Compositions for treating joint or connective tissue diseases, comprising dextran or poloxamer - Patent Application 20070122997

Abstract

To provide pharmaceutical compositions for treating joint or connective tissue diseases, compositions for cartilage regeneration, or pharmaceutical compositions for treating inflammatory diseases.SOLUTION: Disclosed is a composition for preventing or treating a joint disease or connective tissue disease containing dextran, poloxamer or a mixture thereof as the effective ingredients.EFFECT: The composition of the invention stays in the joint or connective tissue for a long time due to the shock-absorbing effect, coating effect or anti-inflammation effect, and relieves the shock, covers a damaged portion in a specific manner thereto, or reduces inflammation of an adhered portion. Thus, the composition may be effectively used for the treatment of the joint or connective tissue disease, or for the cartilage regeneration.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present invention relates to a composition for treating joint or connective tissue diseases, cartilage regeneration, or inflammatory diseases, which comprises dextran, poloxamer, or a mixture thereof. [Background technology]

[0002] Joints are the areas where bones connect and are made up of cartilage, joint capsules, synovial membranes, ligaments, tendons, muscles, etc., which allow smooth movement between the bones and absorb the shock generated by movement. Arthritis refers to functional abnormalities such as inflammation in the joints caused by various causes. Connective tissue is tissue that connects tissues in animals to form organs, and examples include cartilage, intervertebral discs, tendons, ligaments, bones, skin, adipose tissue, blood vessels, and intestinal tissue.

[0003] Medications used to treat arthritis can be broadly classified based on their primary mechanism of action: reducing inflammation, slowing disease progression, and reducing concentrations of metabolic products such as uric acid. Many arthritis medications work by reducing inflammation. Inflammation is a pathological process that causes pain, swelling, heat, redness, and stiffness. Drugs that rapidly relieve inflammation include nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and steroidal anti-inflammatory drugs (e.g., cortisone). However, these anti-inflammatory and anti-inflammatory drugs primarily function to relieve pain rather than treat the disease, and long-term use of these drugs can lead to numerous complications. Steroidal anti-inflammatory drugs, in particular, only temporarily relieve pain through their strong, temporary anti-inflammatory effects, without addressing the underlying cause of the disease. These drugs can lead to overuse of the joints, which can lead to joint destruction and aggravate the condition, so caution is advised when using them.

[0004] Therefore, conventional treatments used for joint damage such as arthritis are limited in their effectiveness, have obvious toxic side effects, cannot be used sustained for long periods of time, and are therefore limited in their effectiveness. Therefore, there is a pressing need for new and novel treatments or therapeutic agents that overcome the drawbacks of conventional treatments.

[0005] Dextran, a type of polysaccharide, refers to a polymer of D-glucose. It has a structure similar to starch and glycogen, with D-glucose units linked in a linear chain via α-1,6 bonds and occasionally branched via α-1,4 or α-1,3 bonds. Commonly used high-molecular-weight dextrans are known to frequently induce allergic reactions in the body. Dextran has been used as a plasma expander, a blood coagulant (anti-adhesion agent), and a filler using cross-linked dextran, but its use as a therapeutic agent for joint or connective tissue diseases has not been reported. Poloxamer, a nonionic surfactant, is known to be used as an emulsifier, stabilizer, and solubilizer in addition to its role as a surfactant.

[0006] Currently, due to limitations of conventional treatments for joint or connective tissue diseases, namely, limited efficacy, significant toxic side effects, and lack of durability, new treatments or therapeutic agents are needed. However, there is no known research on the treatment of joint or connective tissue diseases using dextrans or poloxamers, particularly low molecular weight dextrans or poloxamers, that do not cause allergic reactions when administered internally. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the present inventors have conducted research into the treatment of joints or connective tissues and have found that dextran, poloxamer, or a mixture thereof does not cause allergic reactions and has a protective effect on joints and connective tissues by providing a buffering effect, a maintenance effect, or an anti-inflammatory effect on damaged areas of joints and connective tissues, thereby completing the present invention.

[0008] An object of the present invention is to provide a pharmaceutical composition for treating joint diseases or connective tissue diseases, a composition for cartilage regeneration, or a pharmaceutical composition for treating inflammatory diseases, which comprises dextran, poloxamer, or a mixture thereof. [Means for solving the problem]

[0009] To achieve the above object, the present invention provides a pharmaceutical composition for treating joint or connective tissue diseases, comprising dextran, poloxamer, or a mixture thereof.

[0010] The present invention also provides a pharmaceutical composition for preventing or treating joint diseases or connective tissue diseases, comprising a mixture of two compounds selected from the group consisting of dextran 1, dextran 5, and poloxamer 188.

[0011] The present invention also provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises dextran, poloxamer, or a mixture thereof as an active ingredient.

[0012] The present invention also provides a composition for cartilage regeneration, which comprises dextran, poloxamer, or a mixture thereof as an active ingredient.

[0013] The present invention also provides a composition for cartilage regeneration, comprising a mixture of two compounds selected from the group consisting of dextran 1, dextran 5, and poloxamer 188. [Effects of the Invention]

[0014] The composition of the present invention has a buffering effect, a coating effect, or an anti-inflammatory effect, and can remain at the damaged area of ​​the joint or connective tissue for a long time to cushion the impact, specifically wrap the damaged area, and reduce inflammation at the adhesion site, so it can be effectively used for the treatment of joint diseases, connective tissue diseases, or cartilage regeneration. [Brief explanation of the drawings]

[0015] [Figure 1] This figure shows the results of examining the cell proliferation ability of normal chondrocytes 24 hours after treatment with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001 compared to the negative control group (nc)). [Figure 2] This figure shows the results of examining the cell proliferation ability of normal chondrocytes 48 hours after treatment with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001 compared to the negative control group (nc)). [Figure 3] This figure shows the results of examining the cell proliferation ability of chondrocytes in an in vitro model of osteoarthritis induced by rhIL-1α treatment, 24 hours after treatment with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001, respectively, compared to the inflammation-induced positive control group (pc)). [Figure 4]This figure shows the results of examining the cell proliferation ability of chondrocytes in an in vitro model of osteoarthritis induced by rhIL-1α treatment, 48 hours after treatment with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001, respectively, compared to the inflammation-induced positive control group (pc)). [Figure 5] This figure shows the results of examining the IL-10 / IL-6 gene expression ratio in chondrocytes of an in vitro osteoarthritis model treated with rhIL-1α, treated with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001, respectively, compared to the inflammation-induced positive control group (pc)). [Figure 6] This figure shows the results of examining the MMP-3 gene expression level in chondrocytes of an in vitro model of osteoarthritis induced by rhIL-1α treatment, treated with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001, respectively, compared to the inflammation-induced positive control group (pc)). [Figure 7] This figure shows the results of examining the MMP-13 gene expression level in chondrocytes of an in vitro model of osteoarthritis treated with rhIL-1α, treated with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001, respectively, compared to the inflammation-induced positive control group (pc)). [Figure 8] This figure shows the results of examining the amount of type II collagen produced by treatment of chondrocytes in an in vitro model of osteoarthritis induced by rhIL-1α treatment with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001, respectively, compared to the inflammation-induced positive control group (pc)). [Figure 9]This figure shows the results of examining the amount of aggrecan produced by treating chondrocytes in an in vitro model of osteoarthritis with rhIL-1α with dextran, poloxamer, or a mixture of these (* / ** / *** indicate significance levels of p<0.05 / p<0.01 / p<0.001, respectively, compared to the inflammation-induced positive control group (pc)). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. The present invention provides a pharmaceutical composition for treating joint or connective tissue diseases, comprising dextran, poloxamer, or a mixture thereof. The present invention also provides a method for preventing or treating joint or connective tissue diseases, comprising administering dextran, poloxamer, or a mixture thereof to an individual in need thereof.

