injections for knee osteoarthritis and whose synovial fluid had been collected. This case involved dabigatran administration, which was initiated for the treatment of another disease. In this case, dabigatran administration was temporarily suspended from 9 to 16 weeks after initiation, and this effect can be seen in the synovial fluid collected from 10 to 16 weeks.

[0015] Hereinafter, embodiments of the present invention will be described. Unless otherwise specified, the term "present embodiment" refers to all or any of the embodiments described herein. The first embodiment is an inhibitor of cartilage degeneration in osteoarthritic joints, containing a fibrin formation inhibitor as an active ingredient (hereinafter also referred to as "cartilage degeneration inhibitor according to the present embodiment"). The "joint" of osteoarthritic joints in this embodiment is not particularly limited and encompasses all synovial joints in the body, such as knee joints, hip joints, and finger joints. For example, the inside of osteoarthritis knee joints are subjected to heavy loads, leading to cartilage degeneration and loss. This cartilage degeneration and loss contributes to knee joint deformity, and it is believed that inhibiting the progression of cartilage degeneration can slow the progression of knee osteoarthritis. Here, fibrin refers to a protein (fibrin polymer) formed by the polymerization of fibrin monomers with a molecular weight of approximately 340 kDa into a mesh-like fibrous structure.

[0016] It is known that fibrin (fibrin polymer) is formed within the joints of osteoarthritis (Non-Patent Document 6). It is also known that fibronectin, which is expressed at extremely low levels in normal cartilage, is expressed at high levels in degenerated cartilage in osteoarthritis (Non-Patent Document 7). It has also been reported that fibronectin covalently binds to fibrin through the action of coagulation factor XIII during blood clotting (Non-Patent Document 9). Based on these findings, it is thought that fibrin accumulates in degenerated cartilage in osteoarthritis by binding to locally produced fibronectin. Meanwhile, the inventors have found that tPA, which is barely expressed in normal cartilage, is induced in degenerated articular cartilage in osteoarthritis and present in large amounts within the tissue. The enzymatic activity of tPA is generally inhibited by plasminogen activator inhibitor-1 (PAI-1), but binding to fibrin reduces its susceptibility to PAI-1 inhibition, resulting in potent enzymatic activity (Non-Patent Document 8). The inventors have found that plasmin activity is strongly induced in articular cartilage tissue at degenerated sites in patients with osteoarthritis. Based on the above findings, we hypothesize that this is due to the production of plasmin from plasminogen in the synovial fluid by tPA, which binds to fibrin and exhibits strong enzymatic activity at degenerated cartilage sites. Plasmin directly degrades aggrecan, a major component of cartilage, and activates MMP-1 and MMP-13, which degrade type II collagen. Therefore, if this hypothesis is correct, inhibiting intra-articular fibrin formation would also inhibit plasmin production at degenerated cartilage sites, thereby preventing cartilage degeneration. In vivo, fibrin is primarily produced by thrombin. Therefore, we examined synovial fluid before and after administration of thrombin inhibitors in two patients with osteoarthritis of the knee. We found that administration of thrombin inhibitors reduced plasmin production and the total amount of cartilage matrix degeneration products, confirming that cartilage degeneration and loss in osteoarthritis of the knee were inhibited (see Examples for details).

[0017] From the above, it has been shown that the cartilage degeneration inhibitor of this embodiment, which contains the active ingredient, a fibrin formation inhibitor, inhibits the progression of degeneration and loss of articular cartilage in osteoarthritis, and as a result, exhibits the effect of suppressing the progression of osteoarthritis.

