KR101425303B1 - Pharmaceutical composition comprising isoflavones for preventing and treating arthritis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating arthritis containing 2-deoxyglucose as an active ingredient, and moreover, the pharmaceutical composition can be effectively effective for suppressing COX-2 and PGE2 expression and thus being effective against inflammation.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing and treating arthritis comprising isoflavones,

The present invention relates to a pharmaceutical composition for the prevention and treatment of arthritis, and more particularly, to a pharmaceutical composition having less side effects when applied in vivo than existing synthetic pharmaceutical compositions and having excellent arthritis prevention and treatment effects.

Arthritis is a disease caused by inflammatory changes in the joints caused by various causes such as bacteria and trauma. The incidence of arthritis is estimated to be about 30% of the population over 60 years old. According to foreign statistics, It is known as a disease that is. In addition, recent research by the Korea Institute for Health and Social Affairs has reported that, in Korea, arthritis has the highest incidence of diabetes and hypertension after 50 years of age. Therefore, many researchers expect arthritis to arise as the most important disease that can interfere with the quality of life of mankind in the 21st century along with cancer and cardiovascular disease.

On the other hand, arthritis can be classified into non-inflammatory arthritis and inflammatory arthritis. Non-inflammatory arthritis is an osteoarthritis, and inflammatory arthritis is rheumatoid arthritis.

Treatment of these diseases includes surgical treatment such as pharmacotherapy of nonsteroidal anti-inflammatory drugs (NSAIDs), steroid anti-inflammatory drugs, immunomodulators, and physical therapy such as rehabilitation medicine and artificial joint surgery.

Such conventional treatment methods are generally more effective than those in which sufficient therapeutic effects can not be obtained, and in general, they are caused by long-term disorders, hypersensitivity or medication treatment, and other diseases (such as steroid osteoporosis or interstitial pneumonia) Adverse effects are recognized and cause pain to the patient. In addition, because the cause of the onset is unclear, fundamental treatment is difficult. Depending on the severity of the symptoms, there are many cases where a simple treatment is not performed by the use of a hood or a proper treatment method.

In such a situation, there is a need for a treatment method which can be easily used at home or in the field of medical treatment for the prevention and treatment of the occurrence or progression of these diseases, and which has no side effects and can be used for a long term (long term continuous use).

The purpose of treating arthritis is not only to reduce joint pain and inflammation, to prevent joint deformation, but also to fundamentally elucidate the mechanism of intracellular action that triggers arthritis and to control its mechanism. Treatment of degenerative arthritis, which is currently being used clinically, can be performed by using drugs (analgesics, steroids, nonsteroidal anti-inflammatory drugs) or cartilage protectants (hyaluronic acid, glucosamine, chondroitin, etc.) Tibial proximal osteotomy, joint replacement, and total knee arthroplasty). However, these methods are not directly related to the removal of underlying causes such as chondrocyte degeneration or control of MMP activity and synthesis. In other words, the drug treatment agent only has the effect of nonspecifically alleviating the pain or the inflammatory reaction itself, and the cartilage protecting agent only serves to protect the joints by nourishing the cartilage cells or mitigating the impact. In addition, these drugs should be used with caution because they exhibit side effects such as weight gain, hypertension induction, and peptic ulcer induction. Recently, it has been urgently required to develop a new therapeutic agent which has a relatively low side effect when applied in vivo and has an effect of alleviating symptoms of arthritis.

Patent Document 10-2011-0139292 (public date: December 28, 2011) [Patent Document 1] Japanese Patent Application Laid-Open No. 10-2008-0043763 (published on May 19, 2008)

The present invention provides a pharmaceutical composition for prevention and treatment of arthritis, which has a relatively low side effect and can be used for a long period of time in vivo, and has an effect of alleviating symptoms of arthritis.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating arthritis, which comprises 2-deoxyglucose and isoflavone.

According to a preferred embodiment of the present invention, the isoflavone is selected from the group consisting of soybean, chick pea, red clover, subterranean clover, ground pea, milk vetch, marama bean, sword bean, jack bean, seaside sword bean, caraobean, cluster bean, There are many different types of beans such as balu, hyacinth bean, grass pea, Indian vetch, garden pea, djenko bean, goa bean, beans, yam bean, broad bean, earth pea, lentil, jumping bean, alfalfa, velet bean, African locust bean ), Inga, cyprus vetch, yebnut, tallow tree, polynesian chestnut, kudze root (Puerari) a lobata, oil bean tree, mesquite, tamarind, fenugreek, Indian liquorice, ground nut, defatted soy flake (soy flake, soy flouver, and soy meal. The soy flour may be selected from the group consisting of flake, soy flouver and soy meal.