[0017] In the present invention, "dextran" refers to a polymer of D-glucose, a type of polysaccharide. Dextran has a structure similar to starch and glycogen, with D-glucose units linked in a linear chain via α-1,6 bonds and branched at intervals via α-1,4 or α-1,3 bonds. Dextran can be used as a plasma expander or blood coagulant, and dextran with different molecular weights can be used depending on the purpose. In the present invention, the dextran may have an average molecular weight of 500 to 10,000 Da, preferably 500 to 8,000 Da, and more preferably 1,000 to 5,000 Da. Dextran is named according to its molecular weight, such as "dextran 1" when the average molecular weight is 1,000 Da, "dextran 5" when the average molecular weight is 5,000 Da, and "dextran 10" when the average molecular weight is 10,000 Da.

[0018] Generally, dextrans of 8,000 Da or more are known to pose a high risk of causing allergic reactions when administered to the human body. In particular, the allergic reaction can lead to anaphylaxis, a sudden systemic reaction caused by an antigen-antibody immune response, which is extremely dangerous. However, the use of low-molecular-weight dextrans, preferably 1,000 Da to 5,000 Da dextrans, according to the present invention has the advantage of minimizing allergic reactions and prolonging the therapeutic effect of joint or connective tissue diseases because they are degraded more slowly in the body than known substances (e.g., hyaluronic acid).

[0019] In addition, the low molecular weight dextrans of the present invention are not only less likely to cause allergic reactions than dextrans of 8,000 Da or more, but also have the characteristics of having a rapid onset of action and being able to be distributed over a wide area. The present invention provides preferred examples using dextran 1 and dextran 5. These can be used in combination with dextran 1 and dextran 5, and can also be used in combination with poloxamer 188.

[0020] In the present invention, two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188 may be mixed in a volume ratio of 1:1.

[0021] In the present invention, the concentration of the dextran is expressed as a percentage of mass (g) concentration (w / v) in 100 cc of solvent. For example, when Dextran 1 is contained in the composition as the sole active ingredient, it may be contained at a concentration of 2 to 40 (w / v)%, and when Dextran 5 is contained in the composition as the sole active ingredient, Dextran 5 may be contained at a concentration of 2 to 30 (w / v)%.

[0022] Furthermore, when dextran is contained in the composition as a mixture of dextran 1 or 5, or a mixture of dextran and poloxamer, the concentration of dextran may vary depending on the mixture. For example, when a mixture of dextran 5 and dextran 1 is contained in the composition, the concentration of dextran 5 may be 2 to 40 (w / v)% and the concentration of dextran 1 may be 2.5 to 40 (w / v)%, or the concentration of dextran 5 may be 4 to 40 (w / v)% and the concentration of dextran 1 may be 5 to 40 (w / v)%.

[0023] When dextran is present in a composition at a high concentration, the water content of the polysaccharide can cause excessive viscosity increase, making it difficult to formulate. Therefore, dextran has generally been used at a concentration of 5% or less. However, the compositions of the present invention contain dextran at a concentration of at least 2% to a maximum of 40% (w / v), depending on the molecular weight, thereby maximizing the therapeutic effect on joint and connective tissue diseases. To increase the solubility or stabilize the dextran, the compositions of the present invention can further contain additives, which can include sugar alcohols, monosaccharides, or divalent cations, such as CaCl2.

[0024] In the present invention, "poloxamer" is a nonionic triblock copolymer consisting of hydrophobic propylene oxide at the center and hydrophilic ethylene oxide at both ends. It is temperature-sensitive and can be converted into a sol or gel depending on the concentration and temperature. The properties of poloxamer change depending on the ratio of polyoxypropylene and polyoxyethylene. In addition to being used as a surfactant, poloxamer is also used as an emulsifier, stabilizer, solubilizer, etc.

[0025] In the present invention, the poloxamer can have an average molecular weight of 100 to 20,000 Da.

[0026] The poloxamers include poloxamer 101, poloxamer 105, poloxamer 105 benzoate, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 182 dibenzoate, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer 217, poloxamer 231, poloxamer 232, poloxamer 233, poloxamer 234, poloxamer 235, poloxamer 236, poloxamer 237, poloxamer 238, poloxamer 239, poloxamer 240, poloxamer 241, poloxamer 242, poloxamer 243, poloxamer 244, poloxamer 245, poloxamer 246, poloxamer 247, poloxamer 248, poloxamer 249, poloxamer 250, poloxamer 251, poloxamer 252, poloxamer 253, poloxamer 254, poloxamer 255, poloxamer 256, poloxamer 257, poloxamer 258, poloxamer 259, poloxamer 260, poloxamer 261, poloxamer 262, poloxamer 26 Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403 and Poloxamer 407, preferably including, but not limited to, Poloxamer 188.

[0027] In the present invention, the concentration of the poloxamer is expressed as a percentage of the mass (g) concentration (w / v) in 100 cc of solvent, and may be contained in the composition at a concentration of 1 to 50 (w / v)% or 2 to 30 (w / v)%. For example, when poloxamer 188 is contained as the sole active ingredient in the composition, it may be contained in the composition at a concentration of 1 to 15 (w / v)% or 1 to 10 (w / v)%.

[0028] To increase the solubility or stabilize the poloxamer, the composition of the present invention may further contain additives such as sugar alcohols, monosaccharides, and divalent cations, or may contain sterile water or saline without any additional additives.

[0029] In the present invention, the pharmaceutical composition may contain a mixture of dextran and poloxamer. In particular, when poloxamer 188 is mixed with dextran, it may be mixed with dextran 1 or dextran 5. For example, when mixed with dextran 5, the concentration of dextran 5 may be 2 to 40 (w / v)% and the concentration of poloxamer 188 may be 1 to 20 (w / v)%, or the concentration of dextran 5 may be 4 to 40 (w / v)% and the concentration of poloxamer 188 may be 2 to 10 (w / v)%.

[0030] Furthermore, for example, when mixed with dextran 1, the concentration of the dextran 1 may be 2.5 to 40 (w / v)% and the concentration of the poloxamer may be 1 to 5 (w / v)%, or the concentration of the dextran 1 may be 45 to 55 (w / v)% and the concentration of the poloxamer 188 may be 1 to 2 (w / v)%.

[0031] Poloxamer 188 can be toxic when present in a composition at high concentrations, for example, exceeding 25% (w / v). When mixed with dextran, the concentration should be appropriately adjusted before use.

[0032] As an example, the dextran:poloxamer in the pharmaceutical composition of the present invention may be, for example, in the ratio (w / v) % of 5:0.05, 5:0.5, 5:5, 5:10, 5:20, 5:30, 5:40, 5:50, 5:100, 5:150, 5:200, 10:0.1, 10:1, 10:10, 10:20, 10:30, 10:40, 10:50, 10:100, 10:150, 10:300, 10:400, 50:0.5, 50:1, 50:5, 50:10, 50:20, 50:30, 50:40, 50:50, or, for example, 1:0.01 to 1:40. An exemplary combination is 1 (w / v)% poloxamer 188 and 1% dextran. When 40% (w / v) of Poloxamer 188 is mixed with 20% (w / v) of Dextran 5, the ratio is 1:40, and when 20% (w / v) of Poloxamer 188 is mixed with 2% (w / v) of Dextran 5, the ratio is 1:0.1, so the composition may contain 1:0.1 to 1:40. Here, the average molecular weight of the dextran may be 1,000 Da or 5,000 Da, and the average molecular weight of the poloxamer may be 8,500 Da. In addition, in one embodiment of the present invention, when a mixture of two components selected from the group consisting of Dextran 1, Dextran 5, and Poloxamer 188 is included, the respective components may be mixed in a volume ratio of 1:1 and included in the composition.

[0033] In one embodiment of the present invention, the pharmaceutical composition of the present invention comprises a mixture of dextran 1 and dextran 5. The ratio of dextran 1 to dextran 5 in the pharmaceutical composition may be, for example, 5:0.05, 5:0.5, 5:5, 5:10, 5:20, 5:30, 5:40, 5:50, 5:100, 10:0.1, 10:1, 10:10, 10:20, 10:30, 10:40, 10:50, 10:100, 10:150, 10:200, 50:0.5, 50:1, 50:5, 50:10, 50:20, 50:30, 50:40, 50:50, or 50:1000, for example, 1:0.01 to 1:20. An exemplary combination is dextran 1. When Dextran 2.5% (w / v) is mixed with Dextran 5 40% (w / v), the ratio is 1:16, and when Dextran 1 40% (w / v) is mixed with Dextran 5 2% (w / v), the ratio is 1:0.05, so the range is 1:0.05 to 1:16.

