JP2014101329A - Antiinflammatory agents - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide novel antiinflammatory agents useful as antiinflammatory agents whose inflammatory cytokine expression inhibitory action and prostaglandin production inhibitory action are sufficiently strong.SOLUTION: An antiinflammatory agent comprises a methoxychalkone represented by the specified general formula as an active ingredient. (In the formula, Rand Reach represent a hydrogen atom or a methyl group.)

Description

  The present invention relates to a drug that suppresses inflammatory cytokine expression and prostaglandin production and has an anti-inflammatory effect.

At the time of invasion of pathogenic bacteria, inflammatory cytokines such as IL-1β and TNFα are produced and induce inflammatory mediators such as prostaglandin E ₂ (PGE ₂ ), due to increased vascular permeability and leukocyte infiltration, Growth of pathogenic bacteria is suppressed. Thus, while proper inflammatory response plays an important role in defense of the body, excessive inflammatory response results in damage to self-tissue, exacerbating fever, swelling, redness and pain.

  Steroidal anti-inflammatory drugs are known as drugs that suppress the production of inflammatory cytokines, but these drugs have high anti-inflammatory effects, but have side effects such as decreased immune function, opportunistic infection, and decreased adrenal function. For this reason, steroidal anti-inflammatory agents have a drawback that they cannot be used for a long time. In addition, non-steroidal anti-inflammatory agents often exhibit a mild anti-inflammatory action, and do not necessarily have a sufficient effect on all inflammations. Therefore, development of a material with few side effects and excellent anti-inflammatory action is demanded.

On the other hand, chalcone is a natural component contained in fruits, vegetables, tea, etc. Among chalcone and its methyl-substituted compounds, compounds having antibacterial properties, antibacterial properties, antitumor properties, anti-inflammatory properties, etc. are known. (Patent Documents 1 to 4, Non-Patent Document 1).
However, it has not been known so far that 3,4-dimethoxychalcone suppresses inflammatory cytokine expression and prostaglandin production and has an anti-inflammatory effect in chalcones.

JP 2008-120720 A International Publication No. 2007/052330 JP 2007-1898 A JP-T-2003-528842

European Journal of Medicinal Chemistry 42 (2007) 125-137

  The present invention relates to providing a novel anti-inflammatory agent that has a sufficiently strong inflammatory cytokine expression-suppressing effect and prostaglandin production-suppressing effect and is useful as an anti-inflammatory agent.

  The present inventors have shown that the methoxychalcone represented by the following general formula (1) has a prostaglandin production inhibitory action and an inflammatory cytokine expression inhibitory action, and further an ultraviolet-induced inflammatory cytokine expression inhibitory action, and an anti-inflammatory agent As found useful.

That is, the present invention relates to the following 1) to 3).
1) An anti-inflammatory agent containing methoxychalcone represented by the following general formula (1) as an active ingredient.
2) Inflammatory cytokine expression inhibitor containing methoxychalcone represented by the following general formula (1) as an active ingredient.
3) A prostaglandin production inhibitor containing methoxychalcone represented by the following general formula (1) as an active ingredient.

(In the above formula, R ¹ and R ² each represent a hydrogen atom or a methyl group.)

The anti-inflammatory agent, inflammatory cytokine expression inhibitor and prostaglandin production inhibitor of the present invention can suppress the expression of inflammatory cytokines such as TNFα and prostaglandin production such as IL-1β-induced PGE ₂ . As a result, it is effective in suppressing various skin inflammations such as dermatitis such as erythema and edema, contact dermatitis, atopic dermatitis, psoriasis caused by ultraviolet exposure to the skin. The anti-inflammatory agent, inflammatory cytokine expression inhibitor and prostaglandin production inhibitor of the present invention are not limited to dermatitis, bronchitis, asthma, allergic rhinitis, ventilation, nephritis, colitis, arthritis, periodontal It can be widely used for the prevention and treatment of diseases, hay fever, autoimmune diseases, chronic inflammation associated with obesity and the like.

