KR20030084271A - Composition containing alpha-viniferin for prevention and therapy of arthritic disease - Google Patents

Abstract

PURPOSE: A composition containing alpha-viniferin from Carex humilis Leyss. as a main component is provided. The alpha-viniferin inhibits COX-2 activity and TNF release at the same time, but not COX-1 and is thus effectively used in treatment of various inflammatory disorders associated with COX-2 and TNF, containing arthritis. CONSTITUTION: A pharmaceutical composition for prevention and treatment of arthritis contains alpha-viniferin induced from Carex humilis Leyss. and a pharmaceutically acceptable carrier. The composition is formulated into an oral formulation such as tablets or capsules, an external formulation, a suppository or a sterile injectable solution. The effective dose of the composition is 2 to 20mg/kg body weight.

Description

Composition containing alpha-viniferin for prevention and therapy of arthritic disease

The present invention relates to a composition for preventing and treating arthritis, including alphaviniferin.

Arthritis is a general term for diseases involving pain, stiffness, and inflammation in the joint area. Rheumatoid arthritis caused by inflammation of the joint synovial membrane and osteoarthritis caused by degeneration of articular cartilage are well known (Rullan, E., et al. , Curr. Rheumatol.Rep., 3 : 445-52, 2001). Arthritis is a chronic inflammatory disease in which the onset and progression of the disease gradually progresses, and the relief and deterioration are repeated. Pain and swelling in the joint area are present. In addition, chronic progression causes calcium to escape from joints and cartilage breaks down, resulting in joint deformation and subcutaneous nodules (Lee, DM, et al., Lancet, 358 : 903-11, 2001).

Rheumatoid arthritis is a pathophysiological phenomenon characterized by the formation of inflammatory tissue called pannus by synovial macrophages, polymorphonuclear leukocytes, fibroblasts and synovial cells, which are secreted from the inflammatory tissues. Matrix metalloprotease, collagenase, and the like are known to damage collagen and proteoglycan in joint tissues and damage cartilage and connective tissue (Martel-Pelletier, J.). , et al. , Best Pract.Res. Clin.Rheumatol., 15 : 805-29, 2001; Close, DR, Ann.Rheum.Dis . , 60 : iii62-7, 2001; Cunnane, G., et al. , Arthritis Rheum., 44 : 1744-53, 2001). The pathogenesis of rheumatoid arthritis is not yet known, but recent studies have shown that inflammatory cytokines are present in high levels in the synovial fluid of patients with rheumatoid arthritis (Feldmann, M., et al., Curr. Dir Autoimmun., 3 : 188-99, 2001; Kavanaugh, A., Arthritis Rheum. , 47 : 87-92, 2002). These cytokines are responsible for inflammatory reactions in the joint area, including promoting the release of proteases from cells. It is known to continuously intensify (Ishiguro, N., et al., Arthritis Rheum. , 44 : 2503-11, 2001).

Conventionally, nonsteroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, immunosuppressants, immunomodulators, gold compounds, penicylamine preparations or antimalarial preparations have been used as pharmacotherapy for rheumatoid arthritis (Schuna, AA, J). Am. Pharm.Assoc (Wash), 38 : 728-35, 1998; Haagsma, CJ, Drugs Aging, 13 : 281-9, 1998; O'Dell, JR, Rheum.Dis.Clin.North Am., 24 : 465-77, 1998). However, nonsteroidal anti-inflammatory drugs that have been used for inflammatory diseases cause gastrointestinal disorders, and steroidal anti-inflammatory drugs that have been used for the treatment of chronic inflammatory diseases such as arthritis as strong immunosuppressive drugs have long-term side effects. (Straus, WL, et al., Gastroenterol. Clin. North Am. , 30 : 895-920, 2001; Da Silva, JA, et al., Rheum. Dis. Clin. North Am. , 26 : 859-80, 2000). On the other hand, other drugs also have the effect of temporarily alleviating the symptoms of arthritis, there was a problem that can not obtain the fundamental arthritis treatment effect.