[0018] In this embodiment, the fibrin formation inhibitor may inhibit either the formation of fibrin monomers or the formation of mesh-like fibrin fibers from fibrin monomers. Examples of the fibrin formation inhibitor include, but are not limited to, thrombin inhibitors that inhibit the formation of fibrin monomers from fibrinogen (e.g., dabigatran etexilate methanesulfonate (Pradaxa®), argatroban hydrate (Novastan HI, Slonnon HI®, argatroban HI), and hirudin), as well as coagulation factor Xa inhibitors (e.g., rivaroxaban (Xarelto®), apixaban (Eliquis®), edoxaban tosilate hydrate (Lixiaxia®), and the like). Na (registered trademark)), fondaparinux sodium (AriXtra (registered trademark)), etc.), warfarin potassium (warfarin, warfarin K), heparin (e.g., heparin sodium (Hepafild (registered trademark), HepaFlush (registered trademark), heparin Na, heparin sodium), heparin calcium (heparin Ca, heparin calcium), etc.), low molecular weight heparin (e.g., enoxaparin sodium (Clexane (registered trademark)), parnaparin sodium (Parnaparin Na, RhoHepa (registered trademark)), dalteparin sodium (Dalteparin Na, Fragmin (registered trademark)), danaparoid sodium (Orgaran (registered trademark)), etc.), dried human antithrombin III concentrate (Neuart, Donketsu Nonthron (registered trademark)), antithrombin gamma (Acoalan (registered trademark)), dried human activated protein C concentrate (Anact C (registered trademark)), thrombomodulin alpha (Recomodulin (registered trademark)), batroxobin (Defibrase (registered trademark)).

[0019] The cartilage degeneration inhibitor according to this embodiment may be provided as a pharmaceutical product or pharmaceutical composition (hereinafter also referred to as "the pharmaceutical composition according to this embodiment, etc."). The cartilage degeneration inhibitor according to this embodiment may contain one or more fibrin formation inhibitors as active ingredients. Furthermore, the cartilage degeneration inhibitor according to this embodiment may contain, in addition to the fibrin formation inhibitor as an active ingredient, other ingredients that are effective in improving knee osteoarthritis. Alternatively, the cartilage degeneration inhibitor according to this embodiment may be administered in combination with the other ingredients as a different formulation.

[0020] When administering the pharmaceutical composition according to this embodiment to a subject, the route of administration is not limited as long as the progression of cartilage degeneration in osteoarthritis is suppressed, and examples thereof include oral administration and parenteral administration (intravenous administration, subcutaneous administration, intra-articular administration, transdermal administration, etc.). Intra-articular administration is preferred to minimize side effects (e.g., bleeding) that may occur with systemic administration. Examples of administration methods in this case include administering a sustained-release formulation into the joint.

[0021] Dosage forms of the pharmaceutical composition according to this embodiment include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, and injections. These preparations are prepared according to conventional methods. Liquid preparations may be dissolved or suspended in water or other suitable solvents before use. Tablets and granules may also be coated by known methods. Injections are prepared by dissolving the active ingredient in water, but may also be dissolved in saline or glucose solution as needed, and buffers and preservatives may be added.

[0022] Preparations for oral or parenteral administration are provided in any dosage form. Examples of dosage forms include pharmaceutical compositions in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or solutions, and pharmaceutical compositions for parenteral administration in the form of injections for intraarticular, intravenous, intramuscular, or subcutaneous administration, drip infusions, sustained-release preparations, transdermal absorption preparations, transmucosal absorption preparations, nasal drops, inhalants, suppositories, and the like. Injections and drip infusions can also be prepared in a powdered dosage form, such as a lyophilized form, and dissolved in an appropriate aqueous medium, such as physiological saline, before use.

[0023] The type of formulation additives used in the production of the pharmaceutical compositions and the like according to this embodiment, the ratio of the formulation additives to the active ingredient, and the production method of the therapeutic composition and the preventive composition can be appropriately selected by those skilled in the art depending on the form of the composition. The formulation additives can be inorganic or organic substances, or solid or liquid substances, and can generally be blended in an amount of 1 to 90% by weight based on the weight of the active ingredient. Specific examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, Veegum, titanium oxide, sorbitan fatty acid esters, sodium lauryl sulfate, glycerin, fatty acid glycerin esters, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, wax, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, and water.