It is a figure which shows the result of having measured IL-1 (beta) induction prostaglandin production suppression. It is a figure which shows the result of having measured IL-1 (beta) induction inflammatory cytokine expression suppression. It is a figure which shows the result of having measured UVB induction inflammatory cytokine expression suppression.

  As used herein, “non-therapeutic” is a concept that does not include medical practice, that is, does not include methods for surgery, treatment or diagnosis of humans.

The active ingredient of the anti-inflammatory agent, inflammatory cytokine expression inhibitor or prostaglandin production inhibitor of the present invention is methoxychalcone represented by the above general formula (1) (hereinafter also simply referred to as “methoxychalcone”). . R ¹ and R ² in the general formula (1) each represent a hydrogen atom or a methyl group, and preferably 4′-hydroxy-3,4,2 wherein R ¹ and R ² are methyl groups as shown below. ', 6'-tetramethoxy chalcone (formula (2), R ¹ is a methyl group, 4 R ² is a hydrogen atom', 6'-dihydroxy--3,4,2'- trimethoxy chalcone (formula (3) 2 ′, 4 ′, 6′-trihydroxy-3,4-dimethoxychalcone formula (4), wherein R ¹ and R ² are hydrogen atoms, preferably 4′-hydroxy-3,4,2 ', 6''-tetramethoxychalcone and 4', 6'-dihydroxy-3,4,2'-trimethoxychalcone. These compounds may be used alone or in combination of two or more. .

  The methoxychalcone used in the present invention is not particularly limited, but for example, a product obtained by acid hydrolysis of the sugar part of methyl hesperidin known as a food additive or the like, or a production method such as an aldol condensation reaction of acetophenones and benzaldehydes (Refer to Sakiyu, Nippon Kagaku Kagaku, 1958, 79 (6), 736-740).

As shown in Examples below, methoxychalcone has an IL-1β-induced PGE ₂ production inhibitory effect and an inflammatory cytokine expression inhibitory effect such as TNFα in an in vitro evaluation system. In particular, 4′-hydroxy-3,4,2 ′, 6′-tetramethoxychalcone and 4 ′, 6′-dihydroxy-3,4,2′-trimethoxychalcone strongly inhibits IL-1β-induced PGE ₂ production. It has an action and an inflammatory cytokine expression action.

Here, prostaglandin E ₂ (PGE ₂ ) enhances the pain-inducing action and vascular permeability enhancing action of bradykinin and inflammatory exudation by bradykinin and histamine through its production. Therefore, the inflammatory reaction can be suppressed by suppressing the production of prostaglandin E ₂ and suppressing the action of inflammatory cytokines such as TNFα.

  Therefore, the methoxy chalcone is used as a pharmaceutical, a quasi-drug, a cosmetic, a supplement, a food or a feed for suppressing various inflammations, for example, skin inflammation induced by UV exposure, or for blending with these. Useful as a material or formulation. The use can be in humans or non-human animals, or specimens derived therefrom, and can be therapeutic or non-therapeutic.

  That is, the methoxychalcone can be used as an anti-inflammatory agent, a prostaglandin production inhibitor, or an inflammatory cytokine production inhibitor, and can further be used to produce these agents.

  The dosage forms of the above pharmaceutical products (including quasi drugs or supplements) are tablets, capsules, granules, powders, syrups, intravenous injections, intramuscular injections, suppositories, inhalants, transdermal absorption agents, Eye drops, nasal drops, poultices, poultices, ointments, lotions, creams, tinctures, liniments, suspensions, lotions, bops, pastes, gels, sprays, aerosols or oils, oral preparations (dentifrices) , Liquid dentifrice, mouthwash, gingival massage cream, oral ointment, gargle tablets, troches, throat lozenges, etc.). As an administration form when used as a pharmaceutical, quasi-drug, or supplement, it can be used by any route such as oral administration, parenteral administration, or topical administration. The target site for inflammation may be any of skin, large intestine, joint, oral cavity, and other various organs.

