KR102316719B1 - Compositions for treating joint or connective tissue disease comprising dextran or poloxamer - Google Patents

Abstract

The present invention relates to a composition for treating a joint disease or a connective tissue disease comprising dextran, a poloxamer, or a mixture thereof, or a composition for treating a joint disease or a connective tissue disease comprising stem cells in the composition. The pharmaceutical composition of the present invention can stay for a long time in the damaged area of the joint or connective tissue by the buffering effect, the coating effect or the anti-inflammatory effect to relieve the impact and reduce the inflammation of the damaged area or the adhesion area specifically, It can be usefully used for the treatment of joint diseases or connective tissue diseases.

Description

Composition for treating joint or connective tissue disease comprising dextran or poloxamer TECHNICAL FIELD

The present invention relates to a composition for treating a joint disease or a connective tissue disease comprising dextran, a poloxamer, or a mixture thereof, or a composition for treating a joint disease or a connective tissue disease comprising stem cells in the composition.

A joint is a part where bones meet, and is composed of cartilage, joint capsule, synovial membrane, ligaments, tendons, and muscles so that the bones can move smoothly between them. Arthritis is a functional abnormality accompanied by inflammation in the joints due to various causes. These joint diseases are chronic joint rheumatism, which is understood to be caused by autoimmunity, infectious arthritis caused by bacterial infection, and joint cartilage or It can be largely divided into osteoarthritis, in which bone degeneration or destruction occurs, and crystalline arthritis in which soluble metabolites are deposited as crystals in the connective tissue around the joint.

Connective tissue is also referred to as connective tissue, and is a tissue that forms organs by connecting tissues in an animal, for example, intervertebral discs, skin, tendons, ligaments, and the like.

Drugs used to treat arthritis can be roughly classified based on their main mechanism of action: reduction of inflammation, delay of disease progression, and reduction of the concentration of metabolic products such as uric acid. Inflammation is a pathological process that causes pain, swelling, heat, redness, stiffness, etc., and drugs that rapidly relieve inflammation include nonsteroidal anti-inflammatory drugs including aspirin and steroidal anti-inflammatory drugs including cortisone. However, the main action of these anti-inflammatory and anti-inflammatory drugs is to relieve pain rather than to treat the disease, and when the drug is taken for a long time, many complications occur. In particular, steroidal anti-inflammatory drugs have the potential to induce excessive use of the joint by simply temporarily reducing pain due to its strong and temporary anti-inflammatory action, regardless of the cause of the disease, which destroys the joint and aggravates the disorder. Therefore, caution is required when using it.

Therefore, conventional treatments used for joint damage such as arthritis have limited effectiveness, are accompanied by obvious toxic side effects, and their effectiveness is limited because they cannot be used continuously for a long period of time. Or, there is an urgent need for a therapeutic agent.

On the other hand, dextran, which refers to a polymer of D-glucose as one of polysaccharides, has a structure similar to starch or glycogen, and D-glucose leads to a straight chain shape through α-1,6 bonds and α-1 ,4 bonds are branched. This is in contrast to starch or glycogen having α-1,4 bonds and branching points with α-1,6 bonds. In addition, the existence of α-1,2 bonds and α-1,3 bonds is also known, but the amount and type differ depending on the origin of the dextran. High molecular weight dextran is known to frequently cause allergic reactions in the body. Dextran has been used as a blood extender, an anticoagulant (anti-adhesion agent), and a filler using cross-linked dextran, but it has not been reported to be used as a treatment for joint disease or connective tissue disease. Poloxamer refers to a nonionic surfactant, and is known to be used as an emulsifier, stabilizer, solubilizer, etc. in addition to surfactants.

Due to the limited effectiveness, obvious toxic side effects, and lack of persistence, which are the limitations of current treatments for joint disease or connective tissue disease, a new treatment or therapeutic agent is needed. In particular, when administered in the body, dextran or Poloxamers, particularly low molecular weight dextran or studies on the treatment of connective tissue diseases using dextran or poloxamer is not known.

Accordingly, the present inventors have conducted research for the treatment of joints or connective tissue, and as a result, dextran, poloxamer, or a mixture thereof does not cause an allergic reaction, and has a buffering effect, preservation effect or anti-inflammatory effect on damaged joints and connective tissue. The present invention was completed by confirming that there is an effect of protecting joints and connective tissues.

It is an object of the present invention to provide a pharmaceutical composition for treating joint disease or connective tissue disease, including dextran, poloxamer, or a mixture thereof.

An object of the present invention is stem cells; To provide a pharmaceutical composition for preventing or treating joint disease or connective tissue disease, including dextran, poloxamer, or a mixture thereof.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating joint disease or connective tissue disease, comprising dextran, poloxamer, or a mixture thereof.

In addition, the present invention is stem cells; It provides a pharmaceutical composition for preventing or treating joint disease or connective tissue disease, including dextran, poloxamer, or a mixture thereof.

In addition, the present invention provides a pharmaceutical composition for preventing or treating joint disease or connective tissue disease, comprising a mixture of two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188 do.

In addition, the present invention is a stem cell; It provides a pharmaceutical composition for preventing or treating joint disease or connective tissue disease, comprising a mixture of two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188.

The pharmaceutical composition of the present invention can stay for a long time in the damaged area of the joint or connective tissue by the buffering effect, the coating effect or the anti-inflammatory effect to relieve the impact and reduce the inflammation of the damaged area or the adhesion area specifically, It can be usefully used for the treatment of joint diseases or connective tissue diseases.

1 is a diagram showing the results of confirming the cell proliferation ability of chondrocytes 24 hours after treatment with dextran, poloxamer, or a mixture thereof for normal chondrocytes (*/**/*** denotes each Significance of p<0.05/p<0.01/p<0.001 compared to the negative control group (nc)).
2 is a diagram showing the results of confirming the cell proliferation ability of chondrocytes 48 hours after treatment with dextran, poloxamer, or a mixture thereof for normal chondrocytes (*/**/*** denotes each Significance of p<0.05/p<0.01/p<0.001 compared to the negative control group (nc)).
3 is a diagram showing the results of confirming the cell proliferation ability of chondrocytes 24 hours after treatment with dextran, poloxamer, or a mixture thereof for chondrocytes in an in vitro model of osteoarthritis according to rhIL-1α treatment ( */**/*** indicates the significance of p<0.05/p<0.01/p<0.001 compared to the induction positive control (pc), respectively).
4 is a diagram showing the results of confirming the cell proliferation ability of chondrocytes 48 hours after treatment with dextran, poloxamer, or a mixture thereof for chondrocytes in an in vitro model of osteoarthritis according to rhIL-1α treatment ( */**/*** indicates the significance of p<0.05/p<0.01/p<0.001 compared to the induction positive control (pc), respectively).
5 is a diagram showing the results of confirming the expression level of MMP-3 gene according to treatment with dextran, poloxamer, or a mixture thereof in chondrocytes of an in vitro model of osteoarthritis according to rhIL-1α treatment; (*/**/*** indicates the significance of p<0.05/p<0.01/p<0.001 compared to the induction positive control (pc), respectively).
6 is a diagram showing the results of confirming the expression level of the MMP-13 gene according to treatment with dextran, poloxamer, or a mixture thereof in chondrocytes of an in vitro model of osteoarthritis according to rhIL-1α treatment. (*/**/*** indicates the significance of p<0.05/p<0.01/p<0.001 compared to the induction positive control (pc), respectively).
7 shows the results of confirming the IL-10/IL-6 gene expression ratio according to the treatment of dextran, poloxamer, or a mixture thereof for chondrocytes in an in vitro model of osteoarthritis following rhIL-1α treatment; (*/**/*** indicates the significance of p<0.05/p<0.01/p<0.001 compared to the positive control for inflammation (pc), respectively).
8 is a diagram showing the results of confirming the amount of type 2 collagen produced by treatment with dextran, poloxamer, or a mixture thereof in chondrocytes in an in vitro model of osteoarthritis according to rhIL-1α treatment (*/ **/*** indicates the significance of p<0.05/p<0.01/p<0.001 compared to the induction positive control (pc), respectively).
9 is a diagram showing the results of confirming the amount of agrican produced by treatment with dextran, poloxamer, or a mixture thereof for chondrocytes in an in vitro model of osteoarthritis according to rhIL-1α treatment (*/ **/*** indicates the significance of p<0.05/p<0.01/p<0.001 compared to the induction positive control (pc), respectively).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for treating joint disease or connective tissue disease, comprising dextran, poloxamer, or a mixture thereof.