[0024] To prepare solid formulations for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, and anhydrous silicic acid to form a powder, or, if necessary, with binders such as sucrose, hydroxypropyl cellulose, and polyvinylpyrrolidone, and disintegrants such as carboxymethylcellulose and calcium carboxymethylcellulose, followed by wet or dry granulation to form granules. To prepare tablets, these powders and granules can be compressed as is, or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets can be coated with enteric-coated bases such as hydroxypropylmethylcellulose phthalate and methacrylic acid-methyl methacrylate polymer to form enteric-coated formulations, or coated with ethylcellulose, carnauba wax, and hydrogenated oil to form sustained-release formulations. To prepare capsules, the powder or granules can be filled into hard capsules, or the active ingredient can be dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, or the like and then coated with a gelatin membrane to form soft capsules.

[0025] To prepare an injection, the active ingredient may be dissolved in distilled water for injection, if necessary, together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc., and an isotonic agent such as sodium chloride or glucose, and then sterile filtered and filled into an ampule, or mannitol, dextrin, cyclodextrin, gelatin, etc. may be added, followed by vacuum freeze-drying to produce an injection that is dissolved just before use. Alternatively, the active ingredient may be emulsified in water with lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. to produce an emulsion for injection.

[0026] The pharmaceutical compositions of the present invention may be formulated as sustained-release formulations, such as implants or microencapsulated delivery systems, using pharmaceutically acceptable carriers that can prevent immediate elimination from the body. Such carriers include biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Liposomes can be prepared as lipid compositions containing, but not limited to, phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanol (PEG-PE), filtered through a filter with an appropriate pore size to obtain a suitable size, and then purified by reverse-phase evaporation.

[0027] The dosage and frequency of administration of the compositions and the like according to the present embodiment are not particularly limited and can be appropriately selected at the discretion of a physician depending on conditions such as the purpose of treatment and prevention, the type of disease, and the patient's weight and age. Generally, the daily oral dose for adults is, for example, about 0.01 to 1000 mg (weight of active ingredient), and can be administered once a day or in divided doses, daily or every few days. When used as an injection, it is desirable to administer, for example, 0.001 to 100 mg (weight of active ingredient) per day to an adult, either continuously or intermittently.

[0028] The pharmaceutical composition according to this embodiment may be provided in the form of a kit together with instructions for administration, etc. The drug contained in the kit is supplied in a container made of a material that maintains the activity of the drug for a long period of time, does not adsorb to the inside of the container, and does not alter the components. For example, a sealed glass ampoule may contain a buffer sealed in the presence of a neutral, non-reactive gas such as nitrogen gas. The kit may also include instructions for use, or may be provided in a form that is available online, etc.

[0029] The second embodiment is a method for suppressing cartilage degeneration in osteoarthritic joints, comprising administering a pharmaceutical composition or the like according to this embodiment (i.e., a drug or pharmaceutical composition containing a fibrin formation inhibitor as an active ingredient) to a subject (i.e., a subject in which cartilage degeneration is to be suppressed). The second embodiment also includes a method for suppressing the progression of osteoarthritis, comprising administering a pharmaceutical composition or the like according to this embodiment to a subject. The subject of the method for suppressing cartilage degeneration according to the second embodiment is not particularly limited and may be, for example, any animal classified as a mammal, including humans as well as non-human animals such as pet animals such as dogs, cats, rabbits, and ferrets, and livestock animals such as cows, pigs, sheep, and horses, with humans being a particularly preferred subject.

[0030] The disclosures of all documents cited herein are incorporated by reference in their entirety. Furthermore, throughout this specification, when the singular forms "a," "an," and "the" are used, they include the plural as well as the singular, unless the context clearly dictates otherwise. Furthermore, in this specification, the terms "about" and "approximately" refer to a numerical range of ±10%. The present invention will be further described below with reference to examples, but these examples are merely illustrative of embodiments of the present invention and are not intended to limit the scope of the present invention.

