KR20200015671A - Compositions for treating joint or connective tissue disease comprising dextran or poloxamer - Google Patents

Abstract

The present invention relates to: a composition for treating joint diseases or connective tissue diseases, containing dextran, poloxamer or a mixture thereof; or a composition for treating joint diseases or connective tissue diseases further comprising stem cells in the composition. A pharmaceutical composition of the present invention enables, by means of buffering, coating or anti-inflammatory effects, impacts to be relieved, a damaged area to be specifically encompassed, and inflammation of an adhered area to be reduced while staying for a long time in the damaged area of joints or connective tissues, thereby being able to be usefully used for treating joint diseases and connective tissue diseases.

Description

Compositions for treating joint disease or connective tissue disease comprising dextran or poloxamer

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

Joints are areas where bones meet, and are composed of cartilage, articular capsule, synovial membrane, ligaments, tendons, and muscles to smoothly move between bones and bones, and absorb shocks generated by movement. Arthritis is caused by various causes of dysfunction, including inflammation in the joints, these joint diseases are thought to be caused by autoimmune chronic joint rheumatism, infectious arthritis caused by bacterial infection, joint cartilage or Osteoarthritis, which causes degeneration or destruction of bone, and crystalline arthritis, in which soluble metabolites are deposited as crystals in connective tissue around joints, can be broadly classified.

Connective tissue, also called connective tissue, is a tissue that connects tissues in an animal to form organs, for example, an intervertebral disc, skin, tendons, ligaments, and the like.

Drugs used to treat arthritis can be roughly based on the main mechanism of action: decreased inflammation, delayed disease progression, and decreased metabolic acid concentrations, such as uric acid. Many arthritis drugs reduce inflammation. Inflammation is a pathological process that causes pain, swelling, heat, redness, and stiffness. Drugs that relieve inflammation rapidly include nonsteroidal anti-inflammatory drugs, including aspirin, and steroidal anti-inflammatory drugs, including cortisone. However, these anti-inflammatory and anti-inflammatory drugs are the main action to relieve pain rather than the treatment of the disease and many complications occur when taking the drug for a long time. In particular, steroidal anti-inflammatory drugs are irrelevant to the causative agent of the disease and may lead to overuse of the joint simply by temporarily reducing pain due to strong and temporary anti-inflammatory action, which can destroy joints and worsen the disorder. Therefore, use caution.

Therefore, conventional therapies used for joint damage such as arthritis have limited efficacy, have obvious toxic side effects, and their effectiveness is limited because they cannot be used continuously for a long time, and thus new novel therapies that overcome the disadvantages of existing therapies. Or treatment is urgently needed

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

There is a need for new therapies or therapeutic agents due to the limited effectiveness, obvious toxic side effects, and lack of persistence of the current therapies for joint or connective tissue diseases, especially dextran that does not cause allergic reactions when administered to the body, or The study of the treatment of joint disease or connective tissue disease using poloxamers, especially low molecular weight dextran or poloxamer, is not known.

Accordingly, the present inventors have conducted research for the treatment of joints or connective tissues, and as a result, dextran, poloxamer, or a mixture thereof does not cause an allergic reaction, and due to buffering effect, preservation effect, or anti-inflammatory effect on joint and connective tissue damages. 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 mixtures thereof.

An object of the present invention is a stem cell; It is to provide a pharmaceutical composition for the prevention or treatment of joint diseases or connective tissue diseases, 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, including dextran, poloxamer or a mixture thereof.

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

The present invention also provides a pharmaceutical composition for the prevention or treatment of joint disease or connective tissue disease, including a mixture of two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188. do.

The present invention also stem cells; It provides a pharmaceutical composition for the prevention or treatment of joint disease or connective tissue disease, including a mixture of two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188.

Since the pharmaceutical composition of the present invention can long-lasting damage to joints or connective tissues by buffering effect, coating effect, or anti-inflammatory effect, alleviates impact, specifically wraps damaged areas or reduces inflammation of adhered areas, 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 (* / ** / *** are respectively) P <0.05 / p <0.01 / p <0.001 relative to the negative control (nc)).
Figure 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 (* / ** / *** are respectively) P <0.05 / p <0.01 / p <0.001 relative to the negative control (nc)).
3 is a diagram showing the results of confirming the cell proliferation capacity of chondrocytes 24 hours after treatment with dextran, poloxamer or a mixture thereof for chondrocytes of osteoarthritis in vitro model according to rhIL-1α treatment ( * / ** / *** indicate the significance of p <0.05 / p <0.01 / p <0.001 relative to the pro-inflammatory 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 of the osteoarthritis in vitro model according to rhIL-1α treatment ( * / ** / *** indicate the significance of p <0.05 / p <0.01 / p <0.001 relative to the pro-inflammatory positive control (pc), respectively).
5 is a diagram showing the results of confirming the expression level of MMP-3 gene by dextran, poloxamer or mixtures thereof for chondrocytes of osteoarthritis in vitro model according to rhIL-1α treatment. (* / ** / *** represents the significance of p <0.05 / p <0.01 / p <0.001 compared to the pro-inflammatory positive control (pc), respectively).
6 is a diagram showing the results of confirming the expression level of MMP-13 gene by the treatment of dextran (poloxamer) or a mixture thereof for chondrocytes in osteoarthritis in vitro model according to rhIL-1α treatment. (* / ** / *** indicates the significance of p <0.05 / p <0.01 / p <0.001 relative to the pro-inflammatory positive control (pc), respectively).
Figure 7 shows the results of confirming the IL-10 / IL-6 gene expression ratio according to the treatment of dextran, poloxamer or mixtures of chondrocytes in the osteoarthritis in vitro model according to rhIL-1α treatment (* / ** / *** shows the significance of p <0.05 / p <0.01 / p <0.001 relative to the pro-inflammatory positive control (pc), respectively).
8 is a diagram showing the results of confirming the type 2 collagen production amount according to the treatment of dextran, poloxamer or mixtures of chondrocytes in the osteoarthritis in vitro model according to rhIL-1α treatment (* / ** / *** indicate the significance of p <0.05 / p <0.01 / p <0.001 relative to the pro-inflammatory positive control (pc), respectively).
9 is a diagram showing the results of confirming the amount of aggrican production according to the treatment of dextran, poloxamer or mixtures of chondrocytes in the osteoarthritis in vitro model according to rhIL-1α treatment (* / ** / *** indicate the significance of p <0.05 / p <0.01 / p <0.001 relative to the pro-inflammatory 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, including 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 starch or glycogen, and D-glucose leads to a straight chain shape with α-1,6 bonds, and α-1,4 bonds are branched in several places. Dextran can be used as a blood extender and blood coagulation inhibitor, and may be used by varying the molecular weight of dextran according to the purpose. In the present invention, the dextran may have a molecular weight of 500 to 10,000 Da, preferably 500 to 8,000 Da, more preferably 1,000 to 5,000 Da. Dextran is used according to the molecular weight, such as 'dextran 1' when the molecular weight is 1,000 Da, 'dextran 5' when the 5,000 Da, and 'dextran 10' when the molecular weight is 10,000 Da.