To prepare pharmaceutical preparations of such various dosage forms, the above methoxychalcone alone or other pharmaceutically acceptable excipients, binders, extenders, disintegrants, surfactants, lubricants Swelling agent, dispersing agent, buffering agent, preservative, flavoring agent, flavoring agent, coating agent, carrier, diluent, osmotic pressure adjusting agent, fluidity promoter, absorption aid, pH adjusting agent, emulsifier, preservative, stable Agent, antioxidant, colorant, UV absorber, wetting agent, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, flavoring agent, flavoring agent, extender, surfactant, dispersant , Buffering agents, preservatives, fixing agents, fragrances, coating agents and the like can be used in appropriate combinations.
In addition, known medicinal ingredients can be appropriately mixed in the preparation. Examples of such components include vitamins such as vitamins C and E, amino acids and peptides and derivatives thereof, nucleic acids and derivatives thereof, saccharides and derivatives thereof, and other carotenoids, soybean isoflavones, catechins, and antioxidants such as chlorogenic acid. Etc.

  The content of methoxychalcone in the above preparation is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, based on the total mass of the preparation. And preferably it is 90 mass% or less, More preferably, it is 60 mass% or less. Preferably it is 0.001-90 mass%, More preferably, it is 0.01-60 mass%.

  When the above methoxychalcone is used as a pharmaceutical or in combination with a pharmaceutical, the dosage may vary according to the subject's condition, weight, sex, age or other factors, but per adult per oral administration The daily dose is usually preferably 0.001 g or more, particularly preferably 0.01 g or more, as methoxychalcone. And it is preferably 10 g or less, particularly preferably 3 g or less. Preferably it is 0.001-10g, Most preferably, it is 0.001-100g. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to divide once to several times a day, and to administer continuously for several weeks to several months.

  When the methoxychalcone is used as a quasi-drug or cosmetic, it can be in the form of an external preparation for skin, a cleaning agent, a makeup cosmetic, etc., depending on the method of use, lotion, emulsion, gel, cream, ointment It can be provided in various dosage forms such as agents, powders and granules. Such quasi-drugs and cosmetics in various dosage forms are formulated into quasi-drugs, skin cosmetics, cleansing agents, etc., as long as they do not affect the effects of the methoxychalcone alone. It can be adjusted by blending various components, medicinal components and the like. For example, various oils, resins, surfactants, gelling agents, preservatives, antioxidants, solvents, alcohol, water, chelating agents, thickeners, UV absorbers, emulsion stabilizers, pH adjusters, dyes, powders , Fragrances, solubilizers, cleaning agents, plant extracts, moisturizing ingredients such as sodium hyaluronate, skin antiaging agents, whitening agents, and the like.

  In the above preparation, the content of methoxychalcone in the total amount is usually preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, and further preferably 0.01% by mass or more. And preferably it is 20 mass% or less, More preferably, it is 10 mass% or less, More preferably, it is 5 mass% or less. Moreover, Preferably it is 0.0001-20 mass%, More preferably, it is 0.001-10 mass%, More preferably, it is 0.01-5 mass%.

  Various foods containing the methoxychalcone include, in addition to general foods and drinks, skin such as erythema and edema of the skin, wrinkles and sagging, spots and freckles, skin elasticity, etc. Functional foods such as beauty foods, foods for the sick, functional nutritional foods or foods for specified health use are included as necessary, with the concept of effects associated with anti-inflammation, such as reducing or suppressing damage to foods. The

  The form of the food product can be solid, semi-solid or liquid. Examples of food include confectionery such as breads, noodles, cookies, jelly, dairy products, frozen foods, instant foods, processed starch products, processed meat products, other processed foods, coffee beverages, soups, Examples include seasonings, dietary supplements, and the like, and raw materials thereof. Further, like the above-mentioned preparation for oral administration, it may be in tablet form, pill form, capsule form, liquid form, syrup form, powder form, granule form and the like.

  Such foods include methoxychalcone, other food and drink materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, colorants, UV absorbers, antioxidants, moisturizers, A sticking agent, a sticking agent, a dispersing agent, a wetting agent and the like can be blended and combined as appropriate.

