KR20230000997A - Biguanide compound-based injection therapeutic composition for joint disease treatment - Google Patents

Abstract

The present invention relates to an injectable and oral therapeutic composition for treating joint diseases based on a biguanide compound. SD282, a novel compound of the present invention, was confirmed to reduce the degree of pain caused by osteoarthritis and to protect cartilage damage when administered by injection or oral administration to rats with induced osteoarthritis. In addition, it was confirmed that the expression of IL-1β, IL-6, and TNF-α, which are inflammatory factors, and the expression of MCP-1, which is a bone disease factor, are suppressed. In addition, it was confirmed that cartilage damage induced by arthritis was suppressed by increasing the expression of IL-10, which is an anti-inflammatory factor, TIMP1 and TIMP3, which are cartilage regeneration factors, and SOX9 and Aggrecan, which are cartilage formation markers.

Description

Biguanide compound-based injection therapeutic composition for joint disease treatment

The present invention relates to an injectable and oral therapeutic composition for treating joint diseases based on a biguanide compound.

Osteoarthritis is a type of arthritis, also referred to as degenerative arthritis, and refers to arthritis caused by degenerative changes in cartilage and surrounding bones in synovial joints. That is, osteoarthritis is a disease characterized by gradual loss of articular cartilage, hypertrophy of bones located below the cartilage, bone formation at the joint edge, and non-specific synovial inflammation. Osteoarthritis is a disease caused by damage to cartilage due to aging or excessive physical pressure (eg, obesity, trauma, etc.). Therefore, osteoarthritis shows severe pain and movement disorders in joints that receive a lot of weight, that is, the knee (knee) joint and the hip (hip) joint.

Osteoarthritis is a cartilage change stage that causes swelling due to an increase in water content in the cartilage (stage 1), and a fibrillation stage (stage 2) in which the cartilage surface is cracked and torn as the cartilage is destroyed, revealing the bone and narrowing the joint cavity. , Chondrocytes begin to produce cartilage to restore cartilage, but since cartilage destruction occurs faster than cartilage production, overall cartilage is reduced (Stage 3), bone changes that result in bone deformity and dysfunction Step (step 4), and the joint soft tissue change step (step 5) in which the soft tissue becomes thicker.

Rheumatoid arthritis, which belongs to a different classification from osteoarthritis, is a chronic autoimmune disease characterized by inflammation and proliferation of synovial cells. Unlike osteoarthritis, osteoporosis and bone erosion of bones around joints occur. In rheumatoid arthritis, inflammation of the synovial membrane spreads to the joint capsule, ligament, and tendon (stage 1), and the gradual destruction of joint cartilage narrows the joint space. The joint membrane and ligament tension is lost (step 2), the inflammation invades the bone and partial erosion of the bone occurs (step 3), and the joint function is lost (step 4). Therefore, osteoarthritis and rheumatoid arthritis have completely different causes and progression stages, and treatment methods for them are also different.

Currently, drugs such as acetaminophen, tramadol, nonsteroidal antiinflammatory drugs (NSAIDs), diacerin, and glucosamine are used for the treatment of osteoarthritis. Among these, non-steroidal anti-inflammatory drugs have been pointed out as problems with gastrointestinal side effects such as gastric and duodenal ulcers. Therefore, when these drugs are administered to patients with osteoarthritis who have risk factors for gastrointestinal side effects, cytoprotective agents such as rebamipide, H2-receptor antagonists such as cimetidine and ranitidine, and proton pump inhibitors such as omeprazole are used. prescribed at the same time.