In the present invention, “dextran” refers to a polymer of D-glucose, which is one of polysaccharides. Dextran has a structure similar to that of starch or glycogen, and D-glucose leads to a straight chain shape with α-1,6 bonds, and α-1,4 bonds are branched here and there. Dextran can be used as a blood extender and blood clotting agent, and the molecular weight of dextran can be varied depending on the purpose. In the present invention, the dextran may have a molecular weight of 500 to 10,000Da, preferably 500 to 8,000Da, more preferably 1,000 to 5,000Da. Depending on the molecular weight, 'dextran 1' when the molecular weight is 1,000 Da, 'dextran 5' when the molecular weight is 5,000 Da, and 'dextran 10' when the molecular weight is 10,000 Da, etc.

In general, dextran of 8,000 Da or more is known to cause an allergic reaction when administered to the human body. In particular, the allergic reaction may cause anaphylaxis, which is a rapid systemic reaction caused by an antigen-antibody immune response, which may be quite dangerous. However, when using the low molecular weight dextran according to the present invention, preferably dextran of 1,000 Da to 5,000 Da, there is an advantage in that allergic reactions can be minimized, and compared to known substances (eg, hyaluronic acid) in the body Since the rate of decomposition is slow, there is an advantage that the therapeutic effect of joint disease or connective tissue disease can be maintained for a long time.

In addition, the low-molecular-weight dextran according to the present invention does not have an allergic reaction compared to dextran of 8,000 Da or more, and has a fast onset of action time and can be spread over a wide range.

The present invention provides examples using dextran 1 and dextran 5 as preferred examples. They can be used in combination with dextran 1 and dextran 5, and can also be used in combination with poloxamer 188.

In the present invention, the concentration of dextran is expressed as a percentage of the concentration (w/v) by mass (g) in 100cc of solvent, for example, when dextran 1 is included in the composition as a single active ingredient, 2 to 40 (w/v) )%, and when dextran 5 is included in the composition as a single active ingredient, dextran 5 may be included in a concentration of 2 to 30 (w/v)%.

In addition, when dextran is included in the composition as a mixture of dextran 1 or 5 or a mixture of dextran and poloxamer, the concentration of dextran may be different depending on the mixing object. For example, when a mixture of dextran 5 and dextran 1 is included in the composition, the concentration of dextran 5 is 2 to 40 (w/v) %, and the concentration of dextran 1 is 2.5 to 40 (w/v) % Alternatively, 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) %.

When dextran is present in a high concentration in the composition, since the viscosity of the polysaccharide is excessively increased due to the water-containing nature, it is difficult to form an appearance, so dextran has been generally used at a concentration of 5% or less. However, since dextran in the composition of the present invention is included in a concentration of at least 2 to 40 (w/v)% depending on the molecular weight, the therapeutic effect of joint disease or connective tissue disease can be maximized. In order to increase or stabilize the solubility of dextran, the composition of the present invention may further include an additive, wherein the additive is a sugar including sorbitol, xylitol, erythritol, lactitol, mannitol and maltitol and mixtures thereof. Alcohols, or erythrose, erythrulose, threose, arabinose, ribose, ribulose, wood sugar, xylulose , lyxose, allose, altrose, psicose, galactose, gulose, idose, mannose, sorbo Monosaccharides including sorbose, talose, tagatose, sedoheptulose, mannoheptulose and mixtures thereof, or _divalent including CaCl 2 It may contain cations.

In the present invention, "poloxamer" is a nonionic triblock copolymer in which hydrophilic ethylene oxide is bonded to both ends with a hydrophobic propylene oxide as the center, and has a temperature-sensitive characteristic, so that the sol ( Sol) and gel can be converted, and the properties of poloxamers vary depending on the ratio of polyoxypropylene and polyoxyethylene.Poloxamers are used as emulsifiers, stabilizers, and solubilizers in addition to surfactants.

In the present invention, the poloxamer may have an average molecular weight of 100 to 20,000 Da.

The poloxamer is poloxamer 101, poloxamer 105, poloxamer 105 benzoate, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 182 dibenzoate, poloxamer Poloxamer 183, Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 212, Poloxamer 215, Poloxamer 217, Poloxamer 231, 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 and preferably poloxamer 188, but is not limited thereto.

In the present invention, the concentration of the poloxamer is a percentage of the mass (g) concentration (w/v) in 100cc of the solvent, a concentration of 1 to 50 (w/v)%, a concentration of 2 to 30 (w/v)% may be characterized as included in the composition, for example, poloxamer 188 is included in the composition at a concentration of 1 to 15 (w/v)% or 1 to 10 (w/v)% when included as the sole active ingredient in the composition. can

In order to increase or stabilize the solubility of the poloxamer, the composition of the present invention may further include additives such as sugar alcohols, monosaccharides, and divalent cations, and sterile water or physiological saline is included in the composition without additional additives may be

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

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

Poloxamer 188 may exhibit toxicity when included in the composition at a high concentration, for example, exceeding 25 (w/v)%, and may be used by appropriately adjusting the concentration when mixed with dextran.

In one embodiment, the dextran:poloxamer in the pharmaceutical composition of the present invention is, for example, in a (w/v)% ratio 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, such as 1:0.01 to 1:40, with 1 (w/v)% poloxamer 188 in an exemplary combination When mixing dextran 1 40 (w/v)% 1:40, when mixing poloxamer 188 20 (w/v)% and dextran 5 2 (w/v)%, a ratio of 1:0.1 reflects Thus, it may be included in a ratio of 1:0.1 to 1:40. Here, the average molecular weight of the dextran may be 1,000Da or 5,000Da, and the average molecular weight of the poloxamer may include poloxamer 188, which is 8,500Da. In addition, as an embodiment of the present invention, when a mixture of two types selected from the group consisting of dextran 1, dextran 5 and poloxamer 188 is included, each component is mixed in a volume ratio of 1:1 to add to the composition. may be included.