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

In addition, the low molecular weight dextran according to the present invention is not allergic than the dextran of 8,000 Da or more, as well as the onset of the operating time is fast, it has a feature that can be spread over a wide range.

The present invention provides examples using Dextran 1 and Dextran 5 as preferred examples. These may be used in combination with Dextran 1 and Dextran 5 and may also be used in combination with Poloxamer 188.

In the present invention, the concentration of dextran is expressed as a percentage of mass (g) concentration (w / v) in 100 cc of solvent, such as 2 to 40 (w / v) when dextran 1 is included in the composition as a single active ingredient. It may be included at a concentration of)%, and when dextran 5 is included in the composition as a single active ingredient, dextran 5 may be included at 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, a mixture of dextran and poloxamer, the concentration of dextran may be different depending on the object of mixing. For example, when a mixture of dextran 5 and dextran 1 is included in the composition, the concentration of dextran 5 is 2-40 (w / v)%, and the concentration of dextran 1 is 2.5-40 (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, dextran has been generally used at a concentration of 5% or less because of the disadvantage of making it difficult to form a property due to excessive increase in viscosity due to the water-containing properties of polysaccharides. However, since dextran is included in the composition of the present invention at a concentration of at least 2 to at most 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 such dextran solubility, the composition of the present invention may further include an additive, and the additive may include sugars including sorbitol, xylitol, erythritol, lactitol, mannitol and maltitol, and mixtures thereof. Alcohols, or erythrose, erythrulose, threose, arabinose, ribose, ribulose, wood, xylulose , Lyxose, allose, altrose, psicose, galactose, gulose, idose, mannose, sorbo Monosaccharides comprising sorbose, talose, tagatose, sedoheptulose, mannoheptulose and mixtures thereof, or _divalent including CaCl ₂ Cations may be included.

In the present invention, "poloxamer" is a nonionic triblock copolymer in which hydrophilic ethylene oxide is bonded to both ends of hydrophobic propylene oxide, and has a characteristic of temperature sensitivity. The conversion of sol and gel is possible, and the properties of poloxamer vary depending on the ratio of polyoxypropylene and polyoxyethylene Poloxamer is used as an emulsifier, stabilizer, and dissolution aid in addition to surfactants.

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

The poloxamer 101, poloxamer 105, poloxamer 105, poloxamer 105 benzoate, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 182 dibenzoate, polox Sammer 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, but not limited to, poloxamer 188.

In the present invention, the concentration of the poloxamer is a percentage of the mass (g) concentration (w / v) in 100 cc of solvent, the concentration of 1 to 50 (w / v)%, the concentration of 2 to 30 (w / v)% It may be characterized in that it is 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 be.

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, divalent cations, and sterile water or physiological saline may be included in the composition without additional additives. It may be.

In the present invention, the pharmaceutical composition may include a mixture of dextran and poloxamer. In particular, when poloxamer 188 is used in combination 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 poloxamer may be 1 to 5 (w / v)%, or 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 be toxic when included in the composition at high concentrations, such as greater than 25 (w / v)%, and may be used at appropriate concentrations 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, poloxamer 188 1 (w / v)% and Reflects a ratio of 1:40 when mixing dextran 1 40 (w / v)% and 1: 0.1 when mixing 20 (w / v)% of poloxamer 188 and 2 (w / v)% of dextran 5 1: 0.1 to 1:40 may be included. 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 include poloxamer 188, which is 8,500 Da. In addition, when an embodiment of the present invention includes two mixtures selected from the group consisting of dextran 1, dextran 5 and poloxamer 188, each component is mixed in a volume ratio of 1: 1 to the composition May be included.

Also in one embodiment of the present invention, the pharmaceutical composition of the present invention comprises a mixture of dextran 1 and dextran 5, wherein dextran 1: dextran 5 in the pharmaceutical composition is for example (w / v)% As ratio 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, dextran 1 2.5 (w / v)% and Reflects a ratio of 1:16 when mixing dextran 5 40 (w / v)% and 1: 0.05 when mixing dextran 1 40 (w / v)% and dextran 5 2 (w / v)% And 1: 0.05 to 1:16.

When the composition of the present invention comprises a mixture of dextran and poloxamer, the hydrophobic component of the poloxamer may enhance the solubility of dextran or the concomitant drug, and the polyethylene glycol (PEG), a hydrophilic component of poloxamer, preserves the cell membrane. It can preserve the tissue by preventing adhesion. However, poloxamers can be easily diluted by body fluids, and can be easily absorbed by body fluids. Thus, poloxamers can increase stability in the body by mixing with dextran. Therefore, in the case of the mixture of dextran and poloxamer, the viscosity remains longer than that of dextran alone or poloxamer alone to cover the damaged joint or connective tissue area, and prevents the pathological adhesion of the tissues, Reducing inflammatory findings facilitates the supply of nutrients and promotes tissue regeneration. The effect can be induced by the cushioning effect or the coating or anti-inflammatory effect of the dextran and poloxamer mixtures.