Accordingly, the inventors of the present invention, while searching for a material capable of effectively preventing or treating osteoarthritis, confirmed that a newly synthesized novel compound can effectively treat osteoarthritis, thereby completing the present invention.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating bone disease comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:

[Formula 1]

Another object of the present invention is to provide a food composition for preventing or improving bone disease comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating bone disease comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:

[Formula 1]

In addition, the present invention provides a food composition for preventing or improving bone disease comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

SD282, a novel compound of the present invention, was confirmed to reduce pain caused by osteoarthritis and protect cartilage damage when injected or orally administered to rats induced with osteoarthritis. In addition, it was confirmed that the expression of IL-1β, IL-6 and TNF-α, which are inflammatory factors, and the expression of MCP-1, which is a bone disease factor, were suppressed. In addition, it was confirmed that the anti-inflammatory factor IL-10, the cartilage regeneration factors TIMP1 and TIMP3, and the cartilage formation markers SOX9 and Aggrecan were suppressed to inhibit cartilage damage induced by arthritis, which is useful for related industries. available.

1 is a diagram showing the results of measuring the degree of arthritic pain improvement according to the SD282 treatment of the present invention by measuring seconds and weight until the time to endure when a certain amount of pain was applied (A: from the time of induction of osteoarthritis, B: from the start of osteoarthritis Average of pain results up to the end of day 25).
Figure 2 is an osteoarthritis animal model according to the SD282 treatment of the present invention, using the principle of applying less force to the painful leg. right hind rimb), and SD282 administration compared to the vehicle, confirming that the balance of both feet was well maintained and statistically significant (A: from the time of osteoarthritis induction, B: from the start of osteoarthritis to the end of osteoarthritis on the 25th day).
Figure 3 is a diagram confirming the degree of cartilage damage in an osteoarthritis animal model according to the SD282 treatment of the present invention by Safranin O staining.
Figure 4 quantifies the degree of cartilage damage in an osteoarthritis animal model according to the SD282 treatment of the present invention.
Figure 5 is a diagram confirming the infiltration of inflammatory factors in the synovial tissue of an animal model of osteoarthritis according to the SD282 treatment of the present invention by immunohistochemical staining (A: immunohistochemical staining result, B: quantification of the degree of inflammatory factor).
6 is a diagram showing the quantification of IL-6 expression according to IL-1β treatment in SD282 of the present invention in a chondrocyte cell line of an osteoarthritis patient.
7 is a diagram showing the quantification of MCP-1 expression in chondrocyte cell lines of osteoarthritis patients treated with SD282 according to the present invention (A: expression confirmed by IL-1β treatment, B: expression by IL-1β and IL-17 treatment) Confirm).
8 is a diagram showing the quantification of the expression of IL-10, an anti-inflammatory factor, in cartilage cell lines of osteoarthritis patients treated with SD282 according to the present invention (A: expression confirmed by IL-1β treatment, B: IL-1β and IL-17 expression confirmation per treatment).
9 is a diagram showing the quantification of the expression of TIMP1 and TIMP3, which are cartilage regeneration factors, in cartilage cell lines of osteoarthritis patients treated with SD282 according to the present invention (A: TIMP1 expression confirmed, B: TIMP3 expression confirmed).
10 is a diagram showing the quantification of the expression of SOX9 and Aggrecan, which are chondrogenic markers, in osteoarthritis patient chondrocyte cell lines treated with SD282 according to the present invention (A: confirmed expression by IL-1β treatment, B: IL-1β and IL-17 expression confirmation per treatment).
11 is a diagram confirming the pain control effect in an osteoarthritis animal model according to oral administration of SD282 of the present invention (A: Confirmation of improvement in Paw withdrawal latency, B: Confirmation of improvement in Paw withdrawal threshold, C: Confirmation of improvement in Weight on right hind rimb ).

The present invention provides a pharmaceutical composition for preventing or treating bone disease comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:

More preferably, the compound of Formula 1 is SD282 (N-Ethyl-4-fluorophenyl biguanide), which may be a derivative of biguanide.

