Compositions for treating joint or connective tissue diseases comprising dextran or poloxamer - Patent Application 20070229333

Abstract

The present invention relates to a composition for treating joint diseases or connective tissue diseases, comprising dextran, poloxamer, or a mixture thereof, or a composition for cartilage regeneration, comprising dextran, poloxamer, or a mixture thereof. The composition of the present invention has a buffering effect, a coating effect, or an anti-inflammatory effect, and can remain at the site of injury in the joint or connective tissue for a long time to cushion the impact, specifically cover the injury, or reduce inflammation at the site of adhesion, making it effective for treating joint diseases or connective tissue diseases or for cartilage regeneration.

Description

[Technical field]

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

[0002] A joint is a site where bones connect to each other, and is made up of cartilage, joint capsule, synovial membrane, ligaments, tendons, muscles, etc., to allow smooth movement between the bones, and acts to absorb shocks generated by movement. Arthritis refers to the occurrence of functional abnormalities accompanied by inflammation in the joints due to various causes. Connective tissue is a tissue that connects tissues in animals to form organs, and examples of this include cartilage, intervertebral discs, tendons, ligaments, bones, skin, adipose tissue, blood vessels, and intestinal tissue.

[0003] Drugs used to treat arthritis can be broadly classified based on their main mechanism of action, such as reducing inflammation, delaying disease progression, and reducing the concentration of metabolic products such as uric acid, and many arthritis drugs work to reduce inflammation. Inflammation is a pathological process that causes pain, swelling, heat, redness, stiffness, etc., and drugs that quickly relieve inflammation include non-steroidal anti-inflammatory drugs such as aspirin and steroidal anti-inflammatory drugs such as cortisone. However, these anti-inflammatory and anti-inflammatory drugs mainly work to relieve pain rather than treat the disease, and many complications occur when the drugs are taken for a long period of time. In particular, steroidal anti-inflammatory drugs are not related to treating the cause of the disease, but simply temporarily relieve pain through their strong and temporary anti-inflammatory effect, which can lead to overuse of the joints, which can destroy the joints and worsen the disorder, so caution is required 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. There is a strong need for new novel treatments or therapeutic agents that overcome the shortcomings of conventional treatments.

[0005] On the other hand, dextran is a type of polysaccharide, which refers to a polymer of D-glucose and has a structure similar to starch and glycogen, with D-glucose linked in a straight chain by α-1,6 bonds and branched at places by α-1,4 or α-1,3 bonds. High molecular weight dextran, which is commonly used, is known to often cause allergic reactions in the body. Dextran has been used as a plasma expander, blood coagulant (anti-adhesion agent), and filler using cross-linked dextran, but there have been no reports of its use as a therapeutic agent for joint diseases or connective tissue diseases. Poloxamer refers to a non-ionic surfactant, and is known to be used as an emulsifier, stabilizer, and solubilizer in addition to surfactants.

[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, and there is currently no known research into the treatment of joint or connective tissue diseases using dextrans or poloxamers, particularly low molecular weight dextrans or poloxamers, that do not cause an allergic reaction 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 confirmed that dextran, poloxamer or a mixture thereof does not cause allergic reactions and has a buffering effect, a retaining effect or an anti-inflammatory effect on the damaged parts of joints and connective tissues, thereby protecting the joints and connective tissues. Based on this, the present invention has been completed.

[0008] The object of the present invention is to provide a pharmaceutical composition for the treatment of joint diseases or connective tissue diseases, for cartilage regeneration or for the treatment of inflammatory diseases, comprising 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 a joint disease or connective tissue disease, comprising a mixture of two 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 an inflammatory disease, comprising 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 selected from the group consisting of dextran 1, dextran 5 and poloxamer 188. Effect of the Invention

[0014] The composition of the present invention can be effectively used for the treatment of joint diseases, connective tissue diseases, or cartilage regeneration, since it remains at the damaged part of the joint or connective tissue for a long time due to its buffering effect, coating effect, or anti-inflammatory effect, cushions the impact, wraps the damaged part specifically, and reduces inflammation at the adhesion site. [Brief description of the drawings]