[0031] 1. Protein Extraction Method from Cartilage Tissue. For the analysis of cartilage proteins, cartilage tissue (1-3 g wet weight) was collected at the time of surgery from patients with end-stage or advanced knee osteoarthritis scheduled for total joint replacement. Patients had medial-type osteoarthritis, with preoperative plain radiographs indicating Kellgren-Lawence grade 3 or 4. Osteoarthritis was diagnosed according to established criteria (Altman et al., Arthritis Rheum, 29:1039-1049, 1986). Patients suspected of inflammatory arthritis based on blood test results were excluded. Cartilage samples were collected from the proximal articular surface of the tibia in each knee joint. Cartilage samples were collected from areas with no visible degeneration (non-degenerated areas) and areas with obvious degeneration (degenerated areas). Using a scalpel, the entire cartilage layer was removed, beginning just above the subchondral bone. The obtained cartilage tissue was thoroughly washed with ice-cold phosphate-buffered saline (PBS, pH 7.4) immediately after collection, and excess water was removed. The tissue was then sealed in a plastic bag and stored at -80°C until analysis.

[0032] Protein extraction from cartilage tissue was performed as follows. All extraction procedures were performed at 4°C or on ice. Cartilage tissue was minced with a scalpel, its wet weight was measured, and then 5 volumes (v / w) of PBS was added and homogenized using a Polytron homogenizer. Extraction was then continued in the same buffer (PBS) with gentle shaking. After 2–3 hours of shaking, the PBS was removed and replaced with PBS containing 0.1% Triton X-100 (PBS-T), a volume 5 times (v / w) that of the cartilage tissue. Extraction was continued in this buffer overnight with gentle shaking. The next day, the PBS-T was removed, and 5 volumes (v / w) of guanidine buffer (6 M Gdn-HCl, 50 mM Tris-HCl, pH 7.4) was added to the cartilage tissue. Extraction was continued with gentle shaking. After 48 hours, the guanidine buffer was collected and replaced with PBS by dialysis for 72 hours using a Slide-A-Lyzer Cassette (pore size 2 kDa, ThermoFischer Scientific).

[0033] At each stage of the extraction, the buffer containing the extract was centrifuged, and the supernatant was filtered through a 0.22 μm pore-size polyethersulfone filter, dispensed, and stored at -80°C until analysis. The amount of protein extracted from cartilage tissue was calculated by measuring the amount of protein extracted in each of the three buffers used for extraction (PBS buffer, PBS-T buffer, and guanidine buffer) and adding these amounts together. The enzyme activity of the extracted protein was also calculated by measuring the activity in each of the three buffers and adding these amounts together.

[0034] 2. Increased Plasmin Activity in Degenerated Articular Cartilage in Osteoarthritis of the Knee 2-1. Measurement of Plasmin Activity in Articular Cartilage Tissue In seven cases of osteoarthritis of the knee, articular cartilage samples were collected from macroscopically non-degenerated and degenerated areas of the proximal tibia articular surface during total joint replacement surgery. Protein was extracted from each sample using the method described in "1. Protein Extraction Method from Cartilage Tissue," and the activity of plasmin, a type of serine protease, was measured. Measurements were performed using a commercially available activity measurement kit (SensoLyte Rh110 Plasmin Assay Kit, AnaSpec) according to the protocol provided with the kit. The results revealed that plasmin activity was increased in the degenerated cartilage tissue compared to the non-degenerated areas in all seven cases (Figure 1).