According to another preferred embodiment of the present invention, the isoflavone may be at least one selected from the group consisting of daidzein, genistein, glycitein, daidzin, genistin, glycitin and glycosides thereof.

According to another preferred embodiment of the present invention, the isoflavone may be genistein.

According to another preferred embodiment of the present invention, the pharmaceutical composition may contain 0.1 to 10 parts by weight of isoflavone based on 100 parts by weight of 2-deoxyglucose.

In addition, the pharmaceutical composition of the present invention can prevent and treat degenerative arthritis or rheumatoid arthritis.

The pharmaceutical composition of the present invention provides a pharmaceutical composition for prevention and treatment of arthritis, which has less side effects than conventional synthetic drug therapies and has an effect of alleviating symptoms of arthritis.

Fig. 1 shows the result of Western blot analysis as measured in Experimental Example 1, in which 5 mM 2-deoxyglucose and 2 μM SOV, 0.5 μM PAO or 40 μM genistein (GN) were treated.
FIG. 2 shows the results of Western blot analysis of 5 mM 2-deoxyglucose and / or 2 μM SOV or 40 μM genistein (GN) in Experimental Example 1.
Figure 3 is a result of quantifying the Western blot analysis of Figure 2 with an image J program.
FIG. 4 shows the result of Western blot analysis as measured in Experimental Example 1, in which 5 mM 2-deoxyglucose and 40 μM genistein (GN) or 10 μM PP2 were treated.
FIG. 5 shows the result of Western blot analysis as measured in Experimental Example 1, in which 5 mM 2-deoxyglucose and / or 1, 2, and 5 μM SOV were treated alone or in combination.
FIG. 6 shows results of Western blot analysis as measured in Experimental Example 1, in which 5 mM of 2-deoxyglucose and / or 0.2, 0.5, and 1 μM PAO were treated alone or in combination.
FIG. 7 shows the result of Western blot analysis as measured in Experimental Example 1, in which 5 mM of 2-deoxyglucose and / or 10, 20, and 40 μM genistein (GN) were treated alone or in combination.
FIG. 8 shows PGE2 test results of Example 1 and Comparative Examples 1 to 3 measured in Experimental Example 2. FIG.

Hereinafter, the present invention will be described in more detail.

As described above, conventional medicines have only an effect of nonspecifically relieving the pain or the inflammatory reaction itself, and the cartilage protecting agent only serves to protect joints by supplying nutrients to cartilage cells or mitigating impacts. In addition, these drugs should be cautious of choice because they also show side effects such as weight gain, hypertension induction, and peptic ulcer induction.

Therefore, in order to ultimately treat arthritis, it is necessary to elucidate the mechanism of decreasing the production of arthritic substance in chondrocytes, the signaling process of chondrocyte involved in causing inflammatory reaction, the regulation of gene expression, .

The present invention solves the above-mentioned problems by providing a pharmaceutical composition for the prevention and treatment of arthritis including 2-deoxyglucose and isoflavone. In addition, the pharmaceutical composition for prevention and treatment of joint name of the present invention can provide a pharmaceutical composition having less side effects than conventional synthetic drug treatment agents and excellent in the effect of alleviating symptoms of arthritis.

The present invention provides a pharmaceutical composition for preventing and / or treating arthritis comprising 2-deoxyglucose and isoflavone.

The content of 2-deacylated glucose and isoflavone contained in the pharmaceutical composition of the present invention is not particularly limited as long as it has an activity to prevent and / or treat arthritis, but is preferably 0.1 to 10 Parts by weight of isoflavones.

The type of isoflavone contained in the pharmaceutical composition of the present invention is not particularly limited, but may be any commercially available or obtainable isoflavone, and may be those separated from legumes or processed products thereof, Is used as a seed bean, a chick pea, a red clover, a subterranean clover, a ground pea, a milk vetch, a marama bean, , Sword bean, jack bean, seaside sword bean, caraobean, cluster bean, balu, hyacinth bean, Some examples include grass pea, Indian vetch, garden pea, djenko bean, goa bean, yam bean, broad bean, peanut, earth pea, lentil, jumping bean, alfalfa, velvet Beans, velvet beans, African locust beans, inga, cyprus vetch, yebnut, tallow tree, polynesian chestnut, It is also possible to use kudze root (Pueraria lobata), oil bean tree, mesquite, tamarind, fenugreek, Indian liquorice, ground nut, Defatted soy flakes, soy flouver, and soy meal may be used as the soybean flour.

