JP6985135B2 - Regulatory T cell differentiation inhibitor and immunomodulatory composition - Google Patents

Description

The present invention relates to a regulatory T cell differentiation inhibitor and an immunomodulatory composition.

The body's immune system protects the body from a wide variety of pathogens. Helper T cells play the role of the control tower. Helper T cells are born in the thymus, go out of the thymus (periphery), circulate in the body, and are divided into three types of effector T cells, Th1, Th2, and Th17, depending on the type of pathogen that has invaded the body. And induces the optimal immune response to eliminate each pathogen. Further, among the helper T cells, there are T cells (regulatory T cells: Treg) that suppress the effector T cells and appropriately control the immune response. Regulatory T cells suppress the immune response by various mechanisms and play an important role in maintaining homeostasis of the living body. Therefore, abnormalities (decreased) of regulatory T cells lead to excessive activation of effector T cells, leading to autoimmune diseases and allergic diseases. That is, it can be said that an autoimmune disease such as rheumatism, an inflammatory disease such as atopic dermatitis, and an allergic disease such as pollinosis are a state of hyperimmunity or a state in which immune tolerance is broken. Therefore, the enhancement of regulatory T cells is considered to be effective in the treatment of the above-mentioned diseases.

On the other hand, it is known that in cancer tissues, the proportion of regulatory T cells is increasing, resulting in an immune-unresponsive state in which the immune response is stopped (Non-Patent Documents 1 and 2). That is, in the body of a cancer patient, immunity is suppressed by an abnormally increased number of regulatory T cells, and the immune system in the body does not function normally. Therefore, if the abnormally increased regulatory T cells in the body of a cancer patient can be reduced, that is, if the differentiation of regulatory T cells can be suppressed, the immunosuppressive environment is improved and immunity functions normally. However, it is thought that it can suppress infections such as viruses, bacteria, and parasites, and can be expected to have anticancer effects.

Nivolumab is known as a drug developed with this concept. Nivolumab is a drug that improves the immunosuppressive environment by blocking PD-1, which is an immunosuppressive factor, with a specific antibody (anti-PD-1 antibody), and activates autoimmunity to exert anticancer effects. Is. In addition, a drug aimed at antitumor effect by killing CCR4 antibody-positive regulatory T cells and suppressing their action (mogamulizumab: anti-CCR4 antibody) (drug for CCR4-positive adult T-cell leukemia / lymphoma) is also known. There is.

Wada J., et al., Anticancer Res., 28, 2401-2408, 2008 Wada J., et al., Anticancer Res., 30, 3747-3757, 2010

It is an object of the present invention to provide an immunomodulatory composition which has an action of suppressing the differentiation of regulatory T cells and is useful as an immunostimulatory composition.

As a result of diligent studies to solve the above problems, the compound represented by the following formula (I) (hereinafter, also simply referred to as "Compound I") has an action of suppressing the differentiation of regulatory T cells, that is, It has been found that it has an effect of suppressing the proliferation of regulatory T cells. Therefore, by suppressing the function of regulatory T cells in the body using the compound I, the suppressed immune response can be activated, and as a result, infection suppression of viruses, bacteria, parasites, etc., and cancer prevention can be achieved. We have completed the present invention with the conviction that it can be effectively effective in preventing recurrence.

（Ｉ−２）上記（Ｉ）で示される化合物が、下式（II）または（III）で示される立体異性体のいずれか少なくとも１つである、（Ｉ−１）に記載する制御性Ｔ細胞分化抑制剤：

The present invention has the following embodiments.
(I) Regulatory T cell differentiation inhibitor (I-1) A regulatory T cell differentiation inhibitor containing the compound represented by the following formula (I), a salt thereof, or a solvate thereof as an active ingredient:

(I-2) The regulatory T according to (I-1), wherein the compound represented by the above (I) is at least one of the stereoisomers represented by the following formula (II) or (III). Cell differentiation inhibitor:

(II) Immunoregulatory Composition (II-1) An immunomodulatory composition containing the regulatory T cell differentiation inhibitor according to (I-1) or (I-2) above.
Here, the description "regulatory T cell differentiation inhibitor described in (I-1) or (I-2)" refers to "a compound represented by the formula (I) described in (I-1), the same thereof. It can be paraphrased as "at least one of a salt or a solvate thereof, or a stereoisomer represented by the formula (II) or (III) described in (I-2)" (in the corresponding claim 2). The same). That is, the immunomodulatory composition contains at least one of compound (I) or compounds (II) and (III) as an active ingredient regardless of whether or not the composition is used as a regulatory T cell differentiation inhibitor. Contains the composition contained as.
(II-2) The composition for immunomodulation according to (II-1), which has an immunostimulatory action. That is, the immunomodulatory composition of the present invention can be said to be an immunostimulatory composition.
(II-3) The immunomodulatory composition according to (II-1) or (II-2), which is a food or drink, a pharmaceutical product, a quasi drug, a cosmetic product, or a feed.