In addition, various vitamins, amino acids and peptides and derivatives thereof, nucleic acids and derivatives thereof, saccharides and derivatives thereof, carotenoids, soybean isoflavones, catechins, antioxidant components such as chlorogenic acid, and the like can be appropriately blended.
The content of methoxychalcone in the food or drink varies depending on the use form, but is usually preferably 0.0001% by mass or more, and more preferably 0.001% by mass or more. And preferably it is 50 mass% or less, More preferably, it is 10 mass% or less. Moreover, Preferably it is 0.0001-50 mass%, More preferably, it is 0.001-10 mass%.

  Examples of the feed include feed for small animals used for rabbits, rats, mice and the like, feed for pet foods used for dogs, cats, small birds, squirrels and the like, and can be prepared in the same form as the above foods.

  In general, the content of the methoxychalcone in the food or feed is preferably 0.0001% by mass or more, more preferably 0.001% by mass or more. And preferably it is 10 mass% or less, More preferably, it is 1 mass% or less. Preferably it is 0.0001-10 mass%, More preferably, it is 0.001-mass%.

With respect to the above-described embodiment, the following aspects are disclosed in the present invention.
<1> An anti-inflammatory agent comprising methoxychalcone represented by the general formula (1) as an active ingredient.
<2> An inflammatory cytokine production inhibitor comprising methoxychalcone represented by the general formula (1) as an active ingredient.
<3> A prostaglandin production inhibitor containing methoxychalcone represented by the general formula (1) as an active ingredient.
<4> The agent according to any one of <1> to <3>, which reduces or suppresses inflammation induced by ultraviolet exposure.
<5> Use of methoxychalcone represented by the above general formula (1) for producing an anti-inflammatory agent.
<6> Use of methoxychalcone represented by the above general formula (1) for producing an inflammatory cytokine production inhibitor.
<7> Use of methoxychalcone represented by the above general formula (1) for producing a prostaglandin production inhibitor.
<8> A methoxychalcone represented by the above general formula (1) for use in anti-inflammation.
<9> A methoxychalcone represented by the above general formula (1) for use in suppressing inflammatory cytokine production.
<10> A methoxychalcone represented by the above general formula (1) for use in inhibiting prostaglandin production.
<11> The use according to any one of <8> to <10>, which is a non-therapeutic use.
<12> An anti-inflammatory method in which the methoxychalcone represented by the general formula (1) is administered to a human or an animal.
<13> A method for suppressing inflammatory cytokine production, wherein the methoxychalcone represented by the general formula (1) is administered to a human or an animal.
<14> A method for inhibiting prostaglandin production, comprising administering the methoxychalcone represented by the general formula (1) to a human or an animal.
<15> The method according to any one of <12> to <14>, wherein the inflammation induced by ultraviolet exposure is reduced or suppressed.
<16> The method according to any one of <12> to <15>, which is a non-therapeutic method.

Example 1
(Preparation of methoxychalcone)
Each methoxychalcone was prepared using the above-mentioned production method (see Sakiba, Nippon Kagaku Kagaku, 1958, 79 (6), 736-740).

(I) Preparation of 4'-hydroxy-3,4,2 ', 6'-tetramethoxychalcone and 4', 6'-dihydroxy-3,4,2'-trimethoxychalcone Methyl hesperidin (food additive grade) 5.0 g (trade name, manufactured by Alps Pharmaceutical Co., Ltd.) was dissolved in 250 mL of 50% EtOH, 15 mL of concentrated hydrochloric acid was added, and the mixture was heated to reflux for 16 hours. After cooling, the mixture was neutralized with an aqueous sodium hydroxide solution. Ethanol was distilled off with an evaporator, followed by extraction with ethyl acetate, washing, drying, filtration and concentration to obtain a crude product. This was fractionated by silica gel column chromatography (hexane-ethyl acetate-methanol). Furthermore, the objective fraction was recrystallized, and 156 mg of 4′-hydroxy-3,4,2 ′, 6′-tetramethoxychalcone (methoxychalcone 1) and 4 ′, 6′-dihydroxy-3,4,2 112 mg of '-trimethoxychalcone (methoxychalcone 2) was isolated.