The separation of isoflavones from one or more selected from the above plant groups may be carried out using any of the solvents used for the extraction of substances from natural materials without particular limitation, and water, C ₁ to C ₅ alcohols, ethyl acetate, acetone And acetonitrile may be used.

The type of isoflavone contained in the pharmaceutical composition of the present invention is not particularly limited, but preferably is selected from the group consisting of daidzein, genistein, glycitein, daidzin, genistin, glycitin and glycosides thereof , And more preferably one or more selected from the group consisting of genistein.

Furthermore, the content of 2-deacylated glucose and isoflavone in the pharmaceutical composition of the present invention is not particularly limited as long as it has activity for preventing and / or treating arthritis, but is preferably 0.1 to 10 parts by weight per 100 parts by weight of 2- More preferably 0.1 to 5 parts by weight, and more preferably 0.2 to 2 parts by weight of isoflavone.

In addition, the pharmaceutical composition of the present invention exerts its effect in the usual inflammation caused in the joints, and is preferably effective in degenerative arthritis or rheumatoid arthritis.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, water for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols And may further contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabel and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The pharmaceutical composition according to the present invention may be formulated into a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and isotonic aqueous solution or suspension is preferable as a typical dosage form for parenteral administration. The injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.

The pharmaceutical composition formulated as described above may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. The term " effective amount " as used herein refers to an amount that shows a preventive or therapeutic effect when administered to a patient. In addition, the pharmaceutical composition according to the present invention may vary depending on the patient's age, sex and body weight, but is generally administered in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, It can be divided into several doses. Further, the dosage of the pharmaceutical composition according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.

Preparation Example  1. Chondrocyte culture

New Zealand white rabbit cartilage slices were purchased and rabbit articular cartilage cells were obtained from the cartilage slices. The rabbit articular cartilage cells were dissected from the chondral slices in an aseptic condition and then stained with 0.2% collagenase type 2 (purchased from Sigma, Calif., USA) for 6 hours in a buffer (DMEM; Dulbecco's modified Eagle's medium purchased from Invitrogen, , 381 U / mg solids). The mixture of the buffer solution, 0.2% collagenase, and cartilage slice was centrifuged at 20O < 0 > C at 230 xg for 10 minutes to obtain chondrocytes. The chondrocytes were cultured in RPMI 1640 supplemented with 10% fetal bovine calf serum (purchased from Invitrogen, CA), 50 ug / ml streptomycin (purchased from Sigma, CA) and 50 units / ml penicillin ). ≪ / RTI >

Then, the resuspended chondrocytes and DMEM were cultured at a density of 5 × 10 ⁴ cells / cm ² in a culture dish, and cultured cells were used in the following experiments.

Example  1. 2- Deoxidized glucose  And isoflavones

2-deoxyglucose and isoflavone on the expression of inflammatory protein COX-2 in cartilage cells. In this example, the isoflavones were treated with genistein.

The chondrocytes cultured in Preparation Example 1 were treated for 24 hours at a concentration of 5 mM 2-deoxyglucose (purchased from Sigma, CA, USA) and 10, 20, 40 μM of genistein (purchased from Biomol, Pennsylvania, USA).

Comparative Example  1. 2- Deoxidized glucose  Single processing

The cells were treated in the same manner as in Example 1 except that the chondrocytes cultured in Preparation Example 1 were treated with 5 mM of 2-deoxyglucose.

Comparative Example  2. Sodium Vanadate  process

Except that the chondrocytes cultured in Preparation Example 1 were treated at a concentration of 5 mM of 2-deoxyglucose and 1, 2, and 5 [mu] M of sodium orthovanadate (SOV; purchased from Sigma Corporation, 1.

Comparative Example  3. Oxidation Phenyl arsine  process

Chondrocytes cultured in Preparation Example 1 were treated with 5 mM of 2-deoxyglucose and treated with phenylarsine oxide (PAO, purchased from Calbiochem, CA, USA) at a concentration of 0.2, 0.5, 1 μM Treated in the same manner as in Example 1.