[0015] [Figure 1] This figure shows the results of confirming 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)). [Diagram 2] This figure shows the results of confirming 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)). [Diagram 3] This figure shows the results of confirming 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 confirming 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)). [Diagram 5] This figure shows the results of confirming the IL-10 / IL-6 gene expression ratio in chondrocytes of an in vitro osteoarthritis model treated with rhIL-1α by 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 6] This figure shows the results of confirming the MMP-3 gene expression level in chondrocytes of an in vitro model of osteoarthritis treated with rhIL-1α by 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 7] This figure shows the results of confirming the MMP-13 gene expression level in chondrocytes of an in vitro model of osteoarthritis treated with rhIL-1α by 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 8] This figure shows the results of confirming the amount of type II collagen produced by treatment of chondrocytes in an in vitro model of osteoarthritis treated 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)). [Figure 9]This figure shows the results of confirming the amount of aggrecan 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)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0017] In the present invention, "dextran" means a polymer of D-glucose, which is a type of polysaccharide. Dextran has a structure similar to starch and glycogen, in which D-glucose is linearly linked to α-1,6 bonds and branched at places with α-1,4 or α-1,3 bonds. Dextran can be used as a plasma expander or blood coagulant, and can be used with different molecular weights depending on the purpose. In the present invention, the dextran can 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] In general, dextran of 8,000 Da or more is known to have a high risk of causing allergic reactions when administered to the human body. In particular, the allergic reaction may cause anaphylaxis, a sudden systemic reaction caused by an antigen-antibody immune reaction, which is very dangerous. However, the use of low molecular weight dextran, preferably 1,000 Da to 5,000 Da dextran, according to the present invention has the advantage of minimizing allergic reactions, and has the advantage of prolonging the therapeutic effect of joint diseases or connective tissue diseases because it is decomposed 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 characterized by not only being free of allergic reactions compared to dextrans of 8,000 Da or more, but also by their rapid onset of action and ability to be distributed over a wide area. As preferred examples, the present invention provides 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 types 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 what is being mixed in. For example, when a mixture of dextran 5 and dextran 1 is contained in the composition, the concentration of the dextran 5 may be 2 to 40 (w / v)% and the concentration of the dextran 1 may be 2.5 to 40 (w / v)%, or the concentration of the dextran 5 may be 4 to 40 (w / v)% and the concentration of the dextran 1 may be 5 to 40 (w / v)%.

[0023] When dextran is present in a composition at a high concentration, the viscosity increases excessively due to the water-containing nature of polysaccharides, making it difficult to formulate a desired shape. Therefore, dextran has generally been used at a concentration of 5% or less. However, dextran is contained in the composition of the present invention at a concentration of at least 2% to a maximum of 40% (w / v) depending on the molecular weight, so that the therapeutic effect of joint diseases or connective tissue diseases can be maximized. In order to increase or stabilize the solubility of such dextran, the composition of the present invention can further contain an additive, which can include sugar alcohols, monosaccharides, or divalent cations including CaCl2.

[0024] In the present invention, "poloxamer" is a non-ionic triblock copolymer consisting of hydrophobic propylene oxide and hydrophilic ethylene oxide at both ends, and has the characteristic of temperature sensitivity, being capable of converting between sol and gel depending on the concentration and temperature, and 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 The poloxamer may be selected from the group consisting of 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 mass (g) concentration (w / v) in 100 cc of solvent, and may be characterized as being 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] In order to increase the solubility or stabilize the poloxamer, the composition of the present invention may further contain additives such as sugar alcohols, monosaccharides, divalent cations, etc., or the composition may contain sterile water or saline without additional additives.

[0029] In the present invention, the pharmaceutical composition can include a mixture of dextran and poloxamer. In particular, when poloxamer 188 is mixed with dextran, it can be mixed with dextran 1 or dextran 5. For example, when mixed with dextran 5, the concentration of dextran 5 can be 2-40(w / v)% and the concentration of poloxamer 188 can be 1-20(w / v)%, or the concentration of dextran 5 can be 4-40(w / v)% and the concentration of poloxamer 188 can be 2-10(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)%, and when mixed with dextran, the concentration must be appropriately adjusted before use.

[0032] As an example, the dextran:poloxamer in the pharmaceutical composition of the present invention may include, for example, as a 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, and 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 40 (w / v), 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, and 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, as an embodiment of the present invention, when a mixture of two 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 addition, as an embodiment of the present invention, the pharmaceutical composition of the present invention includes a mixture of dextran 1 and dextran 5, and the ratio of dextran 1: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, 50:1000, for example, 1:0.01 to 1:20. An exemplary combination is dextran 1. When Dextran 1 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 composition of the present invention contains a mixture of dextran and poloxamer, the hydrophobic component of poloxamer can increase the solubility of dextran or the combined drug, and the hydrophilic component of poloxamer, PEG (polyethylene glycol), can hold cell membranes to prevent adhesion and hold tissue. However, poloxamer has the disadvantage of being easily diluted and absorbed by body fluids, so that its stability in the body can be increased by mixing with dextran. Therefore, in the case of a mixture of dextran and poloxamer, the viscosity lasts longer than when dextran or poloxamer is contained alone, and it can wrap damaged joints or connective tissue sites, preventing pathological adhesion of tissues and reducing inflammation caused by friction, thereby facilitating the supply of nutrients and promoting tissue regeneration. The above effects can be induced by the cushioning effect, coating effect, or anti-inflammatory effect of the mixture of dextran and poloxamer.

[0035] In the present invention, the term "cushion effect" refers to the effect of acting like a cushion to absorb shocks by reducing friction at the site of administration, for example, at a joint, and the term "coating effect" refers to the effect of quickly covering and coating hardened sites, for example, damaged cartilage tissue within a joint, to facilitate movement of the knee joint. These effects reduce inflammatory responses at damaged joint sites.