Compound I, which is an active ingredient of the regulatory T cell differentiation inhibitor and the immunomodulatory composition of the present invention, is a novel compound not published in the literature. Therefore, the present invention relates to a novel compound I as a regulatory T cell differentiation inhibitor and an active ingredient of an immunomodulatory composition, particularly an immunostimulatory composition, based on its regulatory T cell differentiation inhibitory action. It provides new uses for. That is, according to the present invention, it is possible to provide a new immunomodulatory composition containing compound I as an active ingredient, particularly an immunostimulatory composition. Further, the immunomodulatory composition can be widely used as a food or drink, a pharmaceutical product, a quasi drug, a cosmetic product, or a feed.

It is a figure which shows the processing process used when the compound (I) of this invention was isolated and prepared from a plant in Example 1. FIG. The UV spectrum of the peak fraction having a retention time of 27 minutes of the above HPLC chromatogram is shown. ^{1 1} H-NMR of the compound of this invention is shown. ^{The 13} C-NMR of the compound of this invention is shown. The HMQC analysis result of the compound of this invention is shown. Shows the ¹ H- ¹ H COZY analysis results of the compounds of the present invention. The HMBC analysis result of the compound of this invention is shown. Shows the structure of a compound of the invention were assigned from an analysis result such as ¹ H- ¹ H COZY and HMBC correlations. The results of confirming the coordination of the OH groups at the 6'and 8'positions of the compound of the present invention by the correlation of NOESY are shown. This is a result showing that the compound I (1 μg / ml, 5 μg / ml, 10 μg / ml) of the present invention has a regulatory T cell differentiation inhibitory effect in a concentration-dependent manner (Example 2). The results of culturing epidermal cells in the presence or absence (control) of the compound of the present invention (1 μg / ml, 10 μg / ml) and measuring the expression level of profilaggrin mRNA are shown.

(I) Active ingredient of regulatory T cell differentiation inhibitor and immunomodulatory composition The regulatory T cell differentiation inhibitor and immunomodulatory composition of the present invention are the compound represented by the following formula (I), a salt thereof, and the like. In addition, they contain solvates as active ingredients:

The compound represented by the above formula (I) (hereinafter, also referred to as “Compound I”) includes the stereoisomers represented by the following formulas (II) and (III) (hereinafter, “Compound II” and “Compound III”, respectively. Also referred to as). Hereinafter, in the present specification, the term "Compound I" is used as a generic term for Compound II and Compound III.

The regulatory T cell differentiation inhibitor and immunomodulatory composition of the present invention may contain either of the above compounds II or III as an active ingredient, or both compounds may be contained in a mixed state. It may be a thing.

The salt of compound I is not particularly limited as long as it has the action of the present invention (regulatory T cell differentiation inhibitory action), but is preferably safe for living organisms. Specifically, it is preferably a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt is an acid addition formed from an inorganic acid, an organic acid or an inorganic base that can be administered to a living body including humans. Salts or base addition salts can be mentioned. Specifically, the inorganic acid forming the acid addition salt is not limited, but halogen acid or sulfuric acid such as hydrochloric acid and hydrochlorite; organic acids include acetic acid, lactic acid, malic acid, maleic acid, malonic acid and fumal. Carbodies such as acids, succinic acid, adipic acid, propionic acid, glycolic acid, hydroxybutyl acid, salicylic acid, tartaric acid, glutaric acid, and citric acid; phosphonic acid such as phosphoric acid and 2- or 3-glycerophosphate; and methane. A sulfonic acid such as a sulfonic acid can be exemplified. The bases that form the base addition salt are not limited, but are not limited, but are hydroxides of alkali metals (sodium, potassium, lithium) and alkaline earth metals (magnesium, calcium), and ammonia hydroxides (tetramethylammonium hydroxides). , 4th ammonium hydroxide). Also included in the salts of the compounds of the invention are salts formed of pharmaceutically acceptable amines such as ammonia, alkylamines, hydroxyalkylamines, N-methylglucamine, benzylamines, piperidines, and pyrrolidines.