In a recent study, the isozyme of cyclooxygenase (hereinafter abbreviated as "COX"), the target enzyme of nonsteroidal anti-inflammatory drugs, is called cyclooxygenase-1 (hereinafter referred to as "COX-1"). ) And cyclooxygenase-2 (hereinafter abbreviated as "COX-2") are known. COX-1 produces prostanoids that maintain normal physiology of the stomach, intestines, and kidneys, whereas COX-2 amplifies biosynthesis by inflammatory stimuli, which is excessive to maintain the inflammatory response of biological defenses. Produces prostanoids (Eberhart, CE, et al., Gastroenterology, 109 : 285-301, 1995; Feng, L., et al., J. Clin.Invest ., 95 : 1669-75, 1995) . The side effects of nonsteroidal anti-inflammatory drugs are due to the inhibition of COX-1 activity (Brzozowski, T., et al., Microsc. Res. Tech., 53: 343-53, 2001). To improve their side effects, 4- (5- p -tolyl-3-trifluoromethyl-pyrazol-1-yl) -benzene sulfoamide (4- (5- p -tolyl-3-trifluoromethyl-pyrazol -1-yl) -benzene sulfonamide) and 4- (4-methanesulfonyl-phenyl) -3-phenyl -5 H-furan-2-one (4- (4-methanesulfonyl -phenyl) -3-phenyl-5 H -furan-2-one) has been developed and is used clinically as an anti-inflammatory drug under the trade names Celebrax and Vioxx (FitzGerald, GA, et al., N. Engl. J.). Med., 345 : 433-42, 2001; Matheson, AJ, et al., Drugs, 61 : 833-65, 2001).

Meanwhile, the water-soluble receptor of TNF, an inflammatory cytokine involved in the deformation of the joint area known as the specific pathology of arthritis, has been approved by the US FDA as an antiarthritis drug under the trade names Embrel and Remicade. Taylor, PC, et al., Curr. Dir.Autoimmun . , 2 : 83-102, 2000; Alldred, A., Expert.Opin.Pharmacother ., 2 : 1137-48, 2001).

However, the water-soluble receptor of TNF is a protein agent, and when administered via oral, transdermal, etc., there is a characteristic that the activity is lost due to hydrolysis, the route of administration to the patient is limited to intravenous injection. In particular, the prevention and treatment of arthritis generally requires long-term drug administration, so it is very important to diversify the route of administration. Therefore, there is an urgent need for new drug development to search for low molecular weight substances that can prevent and treat arthritis, which can diversify the route of administration.

In order to solve the above problems, the present inventors conducted a study to search for a low molecular weight natural substance exhibiting efficacy of preventing and treating arthritis by simultaneously inhibiting COX-2 activity and TNF release. It was found that the present invention was completed and the present invention was completed. Accordingly, it is an object of the present invention to provide a composition for preventing and treating arthritis.

Figure 1 shows the selective inhibitory effect of alphaviniprine on cyclooxygenase, divided by COX-2 (●) and COX-1 (○), changes in the activity of cyclooxygenase according to the change in the concentration of alphaviniprine. (P <0.001 (*)).

Figure 2 shows the inhibitory effect of alphabinifrine on TNF release (●) and the TNF action (○) (p <0.001 (*)).

Figure 3 shows the inhibitory effect on the arthritis edema in the adjuvant-induced arthritis animal model divided into a control (○), alphabiniferin (●), ketoprofen (△) and dexamethasone (▲).

Figure 4 shows the inhibitory effect on the joint deformation for the animal model of arthritis induced by the adjuvant divided into a control (○), alpha bean perrin (●), ketoprofen (△) and dexamethasone (▲).

The present invention relates to a composition for the prevention and treatment of arthritis, comprising alpha viniferin and a pharmaceutically acceptable carrier.