[0036] Therefore, the composition of the present invention, which induces a buffering effect, a coating effect or an anti-inflammatory effect, is suitable for regenerating or improving damaged tissues, and can be effectively used in the treatment of joint diseases or connective tissue diseases. The above-mentioned effects of the composition of the present invention are similar to those of stem cell therapeutics currently used for the treatment of joint diseases, and can be competitive compared to stem cell therapeutics that require high technical capabilities and costs.

[0037] Meanwhile, the pharmaceutical composition for preventing or treating joint or connective tissue diseases, which contains dextran, poloxamer or a mixture thereof, of the present invention may further contain stem cells to promote the preventive or therapeutic effect of joint or connective tissue diseases as the object of the present invention. The 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 stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat (or fat tissue cells), muscle, nerve, skin, amniotic membrane and placenta (or placental tissue cells), urine, etc., but are not limited thereto. The stem cells may be stem cells or concentrates thereof, stem cell culture fluid or concentrates thereof, stem cell culture secretions or concentrates thereof, or combinations thereof. The dextran or poloxamer in the composition can play a role in specifically delivering stem cells to damaged lesions of joints or connective tissues, and also has the effect of increasing the ability of the stem cells to settle in the delivered lesions and induce regeneration of damaged tissues.

[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 ameliorated or beneficially altered by administration of a composition. The pharmaceutical composition of the present invention may further contain pharma- ceutically acceptable additives, and may be formulated into a unit-dosage formulation suitable for administration into the body of a patient according to a conventional method in the pharmaceutical field.The formulation may also be formulated by mixing with a pharma-ceutically acceptable carrier or medium, such as sterilized water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, or binder, and into a pharma-ceutically approved unit dosage form.

[0040] The pharmaceutical composition may be in a solution or 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 a general administration route such as oral administration, injection, or infusion.

[0042] In the case of oral administration, it may be used as a composition having the above composition, or as a preparation such as a tablet, pill, capsule, gel, or syrup together with a pharma- ceutically acceptable carrier or excipient. However, since it often takes a long time to absorb solid preparations such as refined or powdered preparations, oral administration in the form of a liquid is preferred. In that case, it is preferred to administer it as an aqueous solution together with appropriate additives, such as salts such as sodium chloride, buffers, chelating agents, etc.

[0043] Alternatively, drugs can be administered via targeted drug delivery systems such as liposomes coated with specific antibodies that target diseased joints or connective tissues, allowing the liposomes to be selectively taken up by diseased tissues.

[0044] When the pharmaceutical composition of the present invention is administered in a formulation for local administration, the composition may be dissolved in sterile distilled water to which an appropriate buffer, isotonicity agent, etc. has been added, and then the composition may be directly injected into an individual's joint cavity, connective tissue, intravenously, subcutaneously, intradermally, intra-articularly, or into a muscle, or may be 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 the 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, and furthermore allows for high local concentration levels with little or no systemic release.

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

[0050] In addition, the pharmaceutical composition can further include an anti-inflammatory agent that is widely used in the art. The anti-inflammatory agent can be non-steroidal, steroidal or a combination thereof. Non-limiting examples of non-steroidal anti-inflammatory agents include oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates, such as aspirin, disalcid, benolic acid, trisate, 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, niflum. Acid, and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miloprofen, thioxaprofen, suprofen, aluminoprofen, 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 the 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, fluclorone 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, diflurosone diacetate, fluradrenolone acetonide, medrysone, amcinafide, amcinafide, betamethasone and its esters balance, chloroprednisone, chloroprednisone acetate, clocorterone, 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 pharmaceutical effective amount.The term "pharmaceutical effective amount" means an amount sufficient to treat a disease, and the effective dose level can be determined according to factors including the severity of the disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time, the administration route and excretion rate, the duration of treatment, the drugs used in combination with or used simultaneously with the composition of the present invention, and other factors well known in the medical field.The amount of dextran, poloxamer, or mixture thereof varies in a wide range and is determined according to the individual needs of each particular case.

[0053] The pharmaceutical compositions of the present invention may be administered in combination with known agents for the treatment of joint or connective tissue disorders, and may 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 comprising dextran, poloxamer or mixtures thereof induce long-lasting pain relief in individuals suffering from joint or connective tissue diseases. The use of the pharmaceutical compositions according to the present invention allows the treatment and / or prevention of acute as well as chronic joint or connective tissue diseases, avoiding side effects of major drugs and without excessively invasive techniques.

[0056] In the present invention, the term "connective tissue" refers to tissue that is widely distributed in animal tissues and plays a role in connecting, protecting, and charging cells, organs, and the like, 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, and the like.

