KR20060067900A - A pharmaceutical composition for treating arthritis - Google Patents

Abstract

The invention (a) 20- O -β-D- glucopyranosyl -20 (S) of the following formula (I) as an active ingredient-protocol Panax Oh diol (20- O -β-D-glucopyranosyl -20 (S) - protopanaxadiol) or a therapeutically effective amount thereof; And (b) a pharmaceutically acceptable carrier.

Formula 1

20-O-β-D-glucopyranosyl-20 (S) -protopanaxadiol, compound K, saponin, ginsenosides, arthritis, osteoarthritis

Description

A pharmaceutical composition for treating arthritis

1 is a graph showing the effect of compound K on the expression of matrix metalloproteinase-3 (MMP-3) in chondrocytes treated with IL-1β. In the figure, CpK represents Compound K.

2 is a graph showing the effect of compound K on the expression of MMP-13 in chondrocytes treated with IL-1β.

3 is a graph showing the effect of compound K on the production of nitric oxide (NO) in chondrocytes treated with IL-1β.

4 is a graph showing the results of analyzing the toxicity of Compound K on chondrocytes.

5 is a graph showing the results of experiments confirming whether various types of ginsenosides have an effect on NO production.

6 is a graph showing the results of experiments confirming whether ginsenoside Rb1 has an effect on NO production.

7 is a photograph of Western blotting results showing that compounds K and Rb1 inhibit expression of inducible nitric oxide synthase (iNOS).

FIG. 8 is a photograph of Western blotting results showing the effect of compound K on the activation (ie phosphorylation) of mitogen activated protein kinase (MAP kinase).

The present invention relates to a pharmaceutical composition for treating arthritis.

Arthritis is a generic term for various conditions that cause pain, swelling and limited movement in joints and connective tissue. The specific etiology of arthritis is not clear. On the other hand, most programs of arthritis treatment are currently made through a combination of medication, exercise, rest, joint protection and surgery.

The most frequent forms of arthritis are osteoarthritis, fibromyalgia and rheumatoid arthritis.

Osteoarthritis is a degenerative joint disease characterized by the destruction of articular cartilage. Cartilage functions to cushion the ends of the bones in the joints. The destruction of the cartilage causes the bones to rub against each other, which causes pain and limits movement. Osteoarthritis mainly occurs in weighted joints such as knees, hips, feet and back. Risk factors for osteoarthritis include aging, obesity, joint damage and genetic predisposition. The molecular mechanism for osteoarthritis is likely to be the destructive enzymes secreted by the chondrocytes themselves.

The current treatments for osteoarthritis focus on alleviating pain and improving joint movement. Drugs prescribed for pain relief and inflammatory reactions include aspirin, acetaminophen, ibuprofen and nonsteroidal anti-inflammatory drugs (NSAIDs). In addition, a method of injecting a corticosteroid directly into a joint is also used.

Fibromyalgia, on the other hand, is a pain that affects tissues attached to muscles and bones. For the purpose of treating fibromyalgia, aspirin, acetaminophen, ibuprofen and nonsteroidal anti-inflammatory drugs (NSAIDs) are prescribed for pain relief and inflammatory reactions.

Rheumatoid arthritis is an inflammatory disease as a type of autoimmune disease. Rheumatoid arthritis is diagnosed by examining the presence of rheumatoid factor in the blood, which is found in about 80% of adults with rheumatoid arthritis.

Conventional methods for the treatment of rheumatoid arthritis are focused on the relief of edema, the relief of pain and stiffness, and the maintenance of normal joint function. In addition, drugs used in the treatment of rheumatoid arthritis are mainly nonsteroidal anti-inflammatory drugs (NSAIDs) that can control inflammation, joint pain, stiffness and edema. In addition, drugs such as prednisone, methotrexate, hydroxychloroquine, azulpidine, gold salt and cyclosporin are also used.