Compound I or a salt thereof may be in the form of a solvate. The solvent that can form a solvent with compound I or a salt thereof is not limited, and is, for example, methanol, ethanol, 2-propanol (isopropyl alcohol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine or acetic acid. Organic solvents such as ethyl, or water can be mentioned.

Compound I can also be produced by chemical synthesis, but as another method, as described in Examples described later, Thymelaeaceae Aquilaria filaria Merrill (scientific name: Aquilaria filaria Merrill) is derived from Aquilaria (agarwood). It can also be isolated from the extract.

The agarwood used to isolate compound I is the resin-deposited portion of the A. filaria Merrill material, as described above. It is commercially available and has grades 1 to 6 for commercial purposes. The first-class product with the highest quality as a fragrant wood is a lump made only of resin, and the second-class product is mainly made of resin and some material is seen, and the higher the grade number, the lower the quality. The compound I of the present invention is isolated from a quaternary ginkou (agarwood) as shown in Examples.

The "agarwood extract" used as an isolation material for compound I is an extract obtained by extracting a specific component containing compound I from the above-mentioned agarwood using various solvents. As the extraction solvent used here, either a polar solvent or a non-polar solvent can be used, and these can be mixed and used. As the extraction solvent, water: methanol, ethanol, propanol, butanol and the like may have branches having 1 to 12 carbon atoms. Monohydric alcohol: ethylene glycol, propylene glycol, butylene glycol and the like: polyhydric alcohol: acetone, Ketones such as methyl ethyl ketone: Esters such as methyl acetate and ethyl acetate: Ethers such as tetrahydrofuran and diethyl ether: Polyethers such as polyethylene glycol: Hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride: Hydrocarbons such as hexane, cyclohexane and petroleum ether Hydrocarbons: Aromatic hydrocarbons such as benzene and toluene: pyridines, supercritical carbon dioxide, fats and oils, waxes and other oils can be mentioned. These may be used alone or in combination of two or more.

Since compound I is a lipophilic component, it is preferable to extract the compound using a lipophilic solvent in which the compound can be dissolved and purify the extract to efficiently isolate the compound. Can be done. Examples of the lipophilic solvent in which such a compound can be dissolved include ketones such as acetone and methyl ethyl ketone: esters such as ethyl acetate: isocyanates, ethers such as diethyl ether: dichloromethane, chloroform, carbon halides such as carbon tetrachloride, and the like. A polar aproton solvent can be mentioned. The extract using such a lipophilic solvent can be used as a lipophilic fraction by removing the solvent, but the lipophilic solvent extract may be washed with water and then the lipophilic solvent may be removed to obtain a lipophilic fraction.

Instead of the above-mentioned lipophilic solvent, water: a monohydric lower alcohol having 1 to 3 carbon atoms such as methanol and ethanol: a polyhydric alcohol such as ethylene glycol: a mixture thereof, based on 100 parts by mass of the lipophilic solvent. It is also possible to extract with a lipophilic solvent-hydrophilic solvent mixed solution in which another hydrophilic solvent is added in the range of 60 to 100 parts by volume. The lipophilic solvent and the hydrophilic solvent may be separated into two layers by standing after mixing, and by combining such a lipophilic solvent and a hydrophilic solvent, the target substance is transferred to the lipophilic solvent layer. At the same time, the water-soluble component contained in Jinkou can be transferred to the hydrophilic solvent layer (liquid-liquid distribution). Ethyl acetate is such a lipophilic solvent, and water or methanol is a hydrophilic solvent. A mixture of ethyl acetate and water in a ratio of 4: 5 (volume ratio) is separated into an ethyl acetate layer and an aqueous layer when left to stand after extraction. Therefore, the target component is recovered in the ethyl acetate layer and the water-soluble component is added. By transferring to an aqueous layer, it is possible to easily separate the target component which is lipophilic and the water-soluble component.

More specifically, as shown in Examples described later, an "agarwood extract" can be prepared by obtaining an extraction fraction (AcOEt fraction) with ethyl acetate from agarwood according to the following operation. Of the following operations, the operations not specifically mentioned are carried out at room temperature.
(1) First, shredded, crushed or crushed agarwood (dried product) is extracted by immersing it in methanol overnight at room temperature (cold immersion extraction). The obtained cold-immersed extract is concentrated and dried by an evaporator or the like to obtain an agarwood MeOH extract.
(2) This is distributed to a mixed solvent of ethyl acetate (AcOEt) and water (ethyl acetate: water = 4: 5 (volume ratio)) using a liquid separation funnel (liquid-liquid distribution), and ethyl acetate is 2 Get the AcOEt fraction by re-extracting.