[0057] In the present invention, the causes of "joint disease" or "connective tissue disease" can be largely classified into traumatic, infectious, inflammatory, and degenerative. The composition or therapeutic agent according to the present invention can be used for joint disease and connective tissue disease caused by any cause that damages the joint, and the joint diseases for which the composition of the present invention is expected to be effectively used include osteoarthritis, preferably degenerative arthritis caused by repeated friction, ankylosing spondylitis, psoriatic arthritis, traumatic arthritis, rheumatoid arthritis, patellofemoral pain syndrome, chronic inflammation or arthropathy. In addition, the connective tissue diseases for which the composition of the present invention is expected to be effectively used include connective tissue diseases in which collagen or water loss occurs in cartilage, intervertebral discs, tendons, ligaments, bones, skin, blood vessels, intestinal tissues, etc. due to physical damage or degenerative changes caused by 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 in 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, preferred examples can be provided as follows, which do not show cytotoxicity, effectively show anti-inflammatory effects, suppress gene expression of MMP-3 and MMP-13 proteins that destroy cartilage matrix, promote the synthesis of type II collagen as a cartilage protective effect, and achieve the effect of increasing the amount of aggrecan produced.

[0059] In the present invention, two types 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 a joint disease or a connective tissue disease, comprising a mixture of two 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 dextran 5, which is contained in the composition as a single active ingredient, 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 the dextran 5 can be 2 to 40 (w / v)%, and the concentration of the poloxamer can be 1 to 20 (w / v)%.

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

[0066] The mixture can 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 can 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 an inflammatory disease, and a method for preventing or treating an inflammatory disease using the same.

[0069] Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising 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: 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 an inflammatory disease, and a method for preventing or treating an inflammatory disease, which comprises one or more 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 is possible to show a significantly superior anti-inflammatory effect compared to the anti-inflammatory effect of each component. Specifically, when dextran 1, dextran 5 and poloxamer 188 are administered, it can be shown that the anti-inflammatory effect is excellent 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, in preferred embodiments, dextran 1: 5 (w / v)% and poloxamer 188: 2 (w / v) to 10 (w / v)%, dextran 1: 5 (w / v)% and dextran 5: 4 to 40 (w / v)%, dextran 1: 50 (w / v)% and poloxamer 188: 2 to 10 (w / v)%, dextran 1: 50 (w / v)% and dextran 5: 4 to 40 (w / v)%, poloxamer 188: 2 (w / v)% and dextran 1: The following combinations can be used for anti-inflammatory and therapeutic purposes for inflammatory diseases: 5-50(w / v)% Poloxamer 188 2(w / v)% and Dextran 5 4-40(w / v)%, 10(w / v)% Poloxamer 188 and Dextran 1 5-50(w / v)%, 10(w / v)% Poloxamer 188 and Dextran 5 4-40(w / v)%, 4(w / v)% Dextran 5 and Dextran 1 5-50(w / v)%, 40(w / v)% Dextran 5 and Dextran 1 5-50(w / v)%, or 40(w / v)%, or 40(w / v)% Dextran 5 and Poloxamer 188 2-10(w / v)%.

[0074] Therefore, the mixture of the present invention can be characterized as a mixture of dextran 1 and poloxamer 188, in which 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 being 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 can also be characterized as being a mixture of dextran 5 and poloxamer 188, in which the concentration of the dextran 5 is 2 to 40 (w / v)% and the concentration of the 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 a cartilage matrix, and a method for cartilage regeneration using the same.

[0078] Thus, 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, comprising the step of administering dextran, poloxamer or a mixture thereof to an individual in need of cartilage regeneration.

[0080] In the present invention, a composition for cartilage regeneration or a method for cartilage regeneration is provided, 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] In addition, when a mixture of two or more selected from the group consisting of dextran 1, dextran 5 and poloxamer 188 is administered, in specific examples, the combination may be 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, or 4-40% (w / v)% dextran 5 and 2-10 (w / v)% poloxamer 188. When such combinations are used, the production of collagen or aggrecan is increased, and the expression of MMP-3 or MMP-13 is suppressed, thereby achieving an excellent cartilage regeneration effect.

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

[0084] Moreover, the mixture of the present invention can be characterized as being 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 can also be characterized as being a mixture of dextran 5 and poloxamer 188, in which the concentration of the dextran 5 is 4 to 40 (w / v)% and the concentration of the poloxamer 188 is 2 to 10 (w / v)%.

[0086] In addition, an example that can simultaneously achieve both anti-inflammatory and cartilage regeneration effects and is most suitable for the prevention or treatment of joint diseases or connective tissue diseases is, but is not limited to, when dextran 1, dextran 5, and poloxamer 188 are contained alone, dextran 1 can be 2.5 (w / v)%, dextran 5 can be 2-30 (w / v), and poloxamer 188 can be 1-15 (w / v). When a mixture of two or more selected from the group consisting of dextran 1, dextran 5, and poloxamer 188 is used, dextran 1 can be 5 (w / v)% and dextran 5 can be 4-40 (w / v), dextran 5 can be 4-40 (w / v), and dextran 5 can be 2-10 (w / v), poloxamer 188 can be 2 (w / v)%, and dextran 1 can be 5-40 (w / v).