Alphaviniferin has a structure represented by the following general formula (1), it can be obtained by separating and extracting the components contained in natural substances. The alphaviniferin was first isolated from the roots of Caragana chamlagu belonging to the legume (Legimunosae), and has been reported to have an anti-inflammatory effect against acute inflammation caused by carageenan (Kitanaka, S., et. al ., Chem. Pharm. Bull., 38 : 432-435, 1990). Inhibition of protein kinase C by activity of alphaviniferin (Kulanthaival, P., et al ., Planta Med., 61 : 41-44, 1995), 20-hydroxyexidone (20- antagonism of hydroxyecdysone (Keckeis, K., et al. , Cell.Mol.Life Sci., 57 : 333-336, 2000), inhibitory effect of acetylcholinesterase (Sung, SH, et al. , Biol. Pharm. Bull., 25 : 125-127, 2002). Alphaviniferin used in the present invention was purified from an extract of the root of Carex humilis , a native plant belonging to Cyperacea.

One

On the other hand, the alphabiniferin can be purified by various natural extraction methods (Kitanaka, S., et al., Chem. Pharm. Bull. (Tokyo), 38 : 432-5, 1990; Kulanthaivel, P., et. al., Planta Med., 61: 41-4, 1995), to date no products are actually sold.

The arthritis includes, but is not limited to, rheumatoid arthritis, spondyloarthopathies, gout, osteoarthritis, systemic lupus erythematosus, and juvenile arthritis. arthritis).

The present invention relates to a composition, characterized in that the prevention and treatment of arthritis is achieved by inhibiting the activity of clooxygenase-2.

The present invention relates to a composition, wherein said prevention and treatment of arthritis is achieved by inhibiting TNF release.

The carrier is generally an excipient such as sorbitol; Disintegrants; And binders. The compositions also include preservatives such as methyl paraoxybenzoate or propyl; Flavoring agents such as ethyl vanillin; And an additive component including a colorant such as caramel.

The composition can be administered in a variety of ways, including, but not limited to, oral, intravenous, subcutaneous, topical medication, and the like. In addition, the composition may be prepared in an oral dosage form such as tablets and capsules, respectively, according to a conventional method; External preparations; It can be formulated in the form of suppositories or sterile injectable solutions.

The composition may be administered to a human at a daily dosage of 2 mg / kg to 20 mg / kg body weight, preferably from 3 mg / kg to 9 mg / kg body weight, depending on the indication, symptom or condition of the patient. It may be administered in a daily dose. The pharmaceutical composition comprising alphabinifrine may be administered on a variety of schedules, such as once a day, twice a day, three times a day, and preferably three times a day. However, the dosage range does not limit the scope of the present invention in any aspect.

The present invention relates to a composition, characterized in that alphaviniferin is extracted from the root of the mountain mirror.

Although an Example, an experiment example, and a manufacture example are given to the following and this invention is concretely demonstrated to it, the scope of the present invention is not limited by these.

Example 1 Purification of Alphaviniferin

After the roots of the fresh mountain mirror were chopped, about 3 L of 80% methanol was added per kg, and the extraction process was performed three or more times at room temperature for three days or more. Then, the extraction extract was obtained through filtration and concentration under reduced pressure. About 1 L of water per 100 g of this methanol extract was added and suspended, and extracted three times or more with 1 L of methylene chloride. The aqueous layer was then extracted three more times with 1 L of ethyl acetate. After filtration of the ethyl acetate layer, the soluble material was taken and then concentrated to obtain an extract. The extract was loaded onto a silica gel column (10 x 50 cm) and the active ingredient was eluted with a concentration gradient of methylene chloride and acetone (19: 1 → 5: 1), followed by a Riverspace C-18 column (5 x 50 cm). ), Eluted with a methanol gradient (50% → 20%), loaded onto a silica gel column (3 x 35 cm), and then with a gradient of methylene chloride and ethyl acetate solution (17: 3 → 1: 1). Elution was carried out, and finally loaded on an MCI-gel CHP 20P column (3 × 35 cm) to obtain 3 g of alphabinifrine as an active ingredient with 70% methanol (yield: 3% compared to methanol extract). Purity was greater than 98% using TMS as an internal standard and NMR analysis using a Bruker AM-300 spectrometer.