Compound I is contained in the agarwood extract (AcOEt fraction) obtained by the above method. The method for isolating compound I from such a ginkou extract is not limited, but for example, a method for isolating compound I using gel filtration or molecular sieve chromatography based on the molecular weight (549) of the compound of the present invention, a polarity or a solvent. It is possible to exemplify a method of isolating using adsorption column chromatography (normal phase or reverse phase column chromatography) based on the difference in solubility of the above, and a method of removing impurities by active carbon treatment or the like. , These can be done alone or in combination. In addition, it can be isolated and purified using the activity of compound I of the present invention (for example, the activity of promoting the expression of profilaggrin in epidermal cells shown in the reference example) as an index. A specific method for isolating and obtaining compound I from the agarwood extract (AcOEt fraction) will be described in Examples.

The salt of compound I of the present invention and its solvation can be produced according to a conventional method using the compound I of the present invention prepared by the above method as a raw material.

As will be described later, the compound I of the present invention has an effect of suppressing the differentiation of regulatory T cells, and is therefore useful as an active ingredient of a regulatory T cell differentiation inhibitor, and also cancels immunosuppression and is also used. It is useful as an active ingredient of a composition (immunosuppressive composition) that regulates immunity in the body, such as activating immunity. The route of administration of Compound I, which is used with the expectation of such a purpose or effect, is percutaneous administration such as application as an external composition (external drug, quasi-drug, cosmetic), food and drink such as supplements, or Examples of oral drugs or quasi-drugs include oral administration including oral administration and sublingual administration.

(II) Regulatory T cell differentiation inhibitor The regulatory T cell differentiation inhibitor of the present invention is the above-mentioned compound I and its salt, and a solvate thereof (hereinafter, these are collectively referred to as "compound I and the like". It is characterized by containing (collectively referred to) as an active ingredient.

As mentioned above, regulatory T cells suppress the immune response by various mechanisms and play an important role in maintaining homeostasis of the living body. For example, in tumor tissues, differentiation into regulatory T cells occurs. It is known that it is enhanced and a state of immune non-responsiveness occurs. As shown in Example 2 described later, since compound I and the like can suppress the differentiation of helper T cells into regulatory T cells, immunosuppression due to an increase in regulatory T cells is released and immunization is performed. It can be activated. Therefore, a composition containing an effective amount of Compound I or the like that exerts a regulatory T cell differentiation inhibitory action is useful as a regulatory T cell differentiation inhibitor, as well as an immunosuppressive release composition or an immunostimulatory composition. It is useful as an immunosuppressive composition for substances and the like.

The ratio of compound I or the like contained in the regulatory T cell differentiation inhibitor is not limited as long as the regulatory T cell differentiation inhibitor exerts a regulatory T cell differentiation inhibitory effect, and is 0.1 to 100% by mass. It can be appropriately selected from the range of. The regulatory T cell differentiation inhibitory effect can be evaluated according to the method described in Example 2 described later.

The regulatory T cell differentiation inhibitor of the present invention is a food or drink, a pharmaceutical product, or a pharmaceutical product for controlling regulatory T cell differentiation, which is used as it is or by blending an additive which is permitted in each field as needed. It can be used as a quasi-drug, cosmetics, or feed. Further, the regulatory T cell differentiation inhibitor of the present invention, by adding an appropriate amount thereof, exerts an immunosuppressive release effect or an immunostimulatory effect based on the regulatory T cell differentiation inhibitory effect in foods and drinks, pharmaceuticals, and quasi-drugs. It can be added to goods, cosmetics, or feeds.

Here, the additives include, for example, a base, a carrier, a bulking agent, an excipient, a solvent, a dispersant, an emulsifier, a buffer, and a stable, depending on the form (oral or parenteral) of the regulatory T cell differentiation inhibitor. Agents, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, flavoring agents and the like can be mentioned without limitation. Oral dosage forms such as tablets, pills, powders, powders, granules, liquids, emulsifiers, capsules; or suppositories, injections, drops, ointments, creams, lotions with such additives. It can be prepared in a parenteral dosage form such as.

The administration route when the controllable T cell differentiation inhibitor of the present invention is administered to a living body is not limited, but is, for example, enteral administration such as oral administration, tube feeding, enema administration; intravenous administration, transarterial administration, etc. , Non-enteral administration such as intramuscular administration, subcutaneous administration, intradermal administration, intraperitoneal administration and the like can be adopted.