[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 carried out using Dextran 1 (EP grade, Pharmacosmos), Dextran 5 (pharmaceutical quality, Pharmacosmos), and Poloxamer 188 (cell culture grade, Sigma-Aldrich). For arthritis induction, rhIL-1α (recombinant human IL-1α, Lot No. 200-01A) was purchased from PEPROTECH and used, and indomethacin (Sigma-Aldrich, Lot No. 53-86-1) and sodium hyaluronate (25 mg / 2.5 mL, Aragan Injection (prefilled), Dongkwang Pharm., Specialty Pharmaceuticals) were used as positive control substances.

[0089] Example 1. Preparation of dextran, poloxamer or mixtures thereof and experimental setup 1.1 Cell isolation and culture After isolating the hind leg joints of 3-week-old male SD rats under sterilized conditions, only the cartilage tissue that constitutes the joints was collected and made into single cells, and primary rat chondrocytes were isolated and used. Once the primary cultured cells were stable, they were used for testing, and 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 being used for testing.

[0090] 1.2 Cell culture method The cells isolated from 1.1 above were cultured in an incubator set at a temperature of 37°C, humidity of 95%, and CO2 of 5%, and the temperature and humidity of the culture room were checked 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. During the culture period, when the cell number had grown to 90% or more, the cells were detached with Detached solution (0.25 (w / v)% Trypsin, 0.53 mM EDTA solution (3 mL)), centrifuged at 125 x g for 10 minutes, and the cells were used in the following experiments.

[0091] 1.3 Composition and manufacture of the test group The test groups consisted of a normal control group (NC), an inflammation-induced control group (PC), and groups treated with different concentrations of test substances (single substances or mixtures) 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 system: 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 the present invention were prepared using the following method: Dextran 1 (D) was prepared as a single substance at a concentration of 1-50 (w / v)%, Dextran 5 (T) was prepared as a single substance at a concentration of 1-40 (w / v)%, and Poloxamer 188 (P) was prepared as a single substance at a concentration of 1-35 (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] Dextran 1, 5, and poloxamer 188 were completely dissolved in complete media (Minimum Essential Medium (MEM) medium containing 10% fetal bovine serum, 2 mL L-glutamine, 50 U / mL penicilin, 50 ug / mL streptomycin, etc.) prepared for cell culture, to the appropriate administration concentration (w / v). In order to completely dissolve and homogenize the test substances in the complete media, the solution was stirred slowly with a sterile spatula, stick, magnetic bar, etc. at a cool temperature, especially in the case of poloxamer 188, or was shaken briefly several times without bubbles, until the solution was completely transparent with no floating matter. After the test substances were completely dissolved, they were filtered through a 0.22 μm pore size syringe filter, sealed, and stored in a refrigerator. Further specific single product preparations were carried out in the following manner.

[0097] Preparation of Dextran 1 5% Monohydrate ("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 in this way at a concentration of 0.05 g / mL was filtered using a 0.22 μm pore size syringe filter, stored in a refrigerator, and then used.

[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 in a 1:1 (v:v) ratio and shaken slowly to avoid foaming before use.

[0099] Comparative Example: Preparation of Indomethacin-treated Group (IM) Indomethacin was completely dissolved in DMSO at 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 inhaling complete media into Aragan Injection (prefilled) (sodium hyaluronate 25 mg / 2.5 mL, drug form in a disposable sterile syringe) and diluting it to sodium hyaluronate 25 mg / 3 mL (= sodium hyaluronate 8.33 mg / mL) and using it immediately.

[0101] 1.4 Setting the administration method In the case of primary cultured chondrocytes isolated from rats, the doubling time is about 24 hours, so the test substances were treated when the proliferation and stabilization were appropriate. After culturing the chondrocytes in 48-well or 24-well plates, each test substance was administered once, and one hour before rhIL-1α treatment to induce arthritis, the test solution was administered at 20% (v / v) of the total culture medium volume for each concentration of test substance. 24 hours after rhIL-1α treatment, the culture medium was collected and analytical experiments were carried out. However, when checking the toxicity or proliferation ability of the test substance on the chondrocytes, only the test substance 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 Student's T-test at a p < 0.05 level to show significance.

[0103] Example 2. Cytotoxicity Analysis We confirmed whether the treatment of the test substances changed the proliferation ability of chondrocytes, and performed an analysis of cytotoxicity. The evaluation of cell proliferation ability was performed on untreated chondrocytes not treated with rhIL-1α and on chondrocytes treated with rhIL-1α to induce inflammation. After treating rat chondrocytes with each test substance and culturing them for 24 and 48 hours, MTT analysis (Thiazolyl Blue Tetrazolium Blue; Sigma, M5655) was performed. In the evaluation of cell proliferation ability of the test substances in an in-vitro model of arthritis induced by the inflammation inducer rhIL-1α, rat chondrocytes were treated with each test substance 1 hour before treatment with rhIL-1α, and after culturing them for 24 and 48 hours, MTT analysis (Thiazolyl Blue Tetrazolium Blue; Sigma, M5655) was performed. The results are shown in Figures 1 to 4.