In the present invention, the "cushion effect" means an effect that acts as a cushion to cushion the impact applied by reducing friction in the administered area, for example, the joint, and the "coating effect". ”Means the effect of allowing the knee joint to move comfortably by covering and covering the damaged cartilage tissue in a stiff area, such as a joint, at high speed. These effects can reduce the inflammatory response of the damaged joint site.

Therefore, the composition of the present invention, which induces a buffering effect and a coating effect or an anti-inflammatory effect, is suitable for improving regeneration or damaged tissue, and thus can be effectively used for the treatment of joint disease or connective tissue disease. Such effects of the composition of the present invention is an effect similar to the stem cell therapeutics currently used for the treatment of joint diseases, and may be competitive with stem cell therapeutics requiring high technology and cost.

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

The term "stem cell" in the present invention, refers to a cell having the ability to differentiate into two or more cells while having a self-replicating ability, totipotent stem cells, pluripotent stem cells, It can be classified as a multipotent stem cell.

Stem cells of the invention may be derived from any type of animal, including humans and non-human mammals, and are not limited to whether the stem cells are derived from adults or embryos.

Stem cells of the present invention include embryonic stem cells or adult stem cells, the adult stem cells are 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 adipose tissue cells), muscles, nerves, skin, amniotic membranes 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 cultures or concentrates thereof, culture secretions of stem cells or concentrates thereof, or combinations thereof.

Dextran or poloxamer in the composition may serve to specifically transport stem cells to damaged lesions of joints or connective tissues, and also to induce regeneration of damaged tissues by inducing stem cells to settled lesions. It has a synergistic effect.

Therefore, when the composition of the present invention includes stem cells, it is possible to more effectively treat joint disease or connective tissue disease 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 a joint disease or connective tissue disease by administration of a composition.

In the present invention, "treatment" refers to any action that improves or advantageously 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 in a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field. In addition, the formulation may be pharmaceutically, in combination with a pharmaceutically acceptable carrier or medium, such as sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, excipients, vehicles, preservatives, binders, and the like. It may be formulated by blending in a recognized 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 dissolved in a solvent immediately before administration, but is not limited thereto. It may be manufactured in the most suitable manner in consideration of the situation.

The dosage form of the pharmaceutical composition is not particularly limited, and may be 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 tablets, pills, capsules, gels, syrups, etc. together with a pharmaceutically acceptable carrier and excipient. However, solid preparations such as tablets and powders often take a long time to be absorbed, so oral administration by liquid preparation or the like 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, buffers, chelating agents and the like.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin and soft capsules made of plasticizers such as gelatin and glycerol or sorbitol. Push-fit capsules may include the active ingredient and fillers such as lactose, binders such as starch, lubricants such as talc or magnesium stearate or optionally stabilizers. Soft capsules may be dissolved or suspended in an appropriate liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.

The drug can also be administered in a target drug transport system, such as liposomes coated with specific antibodies that target joint disease or connective tissue. Liposomes can be selectively taken in diseased tissue.

When the pharmaceutical composition of the present invention is administered as a topical formulation, the subject's joint cavity, connective tissue, intravenous, subcutaneous, intradermal, intraarticular, or the like using a suitable buffer, isotonic agent, and the like dissolved in sterile distilled water. It can be injected directly into the muscle, or applied to the skin or in the form of a patch.

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

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

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

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

The pharmaceutical compositions may be stabilizers such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, calcium silicate, polyvinylpyrrolidone and It may further comprise cellulose. The composition may be a lubricant such as magnesium stearate and mineral oil; Wetting agents; Emulsifying and suspending agents; Preservatives such as thimerosal, benzyl alcohol, parabens, methyl- or propylhydroxybenzoate; Antioxidants such as ascorbic acid, dihydroquinone, butylated hydroxytoluene and dithiothritol; And buffers such as sodium monophosphate, dibasic sodium phosphate, sodium benzoate, potassium benzoate, sodium citrate, sodium acetate and sodium tartrate; Chelating agents such as ethylenediaminetetraacetic acid; And isotonicity modifiers such as sodium chloride or dextrose.

The pharmaceutical composition may further comprise an anesthetic. The anesthetics include, but are not limited to, amethokine (tetracaine), lignocaine (lidocaine), xysilocaine, bupivacaine, prilocaine, lopivacaine, benzocaine, mepivocaine, cocaine and Combinations thereof.

The pharmaceutical composition may further comprise an antimicrobial agent. The antimicrobial agents include, but are not limited to, amanpadin hydrochloride, amanpadin sulfate, amikacin, amikacin sulfate, amoglycoside, amoxicillin, ampicillin, amsamycin, vacitracin, beta-lactam, candicidine , Capreomycin, carbenicillin, cephalexin, cephaolidine, cephalotin, cefazoline, cefapirine, cepradine, cephaloglycine, chilomfenicol, chlorhexidine, clohexidine gluconate, chlorhexidine hydrochloride , Chloroxine, Chlorcuraldol, Chlortetracycline, Chlortetracycline Hydrochloride, Ciprofloxacin, Circurin, Clindamycin, Clindamycin Hydrochloride, Clotrimazole, Cloxacillin, Demeclocycline, Diclosacillin, Diiodohydro Loxyquine, doxycycline, ethambutol, ethambutol hydrochloride, e Lithomycin, erythromycin estoleate, ermycin stearate, farnesol, floxacillin, gentamicin, gentamycin sulfate, gramicidine, gseopulbin, haloprogin, haloquinol, hexachlorophene, iminocycline , Iodochlorhydroxyquine, kanamycin, kanamycin sulfate, lincomycin, linenemycin, lineomycin hydrochloride, macrolide, meclocycline, metacycline, metacycline hydrochloride, methenin, methenamin hypofurlate, Methenamin Mandelate, Methicillin, Metonidazole, Myconazole, Myconazole Hydrochloride, Minocycline, Minocycline Hydrochloride, Murphyrosine, Napcillin, Neomycin, Neomycin Sulfate, Nethymycin, Netylmycin Sulfate, Nitropurazone , Norfloxacin, nystatin, octopyrox, oleandomycin, Orcephalosporins, oxacillins, oxytheaclines, oxytetracycline hydrochloride, parachlorometha xylenol, paromomycin, paromomycin sulfate, penicillin, penicillin G, penicillin V, pentamidine, pentamine hydrochloride, pefemidine Neticillin, polymyxin, quinolone, streptomycin sulfate, tetracycline, tobramycin, tolnaftate, triclosan, trifampin, rifamycin, lolitetracycline, silver salt, spectinomycin, spiramycin, strutomycin, Sulfonamides, tetracyclines, tetracyclines, tobramycin, tobramycin sulfates, triclocarbons, triclosan, trimethoprim-sulfamethoxazole, tyrosine, vancomycin and irotricin.

