KR20050055856A - Ap-1 inhibitor comprising costunolide - Google Patents

Abstract

The present invention relates to costunolide, a sesquiterpene lactone compound extracted from the root of Saussurea lappa Clarke, which can be used as an AP-1 inhibitor for treating inflammatory or cancer diseases. In addition, the costunolide of the present invention not only inhibits the expression of inflammation-related factors such as IL-1β, but also inhibits the expression of inflammation-related factors, cell proliferation and natural death of AP-1, a transcriptional regulator that plays an important role in natural death. Since it significantly inhibits activity, it can be usefully used as an AP-1 inhibitor for the treatment of inflammation-related diseases or cancer diseases.

Description

AP-1 inhibitor comprising costunolide

The present invention relates to costunolide, a sesquiterpene lactone compound extracted from the root of Saussurea lappa Clarke, which can be used as an AP-1 inhibitor for treating inflammatory or cancer diseases. will be.

Sesquiterpene lactone is a compound present in many plants belonging to the family Asteraceae and Magnolia and is known to have anticancer activity. Costunolide is a sesquiterpene lactone-based compound that is anticancer (Mori H. et. Al., Cancer Lett., 83 (1-2) , pp171-175, 1994), antiviral activity (Chen HC et. Al. , Antiviral Res. , 27 (1-2) , pp99-109, 1995) and antifungal action (Wedge DE et. Al., Phytochemistry, 53 (7) , pp747-757, 2000; Barrero AF et. Al . , Fitoterapia , 71 (1) , pp60-64, 2000), and also inhibited the killing activity of cytotoxic T lymphocytes and NO production of macrophage. Immunosuppressive effects are also known (Taniguchi M. et. Al., Biosci. Biotechnol. Biochem. , 59 (1) , pp2064-2067, 1995; Matsuda H. et. Al., Bioorg. Med. Chem. , 11 (5) , pp 709-715, 2003).

Inflammatory reactions are associated with various mediators and immune cells of local blood vessels and body fluids when infected with tissues (cells) or external infectious agents (bacteria, fungi, viruses, and various types of allergens). It has a series of complex physiological reactions, including substance secretion, fluid infiltration, cell migration, and tissue destruction, as well as external symptoms such as erythema, edema, fever, and pain. In normal cases, inflammatory reactions restore the function of life by removing external infectious agents and regenerating damaged tissues.However, if the inflammatory response is excessive or persistent due to the absence of antigens or internal substances, the major pathology of the disease (sensitizing disease) , Chronic inflammation) and also become a disorder in the treatment process such as blood transfusion, drug administration, and organ transplantation.

The development of many inflammation-related diseases involves the activation of macrophages and thus the overproduction of inflammation-related factors. Representative inflammation-related factors include nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and prostaglandin E ₂ (PGE ₂ ). They may be involved in the treatment of cells that cause inflammation and infection and abnormalities (eg cancer cells), but they may also destroy tissues in vivo if excessively produced.

Multifunctional cytokines such as TNF-α are not only expressed in normal tissues but also increased in the course of lesions, and play an important role in skin inflammation, particularly during cancer promotion. TNF-α has been reported to be associated with inflammatory skin diseases in humans (Verhoef, J. et al., J. Antimicrob. Chemother., 35 , 1996). Treatment with TNF-α for various inflammatory diseases and allergic symptoms was alleviated. TNF-α also acts as an endogenous cancer promoter by activating neutrophils and increasing hydrogen peroxide production. Therefore, pro-inflammatory molecules (pro) can be inhibited by inhibiting the expression of the cytokine TNF-α, which plays a pivotal role in the inflammatory stage closely related to the oncogenic stage, or inhibiting the production of PGE ₂ due to COX-2 activity inhibition. It is likely that the lesion process accompanied by an increase in inflammatory molecules could be controlled. When treated with antibiotics under bacterial infection, LPS released from the extracellular membranes killing bacteria causes endotoxin shock and, when severely destroyed by this process, does not neutralize the continued production of LPS (Dunn, DL, Surg. Clin.North.Am ., 74 , 1994: Giroir, BP, Crit.Care Med., 21 , 1993: Yamaguchi, Y. et al., J. Reticuloendothel. Soc., 409 , 1982). Diseases caused by excessive production of inflammation-related factors include autoimmune diabetes, such as autoimmune diabetes and rheumatoid arthritis, and allergic inflammatory diseases such as asthma and atopic dermatitis, as well as tissue transplant rejection and sepsis. . Therefore, when such abnormalities occur, there is a need for an effective anti-inflammatory agent that can suppress the excessive production of inflammatory factors.