[0034] When the compositions of the present invention contain a mixture of dextran and poloxamer, the hydrophobic component of the poloxamer can increase the solubility of the dextran or coadministered drug, while the hydrophilic component of the poloxamer, polyethylene glycol (PEG), can hold cell membranes together to prevent adhesions and maintain tissue integrity. However, poloxamer has the disadvantage of being easily diluted and absorbed by body fluids, so mixing it with dextran can increase its stability in the body. Therefore, a mixture of dextran and poloxamer maintains its viscosity longer than a mixture containing dextran or poloxamer alone, allowing it to encase damaged joints or connective tissue sites, preventing pathological tissue adhesions and reducing inflammation caused by friction, thereby facilitating the delivery of nutrients and promoting tissue regeneration. These effects may be induced by the cushioning effect, coating effect, or anti-inflammatory effect of the dextran and poloxamer mixture.

[0035] In the present invention, the term "cushion effect" refers to the effect of acting like a cushion to absorb shock by reducing friction at the site of administration, e.g., a joint, and the term "coating effect" refers to the effect of quickly covering and coating hardened areas, e.g., damaged cartilage tissue within a joint, thereby enabling easier movement of the knee joint. These effects reduce inflammatory responses at the damaged joint site.

[0036] Therefore, the composition of the present invention, which induces buffering, coating, or anti-inflammatory effects, is suitable for regenerating or improving damaged tissues and can be effectively used in the treatment of joint diseases or connective tissue diseases. The effects of the composition of the present invention are similar to those of stem cell therapeutic agents currently used to treat joint diseases, and can be competitive compared to stem cell therapeutic agents that require advanced technology and cost.

[0037] Meanwhile, the pharmaceutical composition for preventing or treating joint or connective tissue diseases containing dextran, poloxamer, or a mixture thereof of the present invention may further contain stem cells to enhance the preventive or therapeutic effect of joint or connective tissue diseases. Stem cells that can be included in the present invention include embryonic stem cells or adult stem cells, and the adult stem cells may be mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells, or amniotic epithelial cells. The stem cells may be derived from, but are not limited to, the umbilical cord, umbilical cord blood, bone marrow, adipose tissue (or adipose tissue cells), muscle, nerve, skin, amniotic membrane and placenta (or placental tissue cells), urine, etc. The stem cells may be stem cells or concentrates thereof, stem cell culture medium or concentrates thereof, stem cell culture secretions or concentrates thereof, or combinations thereof. The dextran or poloxamer in the composition can specifically deliver stem cells to damaged lesions of joints or connective tissues, and also has the effect of increasing the ability of stem cells to settle in the lesions to which they have been delivered and induce regeneration of the damaged tissue.

[0038] In the present invention, "prevention" refers to any action of suppressing or delaying the onset of a joint disease or connective tissue disease by administering a composition.

[0039] In the present invention, "treatment" refers to any action in which the symptoms of a joint or connective tissue disease are improved or beneficially altered by administering a composition. The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable additives and may be formulated into a unit-dose formulation suitable for administration to a patient's body according to a conventional method in the pharmaceutical field. The formulation may be formulated by combining with a pharmaceutically acceptable carrier or vehicle, such as sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, or binder, and blending it into a pharmaceutically acceptable unit dose form.

[0040] The pharmaceutical composition may be in a solution state or a powder state. When the pharmaceutical composition is in a powder state, it can be dissolved in a solvent just before administration. However, the pharmaceutical composition is not limited thereto, and can be prepared in an optimal manner taking into consideration various situations that may occur during the preparation of a drug.

[0041] The administration form of the pharmaceutical composition is not particularly limited, but may be via common administration routes such as oral administration, injection, and infusion.

[0042] For oral administration, the compound may be used as a composition having the above-mentioned composition, or as a formulation such as a tablet, pill, capsule, gel, or syrup together with a pharmaceutically acceptable carrier or excipient. However, since solid preparations such as purified or powdered preparations often take a long time to absorb, oral administration in the form of a liquid is preferred. In such cases, it is preferable to administer the compound as an aqueous solution together with appropriate additives, such as salts such as sodium chloride, buffers, or chelating agents.

[0043] Drugs can also be administered via targeted drug delivery systems, such as liposomes coated with specific antibodies that target joint disease or connective tissue, allowing the liposomes to be selectively taken up by diseased tissue.

[0044] When the pharmaceutical composition of the present invention is administered in a formulation for local administration, it may be dissolved in sterile distilled water to which an appropriate buffer, isotonicity agent, etc. has been added, and then injected directly into an individual's joint cavity, connective tissue, intravenously, subcutaneously, intradermally, intra-articularly, or into a muscle, or applied to the skin, or may be in the form of a patch.

[0045] The individual may be any one selected from mammals including humans, dogs, cats, pigs, horses, cows, sheep, rats, and monkeys, preferably humans.

[0046] In the present invention, the term "intra-articular" refers to intra-articular injection of the pharmaceutical composition of the present invention.

[0047] In the present invention, the term "local administration" refers to a percutaneous injection into or near an inflamed joint. Thus, local administration injection may relate to the epidermis, dermis, muscle or any deep organ.

[0048] The main advantage of local administration is that it selectively limits the analgesic effect to the injured area. Furthermore, local administration allows for high local concentration levels with little or no systemic release.

[0049] The pharmaceutical composition may further comprise a stabilizer, lubricant, buffer, isotonicity adjusting agent, anesthetic, or antibacterial agent.

[0050] In addition, the pharmaceutical composition may further comprise an anti-inflammatory agent commonly used in the art. The anti-inflammatory agent may be non-steroidal, steroidal, or a combination thereof. Non-limiting examples of non-steroidal anti-inflammatory agents include oxicams such as piroxicam, isoxicam, tenoxicam, and sudoxicam; salicylates such as aspirin, disalcid, benorylate, tricate, safaprin, solprin, diflunisal, and fendosal; acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamic acids such as mefenamic acid, meclofenamic acid, flufenamic acid, and niflum. Acid and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and thioprofen; pyrazoles such as phenylbutazone, oxyfenobutazone, feprazone, azapropazone, and trimethasone. Extracts of these nonsteroidal anti-inflammatory agents can also be used.

[0051] Non-limiting examples of such steroidal anti-inflammatory agents include corticosteroids, such as hydrocortisone, hydroxy-triamcinolone, alpha-methyldexamethasone, dexamethasone- Phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, flucortine butyl ester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone , difluorosone diacetate, fluradrenolone, fludrocortisone, difluroson diacetate, fluradrenolone acetonide, medrysone, amcinafide, amcinafide, betamethasone and its ester balance, chloroprednisone, chloroprednisone acetate, clocortelone, clesinolone, dichlorisone, difluprednate, fluroronide, flunisolide, fluoromethalone, fluperolone, fluprenisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone and extracts thereof.

[0052] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease, and the effective dose level can be determined depending on factors including the severity of the disease, the patient's age, weight, health, sex, and sensitivity to the drug, the administration time, administration route and excretion rate, the duration of treatment, and other factors well known in the medical field, including drugs used in combination with or concurrently used with the composition of the present invention. The amount of dextran, poloxamer, or a mixture thereof varies widely and is determined according to the individual needs of each particular case.

[0053] The pharmaceutical composition of the present invention can be administered in combination with known drugs for treating joint or connective tissue diseases, and can be administered simultaneously or sequentially with said therapeutic agents.

[0054] The pharmaceutical composition of the present invention can be administered once or several times at preferred intervals, for example, at weekly intervals.

[0055] According to the present invention, pharmaceutical compositions containing dextran, poloxamer, or a mixture thereof induce long-lasting pain relief in individuals suffering from joint or connective tissue diseases. The use of the pharmaceutical compositions of the present invention avoids the side effects of major drugs, does not require excessively invasive techniques, and allows for the treatment and / or prevention of not only acute but also chronic joint or connective tissue diseases.