Comparative Example  4. Propane phosphatase 2 treatment

Except that the chondrocytes cultured in Preparation Example 1 were treated at a concentration of 5 mM of 2-deoxyglucose and 10 μM of Protein phosphatase 2 (PP2, purchased from Sigma Corporation, USA) 1.

Experimental Example  One. Western Blot  analysis

For quantitative analysis of proteins, western blot analysis of chondrocytes treated in Example 1 and Comparative Examples 1 to 4 was performed.

The western blot analysis was performed using 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40 and 0.1% sodium dodecylsulfate (SDS) (10 g / ml aprotinin, 10 g / ml leupeptin, 10 g / ml pepstatin A and 1 mM 4- (2-aminoethyl) benzenesulfonyl fluoride) and a phosphatase inhibitor (1 mM Na ₃ VO ₄ and 1 mM NaF) was added to prepare whole cell lysate. The lysate was subjected to SDS-polyacrylamide gel electrophoresis to separate the proteins by size and transferred to the nitrocellulose membrane. Anti-COX-2 (purchased from Cayman Chemical, Michigan, USA), anti-GRP94 (purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), an antibody specific for each isolated protein, anti-GADD153 (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), anti-actin (purchased from Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphotyrosine (Purchased from Cell Signaling Technology) and anti-pSrc (purchased from Cell Signaling Technology, Inc., Denver, USA).

The results of the Western blot analysis were quantified with an image J program.

The results of the Western blot analysis are shown in FIGS.

Figure 1 shows the results of treatment with 5 mM 2-deoxyglucose and / or 2 μM SOV, 0.5 μM PAO or 40 μM genistein (GN). As shown in Fig. 1, inflammation induction in chondrocytes combined with 2-deoxyglucose alone and 2-deoxyglucose and 2-deoxyglucose and 2-deoxyglucose (GN) It was confirmed that the expression of COX-2 protein was inhibited. Therefore, it was confirmed that treating 2-deacylated glucose and genistein was effective for inflammation.

FIG. 2 is a further experiment to compare the expression levels of COX-2 according to the treatment with 2-deoxyglucose alone, 2-deoxyglucose and SOV or 2-deoxyglucose and GN complex treatment in FIG. 1, Deoxygenated glucose and / or 2 μM SOV or 40 μM genistein (GN). As shown in FIG. 2, it was confirmed that the expression of COX-2, an inflammation-inducing protein, in cartilage cells treated with 2-deoxyglucose and genistein (GN) . In addition, it was confirmed that the expression of COX-2, an inflammation-inducing protein, was suppressed in chondrocytes treated with 2-deoxyglucose and genistein (GN), rather than with 2-deoxyglucose alone. Therefore, it was confirmed that treating 2-deacylated glucose and genistein was effective for inflammation.

Fig. 3 is a result of quantifying the result of the Western blot analysis of Fig. 2 with an image J program. As shown in FIG. 3, it was confirmed that the expression of COX-2, an inflammation-inducing protein, was remarkably suppressed in chondrocytes treated with 2-deoxyglucose and GN than with 2-deoxyglucose alone treatment. Therefore, it was confirmed that treating 2-deacylated glucose and genistein was more effective for inflammation than 2-deacylated glucose alone.

Figure 4 shows the results of treatment with 5 mM 2-deoxyglucose and / or 40 μM genistein (GN) or 10 μM PP2. As shown in Fig. 4, the expression of COX-2, an inflammation-inducing protein in chondrocytes treated with 2-deoxyglucose and GN, was inhibited more than 2-deoxyglucose alone treatment or 2-deoxyglucose and PP2 complex treatment . Therefore, it was confirmed that treating 2-deacylated glucose and genistein was effective for inflammation.

Figure 5 shows the results of 5 mM 2-deoxyglucose and / or 1, 2, 5 μM SOV alone or in combination. As shown in Fig. 5, in the inhibition of COX-2 expression, the combined treatment of 2-deoxyglucose and PP2 of less than 2 [mu] M had similar effects to the 2-deoxyglucose alone treatment, and 2-deoxyglucose and 2 [ The combined treatment above had a worse effect than the 2-deacylated glucose alone treatment. Therefore, it was confirmed that the combined treatment of 2-deoxyglucose and PP2 was not effective for inflammation.