[0035] 2-2. Mechanism of increased plasmin activity in cartilage degeneration Plasmin is an enzyme that plays a central role in the fibrinolysis (fibrinolysis) reaction, which breaks down clots. Plasmin is produced from plasma plasminogen by plasminogen activators. Two types of plasminogen activators, urokinase (uPA) and tissue-type plasminogen activator (tPA), are known to exist in humans. However, the activity of these plasminogen activators is normally inhibited by endogenous inhibitors, including plasminogen activator inhibitor-1 (PAI-1). However, it is known that tPA, when bound to fibrin, is less susceptible to PAI-1 inhibition, allowing for more efficient plasmin production (Non-Patent Document 8).

[0036] Cartilage samples were collected from macroscopically intact and degenerated areas of cartilage in 17 cases of knee osteoarthritis. Proteins were extracted from each cartilage tissue in three steps using three different buffers, as described in "1. Protein Extraction Method from Cartilage Tissue." The amounts of uPA, tPA, and PAI-1 extracted in the three buffers were then measured by Luminex (Diasorin) using a Human Luminex Assay Kit (R&D Systems). The total amounts were summed to determine the amount of these three proteins extracted from the cartilage tissue. The results showed that all three proteins were extracted in significantly greater amounts from the degenerated cartilage than from the intact cartilage (Fig. 2). In particular, the amount of tPA extracted from the denatured cartilage was greater than that from the intact cartilage in all 17 cases (Fig. 2B). Furthermore, the amount of tPA extracted from the denatured cartilage was, on average, more than 20 times that of uPA (Fig. 2A and B). Next, we measured the enzymatic activities of uPA and tPA in proteins extracted from the non-denatured and denatured cartilage tissues. The enzyme activities of uPA and tPA were measured using the SensoLyte AMC Urokinase (uPA) Activity Assay Kit Fluorimetric and the SensoLyte AMC Tissue-type (tPA) Activity Assay Kit Fluorimetric (both from AnaSpec), respectively, according to the kit's protocol. This measurement was performed on cartilage tissue from 11 cases. No significant difference in uPA activity was observed between the non-denatured and denatured cartilage tissues (Fig. 3A). However, tPA activity was higher than uPA in both the non-denatured and denatured cartilage tissues, and was significantly higher in the denatured cartilage tissues than in the non-denatured cartilage tissues (Fig. 3B). In all 11 cases analyzed, tPA activity was higher in the denatured cartilage tissues than in the non-denatured cartilage tissues (Fig. 3B). These results suggest that plasmin activity in the denatured cartilage tissues may be primarily due to the action of tPA.

[0037] 2-3. Investigation of the Involvement of Fibrin in the Enhancement of Plasmin Activity in Degenerated Cartilage As mentioned above, fibrin deposition in osteoarthritis knee joints and elevated fibronectin expression in degenerated cartilage have been reported (Non-Patent Documents 6 and 7). Furthermore, it has been reported that fibronectin covalently binds to fibrin through the action of coagulation factor XIII during blood clotting (Non-Patent Document 9). Based on these findings, we measured the total amount of fibronectin, fibrin degradation products (FDP) and D-dimer extracted from proteins extracted from cartilage tissue of the 17 cases mentioned above. The average amount of fibronectin extracted from degenerated cartilage was approximately 2.6 μg per gram of wet cartilage tissue (Figure 4A). The average amount of FDP and D-dimer extracted from degenerated cartilage was more than four times that of non-denatured cartilage (Figure 4B). Both results indicated that the amount of FDP and D-dimer extracted from degenerated cartilage was significantly higher than that from non-denatured cartilage. The amounts of fibronectin extracted and the amounts of FDP and D-dimer extracted were measured by Luminex (Diasorin) using a Human Luminex Assay Kit (R&D Systems). These results suggest that fibrin accumulates in areas of cartilage degeneration in osteoarthritis by binding to locally produced fibronectin, and that this fibrin accumulates in areas of cartilage degeneration in osteoarthritis by binding to tPA produced in the same area, inducing tPA activity despite the presence of PAI-1, resulting in the production of plasmin, which in turn leads to the degradation of fibrin.