(Ii) Preparation of 2 ′, 4 ′, 6′-trihydroxy-3,4-dimethoxychalcone 2.0 g of 2 ′, 4 ′, 6′-trihydroxyacetophenone monohydrate was dissolved in 50 mL of THF, and 0 Cooled to ° C. Thereto were added 19 mL of diisopropylethylamine and 3.3 mL of chloromethyl methyl ether, and the mixture was stirred at room temperature for 9.5 hours. After cooling again, water was added and the organic layer was extracted with ethyl acetate, washed, filtered and concentrated to obtain a crude product. This was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain 1.7 g of 2′-hydroxy-4 ′, 6′-bismethoxymethoxyacetophenone.
1 g of 2′-hydroxy-4 ′, 6′-bismethoxymethoxyacetophenone obtained above was dissolved in 20 mL of methanol, and 728 mg of veratraldehyde and 2.9 g of potassium hydroxide were dissolved in 2 mL of water. In addition, the mixture was stirred at room temperature for 55 hours. Thereafter, it was neutralized with hydrochloric acid, extracted with ethyl acetate, washed and dried to obtain a crude product. This was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain 1.1 g of 6′-hydroxy-3,4-dimethoxy-2 ′, 4′-bismethoxymethoxychalcone.
1.1 g of 6′-hydroxy-3,4-dimethoxy-2 ′, 4′-bismethoxymethoxychalcone was dissolved in 60 mL of methanol, 30 mL of 1M hydrochloric acid was added, and the mixture was heated to reflux for 50 minutes. After cooling, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed and dried to obtain a crude product. This was purified by silica gel column chromatography (hexane-ethyl acetate) to obtain 585 mg of 2 ′, 4 ′, 6′-trihydroxy-3,4-dimethoxychalcone (methoxychalcone 3).

Test Example 1: Inhibitory effect on IL-1β-induced prostaglandin production Normal human neonatal foreskin keratinocytes (NHEK) (KURABO) were seeded on a 12-well plate and, when subconfluent, did not contain growth factors (−) medium Instead, the experiment was conducted the next day. The medium was replaced with a (−) medium prepared so that the final concentration of the sample was 20 μM, and pretreated for 16 hours. Thereafter, IL-1β was added so that the final concentration was 1 ng / mL, and the cell supernatant was recovered after 24 hours. The PGE ₂ content in the cell supernatant was determined by PGE ₂ EIA Kit (Cayman). The result is shown in FIG. DMSO was used as a control.

From the result of FIG. 1, PGE ₂ production was induced by IL-1β stimulation, but its suppression was recognized by methoxychalcone treatment. In particular, methoxychalcone 1 and methoxychalcone 2 strongly showed PGE ₂ production inhibitory action.

Test Example 2: IL-1β-Induced Inflammatory Cytokine Expression Inhibitory Effect Normal human neonatal foreskin epidermal keratinocytes (NHEK) (KURABO) were seeded on a 12-well plate, and did not contain growth factors when sub-confluent (−) medium Instead, the experiment was conducted the next day. The medium was replaced with a (−) medium prepared so that the final concentration of the sample was 20 μM, and pretreated for 16 hours. Thereafter, IL-1β was added to a final concentration of 1 ng / mL, and the cells were collected after 4 hours. RNA was extracted from the cells using RNeasy Mini Kit (QIAGEN), 500 μg of cell-extracted RNA was synthesized with High Capacity RNA-to-cDNA Kit (Applied Biosystems), and 20 ng equivalent of total RNA was subjected to real time PCR. IL-1β-induced suppression of inflammatory cytokine expression was measured. The result is shown in FIG. IL1B (Hs01555510_m1) and TNF (Hs00174128_m1) were used as TaqMan Gene Expression Assay (Applied Biosystems) probes that specifically detect the target gene. The target gene expression level was corrected by the expression level of the internal standard gene RPLP0 (Hs99999992_m1). For the analysis, 7500 Real Time PCR System (Applied Biosystems) was used. DMSO was used as a control.