Conventional immunosuppressive drugs are generally accompanied by side effects such as cytotoxicity, infection due to immunosuppression, diabetes, tremor, headache, diarrhea, hypertension, nausea, and renal dysfunction, so the long-term treatment effect is difficult to sustain. . Accordingly, methods of administering or replacing immunosuppressive agents with different mechanisms of action have been attempted, particularly in the field of arthritis, in order to reduce serious side effects and increase immunosuppressive treatment effects. Therefore, it is urgent to develop new immunosuppressive or immunomodulatory therapies that can reduce the side effects of existing immunosuppressants and improve the therapeutic effect, and to discover new immunosuppressant candidates that are safer and have fewer side effects. Accordingly, the inventors of the present invention completed the present invention by confirming that the novel compound SD282 inhibits osteoarthritis and effectively prevents and treats osteoarthritis while reducing the side effects of conventional immunosuppressants.

As used herein, the term "prevention" refers to any action that suppresses symptoms or delays the progression of a specific disease by administering the composition of the present invention.

The term "treatment" used in the present invention refers to all activities that improve or beneficially change the symptoms of a specific disease by administration of the composition of the present invention.

The pharmaceutically acceptable salt may include an acid addition salt formed by a pharmaceutically acceptable free acid, and organic acids and inorganic acids may be used as the free acid. The organic acids include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, aspartic acid, and the like. It may include, but is not limited to. In addition, the inorganic acid may include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like, but is not limited thereto.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. As long as the adjuvant is known in the art, any one may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.

The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention is formulated in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions (injections) according to conventional methods, respectively. It may be used, preferably an injection solution (injection), but is not limited thereto.

The term "injection" used in the present invention is a method of administration for the purpose of local or systemic action by injecting a medicinal solution into a part of the body using a syringe and acupuncture needles, rather than conventional oral administration, suppository administration or external administration. It is a dosing method that can accurately and quickly obtain the effect of the drug and can effectively deliver the drug to the body even when it is impossible to administer the drug orally or in situations where it is difficult to change or absorb the drug due to digestive juices. Injection is largely intradermal There are injections, subcutaneous injections, intramuscular injections, intravenous injections, or arterial injections. Intradermal injection is characterized by slow absorption and visible reaction, and is an injection method that can be used to diagnose or prevent diseases or to check side effects of antibiotics before surgery. Subcutaneous injection is a method of injecting a drug into the subcutaneous connective tissue and is rapidly absorbed, and intramuscular injection is a method of injecting into the muscle tissue of the buttocks or upper arm so that the drug is quickly absorbed by the high blood vessel distribution in the muscle. . Intravenous injection is a method of administering a drug directly into a vein. It is an injection method that allows the drug to act rapidly and administers an accurate amount. , can administer large amounts of drugs. Arterial injection is a method of injecting a drug into an artery flowing into the affected area, and a direct effect of the drug on the affected area can be expected.

On the other hand, as an orthopedic procedure, except for the above injection, intra-articular injection for directly injecting drugs into the joint cavity is being performed, and chondromalacia of the patella, meniscal tear, knee inflammation Alternatively, as a preoperative treatment for arthritis, a drug administration method using joint cavity injection is in the limelight. Joint cavity injection, by directly administering a drug into the joint cavity, can rapidly improve intra-articular pain and induce rapid recovery of cartilage, joint inflammation and bone damage.

Injections for use in the above injections refer to aqueous, water-soluble, oily, suspension, emulsion, and solid (administration after dissolution) formulations, which are formulated for the purpose of injection so that the drug directly acts in the body without passing through the digestive tract. Means a formulated agent. Conditions for the injection include: 1) no excipients, 2) completely sterile, 3) no pyrogens, 4) an osmotic pressure similar to serum osmotic pressure, 5) a pH similar to serum pH, and 6) Any preparation that does not chemically react with components of body tissue can be used as an injection.