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 dextran 1: dextran 5 in the pharmaceutical composition is, for example, (w/v)% 5:0.05, 5:0.5, 5:5, 5:10, 5:20, 5:30, 5:40, 5:50, 5:100, 10:0.1 as a ratio. 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 may be included, for example, 1:0.01 to 1:20, with dextran 1 2.5 (w / v)% in an exemplary combination When dextran 5 40 (w/v)% is mixed 1:16, when dextran 1 40 (w/v)% and dextran 5 2 (w/v)% are mixed, a ratio of 1:0.05 is reflected Thus, it may be included in a ratio of 1:0.05 to 1:16.

When the composition of the present invention includes a mixture of dextran and poloxamer, the hydrophobic component of the poloxamer can enhance the solubility of dextran or a concomitant drug, and the hydrophilic component of the poloxamer, polyethylene glycol (PEG), preserves the cell membrane. And it can preserve the tissue by preventing adhesion. However, since poloxamer can be easily diluted by body fluids and can be easily absorbed into body fluids, it is possible to increase stability in the body by mixing with dextran. Therefore, in the case of a mixture of dextran and poloxamer, the viscosity is maintained longer than when dextran alone or poloxamer alone is included, so that the damaged joint or connective tissue area can be covered. By reducing inflammatory findings, the supply of nutrients is facilitated and tissue regeneration is promoted. The effect may be induced by a cushioning effect or a coating effect or an anti-inflammatory effect of the dextran and poloxamer mixture.

In the present invention, the "cushion effect" means an effect that acts like a cushion that cushions the impact applied by reducing friction in the administered site, for example, the joint, and the "coating effect" ” means the effect of allowing the knee joint to move comfortably by quickly covering and coating the stiffened area, for example, the damaged cartilage tissue in the joint. These effects may reduce the inflammatory response of the damaged joint.

Therefore, the composition of the present invention, which induces a buffering effect and a coating effect or an anti-inflammatory effect, is suitable for regenerating or improving damaged tissue, and thus can be effectively used for the treatment of joint disease or connective tissue disease. The above 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 may have competitiveness compared to stem cell therapeutics that require high technology and cost.

In addition, the present invention is stem cells; It provides a pharmaceutical composition for preventing or treating joint disease or connective tissue disease, including dextran, poloxamer, or a mixture thereof.

As used herein, the term "stem cell" refers to a cell having the ability to differentiate into two or more cells while having the ability to self-renew, totipotent stem cell, pluripotent stem cell, multi It can be classified as a multipotent stem cell.

The stem cells of the present invention may be derived from any type of animal, including humans and non-human mammals, and whether the stem cells are derived from an adult or an embryo, but is not limited thereto.

Stem cells of the present invention include embryonic stem cells or adult stem cells, wherein the adult stem cells are mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and pluripotent stem cells. They may be stem cells or amniotic epithelial cells. The stem cells may be stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat (or adipose tissue cells), muscle, nerve, skin, amniotic membrane and placenta (or placental tissue cells), urine, etc., but is not limited thereto.

The stem cells may be stem cells or a concentrate thereof, a stem cell culture solution or a concentrate thereof, a culture secretion of stem cells or a concentrate thereof, or a combination thereof.

Dextran or poloxamer in the composition can serve to specifically transport stem cells to the damaged lesion of the joint or connective tissue, and also induce regeneration of the damaged tissue by settling in the lesion to which the stem cells are transported. has an elevating effect.

Therefore, when the composition of the present invention includes stem cells, it is possible to more effectively treat joint diseases or connective tissue diseases by transporting and attaching stem cells specifically to damaged tissues.

In the present invention, "prevention" refers to any action that suppresses or delays the onset of joint disease or connective tissue disease by administration of the composition.

In the present invention, "treatment" refers to any action that improves or beneficially changes the symptoms of joint disease or connective tissue disease by administration of the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, and may be formulated into a unit dosage form suitable for administration in a patient's body according to a conventional method in the pharmaceutical field. In addition, the formulation is pharmaceutically acceptable in combination with a pharmaceutically acceptable carrier or medium, for example, sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, or binder, etc. It may be formulated by blending into an acceptable unit dosage form.

The pharmaceutical composition may be in a solution state or a powder state, and when the pharmaceutical composition is in a powder state, it may be used by dissolving it in a solvent immediately before administration, but is not limited thereto. It can be manufactured in the most suitable manner taking into account the circumstances.

The dosage form of the pharmaceutical composition is not particularly limited, but may be administered through a general route of administration, such as oral administration, injection, and administration by infusion.

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, syrup, etc. together with a pharmaceutically acceptable carrier or excipient. However, solid preparations such as tablets or powders often take time to absorb, so oral administration is preferable. In that case, it is preferable to administer as an aqueous solution together with a suitable additive, for example, salts, such as sodium chloride, a buffer, a chelating agent, etc.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin and soft capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient and a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, or optionally a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol.

In addition, the drug can be administered in a target drug delivery system such as liposomes coated with a specific antibody targeting a joint disease or connective tissue. Liposomes can be selectively taken to diseased tissue.

When the pharmaceutical composition of the present invention is administered as a preparation for local administration, an appropriate buffer, isotonic agent, etc. is added and dissolved in sterile distilled water is used in the joint cavity, connective tissue, intravenous, subcutaneous, intradermal, intra-articular or It may be directly injected into a muscle or the like, applied to the skin, or in the form of a patch.

The subject may be any one selected from mammals including humans, dogs, cats, pigs, horses, cattle, sheep, mice, and monkeys, preferably humans.

As used herein, the term "intra-articular" refers to a percutaneous injection of the pharmaceutical composition of the present invention into a joint.

As used herein, the term "local administration" refers to transdermal injection into or near an inflamed joint. Local administration injections are therefore directed to the epidermis, dermis, muscle or any deep organ.

The main advantage of topical administration is that it selectively limits the analgesic effect on the injured area. Moreover, local administration allows for high local concentration levels with little or no systemic release.

The pharmaceutical composition comprises a stabilizer, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, calcium silicate, polyvinylpyrrolidone and It may further comprise cellulose. The composition may contain lubricants such as magnesium stearate and mineral oil; wetting agent; emulsifying and suspending agents; preservatives such as thimerosal, benzyl alcohol, parabens, methyl- or propylhydroxybenzoate; antioxidants such as ascorbic acid, dihydroquinone, butylated hydroxytoluene and dithiothreitol; and buffers such as sodium phosphate monobasic, sodium phosphate dibasic, sodium benzoate, potassium benzoate, sodium citrate, sodium acetate and sodium tartrate; chelating agents such as ethylenediaminetetraacetic acid; and tonicity adjusting agents, such as sodium chloride or dextrose.

The pharmaceutical composition may further include an anesthetic. The anesthetics include, but are not limited to, amethocaine (tetracaine), lignocaine (lidocaine), xylocaine, bupivacaine, prilocaine, ropivacaine, benzocaine, mepivocaine, cocaine and Combinations thereof are included.