Experimental Example 1. Inhibitory effect of COX-2 activity

Enzyme source COX-2 was prepared using a microsome prepared from mouse macrophage Raw264.7 stimulated with lipopolysaccharide (LPS), and enzyme source COX-1 was prepared using a microsome prepared from bovine hyposperm. . Cell line Raw264.7 was suspended in DMEM medium containing 10% FBS and 300 μM aspirin and incubated at 37 ° C. and 5% CO ₂ for 24 hours. Cells were then washed twice with PBS, then DMEM containing LPS (final concentration 10 μg / mL) was added and incubated for 24 hours under the same conditions. Cell Raw264.7 was collected by centrifugation, then suspended in 0.1 M Tris-HCl (pH 8.0) and sonicated to disrupt cell membranes. The supernatant obtained by centrifuging the lysate (enzyme source COX-2) of the cell line Raw264.7 and the homogenate (enzyme source COX-1) of both sperm cells at 10,000 xg for 10 minutes was obtained at 150,000 xg. After centrifugation in time, the precipitated microsomes were used as the enzyme source. Luminometer was measured for 30 seconds in 0.1 M Tris-HCl (pH 8.0) containing 100 μM arachidonic acid, alphaviniferin (0.1 μM to 100 μM), and 25 μM luminol. The enzyme activity of COX was measured. The measurement results are shown in FIG. 1.

In the figure, the x-axis shows the concentration of alphabinifrine (μM), and the y-axis shows the enzyme activity of COX (% of control). As can be seen in FIG. 1, alphaviniferin inhibited COX-2 activity concentration-dependently. Compared with the control (alphaviniferin-treated group) showed a significant inhibitory effect at the sample concentration of 3 μM or more, the concentration of alphabiniferin 50% inhibition of COX-2 was 4.9 μM. However, the inhibitory effect of alphaviniferin on COX-1 showed a significant difference at 50 μM or higher compared to the control group, and showed 44.8 ± 2.1% inhibition rate at 100 μM. Therefore, it was confirmed that alphaviniferin is a substance that selectively inhibits COX-2 activity.

Experimental Example 2 Inhibitory Effect of TNF Release

TNF release of mouse macrophage Raw264.7 stimulated with LPS was measured by ELISA. Cell lines Raw264.7 (2 × 10 4) suspended in DMEM medium containing 10% FBS were dispensed into 96-well culture plates and incubated at 37 ° C., 5% CO ₂ conditions for 24 hours. Cells were then washed twice with PBS, then DMEM containing LPS (final concentration 10 μg / mL) and alphabinifrine (1 μM to 50 μM) was added and incubated for 24 hours under the same conditions. The amount of TNF present in the supernatant obtained by centrifugation at 700 xg for 30 minutes was quantified by ELISA kit.

Cell death of TNF dependent cell line WEHI-164 was measured as an indicator of TNF action. Cell lines WEHI-164 (2 × 10 4) suspended in DMEM medium containing 10% FBS were dispensed into 96-well culture plates, TNF-α (final concentration 3 ng / mL) and alphaviniferin (50 at 12.5 μM). μM) was added and incubated for 48 hours at 37 ° C. and 5% CO ₂ . Cell death of WEHI-164 was measured by the WST-1 colorimetric method. The measurement results are shown in FIG. 2.

In the figure, the x-axis represents the concentration of alphaviniprine (μM), and the y-axis represents the TNF release and action (% of control). The amount or growth inhibition of TNF released when alphaviniprine is treated simultaneously with LPS or TNF The figure shows the relative ratio with the control as 100%. Herein, the control group means the amount of TNF released by the cell line Raw264.7 when only LPS was administered without alphabinifrine in the case of TNF release, and the cell line WEHI-164 when only TNF was administered without alphabiniferin in the case of TNF action. Means growth inhibition. As can be seen in FIG. 2, alphaviniferin inhibited TNF release concentration-dependently. Compared to the control group (LPS-only group) showed a significant TNF release inhibitory effect at a sample concentration of 6.3 μM or more, the alphabiniferin concentration that inhibits TNF release by 50% was 9.8 μM. However, alphaviniferin had no inhibitory effect on TNF action at sample concentration of 12.5-50 μM. Therefore, it was confirmed that alphabinifrine does not affect the action of TNF and that it selectively inhibits the release of TNF.