Conventionally, the development of therapeutic drugs that inhibit the function of enzymes or receptors that are proteins for many diseases, for example, in the treatment of inflammatory diseases including chronic rheumatoid arthritis, cyclooxygenase that synthesizes prostaglandins from arachidonic acid Many nonsteroidal anti-inflammatory agents similar to indomethacin have been developed, targeting 5-lipoxygenase, which synthesizes leukotrienes ( Journal of Pharmaceutical Science , 73 , pp 579-589, 1984). In addition, interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α), known as inflammatory cytokines, are attracting attention as factors for amplifying and exacerbating inflammatory reactions. For example, monoclonal antibodies and small molecule cytokine production inhibitors (Ann. Rep., Med. Chem., 27 , pp209-218, 1992) have been developed for each protein.

However, in diseases caused by quantitative abnormalities of functional protein molecules present in cells or on cell membranes or in cells, the expression level is normalized by suppressing the transcriptional amount of functional molecule genes rather than inhibiting the activity of the functional molecules. It is regarded as a treatment in the real sense, and in the case of autoimmune disease or chronic inflammatory disease including chronic rheumatoid arthritis, in addition to the above-mentioned inflammatory cytokine group or protein mediator derived from arachidonic acid, cell adhesion molecule group or matrix Quantitative abnormalities of many functional proteins, such as the group of metaproteases, are known to be involved (N. Engl., J. Med ., 322 , pp1277-1289, 1990). Although gene expression and protein production of these functional proteins are regulated by a plurality of transcription factors, it is known that a binding sequence (TRE array) of transcription factor AP-1 is commonly present in the promoter region of many genes. It is also reported that the expression of some functional proteins is regulated by the binding of AP-1 to the promoter region.

Activating protein-1 (AP-1) is a transcriptional regulator that plays an important role in the expression of various genes. Inhibition of AP-1 is known to lead to inhibition of proliferation in tumor cells. In addition, AP-1 is known to be involved in the expression of various immune-related genes, in particular it is known to play an important role in the expression of inflammation-related factors.

Accordingly, the present inventors found that the costuolide of the present invention, a sesquiterpene lactone compound extracted from the root of Asteraceae, plays an important role in AP-1, a transcriptional regulator that plays an important role in the expression of inflammation-related factors, cell proliferation and natural death. By confirming that significantly inhibits, the present invention was completed.

It is an object of the present invention to provide a novel use of costunolide as an AP-1 inhibitor for the treatment of inflammatory or cancer diseases. The present invention relates to inflammation in a macrophage model using Costunolide by verifying the inhibitory capacity of the production of IL-1β as a regulator and evaluating the effects on the activities of MARK and AP-1, and identifying the pharmacological mechanism of costunolide against inflammatory and cancer diseases To provide an AP-1 inhibitor for the treatment of inflammatory disease or cancer disease containing as an active ingredient.

In order to achieve the above object, the present invention provides an AP-1 inhibitor for the treatment of inflammatory diseases or cancer diseases containing costunolide as an active ingredient.

The costunolide is commercially available or includes extracts from the roots or leaves of Dandelion, Saussurea lappa Clarke.

In addition, the present invention is AP-1 for the treatment of inflammatory diseases or cancer diseases, characterized in that it is formulated or used in combination with other active substances, such as costunolide and bronchodilators, antihistamines, anti-inflammatory drugs, anticancer agents and antibiotics Provide inhibitors.