The pharmaceutical composition may further comprise an anti-inflammatory agent. The anti-inflammatory agent may be nonsteroidal, steroidal or a combination thereof. Non-limiting examples of nonsteroidal anti-inflammatory agents include oxycams, such as pyroxicam, isoxicam, tenoxycam, sudoxicam; Salicylates such as aspirin, dissalside, benolylate, trilysate, sapapurin, sorbulin, diflunisal and pendosal; Acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isosepak, furofenac, thiopinac, zidomethacin, acematasin, pentiazac, jomepilac, clindac , Oxepinac, felbinac and ketorolac; Phenamate, such as mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid and tolfenamic acid; Propionic acid derivatives such as ibuprofen, naproxen, benoxapropene, flurbiprofen, ketoprofen, phenopropene, fenbufen, indopropene, pyrpropene, carpropene, oxaprozin, Pranopropene, miroprofen, thioxapropene, supropene, aminopropene and thiapropenic; Pyrazoles such as phenylbutazone, oxyphenbutazone, peprazone, azapropazone and trimetazone. Extracts of these nonsteroidal 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, desonini De, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone diacetate, diflucortolone valerate, fluadenolone, fluchlorone acetonide, fludrocortisone, flumeta Son pivalate, Fluorcinolone acetonide, Fluorinoneide, Flucortin butyl ester, Fluorocortolone, Fluprednide (flupredenylidene) acetate, Flulandrenone, Halcinolone, Hydrocortisone acetate, Hydrocortisone Butyrate, Methylprodnisolone, Triamcinolone Acetonide, Khor Tyson, cortodoxone, flucetonide, fludrocortisone, difluoroson diacetate, fluladrenolone, fludrocortisone, difluroson diacetate, fluladrenolone acetonide, medrison, amcinofel, implied Balance of napids, betamethasone and esters thereof, chloroprednisone, chlorprednisone acetate, clocortelone, clecinolone, dichlorisone, diflurednate, fluchloronide, flunisolide, fluorometallon, Fluperololone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednison, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone and extracts thereof do.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease, the effective dose level being the severity of the disease, the age, weight, health, sex, sensitivity of the patient to the drug, the time of administration, the route of administration And factors including the rate of release, duration of treatment, drugs used in combination or coincidental with the compositions of the invention used, and other factors well known in the medical arts. The dosage of dextran, poloxamer, or mixtures thereof varies over a wide range and depends on 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 compositions of the present invention may be administered once or in several divided doses at preferred dosage intervals, such as weekly intervals.

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 composed of the compounds of the present invention formulated in a pharmaceutically acceptable carrier are suitably prepared and placed in suitable containers and indicated for the treatment of the indicated conditions. Appropriate indications indicated in the levels include treatment of joint disease and treatment of connective tissue disease.

According to the present invention, a pharmaceutical composition comprising dextran, poloxamer or mixtures thereof induces long-term pain relief in an individual suffering from joint disease or connective tissue disease. The use of the pharmaceutical compositions according to the 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 the use of too invasive techniques.

In the present invention, "connective tissue" is also known as connective tissue or connective tissue, and refers to a tissue widely distributed in animal tissue and serving to bind, protect, and fill cells, organs, organs, and the like, and is fibrous It is classified as connective tissue, gelatinous tissue, and reticulum tissue, but fibrous connective tissue occupies most of them.

In the present invention, the causes of the occurrence of "joint disease" or "connective tissue disease" can be broadly 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 all causes of damage to the joints, but the joint diseases in which the compositions of the present invention are expected to be used effectively are osteoarthritis, preferably repetitive frictional force. And 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 the case of loss of collagen or water due to degenerative changes due to physical damage or repeated friction of the intervertebral disc, ligament, tendon, skin, and tendon. Disease.

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

One embodiment of the present invention relates to a pharmaceutical composition for the prevention or treatment of 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 in the composition as a single active ingredient dextran 1, the concentration of dextran 1 may be 2 to 40 (w / v)%.

The dextran is included as a single active ingredient in the composition with dextran 5, 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, the concentration of poloxamer may be 1 to 15 (w / v)%.

In the present invention, two species 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 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 Can be)%.

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

In addition, the mixture is a mixture of dextran 1 and poloxamer, 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)%.

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

In addition, one example of the present invention is a stem cell; It relates to a pharmaceutical composition for the prevention or treatment of joint disease or connective tissue disease, including a mixture of two selected from the group consisting of dextran 1, dextran 5 and poloxamer 188.

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

Materials and methods

In the present invention, the experiment is performed using Dextran 1 (EP grade, Pharmacosmos), Dextran 5 (Pharma quality, Pharmacosmos), Poloxamer 188 (Poloxamer 188, cell culture grade, Sigma-Aldrich) In addition, rhIL-1α (recombinant human IL-1α, Lot No. 200-01A) was purchased from PEPROTECH to induce arthritis, and indomethacin (Sigma-Aldrich, Lot No. 53) was used as a positive control. -86-1), sodium hyaluronate (25 mg / 2.5 mL of sodium hyaluronate, Aragan, Dongkwang Pharmaceutical, Specialty Medicine) were used.