[0056] In the present invention, "connective tissue" refers to tissue that is widely distributed in animal tissues and plays a role in connecting, protecting, and charging cells, organs, etc., and is classified into fibrous connective tissue, colloidal tissue, and reticular tissue, with fibrous connective tissue accounting for the majority. For example, the connective tissue of the present invention may include, without limitation, cartilage, intervertebral disc, tendon, ligament, bone, skin, blood vessel, intestinal tissue, etc.

[0057] In the present invention, the causes of "joint diseases" or "connective tissue diseases" can be broadly classified into traumatic, infectious, inflammatory, and degenerative diseases. The compositions or therapeutic agents of the present invention can be used for joint diseases and connective tissue diseases caused by any cause that damages the joints. Joint diseases for which the compositions of the present invention are expected to be effective include osteoarthritis, preferably degenerative arthritis caused by repeated friction, ankylosing spondylitis, psoriatic arthritis, traumatic arthritis, rheumatoid arthritis, patellofemoral pain syndrome, and chronic inflammation or arthropathy. Connective tissue diseases for which the compositions of the present invention are expected to be effective include connective tissue diseases caused by collagen or water loss in cartilage, intervertebral discs, tendons, ligaments, bones, skin, blood vessels, intestinal tissue, etc., due to degenerative changes caused by physical damage or repeated friction. In particular, the composition of the present invention has excellent anti-inflammatory effects and the effects of promoting collagen synthesis and increasing the production of aggrecan, and based on these effects, it can be effectively used for the prevention or treatment of connective tissue diseases, particularly connective tissue diseases associated with inflammation, preferably one or more connective tissue diseases selected from the group consisting of inflammatory bone and joint diseases, inflammatory dermatitis, inflammatory eye diseases, inflammatory myositis, inflammatory gastrointestinal diseases, cartilage diseases, vasculitis, and sprains.

[0058] In the present invention, the following preferred examples can be provided, which do not exhibit cytotoxicity, effectively exhibit anti-inflammatory effects, suppress the expression of MMP-3 and MMP-13 genes, which are proteins that destroy cartilage matrix, promote the synthesis of type II collagen as a cartilage protection effect, and achieve the effect of increasing the amount of aggrecan produced.

[0059] In the present invention, two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188 may be mixed in a volume ratio of 1:1.

[0060] The present invention also relates to a pharmaceutical composition for preventing or treating joint or connective tissue diseases, which comprises a mixture of two compounds selected from the group consisting of dextran 1, dextran 5, and poloxamer 188.

[0061] The dextran is contained in the composition as a single active ingredient in the form of dextran 1, and the concentration of dextran 1 may be 2 to 40 (w / v)%.

[0062] The dextran is contained in the composition as a single active ingredient in the form of dextran 5, and the concentration of dextran 5 may be 2 to 30 (w / v)%.

[0063] The poloxamer may be contained in the composition as a single active ingredient, and the concentration of the poloxamer may be 1 to 15 (w / v)%.

[0064] In one example of the present invention, the mixture is a mixture of dextran 5 and poloxamer, and the concentration of dextran 5 can be 2 to 40 (w / v)%, and the concentration of poloxamer can be 1 to 20 (w / v)%.

[0065] The mixture may be a mixture of dextran 5 and dextran 1, with the concentration of dextran 5 being 2 to 40 (w / v)% and the concentration of dextran 1 being 2.5 to 40 (w / v)%.

[0066] The mixture may be a mixture of dextran 1 and poloxamer, with the concentration of dextran 1 being 2.5 to 40 (w / v)% and the concentration of poloxamer being 1 to 5 (w / v)%.

[0067] The mixture may be a mixture of dextran 1 and poloxamer, with the concentration of dextran 1 being 45 to 55 (w / v)% and the concentration of poloxamer being 1 to 2 (w / v)%.

[0068] Another example of the present invention relates to an anti-inflammatory composition based on the anti-inflammatory effect, a pharmaceutical composition for preventing or treating inflammatory diseases, and a method for preventing or treating inflammatory diseases using the same.

[0069] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises dextran, poloxamer or a mixture thereof as an active ingredient.

[0070] The present invention also provides a method for preventing or treating an inflammatory disease, comprising the step of administering dextran, poloxamer, or a mixture thereof to an individual in need thereof.

[0071] Accordingly, the present invention specifically provides a pharmaceutical composition for preventing or treating inflammatory diseases, and a method for preventing or treating inflammatory diseases, which comprises one or more compounds selected from the group consisting of dextran 1, dextran 5, and poloxamer 188.

[0072] In the present invention, the inflammatory disease may be, but is not limited to, an inflammatory skin disease, an inflammatory eye disease, an inflammatory bone and joint disease, an inflammatory muscle disease, or an inflammatory gastrointestinal disease.

[0073] When a mixture of two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188 is administered, it can exhibit significantly superior anti-inflammatory effects compared to the individual components. Specifically, when dextran 1, dextran 5, and poloxamer 188 are administered, excellent anti-inflammatory effects can be observed over the entire range of dextran 1: 2 and 40 (w / v)%, dextran 5: 2 and 40 (w / v)%, and poloxamer 188: 1 to 10 (w / v)%. For example, preferred examples include 5 (w / v)% dextran 1 and 2 (w / v) to 10 (w / v)% poloxamer 188, 5 (w / v)% dextran 1 and 4 to 40 (w / v)% dextran 5, 50 (w / v)% dextran 1 and 2 to 10 (w / v)% poloxamer 188, 50 (w / v)% dextran 1 and 4 to 40 (w / v)% dextran 5, 2 (w / v)% poloxamer 188 and dextran 1. Combinations of 5-50(w / v)% Poloxamer 188 and 4-40(w / v)% Dextran 5, 2(w / v)% Poloxamer 188 and 4-40(w / v)% Dextran 5, 10(w / v)% Poloxamer 188 and 5-50(w / v)% Dextran 1, 10(w / v)% Poloxamer 188 and 4-40(w / v)% Dextran 5, 4(w / v)% Dextran 5 and 5-50(w / v)% Dextran 1, 40(w / v)% Dextran 5 and 5-50(w / v)% or 40(w / v)% Dextran 5, and 40(w / v)% Dextran 5 and 2-10(w / v)% Poloxamer 188 can be used for anti-inflammatory and therapeutic purposes for inflammatory diseases.

[0074] Therefore, the mixture of the present invention is a mixture of dextran 1 and poloxamer 188, and can be characterized in that the concentration of dextran 1 is 2 to 40 (w / v)% and the concentration of poloxamer 188 is 1 to 10 (w / v)%.

[0075] The mixture of the present invention can also be characterized as a mixture of dextran 1 and dextran 5, in which the concentration of dextran 1 is 2 to 40 (w / v)% and the concentration of dextran 5 is 2 to 40 (w / v)%.

[0076] The mixture of the present invention is a mixture of dextran 5 and poloxamer 188, and can be characterized in that the concentration of dextran 5 is 2 to 40 (w / v)% and the concentration of poloxamer 188 is 1 to 10 (w / v)%.

[0077] Another example of the present invention relates to a composition for cartilage regeneration based on the regenerative effect of cartilage matrix, and a method for cartilage regeneration using the same.

[0078] Accordingly, the present invention provides a composition for cartilage regeneration, which comprises dextran, poloxamer or a mixture thereof as an active ingredient.

[0079] The present invention also provides a method for regenerating cartilage, which comprises the step of administering dextran, poloxamer, or a mixture thereof to an individual in need of cartilage regeneration.

[0080] The present invention provides a composition for cartilage regeneration or a method for cartilage regeneration, which specifically comprises one or more selected from the group consisting of dextran 1, dextran 5, and poloxamer 188, and which can induce an increase in collagen production and an increase in aggrecan production, and reduce the expression level of MMP-3 or MMP-13.

[0081] When one selected from the group consisting of dextran 1, dextran 5, and poloxamer 188 is administered, dextran 1 may be contained at 2 to 40 (w / v)%, poloxamer 188 at 1 to 15 (w / v)%, and dextran 5 at 2 to 30 (w / v)%.