The bronchodilators include terbutaline sulfate, salbutamol, orciprenaline sulfate and ipratropium bromide, and acristatin as an antihistamine. , Ebastine, ketotifen and fexofenadine, anti-inflammatory drugs, acetaminophen, ketoprofen, naproxen, ibuprofen, nabumetone ), And indomethacin, and the like, and anticancer agents include cyclophosphamide, cisplatin, ifosfamide, chlorambucil, cytarabine, and fluorine. Uracil (fluouracil), mecaptopurine, methotrexate, vinblastine, vincristine and vindesine, etc. Include Lin (penicillin), vancomycin (vancomycin), carried on azithromycin (clindamycin), cephalosporins (cephalosporin) and quinolone (quinolones) and the like.

Costunolide of the present invention has little toxicity and side effects, so can be used safely even for long-term use for treatment and prevention purposes.

The pharmaceutical composition as an AP-1 inhibitor of the present invention comprises the costunolide in an amount of 0.02 to 90% by weight based on the total weight of the composition.

In addition, the AP-1 inhibitor for the treatment of inflammatory diseases or cancer diseases, including the costunlide of the present invention may further include excipients commonly used in the art to which the present invention belongs.

AP-1 inhibitors comprising the costunolide of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, and together with methods known in the art of pharmacy, Pharmaceutically acceptable pharmaceutical preparations, for example liquids, powders, granules, tablets, capsules, suspensions, emulsions, syrups, liquid formulations, oral formulations such as aerosols, external preparations such as coating ointments, suppositories, and sterilization Carriers, excipients and diluents which may be formulated in the form of injectable solutions and may be included in the composition comprising costunolide include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrole Reddon, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Costunolide of the present invention is appropriately selected according to the absorption of the active ingredient in the body, the rate of excretion, the age and weight of the patient, sex and condition, the severity of the disease to be treated, but generally 0.001% to 30% in adults It can be administered once or several times a day with a solvent containing an active ingredient, and the amount can be increased or decreased depending on the sex, age and degree of disease of the patient. In addition, the dosage of costunolide may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

Hereinafter, the present invention will be described in detail by the following experimental examples.

However, the following experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following experimental examples.

Experimental Example 1. Inhibitory effect of costunolide on AP-1 transcriptional activity

The effect of costunolide (Wako Pure Chemical Industries, Ltd.) on the transcriptional activity of AP-1 was measured by transient transfection and CAT reporter gene assay. After transfection of the AP-1 reporter vector to the mouse macrophage RAW 264.7 using the DEAE-dextran method, it was treated with costunolide and LPS for 24 hours. The protein was then extracted. The effect of costunolide on AP-1 transcriptional activity was measured by measuring the amount of CAT enzyme expressed from the extracted protein samples using an ELISA kit.

As a result, it was confirmed that costunolide significantly inhibits the activity of AP-1.

Experimental Example 2 Inhibitory Effect of Costunolide on AP-1 DNA Binding

The effect of costunolide on AP-1, a transcriptional regulator known to play an important role in the expression of inflammation-related factors, was measured by gel shift assay. After treatment with costunolide and LPS in RAW 264.7, the nuclear extract was isolated, and then the nuclear extract was reacted with an oligonucleotide labeled with radioisotopes, followed by a nondenaturing polyacrylamide gel. Was separated by electrophoresis, transferred to Whatman 3M cellulose paper, and then photosensitive with X-ray film.

As a result, as shown in FIG. 2, the DNA binding of AP-1 induced by LPS was confirmed to decrease in concentration-dependent manner by costunolide. Therefore, it was confirmed that the inhibition of AP-1 transcriptional activity by costunolide was achieved by inhibiting the DNA binding of AP-1.