Conventional treatments for arthritis (regimens), as described above mainly consists of drugs for mitigating inflammation, but this shows only a limited therapeutic effect. Accordingly, there is a need in the art for a more fundamental arthritis therapeutic agent that can treat arthritis in a different way from inflammation and pain relief.

The present inventors screened various compounds to develop drugs that can treat arthritis more fundamentally than temporary treatments such as inflammation and pain relief. Eventually, specific compounds were effective for expression and production of enzymes and substances related to the development of arthritis. The present invention has been completed by discovering its efficacy.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for treating arthritis.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides (a) 20- O- β-D-glucopyranosyl-20 (S) -protopanaxadiol (20- O- β-D) therapeutically effective amount of -glucopyranosyl-20 (S) -protopanaxadiol) or a prodrug thereof; And (b) provides a pharmaceutical composition for treating arthritis comprising a pharmaceutically acceptable carrier:

Formula 1

The present inventors screened various compounds to develop drugs that can treat arthritis more fundamentally than temporary treatments such as inflammation and pain relief. Eventually, the compound of Formula 1 was expressed in the expression of enzymes and substances related to the development of arthritis and It was found to exert effective efficacy in production. Molecular targets of the effects of the compounds of formula 1 on the treatment of arthritis include: matrix metalloproteinase (MMP) -3, MMP-13, nitric oxide (NO) synthase and mitogen activated protein kinase (MAP kinase) family [p38] MAP kinase (p38 mitogen activated protein kinase), extracellular signal-regulated protein kinase (ERK 1/2) and c-Jun N-terminal kinase (c-Jun N-terminal kinase: JNK)] to be. That is, the compound of formula 1 inhibits the expression of (i) MMP-3 and MMP-13, which are chondrocyte matrixases, (ii) inhibits the production of NO by interfering with the expression of NO synthase, and (i) It is effective in treating arthritis by effectively blocking the increase of MMPs, which are chondrocyte matrixases, and the activation (ie, phosphorylation) of MAP kinase that mediates chondrocyte apoptosis. In addition, since the compound of Formula 1 of the present invention does not affect the expression of a tissue inhibitor of metalloproteinase (TIMP), which is known to antagonize MMPs, it may be a very effective arthritis therapeutic agent.

The compound of the present invention may be represented by Formula 1, wherein the compound of Formula 1 is abbreviated as Compound K in the art. In addition, the compound included as an active ingredient in the pharmaceutical composition of the present invention is not only represented by the formula (1), but also a functional group (for example, -OH group) that can be substituted in the formula (1) according to a conventional method Derivatives prepared by modification should also be construed as being within the scope of the present invention as long as the derivative exhibits efficacy in treating arthritis.

In addition, the active ingredient included in the pharmaceutical composition of the present invention also includes a prodrug of Compound K. Compound K is known as a major metabolite in the body of various saponin components of Panax ginseng . Thus, the term “prodrug of compound K” refers to a compound that can be converted to compound K in the body.

See, eg, Akao T. et al., J. Pharm . Pharmacol . , 50 (10): 1155 (1998) discloses that ginsenoside Rb1 is converted to Compound K in the body.

According to a preferred embodiment of the present invention, the prodrug is saponin isolated from ginseng, more preferably ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3 and the like, even more preferred Preferably ginsenoside Rb1.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraarticular injection, or the like.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

Meanwhile, the pharmaceutical composition of the present invention may further include hyaluronic acid (HA) which is commonly used to treat osteoarthritis. Hyaluronic acid is expected to synergistically increase the therapeutic effect of arthritis of the compositions of the present invention comprising Compound K or a prodrug thereof.

Arthritis that can be treated by the pharmaceutical composition of the present invention is osteoarthritis, fibromyalgia and rheumatoid arthritis, preferably osteoarthritis, most preferably degenerative osteoarthritis.

Unlike the conventional therapeutic agent for arthritis, the pharmaceutical composition of the present invention inhibits the expression of (i) chondrocyte matrix lyases MMP-3 and MMP-13, which are known to cause arthritis development, and (ii) NO synthase. Inhibits the production of NO by interfering with (i) having a fundamental therapeutic mechanism by effectively blocking the increase of MMPs, the chondrocyte matrixase and activation of MAP kinase (ie phosphorylation) that mediates chondrocyte apoptosis. Because of this, it is very effective in treating arthritis.