[0038] 3. Inhibitory Effect of Fibrin Formation Inhibitors on Cartilage Degeneration If the degeneration and loss of cartilage matrix in osteoarthritis is actually caused by plasmin, and if plasmin activity is primarily induced by the action of tPA, then inhibiting fibrin formation in the knee joint of osteoarthritis would suppress plasmin production and prevent the progression of osteoarthritis. During blood coagulation, fibrin is formed by the production of fibrin monomers from fibrinogen by the action of thrombin, and it is thought that intra-articularly induced thrombin is also involved in the formation of fibrin in the knee joint of osteoarthritis. In fact, it has been reported that thrombin-antithrombin III complex (TAT), an indicator of thrombin activity, is highly elevated in synovial fluid of osteoarthritis (Non-Patent Document 10). Therefore, it was thought that inhibiting thrombin activity in the joint would also suppress intra-articular fibrin formation and thus inhibit cartilage degeneration.

[0039] Several anticoagulants that suppress fibrin formation are currently in clinical use, including dabigatran, which directly inhibits the activity of thrombin.

[0040] To investigate whether dabigatran actually inhibits fibrin formation and suppresses cartilage degeneration in osteoarthritis knee joints, we examined changes in synovial fluid following dabigatran administration in two patients (Case 1; male, 52 years old at the time of initiation of treatment; Case 2; female, 80 years old at the time of initiation of treatment) who were newly initiated on dabigatran for the treatment of other diseases during treatment. In each case, dabigatran was administered orally at a dose of 150 mg (Case 1) or 110 mg (Case 2) twice daily, after breakfast and dinner. The concentrations of various factors were measured in synovial fluid samples collected before and after the initiation of dabigatran administration, and their time-dependent changes were examined (Figure 5 (Case 1) and Figure 6 (Case 2)). The following factors were measured in each case: (i) TAT (thrombin-antithrombin III complex), an indicator of thrombin activity; The normal plasma value is 3 ng / ml or less. Measurements were outsourced to SRL. (ii) D-dimer: A type of fibrin degradation product. An indicator of fibrin degradation. The normal plasma value is 1.0 μg / ml or less. Measurements were outsourced to SRL. (iii) PAP (plasmin-α2-antiplasmin complex): An indicator of plasmin activity. The normal plasma value is 0.8 μg / ml or less. Measurements were outsourced to SRL. (iv) MMP-1 (matrix metalloproteinase-1): A type of MMP present in large amounts in synovial fluid that degrades cartilage matrix. Note that synovial fluid is rarely collected from normal joints, so the normal value is unknown. Concentrations were measured using Luminex (Diasorin) with a Human Luminex Assay Kit (R&D Systems). (v) Product of aggrecan concentration and synovial fluid volume The concentration of aggrecan (herein abbreviated as PG because aggrecan is a type of proteoglycan) in synovial fluid was measured using the Aggrecan (PG) Human ELISA Kit (ThermoFisher).Aggrecan is a major component of articular cartilage, and the product of its concentration and synovial fluid volume indicates the total amount of aggrecan released from cartilage within the joint, and is thought to be an index of cartilage degeneration.

[0041] In these cases, TAT levels were elevated in the synovial fluid before oral dabigatran administration was initiated, but TAT levels rapidly decreased with oral dabigatran administration (Figures 5A and 6A). In one of these two cases (Case 2), dabigatran administration was temporarily suspended from Week 9 to Week 16 for dental treatment, and this effect was evident in synovial fluid samples collected from Weeks 10 to 16 after administration. In this case, TAT levels in the synovial fluid clearly increased following the cessation of dabigatran (Week 10) and then decreased again clearly upon resumption of administration (Week 18) (Figure 6A). These results suggest that oral dabigatran indeed suppresses thrombin activity in synovial fluid of patients with knee osteoarthritis.