Example  1. Preparation and Experimental Preparation of Dextran, Poloxamer or Mixtures thereof

1.1 Isolation Culture of Cells

The hind limb joints of three-week-old male SD rats were isolated under sterile conditions, and then only the chondrocytes constituting the joints were collected and single-celled to separate primary rat chondrocytes. When the primary cultured cells were stable, they were used for the test, and at least six animals were isolated at the time of the primary culture. In order to specify the stabilized chondrocytes, the expression level of chondrocyte markers, type 2 collagen or SOX9 gene, after RNA isolation was confirmed using real-time RT-PCR, and then used for testing.

1.2 Cell Culture Methods

Cells isolated at 1.1 were cultured in an incubator set at a temperature of 37 ° C., a humidity of 95%, and a CO _{2 of} 5%. 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. After observing the number of cells during the incubation period, the cells were separated with Detached solution (0.25 (w / v)% Trypsin, 0.53 mM EDTA solution (3 mL)) and centrifuged at 125 X g for 10 minutes. Was separated and used in the following experiment.

1.3 Test group  Configuration and manufacturing

The test group consisted of rhIL-1α 5 ng / mL in which osteoarthritis was induced by treatment with normal control (NC), pro-inflammatory control (PC), and test substance (single or mixture) concentrations. Shin (IM) or sodium hyaluronate (HN) treatment groups consisted of a single concentration. Test groups are shown in Table 1 below.

TABLE 1

 D = dextran 1, T = dextran 5, P = poloxamer 188, NC = normal group, PC = inflammatory mediator (rhIL-1α) administration 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.

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

The test substance used in the present invention was prepared using the following method: Dextran 1 (D) was 1-50 (w / v)%, Dextran 5 (T) reflecting the maximum concentration dissolved in the cell culture. Was prepared in the range of 1 to 40 (w / v)% and poloxamer 188 (P) in the range of 1 to 35 (w / v)%. In the case of preparing a mixture, two kinds of single materials were prepared by mixing in a volume ratio of 1: 1 (v: v), respectively.

Dextran 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 1, 5 and Poloxamer 188 were completely dissolved to the dose concentrations (w / v), respectively. In order to completely dissolve and homogenize the test substance in complete media, at low temperatures, especially cold for Poloxamer 188, stir slowly with a sterile spatula, a stick or a magnetic bar, or shake several times briefly to avoid foaming. It was dissolved until it became completely transparent without floating matter. After the test material was completely dissolved, the resultant was filtered through a 0.22 μm pore size syringe filter, sealed, and refrigerated.

More specifically, monolithic preparation was carried out in the following manner.

Dextran 1 of  5% mono ('D5') manufacturing

When preparing 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. Thus, the test material dissolved at a concentration of 0.05 g / mL was filtered through a 0.22 μm pore size syringe filter was used after refrigerated storage.

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

D5 and P10 were prepared, respectively, filtered and stored in a 0.22 μm pore size syringe filter, and refrigerated, and then mixed in a ratio of 1: 1 (v: v), respectively, and used after shaking to prevent foaming at a low speed.

Comparative example : Indomethacin  Treatment group ( IM ) Produce

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

Comparative example Sodium hyaluronate treatment group ( HN ) Produce

Sodium hyaluronate is inhaled with complete media in araganese (25 mg / 2.5 mL of sodium hyaluronate, in the form of a sterile disposable syringe), and sodium hyaluronate 25 mg / 3 mL (= sodium hyaluronate 8.33 mg / mL) Diluted with and used immediately.

1.4 Set Dosing Method

In the case of ultra-cultured chondrocytes isolated from rats, the doubling time was about 24 hours, and the test material was treated when the growth and stabilization were appropriate. After culturing chondrocytes in 48 wells or 24 well plates, each test substance is administered once, but 20% (v / v) of the total culture volume by concentration of each test substance 1 hour before rhIL-1α treatment for arthritis induction. Test solution was administered to After 24 hours of rhIL-1α treatment, the culture medium was collected and analyzed. However, when confirming the toxicity or proliferation of the test substance to chondrocytes, only the test substance was treated without rhIL-1α.

1.5 Statistical Analysis

All experimental results were compared with normal control group (NC) or pro-inflammatory control group (PC) and student T-test at p <0.05 level.

Example  2. Cytotoxicity Assay

The cytotoxicity analysis was performed by checking whether the proliferation ability of chondrocytes changed according to the treatment of the test substance. Cell proliferation was evaluated in both untreated chondrocytes not treated with rhIL-1α and chondrocytes induced with inflammation by treatment with rhIL-1α, and cultured for 24 hours and 48 hours after treatment with rat chondrocytes. MTT analysis (Thiazolyl Blue Tetrazolium Blue; Sigma, M5655) was performed. In the evaluation of cell proliferation of test substance in rhIL-1α-induced arthritis in-vitro model, each test substance was treated with rat chondrocytes 1 hour before rhIL-1α treatment and 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 FIG. 1, the cell proliferation rate of dextran 1, dextran 5, poloxamer 188, or mixtures thereof in rat knee joint chondrocytes that did not cause inflammation was observed for 24 hours. As a result, most dextran 1, dex Tran 5, Poloxamer 188, and mixtures thereof were found to aid cell proliferation without being toxic to cells. However, in the poloxamer 188 treatment group of the single treatment group, the reduction of some cell proliferation ability was observed in 24 hours culture at concentrations of 25 (w / v)% (P25) and 35 (w / v)% (P35) of poloxamer 188. It was lower than the drug (commercial drug) sodium hyaluronate (HN) administration group and was not suitable for single administration. In addition, the mixture showed a decrease in some cell proliferation ability in the culture for 24 hours, thereby confirming the possibility that poloxamer 188 may cause cytotoxicity when treated at high concentration. In the mixture, the decrease in cell proliferation was lower in the D50 + P35, P5 + T40, P25 + T40, and P35 + T40 experimental groups than the control (sodium hyaluronate) (HN) administration group.