In addition, the above injections may contain solvents, solubilizing agents, buffers, isotonic agents, stabilizers, antioxidants, soothing agents, and suspending agents as additives according to the Ministry of Food and Drug Safety Guidelines for drug additives. "Additives" that can be incorporated into injections are substances other than the active ingredients contained in the formulation, and are used for purposes such as increasing the usefulness of the drug, facilitating formulation, stabilizing the formulation, and improving the appearance. Excipients, stabilizers, preservatives, buffers, corrigents, suspending agents, emulsifiers, fragrances, solubilizing agents, coloring agents, thickeners, etc. may be used as necessary. However, the additive used means that it does not show a direct pharmacological effect at the dosage of the preparation, is safe, and does not change the therapeutic effect of the preparation or interfere with the test, and can be summarized as follows.

1) Improvement of stability, bioavailability, etc.

2) Maintaining the quality of formulations during preservation or use

3) Improvement of drug economy by controlling the physical properties of medicines

The "solvent" that can be incorporated into the above additives is distilled water for injection, 0.9% sodium chloride injection solution, IV injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated IV injection solution, ethanol, propylene glycol , non-volatile oils-sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate, and the like may be incorporated. Sodium benzoate, sodium salicylate, sodium acetate, urea, urethane monoethylacetamide, butazolidine, propylene glycol, tweens, nijuntamide, hexamine, dimethylacetamide, etc. may be incorporated as "dissolution aids". . As "buffers" can be incorporated weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptones, gums, and the like. Sodium chloride may be incorporated as the "isotonic" agent, and as the "stabilizer", sodium bisulfite (NaHSO ₃ ), carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₃ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid, and the like may be incorporated. As the "sulfating agent", sodium bisulfide 0.1%, sodium formaldehyde, sulfoxylates, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite, and the like may be incorporated. Benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, calcium gluconate, etc. may be incorporated as "silencing agent", and Siemsis sodium, sodium alginate, Tween 80, aluminum monostearate, etc. may be incorporated as "suspending agent". there is.

The pharmaceutical composition of the present invention, when formulated, can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected according to the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmacological activity may not appear, and if the concentration exceeds 5,000 μg/ml, toxicity to the human body may be exhibited.

According to one embodiment of the present invention, the compound may relieve joint pain.

According to one embodiment of the present invention, the compound may inhibit cartilage damage.

According to one embodiment of the present invention, the compound may inhibit the expression of an inflammatory factor, and the inflammatory factor may be a factor selected from the group consisting of IL-1β, IL-6, and TNF-α.

According to one embodiment of the present invention, the compound may inhibit the expression of bone disease factor MCP-1 (Monocyte Chemoattractant Protein-1).

According to one embodiment of the present invention, the compound may increase the expression of the anti-inflammatory factor IL-10.

According to one embodiment of the present invention, the compound may increase the expression of cartilage regeneration factor TIMP1 (tissue inhibitor of matrix metalloproteinase 1) or TIMP3 (tissue inhibitor of matrix metalloproteinase 3).

According to one embodiment of the present invention, the compound may increase the expression of cartilage formation marker SOX9 (SRY-Box Transcription Factor 9) or Aggrecan.

According to one embodiment of the present invention, the bone disease may be osteoarthritis, but is not limited thereto.

According to one embodiment of the present invention, SD282 of the present invention may be administered at a concentration of 0.1 to 100 mg/kg, preferably at a concentration of 30 to 50 mg/kg.

In addition, the present invention provides a food composition for preventing or improving bone disease comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

As used herein, the term "improvement" refers to any action that at least reduces a parameter associated with the condition being treated, eg, the severity of a symptom.

In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.

Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agents described above, natural flavoring agents (thaumatin), stevia extracts (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is

In addition, the food composition, in addition to the active ingredient extract, various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like may be contained. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.