Furthermore, the active ingredient used in the pharmaceutical composition of the present invention exhibits the above-mentioned efficacy even at very low concentrations (μM level) (particularly, in the case of the compound of Formula 1), which is very advantageous for the final development as a medicine. Do. In addition, the pharmaceutical composition of the present invention is very safe because it does not show toxicity to the living body as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example  1: Preparation of Compound K

10 g of ginseng (including red ginseng, ginseng or ginseng root) are dissolved in 2 L of citrate buffer (pH 4.0) and then 10 g of naringiase (Sigma, St Louis, MO) or 10 g of peck It was left for 2 days at 38 ° C. with tinase. 2 L of ethyl acetate was added to extract the reaction compound and then evaporated to give 2.8 g of residue. This material was subjected to silica gel column chromatography to stratify and purified by impregnation with chloroform: methanol (9: 1) and chloroform: methanol (6: 1) solution in turn to give compound K (0.28 g). Purity of the purified compound was confirmed by HPLC.

Example  2: Cultivation of Chondrocytes

Chondrocytes were isolated from the joints of patients with degenerative arthritis as follows: Chondrocytes were chopped into pieces and then digested with 0.2% protease type XIV (Sigma, St. Louis, MO) for 1 hour, followed by 0.2% collagenase. Treated with first type IA (Sigma) at 37 ° C. for 3 hours. After treatment, undigested cartilage fragments were removed using a 70 μm nylon sieve. Chondrocytes were washed with PBS and then DMEM containing 10% fetal bovine serum (FBS; Life Technologies), 100 units / ml penicillin-streptomycin (Life Technologies) and 25 g / ml L-ascorbic acid (Sigma) The monolayer culture was maintained for 7 days in medium (Life Technologies Inc., Rockville, MD). All experiments were conducted using either initial or first passage cells.

Example  3: induced by IL-1β MMP -3 expression inhibition effect confirmed

Whether compound K of the present invention inhibits the expression of matrix metalloproteinase-3 (MMP-3) induced by IL-1β was investigated as follows.

Experimental chondrocytes, except the control group, were treated with IL-1β at a concentration of 2 ng / ml, and at the same time, Compound K was added at 0, 0.1, 0.5, 1, 2 and 5 μM, respectively, and then FMEM-free DMEM solution for 48 hours. Incubated at. After incubation, the culture solution was rolled to measure the concentration of MMP-3. MMP-3 was measured using a commercially available ELISA kit (Biosource international), and the measurement method was performed as included in the product.

As shown in FIG. 1, the expression of MMP-3 induced by IL-1β was dose-dependently reduced by Compound K, and the expression of MMP-3 was decreased at 5 μM of Compound K. It can be seen that it is almost completely suppressed.

Example  4: induced by IL-1β MMP -13 expression inhibition effect confirmed

Whether compound K of the present invention inhibits the expression of matrix metalloproteinase-13 (MMP-13) induced by IL-1β was investigated as follows.

Experimental chondrocytes, except the control group, were treated with IL-1β at a concentration of 2 ng / ml, and at the same time, Compound K was added at 0, 0.1, 0.5, 1, 2 and 5 μM, respectively, and then FMEM-free DMEM solution for 48 hours. Incubated at. After incubation, the culture solution was rolled to measure the concentration of MMP-3. MMP-3 was measured using a commercially available ELISA kit (R & D), and the measurement was performed as included in the product.

As can be seen in FIG. 2, expression of MMP-13 induced by IL-1β was dose-dependently reduced by Compound K, and expression of MMP-3 was almost decreased at 5 μM of Compound K. It can be seen that it is completely suppressed.