[0104] As shown in Figure 1, the cell proliferation rate of dextran 1, dextran 5, poloxamer 188, or a mixture of these was observed for 24 hours in knee joint chondrocytes of rats without induced inflammation. As a result, it was confirmed that most of dextran 1, dextran 5, poloxamer 188, and their mixtures were not toxic to the cells and promoted cell proliferation. However, in the case of the poloxamer 188 treatment group, when cultured for 24 hours at a concentration of 25 (w / v)% (P25) or 35 (w / v)% (P35), a partial decrease in cell proliferation ability was observed more than in the control drug (commercial drug) sodium hyaluronate (HN) treatment group, indicating that it is not suitable for single administration. In addition, a partial decrease in cell proliferation ability was observed in these mixtures when cultured for 24 hours, and from this, it was confirmed that poloxamer 188 may induce cytotoxicity when treated at a high concentration. Additionally, 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 rat knee articular chondrocytes without induced inflammation was observed for 48 hours, and the results were similar to those of the 24-hour culture.

[0106] These results confirmed that when treating normal knee joint chondrocytes, increasing the concentration of Dextran 1 (D) to 50% or Dextran 5 (T) to 40% did not inhibit chondrocyte proliferation as compared to commercially available drugs, whereas Poloxamer 188 at a concentration of 25% or more may be toxic to chondrocytes. Also, in the case of a mixture, cell proliferation ability was partially improved compared to Poloxamer 188 alone, but it was confirmed that in order to avoid inducing toxicity, it is preferable to mix Poloxamer 188 at a concentration of 10 (w / v)% or less.

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

[0108] As shown in Figure 3, the osteoarthritis model also showed that poloxamer 188 inhibited cell proliferation at higher concentrations, whereas dextran 1 and dextran 5 did not inhibit cell proliferation even at higher concentrations. In the case of poloxamer 188, inhibition of cell proliferation was confirmed in the P10-only treatment group, but when this was mixed with D5, D50, T4, T40, etc., a cell proliferation effect was observed, and it was confirmed 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 from the 24-hour culture were confirmed, and it was confirmed that the cell proliferation ability of the P10 treatment group was increased by mixing with dextran 1 or dextran 5.

[0109] Considering the above results, 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, it was shown that treatment with a single substance can induce inhibition of cell proliferation in both normal and inflammatory cells. However, it was confirmed that poloxamer 188 can be used without cytotoxicity when mixed with dextran 1 or dextran 5 at 1-10% (w / v).

[0110] Example 3. Confirmation of anti-inflammatory effect through IL-10 / IL-6 expression ratio An experiment was conducted to confirm the anti-inflammatory effect of dextran 1, dextran 5, or poloxamer 188. Specifically, dextran 1 was varied from 2.5 to 50 (w / v)%, dextran 5 was varied from 2 to 40 (w / v), and poloxamer 188 was varied from 1 to 20 (w / v), and these were administered singly or in combination to confirm the IL-10 / IL-6 expression ratio. The inflammatory inducer rhIL-1α was administered to induce inflammation, and the IL-10 / IL-6 expression ratio was confirmed when rhIL-1α and the test substance (single substance or mixture of these) were treated together. It can be determined that the higher the IL-10 / IL-6 expression ratio, the better the 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 to 3b and 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 by the intercalating method using 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 h, using only dextran 1 (D), dextran 5 (T), and poloxamer 188 (P)

[0115] [Table 3b]

[0116] IL-10 / IL-6 gene expression ratio in chondrocytes treated with RhIL-1α for 24 h 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 expression ratio of IL-10 / IL-6 was observed in the groups treated with Dextran 1 (D) alone, except for the 50 (w / v)% treatment group. However, a significant increase was observed 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 administered with Dextran 1 (D) showed a significant increase in the IL-10 / IL-6 expression ratio, and all of the groups administered with Poloxamer 188 (P) and Dextran 5 (T) showed a significant increase, confirming that the mixture has an excellent anti-inflammatory effect.

[0118] Considering that the increase in the IL-10 / IL-6 expression ratio in the 50 (w / v)% dextran 1 administration group was significant, but the increase was not significant, it indicates that the increase in the IL-10 / IL-6 expression ratio in the osteoarthritis model, i.e., the anti-inflammatory effect, is better when it is mixed with poloxamer 188 (P) or dextran 5 (T) than when the concentration of dextran 1 (D) is increased. In particular, the anti-inflammatory effect is significantly better in most cases than the single administration groups 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)%, confirming the superiority of the combined administration of dextran 1 (D), dextran 5 (T), and poloxamer 188 (P).

[0119] In particular, the mixed administration of Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) showed excellent anti-inflammatory effects over 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). It was confirmed that 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. Since MMP-3 and MMP-13 are proteins that destroy cartilage matrix, if the increase in the expression level of the genes, which can be induced by inflammatory stimulation, is effectively suppressed, it can be evaluated as having excellent cartilage protection effect.