The functional food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating bone diseases. In the present invention, 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions. The health functional food of the present invention may contain ordinary food additives, and the suitability as a food additive is determined according to the general rules of the Food Additive Code and General Test Methods approved by the Food and Drug Administration, unless otherwise specified. It is judged according to standards and standards. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations. For example, a health functional food in the form of a tablet is obtained by granulating a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding, or as described above. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a flavoring agent and the like as needed. Among health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture in which the active ingredient of the present invention is mixed with additives such as excipients in a normal hard capsule. It can be prepared by filling the mixture mixed with gelatin in a capsule base. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. The health functional food in the form of a pill can be prepared by molding a mixture obtained by mixing the active ingredient of the present invention with excipients, binders, disintegrants, etc. by a conventionally known method, and can be coated with sucrose or other coating agents if necessary, Alternatively, the surface may be coated with a material such as starch or talc. Health functional food in the form of granules can be prepared in granular form by a conventionally known method of mixing the active ingredient of the present invention with excipients, binders, disintegrants, etc., and may contain flavoring agents, flavoring agents, etc. can

Hereinafter, the present invention will be described in more detail by examples. These examples are merely for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Preparation Example 1> Synthesis of SD282

A novel compound of the present invention, SD282, was synthesized. Specifically, after dissolving an aniline compound (1.0 mmol) in an acetonitrile solvent in a sealed reactor, dicyanodiamide (1.0 mmol) and concentrated sulfuric acid (1.0 mmol) were added, sealed, and stirred at 175 ° C. for 1 hour. After cooling to room temperature to produce a white solid, the solvent was removed and washed with hexane and isopropyl alcohol to synthesize the following compound (SD-282) in the form of a white solid. For the synthesized compounds, each compound was identified through H-NMR analysis, and the results are as follows.

As ^a result of 1H-NMR analysis, the SD282 compound was 1H-NMR (600MHz, DMSO) δ (ppm): 7.36-7.34 (m, 2H), 7.31-7.28 (m, 2H) 7.105 (s, 2H) 6.89 (s, 4H) 3.69 (t, 2H, J=7.2Hz), 1.07 (t, 3H, J=7.2 Hz), MS (m/z): [M+H]+ 224 was obtained.

<Example 1> Osteoarthritis animal model

<1-1> Production of osteoarthritis animal model

In order to produce the osteoarthritis animal model of the present invention, 5-week-old male Wistar rats were bred at 21 to 22 ° C. in a light-dark cycle at 12-hour intervals, and sterilized water and feed were supplied. raised Thereafter, osteoarthritis was induced by administering monosodium iodoacetate (MIA, Sigma, ST. Louis, MO) to the right knee of the rat at a dose of 3 mg/50 μl to induce osteoarthritis. MIA was administered after being dissolved in physiological saline. Thereafter, the rats were classified into a vehicle group administered with no drug and an excipient of the drug; and an SD282 group administered with SD282, a novel compound of the present invention. At a concentration of 25 mg/kg, SD282 was administered by injection (articular injection) until the end of the experiment.

<1-2> Arthritis pain evaluation

In the animal model prepared in Example 1-1, it was investigated whether injection administration (administration into the joint cavity) of SD282 has an inhibitory effect on joint pain. Specifically, pain was measured using a dynamic plantat aesthsiometer (Ugo Basile, Comerio, Italy), and the machine's pain measurement method was to place a net plate on top of the measuring machine and put a rat inside an acrylic animal fixation frame on top of it. Afterwards, the right foot where the drug was injected was stabbed with a measuring machine. After being stabbed, the time taken for the machine to automatically release the foot (s, seconds) and how much weight was applied to the foot (g) were measured to confirm the time and weight that came out at that time. In addition, in order to confirm the change in the weight balance according to the induction of arthritis, the weight on the right hind limb was measured to confirm the weight to body weight.

As a result, as shown in FIG. 1, it was confirmed that the pain response level of the rats injected with SD282 of the present invention was recovered, and as shown in FIG. 2, in the rats injected with SD282, the weight on right hind rimb increased, , it was confirmed that the weight balance increased.