Example  5: Confirmation of the Inhibitory Effect of NO Production Induced by IL-1β

Whether or not compound K of the present invention inhibits NO (nitric oxide) production induced by IL-1β was investigated as follows.

Experimental chondrocytes, except the control group, were treated with IL-1β at a concentration of 2 ng / ml, and at the same time, Compound K was added at 0, 0.1, 0.5, 1, 2 and 5 μM, respectively, and then FMEM-free DMEM solution for 48 hours. Incubated at. After incubation, the culture solution was rolled up and the total NO concentration was measured by a Griess reaction. The reagents used for the Gries reaction include Gries reagent A: 2% sulfanylamide dissolved in 5% phosphoric acid, and Gries reagent B: 0.2% N- (1-naphthyl) -ether dissolved in 5% phosphoric acid. Used as a composition of lene diamine dihydrochloride.

As can be seen in FIG. 3, NO-1 induced by IL-1β was dose-dependently reduced by Compound K and NO production was almost completely inhibited at 5 μM of Compound K. there was.

Example  6: Toxicity of Compound K to Chondrocytes

In order to determine whether the compound K of the present invention is toxic to chondrocytes, the following experiment was performed:

The experiment was divided into eight groups, and only the DMSO solution used to dissolve Compound K was added to the control group, and the second group was treated with IL-1β at a concentration of 2 ng / ml. In the other six groups, compound K was added at 0.1, 0.5, 1, 2, 5, and 10 μM, respectively, and then incubated in DMEM solution without FBS for 48 hours in 96-well plates. After culture, the degree of proliferation of cells was measured by MTT assay.

As can be seen in Figure 4, Compound K of the present invention did not show typical toxicity to chondrocytes up to the concentration used in the above example, 5 μM, and even at 10 μM. Therefore, it can be seen that the effect of the compound K confirmed in the above example is not an effect due to toxicity but an actual effect, and also the human safety of the compound K of the present invention is excellent.

Example  7: Ginsenoside  Identify effects on NO production

In the present invention, whether ginsenosides other than compound K have an effect of inhibiting NO production induced by IL-1β was investigated in the same manner as in Example 5. Rb2, Rg1 and Rg3 were treated at concentrations of 25 μM, 100 μM and 200 μM, respectively. As can be seen in Figure 5, NO-1 induced by IL-1β showed a mode that is slightly inhibited by ginsenosides such as Rb2, Rg1 and Rg3. Therefore, it can be seen that ginsenosides such as Rb2, Rg1 and Rg3 are preferably converted to compound K, their metabolites, in order to exhibit the therapeutic effect of arthritis.

In addition, an experiment for inhibiting NO production was also performed for Rb1, another ginsenoside. As can be seen in Figure 6, the NO production inhibitory ability was hardly observed at low concentrations compound K is effective, but showed a mode of inhibiting NO production from 25 μM to some extent. Therefore, among ginsenosides, it can be seen that Rb1 has the best NO production inhibitory ability.

Example  8: Confirmation of Inhibition of Inducible Nitric Oxide Synthase

It was confirmed by Western blotting whether the NO production inhibitory effect confirmed in the above example was achieved by inhibiting the expression of inducible nitric oxide synthase (iNOS). Chondrocytes were plated in 6-wells at 5 × 10 ⁵ cells / well, then treated with IL-1β, Compound K and Rb1 at respective concentrations for 24 hours, lysed with Lysis buffer and only supernatants collected. Cell lysates (50 μg protein) were separated by 12% SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Blocking with 5% scheme milk and incubated for 1 hour in TBST (Tris-buffered saline containing 0.1 Tween 20), rabbit anti-iNOS polyclonal antibody was added and then incubated for 12 hours in TBST. After incubation for 2 hours with the addition of a secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG), it was visualized by an enhanced chemiluminescence assay (ECL).

As can be seen in Figure 7, it can be seen that compound K inhibits the expression of iNOS, and this inhibitory effect was clearly observed at 5 μM. On the other hand, ginsenoside Rb1 slightly suppressed iNOS expression at a high concentration of 200 μM.