The pharmaceutical composition may further include an antibacterial agent. The antibacterial agents include, but are not limited to, amantadine hydrochloride, amanfadine sulfate, amikacin, amikacin sulfate, amoglycoside, amoxicillin, ampicillin, amsamycin, bacitracin, beta-lactam, candisidine , capreomycin, carbenicillin, cephalexin, cephaloridin, cephalotin, cefazolin, cefapyrin, cepradin, cephaloglycin, thilomphenicol, chlorhexidine, clohexidine gluconate, chlorhexidine hydrochloride , chloroxine, chlorquiraldol, chlortetracycline, chlortetracycline hydrochloride, ciprofloxacin, circulline, clindamycin, clindamycin hydrochloride, clotrimazole, cloxacillin, demeclocycline, diclosacillin, diiodohyde Roxyquine, doxycycline, ethambutol, ethambutol hydrochloride, erythromycin, erythromycin estolate, erchmycin stearate, farnesol, floxacillin, gentamicin, gentamicin sulfate, gramicidin, geofulvin, halopro Gin, haloquinol, hexachlorophene, iminocycline, iodochlorhydroxyquine, kanamycin, kanamycin sulfate, lincomycin, lineomycin, lineomycin hydrochloride, macrolide, meclocycline, metacycline, metacycline Hydrochloride, Methenine, Methenamine Hyfurate, Methenamine Mandelate, Methicillin, Metonidazole, Miconazole, Miconazole Hydrochloride, Minocycline, Minocycline Hydrochloride, Mupyrosine, Nafcilin, Neomycin, Neomycin Sulfate , netimicin, netylmicin sulfate, nitrofurazone, norfloxacin, nystatin, octopirox, oleandomycin, orcephalosporin, oxacillin, oxytheacin, oxytetracycline hydrochloride, parachlorometa xylenol , paromomycin, paromomycin sulfate, penicillin, penicillin G, penicillin V, pentamidine, pentamidine hydrochloride, peneticillin, polymyxin, quinolone, streptomycin sulfate, tetracycline, tobramycin, tolnaftate, Triclosan, Tripampin, Rifamycin, Lolite Tricycline, silver salt, spectinomycin, spiramicin, struptomycin, sulfonamide, tetracycline, tetracycline, tobramycin, tobramycin sulfate, triclocarbon, triclosan, trimethoprim-sulfamethoxazole, tylosine, vancomycin and irotricin.

The pharmaceutical composition may further include an anti-inflammatory agent. The anti-inflammatory agent may be non-steroidal, steroidal, or a combination thereof. Non-limiting examples of non-steroidal anti-inflammatory agents include oxicam such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates such as aspirin, disalside, benorylate, trilysate, sapaprine, solfrin, diflunisal and pendosal; Acetic acid derivatives such as diclofenac, penclofenac, indomethacin, sulindac, tolmethine, isosepac, furofenac, thiopinac, zidomethacin, acematacin, penthiazac, zomepirac, clindanac , oxepinac, pelvinac and ketorolac; phenamates such as mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid and tolfenoic acid; Propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropene, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, thioxaprofen, suprofen, aminoprofen and thiapropenic; pyrazoles such as phenylbutazone, oxyphenbutazone, peprazone, azapropazone and trimetazone. Extracts of these non-steroidal anti-inflammatory agents may also be used.

Non-limiting examples of such steroidal anti-inflammatory agents include corticosteroids such as hydrocortisone, hydroxy-triamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionate, clobetasol valerate, desoni De, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluchlorolone acetonide, fludrocortisone, flumethasone Son pivalate, fluocinolone acetonide, fluocinonide, flucortin butyl ester, fluocortolone, flupredniden (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone Butyrate, methylprodnisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocorisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medrisone, amsinafel, amsinapid, betamethasone and its esters balance, chloroprednisone, chlorprednisone acetate, clocortelone, clecinolone, dichlorisone, diflurprednate, fluchloro Nid, flunisolide, fluorometallone, fluferolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone and extracts thereof.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” refers to an amount sufficient to treat a disease, and an effective dose level includes the severity of the disease, the age, weight, health, sex, sensitivity of the patient to the drug, administration time, and route of administration. and factors including the rate of excretion, duration of treatment, drugs used in combination with or concurrently with the composition of the present invention, and other factors well known in the medical arts. The dosage of dextran, poloxamer, or mixtures thereof varies widely and is determined according to the individual requirements of each particular case.

The pharmaceutical composition of the present invention may be administered in combination with a known agent for the treatment of joint disease or connective tissue disease, and may be administered simultaneously or sequentially with the therapeutic agent.

The pharmaceutical composition of the present invention may be administered once or divided into several doses at a desired administration interval, for example, at intervals of one week.

In addition, if necessary, the composition of the present invention may be provided in a package or administration device containing one or more unit dosage forms containing the active ingredient. Compositions comprising a compound of the present invention formulated in a pharmaceutically acceptable carrier are suitably prepared and placed in an appropriate container and indicated for use in the treatment of the conditions indicated. Appropriate indications indicated on the level include treatment of joint disease and treatment of connective tissue disease.

According to the present invention, a pharmaceutical composition comprising dextran, poloxamer or a mixture thereof induces long-lasting pain relief in a subject suffering from joint disease or connective tissue disease. The use of the pharmaceutical composition according to the present invention avoids major drug side effects and enables the treatment and/or prevention of acute as well as chronic joint disease or connective tissue disease without using too invasive techniques.

In the present invention, "connective tissue" is also known as connective tissue or connective tissue, and refers to a tissue that is widely distributed in animal tissues and serves to connect, protect, and fill cells, organs, organs, etc., and is fibrous It is classified into connective tissue, collagenous tissue, and reticulum tissue, but fibrous connective tissue accounts for most of it.

In the present invention, the cause 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 diseases and connective tissue diseases caused by any cause that causes joint damage, but the joint disease for which the composition of the present invention is expected to be effectively used is osteoarthritis, preferably repetitive frictional force. and the resulting degenerative osteoarthritis, traumatic arthritis, rheumatoid arthritis, patellar-femoral syndrome, chronic inflammation or arthrosis. In addition, the connective tissue disease in which the composition of the present invention is expected to be effectively used is a connective tissue in case of loss of collagen or moisture due to degenerative changes caused by physical damage or repeated friction of intervertebral discs, ligaments, tendons, skin, tendons, etc. include disease.

In the present invention, it effectively exhibits an anti-inflammatory effect without showing cytotoxicity, suppresses the expression of MMP-3 and MMP-13 genes, which are proteins that destroy cartilage matrix, and promotes the synthesis of type 2 collagen by cartilage protection, Preferred examples that can achieve the effect of increasing the amount of glycan can be provided as follows.

An example of the present invention relates to a pharmaceutical composition for preventing or treating joint disease or connective tissue disease, comprising a mixture of two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188. .

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

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

In addition, the poloxamer is included as a single active ingredient in the composition, and the concentration of the poloxamer may be 1 to 15 (w/v)%.

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.

In one embodiment of the present invention, the mixture is a mixture of dextran 5 and poloxamer, the concentration of dextran 5 is 2 to 40 (w/v)%, the concentration of the poloxamer is 1 to 20 (w/v) )%.

In addition, the mixture is a mixture of dextran 5 and dextran 1, the concentration of dextran 5 is 2 to 40 (w / v) %, the concentration of dextran 1 can be 2.5 to 40 (w / v) % have.