As shown in Figure 2, the results of 48 hours of cell proliferation of dextran, poloxamer or a mixture thereof in rat knee joint chondrocytes that did not cause inflammation were confirmed to be similar to the culture of 24 hours.

These results indicate that, when treated with normal knee joint chondrocytes, chondrocyte proliferation was increased by increasing the concentration of dextran 1 (D) by 50% or by increasing the concentration of dextran 5 (T) by 40%. While not inhibited compared to commercial drugs, it was confirmed that poloxamer 188 may be toxic to chondrocytes at a concentration of 25% or more. In addition, although the cell proliferation ability is partially improved compared to the poloxamer 188 alone, the poloxamer 188 is preferably mixed at a concentration of 10 (w / v)% or less in order not to cause toxicity.

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

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

In Figure 4 it was confirmed that the same results as 24 hours culture, the cell proliferation of the P10 treatment group was confirmed to increase by mixing with dextran 1 or dextran 5.

  Combining the above results, it was confirmed that dextran 1 and dextran 5 do not induce most cytotoxicity in normal and inflammatory cells. On the other hand, as the concentration of poloxamer 188 increases, the treatment with a single substance can induce cell proliferation inhibition in both normal and inflammatory cells, but poloxamer 188 is increased to 1 to 10 (w / v)%. When administered in combination with dextran 1 or dextran 5, it was confirmed that it can be used without cytotoxicity.

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

Experiments were conducted to confirm the effects of osteoarthritis treatment by gene expression of markers for osteoarthritis evaluation. Since MMP-3 and MMP-13 are proteins that destroy cartilage substrates, it can be evaluated that the cartilage protection effect is excellent if it can effectively suppress the increase in the amount of gene expression that can be induced through inflammatory stimuli.

Dextran 1 changes from 2.5 to 50 (w / v)%, Dextran 5 from 2 to 40 (w / v)%, Poloxamer 188 from 1 to 20 (w / v)% Was performed and the expression changes of MMP-3 and MMP-13 were confirmed. Specifically, each test substance was treated with rat chondrocytes 1 hour before rhIL-1α treatment and cultured for 24 hours. After removing the supernatant of the cultured cells, the cells were washed with phosphate buffer (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). Diluted to the same concentration for each experimental group. Equally diluted RNA was synthesized by cDNA using PCR (ReverTra AceR qPCR RT Master Mix with gDNA Remover, Toyobo, FSQ-301). Using the synthesized cDNA, the expression of MMP-3 and MMP-13 in 96 well plates was confirmed by real time RT-PCR (n = 3).

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

TABLE 2

After mixing the primer, SyBr green (SYBR Premix Ex Taq, Takara, RR420A) and the template, real-time RT- by intercalating method using real-time RT-PCR (CFX 96 touch, Bio rad, Hercules, CA, USA) 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 which is a housekeeping gene.

3.1 MMP -3 Confirmation of expression change

 Dextran 1, Dextran 5, Poloxamer 188 The results of expression of MMP-3 following single or mixtures thereof are shown in Tables 3A-3B and FIG. 5.

TABLE 3a

MMP-3 Gene Expression of Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) Mononuclear Cells in 24h-treated RhIL-1α

TABLE 3b

MMP-3 Gene Expression in Dextran 1 (D), Dextran 5 (T), or Poloxamer 188 (P) Mixture in Chondrocytes Treated with RhIL-1α for 24h

As shown in Table 3a and Figure 5, Dextran 1 (D) showed a significant MMP-3 reduction effect compared to the inflammation-induced positive control at 2.5 to 40 (w / v)%, Dextran 5 (T) Showed a significant MMP-3 reduction effect from 2 to 20 (w / v)%. On the other hand, poloxamer 188 (P) showed a decreasing effect in 1 to 20 (w / v)% of all soldiers tested.

As shown in Table 3b and FIG. 5, in the mixture experimental group, dextran 1 (D) 5 (w / v)%, poloxamer 188 (P) 2 to 10 (w / v)%, dextran (T) 4% and 40 (w / v)% The significant effect was confirmed in all experimental groups, but in the group treated with dextran 1 (D) at a high concentration up to 50 (w / v)%, only a significant reduction effect was observed in mixing with P2. Therefore, when dextran 1 was mixed with dextran 5 or poloxamer 188, it was confirmed that mixing the concentration below 50 (w / v)% is preferable. Poloxamer 188 showed most significant effects of MMP-3 reduction in the single treatment and mixed treatment groups, but as confirmed in Example 2, the concentration of poloxamer is preferably 10 (w / v)% or less. It was confirmed that the corresponding P2, P5, P10 can induce significant MMP-3 inhibitory effect even in combination with dextran 1 and dextran 5.

3.2 MMP -13 Confirmation of expression change

Dextran 1, Dextran 5, Poloxamer 188 The results of expression of MMP-13 according to single or mixtures thereof are shown in Tables 4A to 4B and FIG. 6.

TABLE 4a

MMP-13 Gene Expression of Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) Mononuclear Cells in 24h-treated RhIL-1α

 TABLE 4b

MMP-13 Gene Expression in Dextran 1 (D), Dextran 5 (T), or Poloxamer 188 (P) Mixtures in Chondrocytes Treated with RhIL-1α for 24h

As can be seen in Table 4a to Table 4b, Figure 6, dextran 1 (D) in the single body experimental group showed a significant MMP-13 gene increase inhibitory effect at 2.5 to 25 (w / v)%, D40 Although 40 (w / v)% of phosphorus was not significant, the increase of MMP-13 gene was suppressed, whereas 50 (w / v)% of D50 increased MMP-13 level. This property is similar in the mixture of dextran 1 (D). Therefore, dextran 1 (D) was confirmed that the most preferred concentration range of 2.5 to 40 (w / v)%, which is also consistent with the results of MMP-3 gene expression. Dextran 5 (T) showed significant inhibitory effect on MMP-13 gene increase from 2 to 40 (w / v)% when administered alone, and Dextran 5 (T) 4 and 40 (w / v)% were dex. Mixing with Tran 1 (D) 5 (w / v)% and Poloxamer 188 (P) 2 and 10 (w / v)% also showed a significant MMP-13 gene growth inhibitory effect.