<Example 2> Articular cartilage protection effect of SD282

It was investigated whether the SD282 of the present invention inhibits joint destruction in an osteoarthritis animal model. Specifically, the osteoarthritis-induced rat of Example 1-2 was humanely sacrificed, and joint tissue was obtained, and the degree of tissue damage was observed by Safranin O staining. Safranin O stains the acidic proteoglycan present in cartilage tissue, and when combined with glucosaminoglucan in cartilage, the staining is orange, and the color is developed in cartilage. It was confirmed that cartilage damage was suppressed in the SD282 injection group compared to the vehicle.

As a result, in the osteoarthritis-induced rats, the OARSI score and Total Mankin score of the joint tissue increased, and cartilage damage was observed. It was confirmed that this was suppressed (Figs. 3 and 4).

<Example 3> Confirmation of immunomodulatory effect of SD282

In order to confirm the effect of SD282 of the present invention on inflammatory factors in synovial tissue in an animal model of osteoarthritis, the joint tissue of the rat of Example 1-2 was prepared as a paraffin block, and each joint section was immunohistochemically stained. It was confirmed by staining. DAB color-positive cells were quantified, specifically, immune-positive cells for IL-1β, IL-6, and TNF-α were identified and quantified.

As a result, as shown in Figure 5, compared to the Vehicle group, it was confirmed that the expression levels of inflammatory cytokines IL-1β, IL-6 and TNF-α were significantly reduced in the synovial tissue of rats treated with SD282.

<Example 4> Confirmation of SD282 effect in osteoarthritis patient cartilage

<4-1> Confirmation of IL-6 inhibitory effect of SD282 in osteoarthritis patient chondrocyte cell line

In order to confirm whether SD282 of the present invention inhibits the expression of IL-6, an inflammatory factor, in chondrocytes of osteoarthritis patients, it was obtained by cartilage during joint replacement surgery of osteoarthritis patients, treated with collagenase, and separated into single cells of chondrocytes. In this experiment, the experiment was conducted with cells up to passage 4 that do not lose chondrocyte properties. Chondrocytes were seeded with 5x10 ⁴ cells in 24 wells. After seeding and starvation, chondrocytes were treated with 20 ng/ml of IL-1β to promote pathogenesis. In addition, treatment with 20 ng/ml of IL-1β + 10 ng/ml of IL-17 results in the addition of cytokine for joint pathogenesis, and treatment with 200, 500, and 1000 μM of SD282, respectively, and incubation for 24 hours in each condition that promotes a more serious etiological state. After that, the expression of IL-6, an inflammatory cytokine, was measured in the obtained supernatant by ELISA.

As a result, as shown in FIG. 6 , it was confirmed that the expression of IL-6 was significantly decreased in the chondrocyte line treated with SD282 compared to the control vehicle group.

<4-2> Confirmation of MCP-1 inhibitory effect of SD282 in chondrocyte cell lines of patients with osteoarthritis

In order to confirm that SD282 of the present invention reduces the expression of MCP-1 (monocyte chemoattractant protein 1), a bone disease factor, compared to the vehicle group, the chondrocyte cell line of Example 4-1 was treated with IL-1β at 20 The treatment was performed at a concentration of ng/ml, and SD282 was treated at 200, 500, and 1000 μM, respectively, to confirm expression of MCP-1. In addition, the above chondrocyte lines were treated with 20 ng/ml of IL-1β and 10 ng/ml of IL-17, and treated with SD282 at the same concentration as above, respectively, and after 24 hours of culture, supernatant was obtained and expression of MCP-1 was evaluated by ELISA confirmed by

As a result, it was confirmed that the expression of MCP-1 induced by IL-1β or IL-1β and IL-17 decreased in a concentration-dependent manner when treated with SD282 compared to the Vehicle group (FIG. 7).