[0122] 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 confirm the changes in the expression of MMP-3 and MMP-13 associated with these. Specifically, each test substance was administered to rat chondrocytes 1 hour before rhIL-1α treatment and then cultured for 24 hours. After removing the supernatant of the cultured cells, the cells were washed with phosphate buffered saline (PBS). RNA was isolated from the washed cells (GeneAll hybrid-R RNA purification kit; GeneAll, 3033522), and the isolated RNA was quantified using nanodrop (Take3 Multi-Volume plate, BioTeK, Instruments, VT, USA), and then 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 the template were mixed, and real-time RT-PCR was performed by the intercalating method using a real-time RT-PCR (CFX 96 touch, Biorad, Hercules, CA, USA). The quantitative results (gene expression levels) obtained by applying the Ct value to the standard curve were expressed as a value 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, or a mixture thereof, are shown in Tables 5a to 5b and FIG.

[0126] [Table 5a]

[0127] In chondrocytes treated with RhIL-1α for 24 hours, the expression level of MMP-3 gene was increased by dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) alone.

[0128] [Table 5b]

[0129] The MMP-3 gene expression level in chondrocytes treated with RhIL-1α for 24 h was evaluated by the effect of 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 reducing effect at 2.5-40 (w / v)% compared to the inflammation-induced positive control group, Dextran 5 (T) showed a significant MMP-3 reducing effect at 2-20 (w / v)% On the other hand, Poloxamer 188 (P) showed a reducing effect at 1-20 (w / v)% in all experimental groups.

[0131] As shown in Table 5b and FIG. 6, in the mixture experimental group, significant effects were confirmed in all experimental groups with dextran 1 (D) 5 (w / v)%, poloxamer 188 (P) 2-10 (w / v)%, and dextran (T) 4% and 40 (w / v)%, but in the group treated with dextran 1 (D) at a high concentration of up to 50 (w / v)%, a significant reduction effect was confirmed only when mixed with P2. Therefore, it was confirmed that 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 effect on MMP-3 in almost all the single treatment and mixed treatment groups, but as confirmed in Example 2, the concentration of poloxamer is preferably 10 (w / v)% or less, and it was confirmed that P2, P5, and P10 corresponding to this can induce a significant MMP-3 inhibitory effect 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, or a mixture thereof, are shown in Tables 6a to 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) alone.

[0135] [Table 6b]

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

[0137] As can be seen from Tables 6a to 6b and FIG. 7, the single substance experimental group showed that Dextran 1(D) had a significant effect of suppressing the increase in MMP-13 gene at 2.5 to 25(w / v)%, and although it was not significant at D40 (40(w / v)%, the increase in MMP-13 gene was suppressed, and at D50 (50(w / v)), the MMP-13 level was rather increased. These characteristics were also observed in the mixture of Dextran 1(D). 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 the MMP-3 gene expression level. When administered alone, dextran 5 (T) at 2 to 40 (w / v)% showed a significant inhibitory effect on the increase of MMP-13 gene, and 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 single experimental group showed an inhibitory effect on the increase of MMP-13 gene at 5-20 (w / v)%, but when administered in a mixture with dextran, it was confirmed that 2 (w / v)% also showed the same effect. Considering the cytotoxicity experimental results in Example 2, it was confirmed that the concentration of poloxamer 188 (P) is preferably 2-10 (w / v)% when administered in a mixture. P2 and P10 corresponding to this induced a significant inhibitory effect on the increase of MMP-13 gene even in combination with dextran 1 and dextran 5, except for the combination of dextran 1 (D) 50 (w / v)%.

[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 a significant MMP-13 inhibitory effect.

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

[0141] In order to confirm the amount of type II collagen and aggrecan protein produced among the index substances for evaluating osteoarthritis, each test substance was treated with chondrocytes 1 hour before treatment with rhIL-1α, and then cultured for 24 hours. Since type II collagen and aggrecan are cartilage matrix components, if the amount of these components produced increases, it can be determined that the cartilage matrix protection effect is excellent.

[0142] Here, the cell supernatant (culture medium) was 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, the 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 type II collagen production following treatment with dextran 1 (D), dextran 5 (T), poloxamer 188 (P), either alone or in combination, are shown in Tables 7a to 7b and FIG.

[0144] [Table 7a]

[0145] The amount of Type II collagen produced by single substances Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) 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 the presence of a mixture of dextran 1 (D), dextran 5 (T) or poloxamer 188 (P).