Therefore, it can be seen that the excellent NO production inhibitory effect of the compound K is achieved by inhibiting the expression of iNOS.

Example  9: TIMP Impact on

The effects of the compounds of the present invention on the expression of TIMP (tissue inhibitor of metalloproteinase) known to antagonize MMPs, which are chondrogenic substrate enzymes, were investigated as follows. Experimental chondrocytes, except the control group, were treated with IL-1β at a concentration of 2 ng / ml, and at the same time, Compound K was added at 0, 0.1, 0.5, 1, 2 and 5 μM, respectively, and then the DMEM solution without FBS for 48 hours. Incubated at. After incubation, the culture solution was rolled to measure the concentration of TIMP-1. The TIMP-1 was measured using a commercially available ELISA kit (R & D), and the measurement was performed according to the protocol included in the product.

Example  10: MAP Kinase  Impact analysis

The effect of the compound of the present invention on the activation of mitogen activated protein kinase (MAP kinase), which is known to increase MMPs, the chondrocyte matrixase, and mediators that mediate chondrocyte apoptosis, was determined by Western blotting. The analysis was as follows. MAP kinases to be investigated include p38 mitogen activated protein kinase, extracellular signal-regulated protein kinase (ERK 1/2), and c-Jun N-terminal kinase (c-Jun N). -terminal kinase: JNK).

The chondrocytes were plated in 6-wells with 5 X 10 ⁵ cells / well, treated with IL-1β 2 ng / ml, Compound K 2 μM, 5 μM for 30 minutes, lysed with Lysis buffer and the supernatant Collected. Cell lysates (50 μg protein) were separated by 12% SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Blocked with 5% scheme milk and incubated for 1 hour in TBST (Tris-buffered saline containing 0.1 Tween 20), followed by the addition of rabbit anti-p38, phospho-p38, ERK, phospho-ERK, JNK and phospho-JNK antibodies Incubated for 12 hours in TBST. After incubation for 2 hours with the addition of a secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG), it was visualized by an enhanced chemiluminescence assay (ECL).

As can be seen in Figure 8, compound K effectively inhibits the activation of the MAP kinase family.

As described in detail above, the present invention provides a pharmaceutical composition for treating arthritis. Unlike the conventional therapeutic agent for arthritis, the pharmaceutical composition of the present invention inhibits the expression of (i) chondrocyte matrix lyases MMP-3 and MMP-13, which are known to cause arthritis development, and (ii) NO synthase. Inhibits the production of NO by interfering with (i) having a fundamental therapeutic mechanism by effectively blocking the increase of MMPs, the chondrocyte matrixase and activation of MAP kinase (ie phosphorylation) that mediates chondrocyte apoptosis. Because of this, it is very effective in treating arthritis. Moreover, since the active ingredient used in the pharmaceutical composition of the present invention exhibits the above-mentioned effects even at very low concentrations (μM level), it is very advantageous to finally develop into a medicine. In addition, the pharmaceutical composition of the present invention is very safe because it does not show toxicity to the living body as an active ingredient.

Claims (6)

(a) as the active ingredient, and then 20- O -β-D- glucopyranosyl -20 (S) of the formula - O protocol Panax diol (20- O -β-D-glucopyranosyl -20 (S) -protopanaxadiol) , or Therapeutically effective amount of a prodrug thereof; And (b) a pharmaceutically acceptable carrier, the pharmaceutical composition for treating arthritis: Formula 1

The pharmaceutical composition for treating arthritis according to claim 1, wherein the prodrug is saponin isolated from Panax ginseng. The pharmaceutical composition for treating arthritis according to claim 2, wherein the saponin is ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3. 4. The pharmaceutical composition for treating arthritis according to claim 3, wherein the saponin is ginsenoside Rb1. The pharmaceutical composition for treating arthritis according to claim 4, wherein the ginsenoside Rb1 is used. The pharmaceutical composition for treating arthritis according to claim 1, wherein the arthritis is osteoarthritis.

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