In the case of poloxamer 188, MMP-13 gene growth was suppressed at 5 to 20 (w / v)% in the experimental group alone, but 2 (w / v)% could also be effective when administered in combination with dextran. It confirmed that there was. In consideration of the cytotoxicity test results in Example 2, it was confirmed that the concentration of poloxamer 188 (P) is preferably 2 to 10 (w / v)% when mixed administration. The corresponding P2 and P10 induced significant MMP-13 gene growth inhibitory effect even in combination with Dextran 1 and Dextran 5 except for the combination of Dextran 1 (D) 50 (w / v)%.

Example  4.IL-10 / IL-6 Expression ratio  Anti-inflammatory effect

In the same manner as in Example 3, the anti-inflammatory effect of the experimental group was confirmed by confirming the IL-10 / IL-6 expression ratio when the rhIL-1α, which is an inflammation-inducing substance, and the test substance (single, mixture thereof) were treated together for 24 h. As the IL-10 / IL-6 expression ratio increases, it can be judged that the anti-inflammatory effect is excellent.

The primers used in the experiments are shown in Table 5 below, and the control group was used as a housekeeping gene, GAPDH. 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 by intercalating method using Real time RT-PCR (CFX 96 touch, Bio rad, Hercules, CA, USA) 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 which is a housekeeping gene.

TABLE 6a

 IL-10 / IL-6 Gene Expression Ratio of Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) Singles in Chondrocytes Treated with RhIL-1α 24h

TABLE 6b

IL-10 / IL-6 Gene Expression Ratio of Dextran 1 (D), Dextran 5 (T), or Poloxamer 188 (P) Mixture in RhIL-1α Treated Chondrocytes

As shown in Table 6a and FIG. 7, in the group treated with dextran 1 (D) alone, which is a single group treatment experiment, the IL-10 / IL-6 expression ratio except for the 50 (w / v)% treatment group. Although no significant increase was observed in the poloxamer 188 (P) 1 to 20 (w / v)% treatment group, the dextran 5 (T) 2 to 20 (w / v)% group showed a significant increase effect. .

As shown in Table 6b and FIG. 7, there was a significant increase in IL-10 / IL-6 expression ratio in all the mixture administration groups of dextran 1 (D), which was a mixture treatment experiment, and showed poloxamer 188 (P). And also in the mixed group with dextran 5 (T) showed a significant increase all confirmed that the anti-inflammatory effect in the mixture is excellent.

Considering that the increase in IL-10 / IL-6 expression ratio was not significant in the 50 (w / v)% administration of dextran 1, IL-10 / in the osteoarthritis model Increasing the IL-6 expression ratio, ie the anti-inflammatory effect, indicates that administration with poloxamer 188 (P) or dextran 5 (T) is preferred, rather than increasing 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 group administered alone, the mixture administration group was found to be remarkably superior, and thus the superiority of the dextran 1 (D), dextran 5 (T), and poloxamer 188 (P) mixed administrations was confirmed. have.

Example  5. Type II Collagen in Osteoarthritis Model and Agrican Increased production of Agrecan

In order to confirm the type 2 collagen and aglycan protein production in the indicator material for osteoarthritis evaluation, each test substance was treated with chondrocytes and cultured for 24 hours before rhIL-1α treatment. Since collagen type 2 and agrican are cartilage substrate compositions, it can be determined that the cartilage substrate protection effect is excellent when the amount of production thereof is increased.

 Here, the cell supernatant (culture solution) was recovered and centrifuged at 13,000 rpm for 10 minutes, and only the supernatant was used for analysis. The centrifuged cell supernatants were analyzed using manuals of Aggrecan ELISA Kit (Rat Aggrecan ELISA Kit, Mybiosource, MBS261073) and type II Collagen (Type II Collagen detection Kit, Multi-Species, Chondrex, 6018). . For accurate analysis, absorbance was measured at 450 nm and 490 nm using an ELISA reader (EPOCH 2 microplate reader, BioTek, Instruments, VT, USA) of the radio wave field, and then the content of type 2 collagen and aglycan according to the calibration curve (ng / mL) was quantified.

5.1 Formation of Type 2 Collagen

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

TABLE 7a

Type II Collagen Production of Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) Single Cells in 24h-treated RhIL-1α

TABLE 7b

Type II Collagen Production of Dextran 1 (D), Dextran 5 (T), or Poloxamer 188 (P) Mixture in RhIL-1α Treated Chondrocytes

As shown in Table 7a to Table 7b, and Figure 8, dextran 1 (D) 5 to 25 (w / v)%, dextran 5 (T) 4 to 40 (w / v)% alone in the inflammation group 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)% increased the type 2 collagen synthesis compared to the pro-inflammatory positive control group (PC) in both alone and in combination with dextran 5 (T) and poloxamer 188 (P). It was not induced. As confirmed in Example 3, dextran 1 (D) 50 (w / v)% alone did not show the effect of inhibiting the expression of MMP-3, MMP-13 increased only poloxamer 188 (P) 2 Dextran 1 (D) alone or mixed up to 40 (w / v)%, considering that it is possible to inhibit the increased expression of MMP-3, MMP-13 only in admixture with to 10 (w / v)% The results show that it is desirable to use. In particular, the type 2 collagen synthesis was significantly higher than the pro-inflammatory control group (PC) when mixed with 5% (dex) 1 (D) 5 (w / v) and 4% (40%) Dextran 5 (T). Increased.