In addition, the mixture may be a mixture of dextran 1 and poloxamer, the concentration of dextran 1 may be 2.5 to 40 (w/v)%, and the concentration of the poloxamer may be 1 to 5 (w/v)%.

In addition, the mixture may be a mixture of dextran 1 and poloxamer, the concentration of dextran 1 may be 45 to 55 (w/v)%, and the concentration of poloxamer may be 1 to 2 (w/v)%.

In addition, an example of the present invention is stem cells; It relates to a pharmaceutical composition for preventing or treating joint disease or connective tissue disease, comprising a mixture of two types selected from the group consisting of dextran 1, dextran 5 and poloxamer 188.

Terms not otherwise defined herein have meanings commonly used in the art to which the present invention pertains.

Materials and Methods

In the present invention, an experiment is performed using dextran 1 (Dextran 1 (EP grade, Pharmacosmos), dextran 5 (Dextran 5, pharmaceutical quality, Pharmacosmos), and poloxamer 188 (Poloxamer 188, cell culture grade, Sigma-Aldrich)) Also, for arthritis induction, rhIL-1α (recombinant human IL-1α, Lot No. 200-01A) was purchased from PEPROTECH and used as a positive control, and indomethacin (Indomethacin, Sigma-Aldrich, Lot No. 53) -86-1), sodium hyaluronate (sodium hyaluronate 25 mg/2.5 mL, Araganju, Dongkwang Pharmaceutical, prescription drugs) were used.

Example 1. Preparation and experimental preparation of dextran, poloxamer or mixtures thereof

1.1 Cell Isolation and Culture

After separating the hind limb joints of 3-week-old male SD rats in a sterile state, only the cartilage tissues constituting the joints were collected and converted into single cells, and primary rat chondrocytes were isolated and used. When the primary cultured cells were stable, they were used for the test, and at least 6 cells were isolated at one time of primary culture. To identify stabilized chondrocytes, after RNA isolation, the expression level of chondrocyte markers, type 2 collagen or SOX9 gene, was confirmed using real-time RT-PCR and then used for testing.

1.2 Cell culture method

The cells isolated in 1.1 were cultured in an incubator set at a temperature of 37° C., a humidity of 95%, and a CO _{2 of} 5%, and the temperature and humidity of the incubation room were checked every 8 hours. Minimum Essential Medium (MEM) medium supplemented with 10% fetal bovine serum, 2 mL L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin was used as the medium. After observing the number of cells during the incubation period, when the cells proliferate more than 90%, separate the cells with a detached solution (0.25 (w/v)% Trypsin, 0.53 mM EDTA solution (3 mL)) and centrifuge the cells at 125 X g for 10 minutes. was isolated and used in the following experiments.

1.3 Composition and Preparation of Test Groups

The test group consisted of treating the osteoarthritis-induced model with rhIL-1α 5 ng/mL by concentration of normal control group (NC), inflammation-inducing control group (PC), and test substance (single or mixture). Shin (IM) or sodium hyaluronate (HN) treatment group consisted of a single concentration. The test group is shown in Table 1 below.

[Table 1]

D = Dextran 1, T = Dextran 5, P = Poloxamer 188, NC = Normal group, PC = Inflammatory substance (rhIL-1α) group, HN = Sodium hyaluronate, IM = Indomethacin, D+P = dextran 1 and poloxamer 188 mixture, D+T = dextran 1 and dextran 5 mixture, P+T = poloxamer 188 and dextran 5 mixture.

Concentrations of the components used in the present invention were indicated according to the following notation method: D10 = 10(w/v)% solution of dextran 1, P10 = 10(w/v)% solution of poloxamer 188, T10 = 10(w/v)% solution of dextran 5

The test substance used in the present invention was prepared using the following method: 1 to 50 (w/v)% of dextran 1 (D), 1 to 50 (w/v)% of dextran 5 (T) reflecting the maximum concentration soluble in the cell culture medium A single product was prepared within the range of 1 to 40 (w/v)%, and poloxamer 188 (P) from 1 to 35 (w/v)%. In the case of preparing a mixture, two types of single substances were prepared by mixing in a volume ratio of 1:1 (v:v), respectively.

Dextran in complete media (Minimum Essential Medium (MEM) medium containing all of 10% fetal bovine serum, 2 mL L-glutamine, 50 U/mL penicilin, 50 ug/mL streptomycin, etc.) 1, 5 and poloxamer 188 were completely dissolved according to the dose concentration (w/v), respectively. In order to completely dissolve and homogenize the test substance in the complete media, it is stirred slowly with a sterile spatula, stick, magnetic bar, etc. at a cool temperature, especially in the case of Poloxamer 188, or shaken briefly several times so as not to form bubbles. Dissolve until completely clear with no floatation. After the test substance was completely dissolved, it was filtered with a 0.22 μm pore size syringe filter, sealed, and stored in the refrigerator.

More specifically, single substance preparation was performed in the following way.

Preparation of 5% single substance ('D5') of dextran 1

When preparing a 5% test solution of dextran 1, 5 g of dextran 1 was aliquoted, completely dissolved in complete media, and the total amount was adjusted to 100 mL to prepare a D5 (w/v)% test solution. As such, the dissolved test substance at a concentration of 0.05 g/mL was filtered using a 0.22 μm pore size syringe filter and stored in a refrigerator before use.

Preparation of a mixture of 5% of dextran 1 and 10% of poloxamer 188 ('D5+P10')

Each of D5 and P10 was prepared, filtered with a 0.22 μm pore size syringe filter, and stored in a refrigerator, and each volume was mixed in a 1:1 (v:v) ratio and shaken at low speed to prevent foaming before use.

Comparative Example: Indomethacin treatment group (IM) preparation

Indomethacin was completely dissolved in DMSO at a concentration of 5 mM, diluted with complete media, filtered with a 0.22 μm pore size syringe filter, and adjusted to have a final concentration of 5 μM when treated with cells.

Comparative Example: Preparation of sodium hyaluronate treated group (HN)

Sodium hyaluronate is sodium hyaluronate 25 mg/3 mL (= sodium hyaluronate 8.33 mg/mL) by inhaling complete media into Araganju (sodium hyaluronate 25 mg/2.5 mL, pharmaceutical form in a sterile disposable syringe). After dilution, it was used immediately.

1.4 Setting the dosing method

In the case of primary cultured chondrocytes isolated from rats, the doubling time was about 24 hours, so the test substance was treated when proliferation and stabilization were appropriate. After culturing chondrocytes in a 48-well or 24-well plate, each test substance is administered once, and 20% (v/v) of the total culture solution by concentration of each test substance 1 hour before rhIL-1α treatment for arthritis induction The test solution was administered so that After 24 hours of rhIL-1α treatment, the culture medium was collected and the assay was performed. However, only the test substance was treated without treatment with rhIL-1α when the toxicity or proliferative ability of the test substance to chondrocytes was confirmed.

1.5 Statistical Analysis

All experimental results were compared and analyzed at p<0.05 level with a normal control group (NC) or an inflammation-inducing positive control group (PC) and student T-test to indicate their significance.