<4-3> Confirmation of IL-10 increasing effect of SD282 in osteoarthritis patient chondrocyte cell line

In order to confirm that SD282 of the present invention increases the expression of IL-10, an anti-inflammatory factor, compared to the Vehicle group, IL-1β was added to the chondrocyte cell line of Example 4-1 at a concentration of 20 ng/ml. and SD282 was treated with 200, 500 and 1000 μM, respectively, and expression of IL-10 was confirmed. In addition, the chondrocyte cell line was treated with 20 ng/ml of IL-1β and 10 ng/ml of IL-17, and 1000 μM of SD282, and the expression of IL-10 was confirmed after culturing for 24 hours.

As a result, as shown in FIG. 8, compared to the Vehicle group, when SD282 was treated, the expression of IL-1β or IL-10 decreased by IL-1β and IL-17 was significantly increased in a concentration-dependent manner. confirmed that.

<4-4> Confirmation of increase in cartilage regeneration factor of SD282 in chondrocyte cell lines of patients with osteoarthritis

In order to determine whether SD282 of the present invention increases the expression of (anabolic pathway) TIMP1 (tissue inhibitor of matrix metalloproteinase 1) and TIMP3 compared to the Vehicle group, the chondrocyte cell line of Example 4-1 In this, 20 ng/ml of IL-1β was treated, and SD282 was treated with 200, 500, and 1000 μM, respectively, and the expression of TIMP1 and TIMP3 was confirmed by real time PCR, respectively.

As a result, as shown in FIG. 9, it was confirmed that the expression of cartilage regeneration factors TIMP1 and TIMP3, which were reduced by treatment with IL-1β, increased significantly in the SD282-treated group compared to the Vehicle group.

<4-5> Confirmation of expression of chondrogenic markers in chondrogenic cell lines of patients with osteoarthritis

It was confirmed whether SD282 of the present invention increases the expression of chondrogenic markers compared to the Vehicle group. Specifically, the expression of SOX9 (SRY-Box Transcription Factor 9), known as a cartilage differentiation marker, and Aggrecan, a cartilage matrix interaction factor, were confirmed. The chondrogenic cell line of Example 4-1 was treated with 20 ng/ml of IL-1β and SD282 at 200, 500, and 1000 μM, respectively, and the expression of chondrogenic markers SOX9 and Aggrecan was confirmed by real-time PCR. In addition, 20 ng/ml of IL-1β and 10 ng/ml of IL-17 were treated, and 500 μM of SD282 was treated to further confirm the expression of SOX9 and Aggrecan, which are chondrogenic markers.

As a result, as shown in FIG. 10, the expression of SOX9 and Aggrecan, which are chondrogenic markers, decreased by treatment with IL-1β or IL-1β and IL-17 in the osteoarthritis patient's chondrocyte cell line compared to the Vehicle group, SD282 It was confirmed that a significant increase was observed in the treated group.

<Example 5> Confirmation of pain control effect of SD282 according to comparison between injection administration (joint cavity administration) and oral administration

It was further confirmed whether SD282 of the present invention controls pain induced by osteoarthritis, and pain control was compared according to the administration method (injection administration (joint cavity administration) and oral administration). Specifically, as in Example 1, SD282 was orally administered at a concentration of 25 mg/kg to an animal model in which arthritis was induced, and then pain was measured using a dynamic plantat aesthsiometer (Ugo Basile, Comerio, Italy). . To measure the pain of the machine, a net plate was placed on the measuring machine, and a rat was placed in an acrylic animal fixing frame on top of the measuring machine, and the right foot was injected with the medicine. After being stabbed, the time taken for the machine to automatically release its foot (s, sec, Paw withdrawal latency) and how much weight it takes to release its foot (g, Paw withdrawal threshold) are measured to check the time and weight that come out at that time. did In addition, in order to confirm the change in the weight balance according to the induction of arthritis, the weight on the right hind limb was measured to confirm the weight to body weight. As a control group, a healthy control group in which arthritis was not induced and a vehicle group in which arthritis was induced and not treated with a drug were used.