[0082] Furthermore, when a mixture of two or more selected from the group consisting of dextran 1, dextran 5, and poloxamer 188 is administered, specific examples include combinations of 5 (w / v)% dextran 1 and 2 (w / v)% poloxamer 188, 5 (w / v)% dextran 1 and 4-40 (w / v)% dextran 5, 50 (w / v)% dextran 1 and 2 (w / v)% poloxamer 188, and 4-40% (w / v)% dextran 5 and 2-10 (w / v)% poloxamer 188. When these combinations are used, the production of collagen or aggrecan is increased, the expression of MMP-3 or MMP-13 is suppressed, and an excellent cartilage regeneration effect can be achieved.

[0083] Therefore, the mixture of the present invention is a mixture of dextran 1 and poloxamer 188, and can be characterized in that the concentration of dextran 1 is 5 to 40 (w / v)% and the concentration of poloxamer 188 is 2 (w / v)%.

[0084] Furthermore, the mixture of the present invention can be characterized as a mixture of dextran 1 and dextran 5, in which the concentration of dextran 1 is 5 (w / v)% and the concentration of dextran 5 is 4 to 40 (w / v)%.

[0085] The mixture of the present invention is a mixture of dextran 5 and poloxamer 188, and can be characterized in that the concentration of dextran 5 is 4 to 40 (w / v)% and the concentration of poloxamer 188 is 2 to 10 (w / v)%.

[0086] Furthermore, examples of compositions that can simultaneously achieve both anti-inflammatory and cartilage regeneration effects and are most suitable for the prevention or treatment of joint diseases or connective tissue diseases include, but are not limited to, 2.5 (w / v)% dextran 1, 2-30 (w / v)% dextran 5, and 1-15 (w / v)% poloxamer 188 when containing dextran 1, dextran 5, and poloxamer 188 alone; and 5-40 (w / v)% dextran 1 and 4-40 (w / v)% dextran 5, or 4-40 (w / v)% and preferably 2-10 (w / v)% dextran 5, 2 (w / v)% poloxamer 188, and 5-40 (w / v)% dextran 1 when using a mixture of two or more selected from the group consisting of dextran 1, dextran 5, and poloxamer 188.

[0087] Terms not specifically defined in this specification have the meanings commonly used in the technical field to which the present invention pertains.

[0088] [Mode for Carrying Out the Invention] material and method In the present invention, experiments were performed using Dextran 1 (EP grade, Pharmacosmos), Dextran 5 (pharmaceutical quality, Pharmacosmos), and Poloxamer 188 (cell culture grade, Sigma-Aldrich). To induce arthritis, recombinant human IL-1α (Lot No. 200-01A) was purchased from PEPROTECH and used. Positive control substances included indomethacin (Sigma-Aldrich, Lot No. 53-86-1) and sodium hyaluronate (25 mg / 2.5 mL, Aragan Injection (prefilled), Dongkwang Pharma, a specialty pharmaceutical company).

[0089] Example 1. Preparation of dextran, poloxamer, or mixtures thereof and experimental setup 1.1 Cell isolation and culture The hind limb joints of 3-week-old male SD rats were isolated under sterile conditions, and only the cartilage tissue that makes up the joint was collected and separated into single cells to isolate primary rat chondrocytes. Once the primary cultured cells were stabilized, they were used for testing. To identify stable chondrocytes, RNA was isolated, and the expression levels of type II collagen and SOX9 genes, which are chondrocyte markers, were confirmed using real-time RT-PCR before use in the test.

[0090] 1.2 Cell culture methods The cells isolated from step 1.1 above were cultured in an incubator set at 37°C, 95% humidity, and 5% CO2. The temperature and humidity of the culture room were monitored every 8 hours. The culture medium used was Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum, 2 mL L-glutamine, 50 U / mL penicillin, and 50 μg / mL streptomycin. When the cell population reached 90% or more during the culture period, the cells were detached with detached solution (0.25 (w / v)% trypsin, 0.53 mM EDTA solution (3 mL)) and centrifuged at 125 x g for 10 minutes. The cells were then used in the following experiments.

[0091] 1.3 Test Group Composition and Manufacturing The test groups were a normal control group (NC), an inflammation-induced control group (PC), and groups treated with various concentrations of test substances (single substance or mixture) in a model in which osteoarthritis was induced with rhIL-1α 5 ng / mL. The positive control groups were treated with indomethacin (IM) or sodium hyaluronate (HN) at a single concentration. The test groups are shown in Table 1 below.

[0092] [Table 1]

[0093] D = Dextran 1, T = Dextran 5, P = Poloxamer 188, NC = normal group, PC = inflammatory inducer (rhIL-1α) administered group, HN = sodium hyaluronate, IM = indomethacin, D+P = mixture of Dextran 1 and Poloxamer 188, D+T = mixture of Dextran 1 and Dextran 5, P+T = mixture of Poloxamer 188 and Dextran 5

[0094] The concentrations of the components used in the present invention are indicated based on the following notation: D10 = 10 (w / v)% solution of Dextran 1, P10 = 10 (w / v)% solution of Poloxamer 188, T10 = 10 (w / v)% solution of Dextran 5.

[0095] The test substances used in this invention were prepared using the following method: Dextran 1 (D) was prepared as a single substance at a concentration ranging from 1 to 50 (w / v)%, Dextran 5 (T) at a concentration ranging from 1 to 40 (w / v), and Poloxamer 188 (P) at a concentration ranging from 1 to 35 (w / v), reflecting the maximum concentration soluble in cell culture medium. When preparing a mixture, the two single substances were mixed at a volume ratio of 1:1 (v:v).

[0096] Dextrans 1 and 5 and poloxamer 188 were completely dissolved to the appropriate administration concentration (w / v) in complete media (Minimum Essential Medium (MEM) containing 10% fetal bovine serum, 2 mL L-glutamine, 50 U / mL penicilin, and 50 μg / mL streptomycin) prepared for cell culture. To completely dissolve and homogenize the test substances in the complete media, the solution was kept cool, especially for poloxamer 188, and was dissolved by gently stirring with a sterile spatula, stick, or magnetic bar at a cool temperature, or by shaking briefly several times to avoid foaming, until the solution was completely transparent and free of suspended solids. After the test substances were completely dissolved, the solution was filtered through a 0.22 μm pore size syringe filter, sealed, and stored refrigerated. Further specific single product preparations were carried out in the following manner.

[0097] Preparation of Dextran 1 5% Monomer ("D5") To prepare a 5% test solution of Dextran 1, 5 g of Dextran 1 was taken and completely dissolved in complete media, and the total volume was adjusted to 100 mL to prepare a D5 (w / v)% test solution. The test substance dissolved at a concentration of 0.05 g / mL was filtered through a 0.22 μm pore size syringe filter and stored in a refrigerator before use.

[0098] Preparation of a mixture of 5% Dextran 1 and 10% Poloxamer 188 ("D5+P10") D5 and P10 were prepared, filtered through a 0.22 μm pore size syringe filter, and stored in a refrigerator. The respective volumes were then mixed at a 1:1 (v:v) ratio and shaken slowly to avoid bubbles before use.

[0099] Comparative Example: Preparation of Indomethacin-treated Group (IM) Indomethacin was completely dissolved in DMSO to a concentration of 5 mM, then diluted with complete media and filtered through a 0.22 μm pore size syringe filter, and the final concentration was adjusted to 5 μM when applied to the cells.

[0100] Comparative Example: Preparation of Sodium Hyaluronate-treated Group (HN) Sodium hyaluronate was prepared by aspirating complete media into Aragan Injection (prefilled) (25 mg / 2.5 mL sodium hyaluronate in a disposable sterile syringe) and diluting it to 25 mg / 3 mL sodium hyaluronate (= 8.33 mg / mL sodium hyaluronate) for immediate use.

[0101] 1.4 Setting the administration method In the case of primary cultured chondrocytes isolated from rats, the doubling time is approximately 24 hours, so test substances were treated at the appropriate time for proliferation and stabilization. After culturing chondrocytes in 48-well or 24-well plates, each test substance was administered once. One hour before treatment with rhIL-1α to induce arthritis, the test solution was administered at 20% (v / v) of the total culture medium volume for each test substance concentration. 24 hours after rhIL-1α treatment, the culture medium was collected and analytical experiments were carried out. However, when examining the toxicity or proliferation ability of a test substance on chondrocytes, the test substance alone was treated without rhIL-1α.