Experimental Example 3 Inhibitory Effect of Costunolide on Phosphorylation of MAPK

Mitogen Activated Protein Kinase (MAPK) is known as a kinase that plays an important role in the activity of AP-1. Therefore, MAPK activity can be used to investigate the mechanism of the inhibitory effect of COTSUNOLIDE on AP-1 activity. The effect of costunolide on was determined. After treatment with costunolide and LPS in RAW 264.7, the protein was separated after 30 minutes, and the degree of phosphorylation of MAPK was measured by western blotting.

As a result, as shown in Figure 3, the costunolide inhibited the phosphorylation of SAPK / JNK and p38 MAPK, it was confirmed that also inhibits the phosphorylation of ERK at high concentrations. Therefore, the inhibitory effect of costunolide on the transcriptional activity of AP-1 was confirmed by inhibiting the MAPK activity by inhibiting the phosphorylation of MAPK.

Experimental Example 4 Inhibitory Effect of Costunolide on the Expression of IL-1β

Since AP-1 is known to be involved in the expression of various inflammation-related factors, the effect of costunolide on the expression of IL-1β, an important inflammation-related factor, was verified. The supernatant was obtained 24 hours after treatment with costunolide 1 hour before inducing activation of macrophage RAW 264.7 using LPS. The amount of IL-1β was measured using an ELISA kit, respectively. In addition, RNA was isolated 6 hours after the treatment of costunolide and LPS in RAW 264.7 to determine the role of costunolide on the expression of IL-1β mRNA. Thereafter, the expression of IL-1β was measured using RT-PCR.

As a result, as shown in Figures 4a and 4b it was confirmed that costunolide significantly inhibits the increase of IL-1β expression by LPS.

Examples of the formulation of the composition are described below, but are not intended to limit the present invention but to explain in detail only.

Formulation Example 1 Preparation of Injection

Costunolide 100 mg

Sodium Metabisulfite 3.0 mg

Methylparaben 0.8 mg

Propylparaben 0.1 mg

Appropriate sterile distilled water for injection

The above ingredients are mixed and made into 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

Costunolide 200 mg

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 3 Preparation of Capsule

Costunolide 100 mg

Lactose 50 mg

Starch 50 mg

Talc 2 mg

Magnesium stearate proper amount

Capsules are prepared by mixing the above ingredients and filling into gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 4 Preparation of Liquid

Costunolide 1000 mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 1000 ml. According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 5 Combination with Other Active Ingredients (Preparation of Tablets)

Costunolide 20 mg

Ketoprofen 200 mg

Lactose 100 mg

Starch 100 mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 6 Preparation of Healthy Food

Costunolide 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

Costunolide 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is mixed with a relatively suitable component for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the costunolide of the present invention not only inhibits expression of inflammation-related factors such as IL-1β, but also AP, a transcriptional regulator that plays an important role in expression of inflammation-related factors, cell proliferation and natural death. Since it significantly inhibits the activity of -1, it can be usefully used as an AP-1 inhibitor for the treatment of inflammatory or cancer diseases.

1 is a diagram showing the effect of costunolide on AP-1 transcriptional activity,

2 is a diagram showing the inhibitory effect of costunolide on the DNA binding of AP-1,

3 is a diagram showing the degree of inhibition of SAPK / JNK and p38 MAPK phosphorylation of costunolide,

Figure 4a is a diagram showing the inhibitory effect of COTSUNOLIDE IL-1β expression by,

Figure 4b is a diagram measuring the expression level of IL-1β using RT-PCR.

Claims (4)

AP-1 inhibitors for the treatment of inflammatory diseases or cancer diseases containing costunolide as an active ingredient. The AP-1 inhibitor according to claim 1, wherein the costunolide is commercially available or extracted from the root or leaves of Dandelion, Saussurea lappa Clarke. An AP-1 inhibitor for the treatment of inflammatory or cancer diseases, characterized in that it is formulated or used in combination with other active substances such as costunolide and bronchodilators, antihistamines, anti-inflammatory analgesics, anticancer agents and antibiotics. The AP-1 inhibitor according to any one of claims 1 to 3, comprising 0.02 to 90% by weight of costunolide of claim 1 relative to the total weight of the composition.