As a result, as shown in FIG. 11, compared to the normal control group, the vehicle group of the animal model administered orally administered by administration method significantly decreased Paw withdrawal latency, Paw withdrawal threshold, and weight on right hind rimb, but oral SD282 In the administered group, Paw withdrawal latency, Paw withdrawal threshold, and weight on right hind rimb were significantly increased, confirming that arthritis-induced pain was improved.

In addition, when comparing the injection administration (joint cavity administration) and the oral administration group by administration method, the injection administration group's Paw withdrawal latency was maintained at 15 seconds (S) 7 to 8 days after drug injection, and the Paw withdrawal threshold was 30 to 35 g, but in the oral administration group, it was confirmed that the Paw withdrawal latency was 10 to 12 seconds (S) and the Paw withdrawal threshold was 20 to 25 g, confirming that the injection administration group was more effective in improving osteoarthritis-induced pain. Therefore, it was confirmed that there is a difference in the treatment effect of osteoarthritis depending on the administration method even for the same novel drug (FIG. 1 and FIG. 11).

Accordingly, it was confirmed that the novel compound of the present invention, SD282, when injected or orally administered to rats induced with osteoarthritis, reduced the degree of pain caused by osteoarthritis and protected cartilage damage. In addition, it was confirmed that the expression of IL-1β, IL-6 and TNF-α, which are inflammatory factors, and the expression of MCP-1, which is a bone disease factor, were inhibited. In addition, it was confirmed that cartilage damage induced by arthritis was inhibited by increasing the expression of anti-inflammatory factor IL-10, cartilage regeneration factors TIMP1 and TIMP3, cartilage formation markers SOX9 and Aggrecan.

Claims (13)

A pharmaceutical composition for preventing or treating bone disease comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:
[Formula 1]

. According to claim 1,
The compound is a pharmaceutical composition for preventing or treating bone diseases, characterized in that to relieve joint pain. According to claim 1,
The compound is a pharmaceutical composition for the prevention or treatment of bone diseases, characterized in that to inhibit cartilage damage. According to claim 1,
The compound is a pharmaceutical composition for preventing or treating bone diseases, characterized in that inhibiting the expression of inflammatory factors. According to claim 4,
The inflammatory factor is a pharmaceutical composition for preventing or treating bone diseases, characterized in that the factor selected from the group consisting of IL-1β, IL-6 and TNF-α. According to claim 1,
The compound is a pharmaceutical composition for preventing or treating bone disease, characterized in that inhibiting the expression of the bone disease factor MCP-1 (Monocyte Chemoattractant Protein-1). According to claim 1,
The compound is a pharmaceutical composition for preventing or treating bone disease, characterized in that increases the expression of the anti-inflammatory factor IL-10. According to claim 1,
The compound is a pharmaceutical composition for preventing or treating bone disease, characterized in that it increases the expression of cartilage regeneration factor TIMP1 (tissue inhibitor of matrix metalloproteinase 1) or TIPM3 (tissue inhibitor of matrix metalloproteinase 3). According to claim 1,
The compound is a pharmaceutical composition for preventing or treating bone disease, characterized in that it increases the expression of SOX9 (SRY-Box Transcription Factor 9) or Aggrecan, a cartilage formation marker. According to claim 1,
The bone disease is a pharmaceutical composition for preventing or treating bone disease, characterized in that osteoarthritis (Osteoarthritis). According to claim 1,
The composition is for preventing or treating bone diseases, characterized in that the formulation is selected from the group consisting of oral formulations such as sterile injectable solutions, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations or suppositories pharmaceutical composition. According to claim 1,
The pharmaceutical composition for preventing or treating bone diseases, characterized in that the composition is administered by a method selected from the group consisting of oral administration, intraarticular injection, intravenous injection, intramuscular injection, subcutaneous injection, and intraperitoneal injection. A food composition for preventing or improving bone disease comprising a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:
[Formula 1]

.

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