[0102] 1.5 Statistical analysis All experimental results were analyzed by comparing with the normal control group (NC) or the inflammation-induced positive control group (PC) using the Student's T-test at a p<0.05 level to indicate significance.

[0103] Example 2. Cytotoxicity analysis We analyzed cytotoxicity to determine whether treatment with the test substances altered the proliferation ability of chondrocytes. Cell proliferation was evaluated using both untreated chondrocytes and chondrocytes treated with rhIL-1α to induce inflammation. Each test substance was treated with rat chondrocytes and cultured for 24 and 48 hours, followed by MTT analysis (Thiazolyl Blue Tetrazolium Blue; Sigma, M5655). To evaluate the cell proliferation ability of the test substances in an in vitro model of arthritis induced by the inflammatory substance rhIL-1α, rat chondrocytes were treated with each test substance 1 hour before treatment with rhIL-1α and cultured for 24 and 48 hours, followed by MTT analysis (Thiazolyl Blue Tetrazolium Blue; Sigma, M5655). The results are shown in Figures 1 to 4.

[0104] As shown in Figure 1, the cell proliferation rate of uninflamed rat knee articular chondrocytes treated with dextran 1, dextran 5, poloxamer 188, or a mixture of these was monitored for 24 hours. Results indicated that most dextran 1, dextran 5, poloxamer 188, and their mixtures were non-toxic to the cells and promoted cell proliferation. However, among the single-treatment groups, the poloxamer 188 treatment group showed a greater decrease in cell proliferation after 24 hours of culture than the control drug (commercially available drug) sodium hyaluronate (HN), indicating that single administration is not suitable. These mixtures also showed a partial decrease in cell proliferation after 24 hours of culture, confirming the possibility that poloxamer 188 may induce cytotoxicity at high concentrations. Furthermore, in the mixture, the D50+P35, P5+T40, P25+T40, and P35+T40mp experimental groups showed a greater decrease in cell proliferation ability than the control drug (commercially available drug) sodium hyaluronate (HN) administered group.

[0105] As shown in Figure 2, the cell proliferation rate of dextran, poloxamer, or a mixture of these in uninflamed rat knee articular chondrocytes was observed for 48 hours, and the results were similar to those of 24-hour culture.

[0106] These results demonstrated that when treating normal knee articular chondrocytes, increasing the concentration of dextran 1 (D) to 50% or dextran 5 (T) to 40% did not inhibit chondrocyte proliferation compared to commercially available drugs, whereas poloxamer 188 at concentrations of 25% or higher can be toxic to chondrocytes. Furthermore, while a mixture of these compounds partially improved cell proliferation compared to poloxamer 188 alone, it was determined that poloxamer 188 should preferably be mixed at a concentration of 10% (w / v) or less to avoid toxicity.

[0107] 3 and 4 show the results of observing the cell proliferation rate of the experimental group treated with rhIL-1α for 24 hours and 48 hours, respectively, in an in vitro osteoarthritis model.

[0108] As shown in Figure 3, the osteoarthritis model also showed that poloxamer 188 inhibited cell proliferation at higher concentrations, while dextran 1 and dextran 5 did not inhibit cell proliferation even at higher concentrations. In the case of poloxamer 188, cell proliferation was confirmed in the P10-only treatment group, but when poloxamer 188 was mixed with D5, D50, T4, T40, etc., a cell proliferation effect was observed, confirming that 1% to 10% (w / v) of poloxamer 188 can be used when mixed with dextran. In FIG. 4, the same results as those obtained with 24-hour culture were confirmed, confirming that the cell proliferation ability of the P10-treated group was increased by mixing with dextran 1 or dextran 5.

[0109] Taking these results together, it was confirmed that dextran 1 and dextran 5 induce almost no cytotoxicity in normal and inflammatory cells. On the other hand, as the concentration of poloxamer 188 increases, treatment with the single substance can induce inhibition of cell proliferation in both normal and inflammatory cells. However, when poloxamer 188 is administered in a mixture with dextran 1 or dextran 5 at 1-10% (w / v), it can be used without cytotoxicity.

[0110] Example 3. Confirmation of anti-inflammatory effect through IL-10 / IL-6 expression ratio Experiments were conducted to confirm the anti-inflammatory effects of dextran 1, dextran 5, and poloxamer 188. Specifically, dextran 1 was varied from 2.5 to 50% (w / v), dextran 5 from 2 to 40% (w / v), and poloxamer 188 from 1 to 20% (w / v), and these were administered singly or in combination to determine the IL-10 / IL-6 expression ratio. To induce inflammation, the inflammatory inducer rhIL-1α was administered, and the IL-10 / IL-6 expression ratio was determined when rhIL-1α and the test substance (single substance or a mixture of these) were administered together. A higher IL-10 / IL-6 expression ratio indicates a more excellent anti-inflammatory effect.

[0111] The primers used in the experiment are shown in Table 2 below, and the housekeeping gene GAPDH was used as a control. The results are shown in Tables 3a and 3b and in FIG. [Table 2]

[0112] The primers, SyBr green (SYBR Premix Ex Taq, Takara, RR420A), and template were mixed, and real-time RT-PCR was performed using the intercalating method in a real-time RT-PCR kit (CFX 96 touch, Bio-Rad, Hercules, CA, USA). The quantitative results (gene expression levels) obtained by applying the Ct values ​​to the standard curve were expressed as values ​​divided by the quantitative results of the housekeeping gene GAPDH.

[0113] [Table 3a]

[0114] IL-10 / IL-6 gene expression ratio in chondrocytes treated with RhIL-1α for 24 hours using dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) alone

[0115] [Table 3b]

[0116] The IL-10 / IL-6 gene expression ratio in chondrocytes treated with RhIL-1α for 24 h, treated with a mixture of dextran 1 (D), dextran 5 (T), or poloxamer 188 (P)

[0117] As shown in Table 3a and Figure 5, in the single-group treatment experiment, no clear increase in the IL-10 / IL-6 expression ratio was confirmed in the groups treated with Dextran 1 (D) alone, except for the 50 (w / v)% treatment group. However, a significant increase was confirmed in the Poloxamer 188 (P) 1-20 (w / v)% treatment group and the Dextran 5 (T) 2-20 (w / v)% treatment group. As shown in Table 3b and Figure 5, in the mixture treatment experiment, all of the groups treated with Dextran 1 (D) showed a significant increase in the IL-10 / IL-6 expression ratio, and all of the groups treated with Poloxamer 188 (P) and Dextran 5 (T) showed a significant increase, confirming the outstanding anti-inflammatory effect of the mixture.

[0118] Although the 50% (w / v) Dextran 1 group showed a significant increase in the IL-10 / IL-6 expression ratio, the increase was not significant. This suggests that the increase in the IL-10 / IL-6 expression ratio, i.e., the anti-inflammatory effect, in the osteoarthritis model is better achieved by administering Dextran 1(D) in combination with Poloxamer 188(P) or Dextran 5(T) rather than by increasing the concentration of Dextran 1(D). In particular, the anti-inflammatory effect was significantly superior in the groups administered Dextran 1(D) at 5 and 50% (w / v), Dextran 5(T) at 4 and 40% (w / v), and Poloxamer 188(P) at 2 and 10% (w / v), confirming the superiority of the combined administration of Dextran 1(D), Dextran 5(T), and Poloxamer 188(P).

[0119] In particular, the administration of a mixture of dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) showed excellent anti-inflammatory effects across the entire range of dextran 1 (D) 5 and 50 (w / v)%, dextran 5 (T) 4 and 40 (w / v)%, and poloxamer 188 (P) 2 and 10 (w / v). All of these combinations showed anti-inflammatory effects, and were therefore effective in treating inflammatory diseases.

[0120] Example 4. Confirmation of cartilage matrix regeneration effect through changes in MMP-3 and MMP-13 gene expression

[0121] An experiment was conducted to confirm the therapeutic effect of osteoarthritis through gene expression of indicator substances for osteoarthritis evaluation. MMP-3 and MMP-13 are proteins that destroy cartilage matrix, so if the increase in the expression level of these genes, which can be induced by inflammatory stimuli, can be effectively suppressed, it can be evaluated as having excellent cartilage protection effects.