[0042] tPA is present in synovial fluid from patients with osteoarthritis, although at lower concentrations than in plasma. Plasminogen is also known to be present in synovial fluid at approximately 30% of the plasma concentration (Non-Patent Document 11). Therefore, if fibrin is produced in the joint of a patient with osteoarthritis, tPA binds to it and generates plasmin from plasminogen, which then degrades fibrin and produces FDP and D-dimer (see also "2-2. Plasmin Activation in Degenerated Cartilage"). In fact, it has been reported that the concentrations of plasmin-α2-antiplasmin complex (PAP) and D-dimer, indicators of plasmin activity, are significantly higher in synovial fluid from patients with osteoarthritis than in plasma from healthy individuals (Non-Patent Document 10; Non-Patent Document 11).

[0043] Based on these findings, we measured PAP and D-dimer levels in the synovial fluid of patients receiving dabigatran. Although these levels were significantly higher than normal plasma values ​​before dabigatran administration, dabigatran administration tended to decrease both levels (Fig. 5B and C, Fig. 6B and C). In particular, D-dimer levels decreased rapidly with dabigatran administration in both patients, prior to the decrease in PAP (Fig. 5B, Fig. 6B). These results suggest that dabigatran suppressed thrombin activity, thereby inhibiting fibrin production in the joints and resulting in a decrease in the concentration of fibrin degradation products, despite continued plasmin production.

[0044] In these cases, the MMP-1 concentration in synovial fluid gradually decreased from 35 weeks (Case 1) or 18 weeks (Case 2) after the start of dabigatran administration (Figures 5D and 6D), and the product of the aggrecan concentration in synovial fluid and the synovial fluid volume also gradually decreased (Figures 5E and 6E). As mentioned above, MMP-1 is an MMP present in relatively large amounts in synovial fluid and has the effect of degenerating cartilage matrix (Non-Patent Document 4). Furthermore, the product of the aggrecan concentration in synovial fluid and the synovial fluid volume is thought to represent the total amount of cartilage matrix degeneration products within the joint. Therefore, the decrease in these values ​​is considered to indicate that continued administration of dabigatran for a certain period of time improved the pathology of knee osteoarthritis and prevented cartilage degeneration and loss.

[0045] In conclusion, in osteoarthritis of the knee, oral administration of dabigatran reduces abnormally elevated thrombin activity in the joint, quickly suppressing fibrin formation, and by maintaining this state for a certain period of time, it is thought that cartilage degeneration and loss are also suppressed.

[0046] The present invention is expected to be widely used in the medical field.

Claims

1. An agent for inhibiting cartilage degeneration in osteoarthritic joints, which comprises a fibrin formation inhibitor as an active ingredient.

2. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is a thrombin inhibitor.

3. The cartilage degeneration inhibitor according to claim 2, wherein the thrombin inhibitor is one or more selected from the group consisting of dabigatran etexilate methanesulfonate, argatroban hydrate and hirudin.

4. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is warfarin potassium.

5. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is a coagulation factor Xa inhibitor.

6. The cartilage degeneration inhibitor according to claim 5, wherein the coagulation factor Xa inhibitor is one or more selected from the group consisting of rivaroxaban, apixaban, edoxaban tosilate hydrate and fondaparinux sodium.

7. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is heparin.

8. The cartilage degeneration inhibitor according to claim 7, wherein the heparin is one or more selected from the group consisting of heparin sodium and heparin calcium.

9. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is low molecular weight heparin.

10. The cartilage degeneration inhibitor according to claim 9, wherein the low molecular weight heparin is any one of enoxaparin sodium, parnaparin sodium, dalteparin sodium, and danaparoid sodium.

11. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is dried concentrated human antithrombin III.

12. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is antithrombin gamma.

13. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is dried concentrated human activated protein C.

14. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is thrombomodulin alpha.

15. The cartilage degeneration inhibitor according to claim 1, wherein the fibrin formation inhibitor is batroxobin.

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