When the regulatory T cell differentiation inhibitor of the present invention is used as it is as a food or drink, a drug, a quasi-drug, a cosmetic or a feed (these are referred to as "food and drink"), the food or drink per day. The dose (ingestion) varies depending on the condition, body weight, purpose, etc. of the subject, but is in the range of 0.1 to 100 mg in terms of the amount of compound I contained in the regulatory T cell differentiation inhibitor. It can be appropriately selected and used. The administration (ingestion) may be performed once a day or divided into a plurality of times (for example, 2 to 5 times).

(III) Immunoregulatory Composition In the present invention, the above-mentioned regulatory T cell differentiation inhibitor containing compound I and the like can be used as the immunomodulatory composition. The immunomodulatory composition targeted by the present invention includes an immunosuppressive release composition and an immunostimulatory composition.

In the present invention, immunosuppression release and immunostimulation are used interchangeably. Both immunosuppression release and immunostimulation mean release of immune system suppression by suppressing the differentiation of helper T cells into regulatory T cells, thereby reducing the immune response (degree and degree and). / Or frequency) can be normalized or activated. Therefore, according to the immunosuppressive composition of the present invention (immunosuppression-releasing composition, immunostimulatory composition), the action of suppressing the differentiation of regulatory T cells such as compound I contained as an active ingredient (participating ingredient). By releasing the suppression of the immune system and activating it based on the above, it becomes possible to improve diseases and physical disorders caused by a decrease in the immune response. Further, according to the immunomodulatory composition of the present invention, by preventing the immune system from being excessively suppressed, it becomes possible to prevent diseases and physical disorders due to a decrease in immune response.

The immunomodulatory compositions of the present invention can be prepared in the form of foods and drinks, pharmaceuticals, quasi-drugs, cosmetics, or feeds. The proportion of compound I or the like contained in the immunomodulatory composition is not limited as long as it exerts an action of releasing the suppression of the immune system based on the action of suppressing the differentiation of regulatory T cells, and is not limited to, for example, 0.1. From the range of ~ 100% by mass, it can be appropriately set according to the purpose of use and the form thereof of the immunomodulatory composition.

The present invention also relates to a method for normalizing or activating the immunity of a subject by administering (ingesting or ingesting) the immunomodulatory composition according to the present invention to the subject. In this method, the differentiation of helper T cells into regulatory T cells is preferably suppressed in the body of the treated subject, and as a result, the immune unresponsive state is suppressed or alleviated. The subject to which the immunomodulatory composition is administered in this method is the same as the administration subject described later. Examples of suitable subjects include pregnant women, lactating women, infants, the elderly, persons with impaired immune function, and the like.

(III-1) Foods and Foods As described above, the immunomodulatory composition according to the present invention can be prepared in the form of foods and drinks. The present invention provides foods and drinks for immunomodulation (for immunosuppression release, immunoactivation). In the present invention, the food or drink (also simply referred to as “food”) may be any food or drink that can be orally ingested, and the form thereof is not particularly limited. It may be liquid, semi-liquid or solid, and includes beverages and sol-like or gel-like forms. Foods and drinks include general foods and drinks (fresh foods, processed foods) as well as functional foods.

Functional food means a food having a certain functionality with respect to a living body, and includes, for example, food for specified health use (including conditional Tokuho [food for specified health use]), food with functional claims, and food with nutritional function. Health functional foods; Special purpose foods; Dietary supplements; Health supplements; Supplements (eg, various dosage forms such as tablets, coated tablets, sugar-coated tablets, capsules and liquids) and so-called health foods such as beauty foods (eg diet foods) Includes food in general. The functional foods of the present invention also include health foods to which a Health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Board) is applied.

Preferred examples of functional foods include special-purpose foods such as foods for the sick, foods for pregnant and lactating women, milk powder for infants, and foods for the elderly. The immunomodulatory composition of the present invention is a food for pregnant or lactating women, and a food for the elderly and a food for the sick for the elderly and the sick whose immune function is deteriorated due to aging, illness or treatment. Is particularly suitable for use as. It is also suitable for use as a food for the frail constitution, whose immune function tends to decrease constitutionally or environmentally. Further, suitable functional foods of the present invention include supplements for pregnant and lactating women, supplements for the elderly, supplements for the sick, and supplements for the frail, for the purpose of enhancing and restoring the immune function.