[0122] Dextran 1, dextran 5, and poloxamer 188 were administered at concentrations ranging from 2.5 to 50% (w / v), 2 to 40% (w / v), and 1 to 20% (w / v), respectively. The concentrations were varied and administered singly or in combination. The changes in MMP-3 and MMP-13 expression were examined. Specifically, rat chondrocytes were treated with each test substance 1 hour before rhIL-1α treatment and then cultured for 24 hours. After removing the culture supernatant, the cells were washed with phosphate buffered saline (PBS). RNA was isolated from the washed cells (GeneAll hybrid-R RNA purification kit; GeneAll, 3033522). The isolated RNA was quantified using a nanodrop (Take3 Multi-Volume plate, BioTek Instruments, VT, USA) and diluted to the same concentration for each experimental group. Equal amounts of diluted RNA were used to synthesize cDNA using PCR (ReverTra AceR qPCR RT Master Mix with gDNA Remover, Toyobo, FSQ-301), and the gene expression of MMP-3 and MMP-13 was confirmed in 96-well plates via real-time RT-PCR using the synthesized cDNA. The primer configurations used in the experiment are shown in Table 4 below, and the housekeeping gene GAPDH was used as a control.

[0123] [Table 4]

[0124] The primers, SyBr green (SYBR Premix Ex Taq, Takara, RR420A), and template were mixed, and real-time RT-PCR was performed using the intercalating method with a real-time RT-PCR kit (CFX 96 touch, Bio-Rad, Hercules, CA, USA). The quantitative results (gene expression levels) obtained by applying the Ct values ​​to the standard curve were expressed as values ​​divided by the quantitative results of the housekeeping gene GAPDH.

[0125] 3.1 Confirmation of changes in MMP-3 expression The results of MMP-3 expression following treatment with Dextran 1, Dextran 5, Poloxamer 188 alone or a mixture thereof are shown in Tables 5a and 5b and FIG.

[0126] [Table 5a]

[0127] MMP-3 gene expression levels in chondrocytes treated with RhIL-1α for 24 hours using dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) alone

[0128] [Table 5b]

[0129] MMP-3 gene expression levels in chondrocytes treated with RhIL-1α for 24 hours, treated with a mixture of dextran 1 (D), dextran 5 (T), or poloxamer 188 (P).

[0130] As shown in Table 5a and Figure 6, Dextran 1 (D) showed a significant MMP-3 reduction effect at 2.5-40 (w / v)% compared to the inflammatory-induced positive control group, Dextran 5 (T) showed a significant MMP-3 reduction effect at 2-20 (w / v)%. On the other hand, Poloxamer 188 (P) showed a reduction effect at 1-20 (w / v)% in all experimental groups.

[0131] As shown in Table 5b and Figure 6, significant effects were observed in all experimental groups treated with 5% (w / v) dextran 1 (D), 2-10% (w / v) poloxamer 188 (P), and 4% and 40% (w / v) dextran (T). However, in the group treated with high concentrations of dextran 1 (D) up to 50% (w / v), only the mixture with P2 showed a significant reduction. Therefore, when dextran 1 is mixed with dextran 5 or poloxamer 188, it is preferable to mix it at a concentration of less than 50% (w / v). Poloxamer 188 showed a significant reduction in MMP-3 in both the single-treatment and mixed-treatment groups. However, as confirmed in Example 2, a poloxamer concentration of 10% (w / v) or less is preferred. It was confirmed that P2, P5, and P10, which correspond to this concentration, were able to induce significant MMP-3 inhibition even in combination with dextran 1 and dextran 5.

[0132] 3.2 Confirmation of changes in MMP-13 expression The results of MMP-13 expression following treatment with Dextran 1, Dextran 5, Poloxamer 188 alone or a mixture thereof are shown in Tables 6a and 6b and FIG.

[0133] [Table 6a]

[0134] In chondrocytes treated with RhIL-1α for 24 hours, the gene expression of MMP-13 was increased by dextran 1 (D), dextran 5 (T), and poloxamer 188 (P).

[0135] [Table 6b]

[0136] The gene expression level of MMP-13 in chondrocytes treated with RhIL-1α for 24 hours, treated with a mixture of dextran 1 (D), dextran 5 (T), or poloxamer 188 (P).

[0137] As can be seen from Tables 6a and 6b and Figure 7, the single-substance experiment showed that Dextran 1(D) significantly inhibited MMP-13 gene expression at concentrations of 2.5 to 25% (w / v). At D40 (40% w / v), MMP-13 gene expression was inhibited, albeit not significantly, and at D50 (50% w / v), MMP-13 levels actually increased. These properties were also observed in Dextran 1(D) mixtures. Therefore, it was confirmed that the most preferable concentration range for Dextran 1(D) was 2.5 to 40% (w / v), which is consistent with the results of MMP-3 gene expression. Dextran 5(T) showed a significant inhibitory effect on the increase of MMP-13 gene when administered alone at 2-40(w / v)%. Dextran 5(T) at 4 and 40(w / v)% also showed a significant inhibitory effect on the increase of MMP-13 gene when mixed with dextran 1(D) at 5(w / v)% and poloxamer 188(P) at 2 and 10(w / v)%.

[0138] In the case of poloxamer 188, the inhibitory effect on MMP-13 gene expression was observed at 5-20% (w / v) in the single-agent experimental group, but when administered in a mixture with dextran, a similar effect was confirmed at 2% (w / v). Considering the cytotoxicity test results in Example 2, it was confirmed that the preferred concentration of poloxamer 188 (P) when administered in a mixture is 2-10% (w / v). Corresponding to this, P2 and P10 also induced a significant inhibitory effect on MMP-13 gene expression when combined with dextran 1 and dextran 5, except for the combination with 50% (w / v) dextran 1 (D).

[0139] Based on the above results, it was confirmed that P2, P5, and P10, even in combination with Dextran 1 and Dextran 5, were able to induce significant MMP-13 inhibitory effects.

[0140] Example 5. Confirmation of increased production of type II collagen and aggrecan in an osteoarthritis model

[0141] To assess the production of type II collagen and aggrecan, which are indicators for assessing osteoarthritis, chondrocytes were treated with each test substance one hour before treatment with rhIL-1α and then cultured for 24 hours. Since type II collagen and aggrecan are components of the cartilage matrix, an increase in their production indicates an excellent cartilage matrix protective effect.

[0142] The cell supernatant (culture medium) was then collected and centrifuged at 13,000 rpm for 10 minutes, and only the supernatant was used for analysis. The centrifuged cell supernatant was used for analysis according to the Aggrecan ELISA Kit (Rat Aggrecan ELISA Kit, Mybiosource, MBS261073) and Type II Collagen (Type II Collagen Detection Kit, Multi-Species, Chondrex, 6018) product manuals. For accurate analysis, absorbance was measured at 450 nm and 490 nm, respectively, using a full-spectrum ELISA reader (EPOCH 2 microplate reader, BioTek Instruments, VT, USA), and the content of Type II collagen and aggrecan (ng / mL) was quantified based on the calibration curve.

[0143] 5.1 Confirmation of type II collagen production The results of the amount of type II collagen produced following treatment with dextran 1 (D), dextran 5 (T), poloxamer 188 (P), or a mixture thereof are shown in Tables 7a to 7b and FIG.

[0144] [Table 7a]

[0145] The amount of Type II collagen produced by dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) alone in chondrocytes treated with RhIL-1α for 24 hours

[0146] [Table 7b]

[0147] Type II collagen production from chondrocytes treated with RhIL-1α for 24 hours in a mixture of dextran 1 (D), dextran 5 (T) or poloxamer 188 (P).