Example 2. Cytotoxicity assay

Cytotoxicity analysis was performed by checking whether the proliferative ability of chondrocytes changes according to the treatment with the test substance. Cell proliferative ability was evaluated in both untreated chondrocytes not treated with rhIL-1α and chondrocytes treated with rhIL-1α to induce inflammation, and cultured for 24 hours and 48 hours after each test substance was treated in rat chondrocytes. After that, MTT analysis (Thiazolyl Blue Tetrazolium Blue; Sigma, M5655) was performed. In the evaluation of the cell proliferative ability of test substances in an in-vitro model of arthritis induced by rhIL-1α, which is an inflammatory substance, each test substance was treated in rat chondrocytes 1 hour before rhIL-1α treatment, and then cultured for 24 hours and 48 hours. MTT analysis (Thiazolyl Blue Tetrazolium Blue; Sigma, M5655) was performed, and the results are shown in FIGS. 1 to 4 .

As shown in Figure 1, as a result of observing the cell proliferation rate of dextran 1, dextran 5, poloxamer 188 or a mixture thereof in rat knee joint chondrocytes that did not induce inflammation for 24 hours, most of dextran 1, dex It was confirmed that Tran 5, poloxamer 188, and mixtures thereof aid cell proliferation without being toxic to cells. However, in the case of the poloxamer 188-treated group among the single treatment groups, a decrease in some cell proliferative ability was contrasted when cultured for 24 hours at a concentration of poloxamer 188 25 (w/v)% (P25) and 35 (w/v)% (P35). It was found to be lower than the drug (marketed drug) sodium hyaluronate (HN) administered group, indicating that it was not suitable for single administration. In addition, in the case of poloxamer 188, it was confirmed that poloxamer 188 may cause cytotoxicity when treated at a high concentration through a decrease in some cell proliferative ability when cultured for 24 hours even in these mixtures. In addition, among the mixtures, the decrease in cell proliferation in the D50+P35, P5+T40, P25+T40, and P35+T40 experimental groups was lower than the control drug (commercially available drug), sodium hyaluronate (HN) administered group.

As shown in FIG. 2 , the cell proliferation rate of dextran, poloxamer or a mixture thereof in non-inflammatory rat knee joint chondrocytes was observed for 48 hours, and results similar to those of 24 hours culture were confirmed.

Through these results, when treating normal knee articular chondrocytes, the proliferation of chondrocytes was not achieved even when the concentration of dextran 1 (D) was increased to 50% or the concentration of dextran 5 (T) was increased to 40%. On the other hand, it was confirmed that poloxamer 188 may be toxic to chondrocytes at a concentration of 25% or more, while not inhibited compared to commercially available drugs. In addition, when composed of a mixture, the cell proliferation ability is partially improved compared to that of poloxamer 188 alone, but in order not to induce toxicity, it was confirmed that poloxamer 188 is preferably mixed at a concentration of 10 (w/v)% or less.

3 and 4 show the results of observing the cell proliferation rate according to the treatment of the experimental group in an in vitro model of osteoarthritis according to the rhIL-1α treatment for 24 hours and 48 hours, respectively.

As shown in FIG. 3 , even in the osteoarthritis model, poloxamer 188 was found to inhibit cell proliferation when the concentration was increased, and dextran 1 and dextran 5 did not inhibit cell proliferation even when the concentration was increased. In the case of poloxamer 188, cell proliferation inhibition was confirmed in the P10 alone treatment group, but when used in combination with D5, D50, T4, T40, etc., the cell proliferation effect was shown. When mixed with dextran, poloxamer 188 It was confirmed that 1% to 10 (w/v)% of

In Figure 4, the same result as the 24 hour culture was confirmed, and it was confirmed that the cell proliferative capacity of the P10-treated group was increased through mixing with dextran 1 or dextran 5.

Combining the above results, it was confirmed that most of dextran 1 and dextran 5 did not induce cytotoxicity in normal and inflammatory cells. On the other hand, it was shown that poloxamer 188 can induce cell proliferation inhibition in both normal cells and inflammation-inducing cells when treated with a single substance as the concentration increases. When administered in combination with dextran 1 or dextran 5, it was confirmed that it can be used without cytotoxicity.

Example 3. Confirmation of MMP-3, MMP-13 gene expression change through real-time RT-PCR

An experiment was performed 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 the cartilage matrix, if it is possible to effectively inhibit the increase in the expression level of the gene that can be induced through inflammatory stimulation, it can be evaluated that the cartilage protection effect is excellent.

Dextran 1 is 2.5 to 50 (w/v)%, Dextran 5 is 2 to 40 (w/v)%, and poloxamer 188 is 1 to 20 (w/v)%, single administration or mixed administration to confirm the expression change of MMP-3 and MMP-13. Specifically, each test substance was treated with 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 using a phosphate buffer solution (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 each Each experimental group was diluted to the same concentration. RNA diluted in the same amount was synthesized as cDNA using PCR (ReverTra AceR qPCR RT Master Mix with gDNA Remover, Toyobo, FSQ-301). Using the synthesized cDNA, gene expression of MMP-3 and MMP-13 was confirmed in a 96-well plate by real-time RT-PCR (n=3).

The composition of the primers used in the experiment is shown in Table 2 below, and GAPDH, a housekeeping gene, was used as a control.

[Table 2]

After mixing the primer, SyBr green (SYBR Premix Ex Taq, Takara, RR420A) and the template, real-time RT-PCR (CFX 96 touch, Bio rad, Hercules, CA, USA) was used to intercalate real-time RT- PCR was performed. The quantitative result (gene expression level) obtained by applying the Ct value to the calibration curve was expressed as the value divided by the quantitative result of GAPDH, a housekeeping gene.

3.1 Confirmation of MMP-3 Expression Changes

Dextran 1, dextran 5, poloxamer 188 alone or a mixture thereof, the expression results of MMP-3 are shown in Tables 3a to 3b and FIG. 5 .

[Table 3a]

Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) MMP-3 gene expression levels in RhIL-1α-treated chondrocytes for 24 h

[Table 3b]

MMP-3 gene expression level of a mixture of dextran 1 (D), dextran 5 (T) or poloxamer 188 (P) in chondrocytes treated with RhIL-1α for 24 h

As shown in Table 3a and FIG. 5, dextran 1 (D) exhibited a significant MMP-3 reduction effect compared to the inflammation-inducing positive control at 2.5 to 40 (w/v)%, and dextran 5 (T) showed a significant MMP-3 reduction effect at 2 to 20 (w/v)%. On the other hand, polosamer 188 (P) exhibited a reduction effect in 1 to 20 (w/v)% of all tested soldiers.

As shown in Table 3b and Figure 5, in the mixture experimental group, dextran 1 (D) 5 (w / v) % and poloxamer 188 (P) 2 to 10 (w / v) %, dextran (T) 4% and A significant effect was confirmed in all experimental groups of 40(w/v)%, but in the group treated with high concentration of dextran 1 (D) 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 the concentration to less than 50 (w/v)%. Poloxamer 188 showed most significantly the MMP-3 reducing effect in the single-treated and mixed-treated groups, but as confirmed in Example 2, the concentration of poloxamer is preferably 10 (w/v)% or less, and thus It was confirmed that the corresponding P2, P5, and P10 can induce a significant MMP-3 inhibitory effect even in combination with dextran 1 and dextran 5.