For poloxamer 188 (P), no increase in type 2 collagen synthesis was observed in 10 (w / v)% and 20 (w / v)% treatment groups except 2 (w / v)% treatment group. As the concentration was increased, the type 2 collagen synthesis was further decreased. However, when poloxamer 188 (P) 2 or 10 (w / v)% is mixed with dextran 1 (D) 5 (w / v)% or dextran 5 (T) 40 (w / v)% Type 2 collagen synthesis increased significantly. These results indicate that dextran 1 (D) and dextran 5 (T) can be treated alone in the synthesis of type 2 collagen of osteoarthritis, but poloxamer 188 (P) alone except 2 (w / v)%. Treatment is not suitable and indicates that a mixed administration of poloxamer 188 (P) and dextran 1 (D) or dextran 5 (T) can effectively induce type 2 collagen synthesis.

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 the single administration. In summary, it was confirmed that it is preferable to administer a mixture of dextran 1, dextran 5 or poloxamer 188 in order to alleviate the inflammatory response in osteoarthritis and promote collagen synthesis.

5.2 Agrican  Confirmation of content quantification

Dextran 1, Dextran 5, Poloxamer 188 Agrican production results of single or mixtures thereof are shown in Tables 8a to 8b, and FIG. 9.

TABLE 8a

Agrican Production of Dextran 1 (D), Dextran 5 (T), and Poloxamer 188 (P) Monomers in Chondrocytes Treated with RhIL-1α for 24h

TABLE 8b

Agrican production of dextran 1 (D), dextran 5 (T), or poloxamer 188 (P) mixtures 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 aglycan production in the 10 to 10 (w / v)% alone treatment group was significantly increased compared to the pro-inflammatory control group (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 no increase in production was induced.

As can be seen in Table 8b and FIG. 9, among the mixed-administered groups, 5% (w / v) of dextran 1 (D) and 2% (w / v) of poloxamer 188 (P), or 5% of dextran 5 (T) ), 4 to 40 (w / v)% of aglycan production was significantly increased compared to pro-inflammatory control (PC), and dexrox 1 (D) 50 (w / v)% of poloxamer Agrican production was significantly increased only in the group administered with 188 (P) 2 (w / v)%. The amount of aglycan production in the mixed doses of dextran 5 (T) 4 to 40 (w / v) and poloxamer 188 (P) 2 to 10 (w / v)% compared to the pro-inflammatory control (PC) It was confirmed that the increase significantly. In particular, dextran 1 (D) 5 (w / v)% and poloxamer 188 (P) 2 (w / v)% are single when mixed with dextran 5 (T) 4 to 40 (w / v)%. It was confirmed that the amount of aglycan production increased significantly compared to the administration.

 Considering the results of type 2 collagen synthesis identified in Example 5.1 above, dextran 1 (D) 5 to 25 (w / v) in order to increase the type 2 collagen synthesis and increase the production of aglycans in the osteoarthritis model ), Dextran 5 (T) 4 (w / v)%, or poloxamer 188 (P) 2 (w / v)% alone, or dextran 1 (D) 5 (w / v)% Dextran 5 (T) 4-40 (w / v)% mixed dose or Dextran 1 (D) 5 (w / v)% mixed poloxamer 188 (P) 2 (w / v)% The case was confirmed to be preferable. Also dextran 5 (T) 4 to 40 (w / v)% is in combination with dextran 1 (D) 5 (w / v)%, especially for dextran 5 40 (w / v)% poloxamer 188 (P) It was confirmed that in combination with 2 to 10 (w / v)% can significantly increase the type 2 collagen synthesis and the production of agrican.

Taken together, the results of dextran 1, dextran 5, and poloxamer 188 may be used to treat osteoarthritis, but not all or a combination thereof exhibits a therapeutic effect on osteoarthritis, and dextran 1, dextran The combination of concentrations of 5 and poloxamer 188 may produce different toxicity and effects, indicating that it is important to derive the appropriate combination.

Claims (14)

Dextran (dextran), poloxamer (poloxamer) or a mixture thereof as an active ingredient, a pharmaceutical composition for the prevention or treatment of joint diseases or connective tissue diseases.
The composition of claim 1, wherein the dextran has a molecular weight of 500 to 10,000 Da.
The composition of claim 1, wherein the poloxamer has a molecular weight of 100 to 20,000 Da.
The composition of claim 1, wherein the dextran is included as a single active ingredient in the composition with dextran 1, and the concentration of dextran 1 is 2 to 40 (w / v)%.
The composition of claim 1, wherein the dextran is included as a single effective ingredient in the composition with dextran 5, and the concentration of dextran 5 is 2 to 30 (w / v)%.
The composition of claim 1, wherein the poloxamer is included as a single active ingredient in the composition, and the concentration of poloxamer is 1 to 15 (w / v)%.
The method of claim 1, wherein the mixture is a mixture of dextran 5 and poloxamer, the concentration of dextran 5 is 2 to 40 (w / v)%, and the concentration of the poloxamer is 1 to 20 (w / v). %.
The method of claim 1, wherein the mixture is a mixture of dextran 5 and dextran 1, wherein the concentration of dextran 5 is 2-40 (w / v)%, and the concentration of dextran 1 is 2.5-40 (w / v)%.
The method of claim 1, wherein the mixture is a mixture of dextran 1 and poloxamer, the concentration of dextran 1 is 2.5 to 40 (w / v)%, the concentration of the poloxamer is 1 to 5 (w / v) Composition, characterized in that%.
The method of claim 1, wherein the mixture is a mixture of dextran 1 and poloxamer, the concentration of dextran 1 is 45 to 55 (w / v)%, the concentration of the poloxamer is 1 to 2 (w / v) Composition, characterized in that%.
The composition of claim 1, wherein the concentration (w / v)% ratio of dextran: poloxamer in the composition is from 1: 0.01 to 1:40.
The composition of claim 8, wherein the concentration (w / v)% ratio of dextran 1 and dextran 5 in the composition is from 1: 0.01 to 1:20.
The poloxamer of claim 1, wherein the poloxamer 101 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, Polox Summer 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 The composition is selected from the group consisting of
Stem Cells;
A pharmaceutical composition for preventing or treating joint disease or connective tissue disease, including dextran, poloxamer, or a mixture thereof.

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