Figure 6 shows the results of 5 mM of 2-deoxyglucose and / or 0.2, 0.5, 1 [mu] M PAO alone or in combination. As shown in Fig. 6, in the inhibition of COX-2 expression, the combined treatment of 2-deoxyglucose and PAO showed similar effects to the 2-deoxyglucose alone treatment, indicating that it was not effective against inflammation.

Figure 7 shows the results of 5 mM 2-deoxyglucose and / or 10, 20, 40 μM genistein (GN) alone or in combination. As shown in Fig. 7, in the suppression of COX-2 expression, the combined treatment of 2-deoxyglucose and GN showed similar effects to that of 2-deoxyglucose alone treatment. However, in the p-tyrosine inhibition, it was confirmed that the combined treatment of 2-deoxyglucose and GN was more effective than the 2-deoxyglucose alone treatment, so that inflammation was more effective than the 2-deoxyglucose alone treatment.

As shown in Figs. 1 to 7, in inhibiting the expression of COX-2, an inflammatory protein in chondrocytes, 2-deoxyglucose alone treatment or 2-deoxyglucose and SOV, 2-deoxyglucose and PAO, It was confirmed that the combined treatment of 2-deoxyglucose and GN was surprisingly more effective than the glucose and PP2 complex treatment.

Experimental Example  2. Irritants PGE2  Expression

In order to examine the effect of PGE2 (Prostaglandin E2) on the expression of inflammation inducer, PGE2 assay was performed on the chondrocytes treated in Example 1 and Comparative Examples 1 to 3. The PGE2 assay was performed in the following manner.

PGE2 is a class of prostaglandins, a pain mediator that directly affects inflammation induction. Various physiological effects of prostaglandin include inflammatory reactions such as rheumatoid arthritis and osteoarthritis, blood pressure suppression, and pain and heat deterioration. do.

Production of PGE2 is carried out by numerical measurements of cells and secreted PGE2 using the PGE2-linked immunosorbent assay kit (purchased from assay designs in Miami, USA). The chondrocytes prepared in Preparation Example 1 were cultured in a standard 96-well microtiter plate at 1 × 10 ⁴ cells / well. The cultured chondrocytes were then used in the same manner as in Example 1 and Comparative Examples 1 to 3 and then used to quantify the amount of PGE2. The PGE2 values were calculated against the standard PGE2 curve. The PGE2 values of the obtained Example 1 and Comparative Examples 1 to 3 are shown in FIG. 8 below.

As can be seen from FIG. 8, the combination treatment of 2-deoxyglucose and SOV or 2-deoxyglucose and PAO had no effect of inhibiting PGE2 more than 2-deoxyglucose alone treatment and 2-deoxyglucose and GN Was effective for PGE2 inhibition and inflammation.

As can be seen from the Examples, Comparative Examples and Experimental Examples, it was found that the addition of 2-deoxyglucose and GN (2-deoxyglucose) and 2-deoxyglucose Were surprisingly effective in inhibiting the expression of inflammatory proteins COX-2 and PGE2 in chondrocytes.

Claims (6)

A pharmaceutical composition for preventing and treating arthritis comprising 2-deoxyglucose and genistein. 2. The method of claim 1, wherein the genistein is selected from the group consisting of soybean, chick pea, red clover, subterranean clover, ground pea, milk vetch, A marama bean, a sword bean, a jack bean, a seaside sword bean, a caraobean, a cluster bean, a balu, Hyacinth bean, grass pea, Indian vetch, garden pea, djenko bean, goa bean, yam bean, Broad bean, earth pea, lentil, jumping bean, alfalfa, velet bean, African locust bean, Inga (bean, bean, such as cyprus vetch, yebnut, tallow tree, polynesian chestnut, kudze root (Pueraria lobata), oil beans Oil bean tree, mesquite, tamarind, fenugreek, Indian liquorice, ground nut, soy flake, soy flour soy flouver, and soy meal. The pharmaceutical composition according to claim 1, wherein the composition is selected from the group consisting of soy flour, soy flour and soy meal. delete delete The pharmaceutical composition for preventing and treating arthritis according to claim 1, wherein the pharmaceutical composition comprises 0.1 to 10 parts by weight of genistein relative to 100 parts by weight of 2-deacidified glucose. The pharmaceutical composition for preventing and treating arthritis according to any one of claims 1, 2, and 5, wherein the arthritis is degenerative arthritis or rheumatoid arthritis.

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