Experimental Example 3. Antiarthritis Efficacy in Animal Models

Sprague-Dawley female rats of 8 weeks old were used as experimental animals. The Mycobacterium butynyl rikum (Mycobacterium butyricum) to Freund's incomplete adjuvant Mycobacterium butynyl rikum the suspension, the right foot of the rat was in (Freund's incomplete adjuvant) was subcutaneously injected by 0.6 mg to induce systemic arthritis model . From day 8 to day 14 (7 days) after injecting mycobacterium butyricum, the same dose (10 mg / kg) of alphabinifrine and ketoprofen and dexamethasone used as positive controls ) Orally once daily. Arthritis edema that progressed to the left foot was measured at 2-3 days intervals using a plethysmometer, and joint deformation induced in the limbs (front and back right and left legs) was measured at weekly intervals by x-ray. The measurement results are shown in FIG. 3.

As can be seen in Figure 3, the control group (group administered only saline used for sample dissolution) was injected with mycobacterium butyricum began to increase the edema of the left foot from day 12. On the other hand, alphaviniprine, a sample, significantly inhibited arthritis edema when administered orally, and the inhibitory effect was similar to that of ketoprofen and dexamethasone used as positive controls.

Meanwhile, limb-induced joint deformation was measured by x-ray and converted into scores. The joint deformity scores were divided into three stages (0; no damage, 1; osteoporosis, 2; osteoporosis and bone erosion at the same time) according to the degree of bone erosion and osteoporosis. Limited to 8 points x 4 (limbs) (Meunier, CJ, et al. , Drug Metab. Dispod., 27 : 26-31, 1999). The measurement results are shown in FIG. 4.

As can be seen in Figure 4, the experimental group administered alpha-viniferin showed a significantly lower joint deformation score compared to the joint deformation of the control group (group administered only saline solution used for sample dissolution). The efficacy of alphaviniferin on joint deformation was similar or stronger than that of ketoprofen and dexamethasone used as positive controls.

Formulation Example 1 Tablet

According to the following composition, each was formulated by a conventional tablet manufacturing method.

Tablet composition

Alphabinifrine 500.0 mg

Lactose 500.0 mg

Talc 5.0 mg

Magnesium Stearate 1.0 mg

Formulation Example 2 Capsule

According to the following composition, a capsule was prepared in the following manner. Capsules were prepared by mixing alphaviniferin with excipients and then filling into gelatin capsules.

Capsule Composition

Alphabinifrine 500.0 mg

Starch 1500 10.0 mg

Magnesium Stearate BP 100.0 mg

Formulation Example 3 Injection

The following components were filled into 2.0 ml ampoules by a conventional method for preparing an injection and sterilized to prepare an injection.

Injectable Composition

Alphabinifrine 50.0 mg

Antioxidant 1.0 mg

Tween 80 1.0 mg

Distilled water for injection to 2.0 ml

While alphaviniferin inhibited COX-2 activity and TNF release simultaneously, it showed selectivity that showed no inhibitory effect on COX-1 activity. Thus, alphaviniferin can be used more effectively in the treatment of various inflammatory diseases involving COX-2 and TNF, including arthritis. In addition, alphabiniferin is a low molecular weight natural substance extracted from plants, and has a characteristic of diversifying the route of administration to patients.

Claims (5)

A composition for the prevention and treatment of arthritis, comprising alpha viniferin and a pharmaceutically acceptable carrier. The composition of claim 1, wherein the prevention and treatment of arthritis is achieved by inhibiting the activity of cyclooxygenase-2. The composition of claim 1, wherein the prevention and treatment of arthritis is achieved by inhibiting TNF release. The composition according to any one of claims 1 to 3, which is formulated in the form of an oral dosage form, external preparation, suppository or sterile injectable solution. The composition according to any one of claims 1 to 3, wherein the alphaviniferin is extracted from a mountain mirror root.