  From the results shown in FIG. 2, IL-1β and TNFα expression was enhanced by IL-1β stimulation, and the expression of methoxychalcone 1 and methoxychalcone 2 was strongly suppressed.

Test Example 3: Inhibitory effect on UVB-induced inflammatory cytokine expression Normal human neonatal foreskin keratinocytes (NHEK) (KURABO) were seeded on a 12-well plate and replaced with a growth medium containing no growth factor when sub-confluent. The experiment was conducted the next day. The medium was replaced with a (−) medium prepared so that the final concentration of the sample was 20 μM, and pretreated for 16 hours. The medium was replaced with PBS, irradiated with UVB (20 mJ; dose of 1 mW / cm ² for 20 seconds), replaced with the medium containing the specimen again, and cells were collected after 8 hours. RNA was extracted from the cells using RNeasy Mini Kit (QIAGEN), 500 μg of cell-extracted RNA was synthesized with High Capacity RNA-to-cDNA Kit (Applied Biosystems), and 20 ng of total RNA was subjected to real time PCR. Inhibition of induced inflammatory cytokine expression was measured. The result is shown in FIG. IL1B (Hs01555510_m1) and TNF (Hs00174128_m1) were used as TaqMan Gene Expression Assay (Applied Biosystems) probes that specifically detect the target gene. The target gene expression level was corrected by the expression level of the internal standard gene ACTB (Hs99999999_m1). For the analysis, 7500 Real Time PCR System (Applied Biosystems) was used. DMSO was used as a control.

  From the results shown in FIG. 3, expression of IL-1β and TNFα was increased by UVB stimulation, and its expression was suppressed by methoxychalcone 1 and methoxychalcone 2 treatment. This revealed that methoxychalcone 1 and methoxychalcone 2 have particularly strong anti-inflammatory effects.

Example 2
Formulation Example 1: Preparation of Tablet A tablet having a diameter of 9 mm and a weight of 200 mg can be prepared by mixing the following components and following a conventional method.

Formulation example 2: Preparation of capsules The following components can be mixed, and capsules can be prepared according to a conventional method.

Formulation Example 3: Preparation of Granule A granule can be prepared according to a conventional method by mixing the following components.

Formulation Example 4: Preparation of cream A cream can be prepared according to a conventional method by mixing the following components.

Formulation Example 5: Preparation of ointment The following ingredients are mixed, and an ointment can be prepared according to a conventional method.

Claims (6)

An anti-inflammatory agent comprising methoxychalcone represented by the following general formula as an active ingredient.
(In the above formula, R ¹ and R ² each represent a hydrogen atom or a methyl group.)   The methoxy chalcone is 4'-hydroxy-3,4,2 ', 6'-tetramethoxy chalcone or 4', 6'-dihydroxy-3,4,2'-trimethoxy chalcone according to claim 1. Anti-inflammatory agent. An inflammatory cytokine expression inhibitor comprising methoxychalcone represented by the following general formula as an active ingredient.
(In the above formula, R ¹ and R ² each represent a hydrogen atom or a methyl group.)   The methoxy chalcone is 4'-hydroxy-3,4,2 ', 6'-tetramethoxy chalcone or 4', 6'-dihydroxy-3,4,2'-trimethoxy chalcone according to claim 3. Inflammatory cytokine expression inhibitor. A prostaglandin production inhibitor containing methoxychalcone represented by the following general formula as an active ingredient.
(In the above formula, R ¹ and R ² each represent a hydrogen atom or a methyl group.)   The methoxy chalcone is 4'-hydroxy-3,4,2 ', 6'-tetramethoxy chalcone or 4', 6'-dihydroxy-3,4,2'-trimethoxy chalcone according to claim 5. Prostaglandin production inhibitor.