[0148] As shown in Tables 7a to 7b and Figure 8, an increase in type II collagen synthesis was observed in the groups administered with 5 to 25 (w / v)% dextran 1 (D) and 4 to 40 (w / v)% dextran 5 (T) alone, compared to the inflammation-induced positive control group (PC). On the other hand, it was shown that 50 (w / v)% 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). As confirmed in Example 4, dextran 1(D) at 50(w / v)% alone does not show any effect of suppressing the increase in expression of MMP-3 and MMP-13, and only a mixture with poloxamer 188(P) at 2-10(w / v)% can suppress the increase in expression of MMP-3 and MMP-13. Considering this, it is preferable to use dextran 1(D) at 40(w / v)% or less alone or in a mixture. In particular, when dextran 1(D) at 5(w / v)% was mixed with dextran 5(T) at 4-40(w / v)%, the synthesis of type II collagen was significantly increased compared to the inflammation-induced positive control group (PC). In the case of Poloxamer 188 (P), no increase in type II collagen synthesis was observed in any of the 10 (w / v) and 20 (w / v)% single treatment groups, except for the 2 (w / v)% treatment group, and it was confirmed that type II collagen synthesis decreased more as the concentration increased. However, when Poloxamer 188 (P) 2 or 10 (w / v)% was mixed 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 indicate that Dextran 1 (D) and Dextran 5 (T) can be used alone in type II collagen synthesis in osteoarthritis, but Poloxamer 188 (P) is not suitable for single treatment except at 2 (w / v)%, and that the mixed 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 and dextran 1 or dextran 5 can significantly increase the gene expression ratio of IL-10 / IL-6 compared to the single administration. Taking these results together, it was confirmed that the administration of 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 aggrecan production following treatment with dextran 1, dextran 5, poloxamer 188, or a mixture thereof, are shown in Tables 8a to 8b and FIG.

[0151] [Table 8a]

[0152] [Table 8b]

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

[0154] As can be seen from Table 6a and Figure 9, the amount of aggrecan produced was significantly increased in the groups treated with Dextran 1 (D) 5-25 (w / v), Dextran 5 (T) 4 (w / v), and Poloxamer 188 (P) 2-10 (w / v)% alone compared to the inflammation-induced positive control group (PC). On the other hand, it was confirmed that the groups treated with Dextran 1 (D) 50 (w / v), Poloxamer 188 (P) 20 (w / v), and Dextran 5 (T) 20-40 (w / v)% alone did not induce an increase in aggrecan production.

[0155] As can be seen in Table 8b and Figure 9, the amount of aggrecan produced was significantly increased in the mixed administration groups of Dextran 1 (D) 5 (w / v)% and Poloxamer 188 (P) 2 (w / v)% or Dextran 5 (T) 4-40 (w / v)% compared to the inflammation-induced positive control group (PC), and the amount of aggrecan produced was significantly increased only in the mixed administration group of Dextran 1 (D) 50 (w / v)% and Poloxamer 188 (P) 2 (w / v)%. It was also confirmed that the amount of aggrecan produced was significantly increased in the mixed administration group of Dextran 5 (T) 4-40 (w / v)% and Poloxamer 188 (P) 2-10 (w / v)% compared to the inflammation-induced positive 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 mixed with dextran 5 (T) 4-40 (w / v)%, the amount of aggrecan produced was significantly increased compared to when administered alone.

[0156] When considering the results of type II collagen synthesis confirmed 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 dextran 1(D) 5-25(w / v)%, dextran 5(T) 4(w / v)%, or poloxamer 188(P) 2(w / v)% alone, or to administer dextran 1(D) 5(w / v)% mixed with dextran 5(T) 4-40(w / v)%, or to administer dextran 1(D) 5(w / v)% mixed with poloxamer 188(P) 2(w / v)%. We also confirmed that dextran 5 (T) 4-40 (w / v)% in combination with dextran 1 (D) 5 (w / v)%, and especially in the case of dextran 5 40 (w / v)%, in combination with poloxamer 188 (P) 2-10 (w / v)%, can significantly increase 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 is not effective in the treatment of osteoarthritis, and that it is important to determine an appropriate combination because toxicity and efficacy may differ depending on the combination of dextran 1, dextran 5 and poloxamer 188 concentrations.

Claims

1. A preventive or therapeutic agent for joint disease or connective tissue disease, comprising dextran, The dextran has an average molecular weight of from 500 to less than 8,000 Da, but not more than 8,000 Da.

2. A composition for preventing or treating a joint or connective tissue disease, comprising dextran, poloxamer 188 and a pharma- ceutical acceptable carrier, The dextran has an average molecular weight of 500 to less than 8,000 Da, but not more than 8,000 Da; and The concentration of the dextran in the composition is 2-40% (w / v); and A composition, wherein the concentration of the poloxamer 188 in the composition is 1-10% (w / v).

3. The method of claim 1 , wherein the connective tissue is cartilage, an intervertebral disc, a tendon, a ligament, a bone, skin, a blood vessel, or an intestinal tissue.

4. The agent according to claim 1, wherein the joint disease or connective tissue disease is at least one selected from the group consisting of osteoarthritis, degenerative arthritis caused by repeated friction, ankylosing spondylitis, psoriatic arthritis, traumatic arthritis, rheumatoid arthritis, patellofemoral pain syndrome, chronic inflammation, and arthropathy.

5. The agent according to claim 1, wherein the connective tissue disease comprises one or more 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.

6. A cartilage regeneration agent comprising dextran, The dextran has an average molecular weight of from 500 to less than 8,000 Da, but not more than 8,000 Da.

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