[0148] As shown in Tables 7a and 7b and Figure 8, an increase in type II collagen synthesis was observed in the groups administered 5-25% (w / v) of Dextran 1 (D) and 4-40% (w / v) of Dextran 5 (T) alone compared to the inflammation-induced positive control group (PC). On the other hand, 50% (w / v) of Dextran 1 (D) alone and in combination with Dextran 5 (T) and Poloxamer 188 (P) did not induce an increase in type II collagen synthesis compared to the inflammation-induced positive control group (PC). This result indicates that dextran 1(D) at 50% (w / v) alone does not inhibit the increased expression of MMP-3 and MMP-13, and that only a mixture with 2-10% (w / v) of poloxamer 188(P) can inhibit the increased expression of MMP-3 and MMP-13, as confirmed in Example 4. Therefore, dextran 1(D) is preferably used alone or in a mixture at 40% (w / v) or less. In particular, the combination of dextran 1(D) at 5% (w / v) with dextran 5(T) at 4-40% (w / v) significantly increased type II collagen synthesis compared to the inflammatory-induced positive control group (PC). Poloxamer 188 (P) alone did not increase type II collagen synthesis in any of the 10% (w / v) and 20% (w / v) treatment groups, except for the 2% (w / v) treatment group. This indicates that type II collagen synthesis decreased with increasing concentration. However, when poloxamer 188 (P) was administered at 2 or 10% (w / v) in combination with dextran 1 (D) 5% (w / v) or dextran 5 (T) 40% (w / v), a significant increase in type II collagen synthesis was observed. These results suggest that dextran 1 (D) and dextran 5 (T) alone are effective in promoting type II collagen synthesis in osteoarthritis, whereas poloxamer 188 (P) alone is not suitable except at 2% (w / v). This suggests that the combined administration of poloxamer 188 (P) with dextran 1 (D) or dextran 5 (T) can effectively induce type II collagen synthesis.

[0149] In the previous Example 3, it was confirmed that the combined administration of poloxamer 188 with dextran 1 or dextran 5 can significantly increase the IL-10 / IL-6 gene expression ratio compared to either alone. Taking these results together, it was confirmed that administering a mixture of dextran 1, dextran 5, or poloxamer 188 is preferable for alleviating inflammatory responses in osteoarthritis and promoting collagen synthesis.

[0150] 5.2 Confirmation of quantitative results of aggrecan content The results of the amount of aggrecan produced following treatment with dextran 1, dextran 5, poloxamer 188 alone or a mixture thereof are shown in Tables 8a and 8b and FIG.

[0151] [Table 8a]

[0152] [Table 8b]

[0153] The amount of aggrecan produced by chondrocytes treated with RhIL-1α for 24 hours was measured using a mixture of dextran 1 (D), dextran 5 (T), and poloxamer 188 (P).

[0154] As shown in Table 6a and Figure 9, the aggrecan production levels were significantly increased in the groups treated with 5-25% (w / v) dextran 1 (D), 4% (w / v) dextran 5 (T), and 2-10% (w / v) poloxamer 188 (P) alone compared with the inflammation-induced positive control group (PC). On the other hand, the groups treated with 50% (w / v) dextran 1 (D), 20% (w / v) poloxamer 188 (P), and 20-40% (w / v) dextran 5 (T) alone failed to induce an increase in aggrecan production.

[0155] As shown in Table 8b and Figure 9, aggrecan production was significantly increased in the groups treated with 5% (w / v) dextran 1 (D) and 2% (w / v) poloxamer 188 (P) or 4-40% (w / v) dextran 5 (T) compared with the inflammatory control group (PC). Aggrecan production was significantly increased only in the group treated with 50% (w / v) dextran 1 (D) and 2% (w / v) poloxamer 188 (P). A significant increase in aggrecan production was also observed in the groups treated with 4-40% (w / v) dextran 5 (T) and 2-10% (w / v) poloxamer 188 (P) compared with the inflammatory control group (PC). In particular, it was confirmed that when dextran 1 (D) 5 (w / v)% and poloxamer 188 (P) 2 (w / v)% were administered in combination with dextran 5 (T) 4-40 (w / v)%, the amount of aggrecan produced significantly increased compared to when administered alone.

[0156] Taking into consideration the results of type II collagen synthesis confirmed previously in Example 5.1, it was confirmed that in order to increase type II collagen synthesis and aggrecan production in an osteoarthritis model, it is preferable to administer 5-25 (w / v)% dextran 1(D), 4 (w / v)% dextran 5(T), or 2 (w / v)% poloxamer 188(P) alone, or to administer 5 (w / v)% dextran 1(D) mixed with 4-40 (w / v)% dextran 5(T), or to administer 5 (w / v)% dextran 1(D) mixed with 2 (w / v)% poloxamer 188(P). Furthermore, we confirmed that dextran 5 (T) 4-40 (w / v)% in combination with dextran 1 (D) 5 (w / v)%, and especially dextran 5 40 (w / v)% in combination with poloxamer 188 (P) 2-10 (w / v)%, significantly increased type II collagen synthesis and aggrecan production.

[0157] Taking the above results into consideration, it can be seen that dextran 1, dextran 5, and poloxamer 188 can be used in the treatment of osteoarthritis, but that a single or complete combination of these will not be effective in treating osteoarthritis. Since toxicity and efficacy may vary depending on the combination of dextran 1, dextran 5, and poloxamer 188 concentrations, it is important to determine the appropriate combination.

Claims

1. A composition for preventing or treating a joint disease or a connective tissue disease, comprising dextran having an average molecular weight of 4,000 to 6,000 Da, The joint or connective tissue disease is (a) a joint disease selected from the group consisting of osteoarthritis, degenerative arthritis, ankylosing spondylitis, psoriatic arthritis, traumatic arthritis, rheumatoid arthritis, patellofemoral pain syndrome, and arthropathy; or (b) a connective tissue disease selected from the group consisting of inflammatory bone and joint diseases, inflammatory myositis, cartilage diseases, and sprains, which connective tissue disease occurs in the intervertebral disc, tendons, or ligaments. A composition for preventing or treating a joint disease or a connective tissue disease, comprising

2. 1. A composition for preventing or treating a joint or connective tissue disease, comprising a mixture of i) dextran having an average molecular weight of 4,000 to 6,000 Da and ii) dextran having an average molecular weight of 800 to 1,200 Da, The joint or connective tissue disease is (a) a joint disease selected from the group consisting of osteoarthritis, degenerative arthritis, ankylosing spondylitis, psoriatic arthritis, traumatic arthritis, rheumatoid arthritis, patellofemoral pain syndrome, and arthropathy; or (b) a connective tissue disease selected from the group consisting of inflammatory bone and joint diseases, inflammatory myositis, cartilage diseases, and sprains, which connective tissue disease occurs in the intervertebral disc, tendons, or ligaments. A composition for preventing or treating a joint disease or a connective tissue disease, comprising

3. A composition for preventing or treating a joint or connective tissue disease, comprising a mixture of dextran having an average molecular weight of 4,000 to 6,000 Da and a poloxamer, The joint or connective tissue disease is (a) a joint disease selected from the group consisting of osteoarthritis, degenerative arthritis, ankylosing spondylitis, psoriatic arthritis, traumatic arthritis, rheumatoid arthritis, patellofemoral pain syndrome, and arthropathy; or (b) a connective tissue disease selected from the group consisting of inflammatory bone and joint diseases, inflammatory myositis, cartilage diseases, and sprains, which connective tissue disease occurs in the intervertebral disc, tendons, or ligaments. A composition for preventing or treating a joint disease or a connective tissue disease, comprising

4. A composition for preventing or treating a joint or connective tissue disease, comprising a mixture of dextran having an average molecular weight of 800 to 1,200 Da and poloxamer, The joint or connective tissue disease is (a) a joint disease selected from the group consisting of osteoarthritis, degenerative arthritis, ankylosing spondylitis, psoriatic arthritis, traumatic arthritis, rheumatoid arthritis, patellofemoral pain syndrome, and arthropathy; or (b) a connective tissue disease selected from the group consisting of inflammatory bone and joint diseases, inflammatory myositis, cartilage diseases, and sprains, which connective tissue disease occurs in the intervertebral disc, tendons, or ligaments. A composition for preventing or treating a joint disease or a connective tissue disease, comprising

5. A composition for cartilage regeneration comprising dextran having an average molecular weight of 4,000 to 6,000 Da.

6. A composition for cartilage regeneration comprising a mixture of the following i) and ii): i) dextran with an average molecular weight of 4,000 to 6,000 Da; ii) Dextran with an average molecular weight of 800 to 1,200 Da.

7. A composition for cartilage regeneration comprising a mixture of dextran having an average molecular weight of 4,000 to 6,000 Da and poloxamer.

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