3.2 Confirmation of MMP-13 Expression Changes

Dextran 1, Dextran 5, Poloxamer 188 The expression results of MMP-13 according to the treatment alone or a mixture thereof are shown in Tables 4a to 4b and FIG. 6 .

[Table 4a]

Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) MMP-13 gene expression levels in RhIL-1α-treated chondrocytes for 24 h

[Table 4b]

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

As can be seen in Tables 4a to 4b and FIG. 6, dextran 1 (D) in the single substance test group exhibited a significant MMP-13 gene increase inhibitory effect at 2.5 to 25 (w/v)%, D40 Although it was not significant at 40(w/v)% of phosphorus, the increase of MMP-13 gene was suppressed, and at 50(w/v)% of D50, the level of MMP-13 was rather increased. These properties were similarly shown in the mixture of dextran 1 (D). Therefore, it was confirmed that 2.5 to 40 (w/v)% of dextran 1(D) is the most preferable concentration range, which is consistent with the MMP-3 gene expression level results. Dextran 5 (T) exhibited a significant inhibitory effect of MMP-13 gene increase in 2 to 40 (w/v)% when administered alone, and Dextran 5 (T) 4 and 40 (w/v)% were dextran 5 (T) 4 and 40 (w/v)% Trans 1 (D) 5 (w / v) % and poloxamer 188 (P) 2 and 10 (w / v) % mixed with 10 (w / v) % also showed a significant MMP-13 gene increase inhibitory effect.

In the case of poloxamer 188, the MMP-13 gene increase inhibitory effect was exhibited at 5 to 20 (w/v)% in the single experimental group, but when administered as a mixture with dextran, 2(w/v)% may also exhibit an effect. confirmed to exist. Considering the results of the cytotoxicity test in Example 2, it was confirmed that the concentration of poloxamer 188(P) is preferably 2 to 10 (w/v)% when mixed administration. P2 and P10 corresponding to these induced significant MMP-13 gene increase inhibitory effects, except for the combination of dextran 1 (D) 50 (w/v)%, even in combination with dextran 1 and dextran 5.

Example 4. Confirmation of anti-inflammatory effect through IL-10/IL-6 expression ratio

The anti-inflammatory effect of the experimental group was confirmed by checking the IL-10/IL-6 expression ratio when rhIL-1α, an inflammatory substance, and a test substance (single substance, a mixture thereof) were treated together in a similar manner to Example 3 for 24 h. As the IL-10/IL-6 expression ratio increases, it can be determined that the anti-inflammatory effect is excellent.

The composition of the primers used in the experiment is shown in Table 5 below, and GAPDH, a housekeeping gene, was used as a control. The results are shown in Tables 6a to 6b and FIG. 7 .

[Table 5]

After mixing the primer, SyBr green (SYBR Premix Ex Taq, Takara, RR420A) and the template, Real Time RT-PCR (CFX 96 touch, Bio rad, Hercules, CA, USA) was used to intercalate Real Time RT-PCR was performed. The quantitative result (gene expression level) obtained by applying the Ct value to the calibration curve was expressed as the value divided by the quantitative result of GAPDH, a housekeeping gene.

[Table 6a]

IL-10 / IL-6 gene expression ratio of dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) in chondrocytes treated with RhIL-1α for 24 h

[Table 6b]

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

As shown in Table 6a and Figure 7, in the group treated with dextran 1 (D), which is a single group treatment experiment, the IL-10/IL-6 expression ratio except for the 50 (w/v)% treatment group was not clearly confirmed, but a significant increase effect was confirmed in the poloxamer 188 (P) 1 to 20 (w/v)% treatment group, and dextran 5 (T) 2 to 20 (w/v)% group .

As shown in Table 6b and Figure 7, a significant increase in IL-10/IL-6 expression ratio was observed in both the mixture administration group of dextran 1 (D), a mixture treatment experiment, and poloxamer 188 (P) and dextran 5 (T) also showed a significant increase in the mixed administration group, confirming that the anti-inflammatory effect in the mixture was excellent.

Although the increase in IL-10/IL-6 expression ratio was significant in the 50(w/v)% administration group of dextran 1, considering that the increase did not stand out, IL-10/IL-6 in the osteoarthritis model The increase in IL-6 expression ratio, ie, anti-inflammatory effect, indicates that it is preferable to administer in combination with poloxamer 188 (P) or dextran 5 (T) rather than increase the concentration of dextran 1 (D). In particular, the anti-inflammatory effect is dextran 1 (D) 5 and 50 (w/v)%, dextran 5 (T) 4 and 40 (w/v)%, poloxamer 188 (P) 2 and 10 (w/v) % compared to the single administration group, most of these mixture administration groups were significantly superior, so it was possible to confirm the superiority of the combined administration of dextran 1 (D), dextran 5 (T), and poloxamer 188 (P). have.

Example 5. Type II Collagen and agrican Confirmation of increased production of (Aggrecan)

To check the production of type 2 collagen and agrican protein among the indicator substances for osteoarthritis evaluation, each test substance was treated with chondrocytes 1 hour before rhIL-1α treatment, and then cultured for 24 hours. Since type 2 collagen and agrican are cartilage matrix compositions, it can be determined that the cartilage matrix protective effect is excellent when the amount of their production is increased.

Here, the cell supernatant (culture solution) was recovered, centrifuged at 13,000 rpm for 10 minutes, and only the supernatant was used for analysis. The centrifuged cell supernatant was used for analysis referring to the manual of Aggrecan ELISA Kit (Rat Aggrecan ELISA Kit, Mybiosource, MBS261073) and type II Collagen (Type II Collagen detection Kit, Multi-Species, Chondrex, 6018) products. . For accurate analysis, absorbance was measured at 450 nm and 490 nm, respectively, using an ELISA reader (EPOCH 2 microplate reader, BioTek, Instruments, VT, USA) of the electromagnetic wave band, and the content of type 2 collagen and agrican (ng /mL) was quantified.

5.1 Confirmation of type 2 collagen production

Dextran 1 (D), dextran 5 (T), poloxamer 188 (P) alone or a mixture thereof, the results of the production of type 2 collagen are shown in Tables 7a to 7b and FIG. 8 .

[Table 7a]

Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) Type II Collagen production amount in chondrocytes treated with RhIL-1α for 24 h

[Table 7b]

Amount of Type II Collagen produced by a mixture of dextran 1 (D), dextran 5 (T) or poloxamer 188 (P) in chondrocytes treated with RhIL-1α for 24 h

As shown in Tables 7a to 7b, and FIG. 8, dextran 1 (D) 5 to 25 (w/v)%, dextran 5 (T) 4 to 40 (w/v)% inflammation in the group alone An increase in type 2 collagen synthesis was observed compared to the induced positive control (PC). On the other hand, dextran 1 (D) 50 (w/v)% showed an increase in type 2 collagen synthesis compared to the inflammation-inducing positive control (PC) in both groups administered alone and in combination with dextran 5 (T) and poloxamer 188 (P). did not appear to be induced. This is, as confirmed in Example 3, dextran 1 (D) 50 (w/v)% alone does not show the effect of inhibiting the increase in expression of MMP-3 and MMP-13, only poloxamer 188 (P) 2 Considering that it is possible to suppress the increase in expression of MMP-3 and MMP-13 only in a mixture with to 10 (w/v)%, dextran 1 (D) is alone or mixed in 40 (w/v)% or less The results show that it is desirable to use In particular, when mixed with dextran 1 (D) 5 (w/v)% and dextran 5 (T) 4 to 40 (w/v)%, the synthesis of type 2 collagen was significant compared to the inflammation-inducing positive control (PC). increased significantly.

In the case of poloxamer 188(P), no effect of increasing type 2 collagen synthesis was observed in the 10(w/v)% and 20(w/v)% alone treatment groups except for the 2(w/v)% treatment group. , it was confirmed that as the concentration increased, the synthesis of type 2 collagen was further decreased. However, when 2 or 10 (w/v)% of poloxamer 188 (P) is mixed and administered with dextran 1 (D) 5 (w/v)% or dextran 5 (T) 40 (w/v)% showed a significant increase in the synthesis of type 2 collagen. These results show that dextran 1 (D) and dextran 5 (T) can be treated alone in the synthesis of type 2 collagen in osteoarthritis, but poloxamer 188 (P) is alone except for 2 (w/v)% The treatment is not suitable, indicating that mixed administration of poloxamer 188 (P) and dextran 1 (D) or dextran 5 (T) can effectively induce type 2 collagen synthesis.

Previously in Example 4, it was confirmed that the mixed administration of poloxamer 188 and dextran 1 or dextran 5 can significantly increase the gene expression ratio of IL-10/IL-6 compared to single administration. Taken together, it was confirmed that administration of a mixture of dextran 1, dextran 5, or poloxamer 188 is preferable in order to relieve the inflammatory response in osteoarthritis and at the same time promote collagen synthesis.

5.2 Confirmation of agrican content quantification results

Dextran 1, Dextran 5, Poloxamer 188 The results of the agrican production amount according to the treatment alone or a mixture thereof are shown in Tables 8a to 8b, and FIG. 9 .

[Table 8a]

Dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) agrican production amount in chondrocytes treated with RhIL-1α for 24 h

[Table 8b]

Agrican production of a mixture of dextran 1 (D), dextran 5 (T) or poloxamer 188 (P) in chondrocytes treated with RhIL-1α for 24 h

As can be seen in Table 8a and Figure 9, dextran 1 (D) 5 to 25 (w / v)%, dextran 5 (T) 4 (w / v)%, poloxamer 188 (P) 2 It was confirmed that the amount of agrican production in the group treated with to 10 (w/v)% alone was significantly increased compared to the positive inflammation-inducing control (PC). On the other hand, dextran 1 (D) 50 (w / v)%, poloxamer 188 (P) 20 (w / v)%, dextran 5 (T) 20 to 40 (w / v)% alone administration group agrican It was confirmed that it did not induce an increase in production.

As can be seen in Table 8b and Figure 9, in the mixed administration group, dextran 1 (D) 5 (w/v)% and poloxamer 188 (P) 2 (w/v)%, or dextran 5 (T ) In the mixed administration group of 4 to 40 (w/v)%, the amount of agrican production was significantly increased compared to the positive control for inflammation (PC), and dextran 1 (D) 50 (w/v)% and poloxamer 188 (P) 2(w/v)% and only the mixed administration group increased the amount of agrican significantly. Even in the mixed administration group between 4 to 40 (w/v)% of dextran 5 (T) and 2 to 10 (w/v)% of poloxamer 188 (P), agrican production was lower than that of the positive control for inflammation (PC). It was confirmed that there was a significant increase. In particular, when dextran 1 (D) 5 (w/v)% and poloxamer 188 (P) 2 (w/v)% are administered in combination with dextran 5 (T) 4 to 40 (w/v)%, a single It was confirmed that the amount of agrican production significantly increased compared to the administration.

When considering the results of the synthesis of type 2 collagen confirmed in Example 5.1 above, in order to increase the synthesis of type 2 collagen and increase the production of agrican in the osteoarthritis model, dextran 1 (D) 5 to 25 (w/v) )%, dextran 5 (T) 4 (w/v)%, or poloxamer 188 (P) 2 (w/v)% alone, or dextran 1 (D) 5 (w/v)% Mixed administration with dextran 5 (T) 4 to 40 (w/v)%, or mixed administration with dextran 1 (D) 5 (w/v)% with poloxamer 188 (P) 2 (w/v)% It was confirmed that it is preferable to do so. Also dextran 5 (T) 4 to 40 (w/v)% is poloxamer 188 in combination with dextran 1 (D) 5 (w/v)%, especially in the case of dextran 5 40 (w/v)% (P) It was confirmed that the combination with 2 to 10 (w/v)% could significantly increase the type 2 collagen synthesis and the production of agrican.

Summarizing the above results, in the treatment of osteoarthritis, dextran 1, dextran 5 and poloxamer 188 can be used, but these alone or all combinations do not show a therapeutic effect on osteoarthritis, and dextran 1, dextran 5 and poloxamer 188 may have different toxicity and effects depending on the concentration combination, so it can be confirmed that it is important to derive an appropriate combination thereof.

Claims (8)

dextran and poloxamer having an average molecular weight of 800 to 1200 Da,
i) comprising the dextran at a concentration of 2.5 to 40 (w/v)% and the poloxamer at a concentration of 1 to 5 (w/v)%; or
ii) the dextran in a concentration of 45 to 55 (w/v)% and the poloxamer in a concentration of 1 to 2 (w/v)%; A pharmaceutical composition for the prevention or treatment of joint disease or connective tissue disease caused by at least one site selected from the group consisting of joint disease or cartilage, intervertebral disc, ligament, skin, bone and tendon.
According to claim 1, wherein the poloxamer is 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 234, poloxamer 235, poloxamer 237, poloxamer 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 Which is selected from the group consisting of, a pharmaceutical composition.
The pharmaceutical composition of claim 1, wherein the joint disease or connective tissue disease is traumatic, infectious, inflammatory, or degenerative.
According to claim 3, wherein the joint disease is osteoarthritis, degenerative arthritis, traumatic arthritis, rheumatoid arthritis, patellar-femoral syndrome or chronic inflammatory arthrosis, the pharmaceutical composition.
The pharmaceutical composition according to claim 3, wherein the joint disease or connective tissue disease is a degenerative disease.
It contains dextran 1 and poloxamer having an average molecular weight of 1000 Da as active ingredients,
i) containing the dextran 1 at a concentration of 2.5 to 40 (w/v)% and the poloxamer at a concentration of 1 to 5 (w/v)%; or
ii) the dextran 1 at a concentration of 45 to 55 (w/v)% and the poloxamer at a concentration of 1 to 2 (w/v)%; A pharmaceutical composition for the prevention or treatment of joint disease or connective tissue disease caused by at least one site selected from the group consisting of joint disease or cartilage, intervertebral disc, ligament, skin, bone and tendon.
7. The pharmaceutical composition of claim 6, wherein the joint disease or connective tissue disease is traumatic, infectious, inflammatory, or degenerative.
Stem Cells;
Claims 1 to 7, comprising the composition of any one of claims 1 to 7, joint disease, or cartilage, intervertebral disc, ligament, skin, bone, and a pharmaceutical for the prevention or treatment of connective tissue disease caused in one or more sites selected from the group consisting of tendons. enemy composition.

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