The functional food of the present invention is useful for normalizing or activating immunity. Therefore, the functional food of the present invention may be one that claims to have the effect of normalizing or activating the immune function of a living body. In particular, foods for specified health use, conditional tokuho [foods for specified health use], or foods with functional claims have an immunonormalization or activation effect, or an action mechanism (regulatory T cells) that bring about immunonormalization or activation. (Differentiation suppression) may be described or displayed. It should be noted that the description or labeling to the effect that it has an immunonormalizing or activating effect may be approved for labeling based on the provisions stipulated in the health functional food system. For example, in the functional food of the present invention, "enhance the ability to protect against infection", "make it difficult to catch a cold", "enhance the resistance of the body", "contribute to the normal function of the immune system", and "healthy". "Maintaining a good immune function" is conceivable.

The functional food of the present invention may be in the form of a solid preparation such as tablets, granules, powders, pills or capsules, a liquid preparation such as a liquid, a suspending agent or a syrup, or a preparation such as a gel. It may be in the shape of a general food or drink.

The food and drink for immunomodulation of the present invention can be prepared by blending the above-mentioned compound I and the like into the food and drink by any suitable method available to those skilled in the art. For example, the above-mentioned regulatory T cell differentiation inhibitor may be prepared in the form of liquid, gel, solid, powder or granule, and then blended into food or drink. Alternatively, compound I or the like itself may be directly mixed or dissolved in the raw material of food or drink. Further, after adding an appropriate excipient or the like to compound I or the like of the present invention, it may be molded into the shape of a pharmaceutical product such as a tablet or a capsule.

(III-2) Pharmaceuticals or Quasi-drugs The immunomodulatory composition according to the present invention can be prepared in the form of pharmaceuticals or quasi-drugs. The present invention provides a drug or a quasi-drug for immunomodulation (for immunosuppression release, immunostimulation).

In the present invention, the pharmaceutical product or quasi-drug contains compound I or the like as an active ingredient, and if necessary, contains a carrier, an excipient, a binder and / or a diluent according to the form thereof. These can be administered orally or parenterally, and when administered orally, they can have the form of granules, powders, tablets, pills, capsules, syrups and the like. Further, examples of the administration form for parenteral use include, but are not limited to, external preparations such as injections, infusions, and nasal administration preparations.

The pharmaceutical product or quasi-drug of the present invention preferably exerts an immunonormalizing or activating effect. The pharmaceutical product or quasi-drug of the present invention may be a drug or quasi-drug for another therapeutic use containing other pharmacological substances mixed with the compound I of the present invention or the like.

The dose of the drug or quasi-drug of the present invention varies depending on the age and body weight of the subject to be administered, the route of administration, and the number of administrations, and can be appropriately set and adjusted at the discretion of those skilled in the art. For example, in the case of oral administration, the daily dose may be appropriately selected from the range of 0.1 to 100 mg in terms of the amount of compound I contained in a drug or quasi-drug. Can be used. The administration may be performed once a day or divided into a plurality of times (for example, 2 to 5 times). In the case of parenteral administration, as an example of the dose, the amount of the compound I of the present invention is about 0.1 to 20 mg per ^{1 cm 2 of the skin, and the frequency is about 1 to several times a day.}

The subject to which the pharmaceutical product or quasi-drug of the present invention is administered is a mammal including humans, livestock, pet animals, experimental (test) animals and the like. In particular, mammals whose immune function has deteriorated due to aging, illness, treatment, etc., and mammals whose immune function tends to decrease constitutionally or environmentally are preferable as targets for administering the pharmaceutical product or quasi-drug of the present invention.

(III-3) Cosmetics The immunomodulatory composition according to the present invention can be prepared in the form of cosmetics. The present invention provides cosmetics for immunomodulation (for immunosuppression release, immunostimulation).

Dosage forms of cosmetics include ointments, liquids, sprays, plasters, oils and fats, powders and the like. Whitening cosmetics include basic cosmetics such as lotions, milky lotions, creams, and facial masks, makeup bases, sunscreens, foundations, makeup cosmetics such as face powders, and cleaning agents such as shampoos and face wash. The blending amount of the compound I in these cosmetics can be appropriately selected depending on the substrate, but is 0.00001% to 20% by mass, preferably 0.0001% to 5% by mass, and more preferably 0.001 to 3% by mass. Is.

As the other ingredients constituting the cosmetics, the ingredients to be blended in cosmetics such as ordinary cosmetics, quasi-drugs, and pharmaceuticals can be blended according to the dosage form and the like. Such components include preservatives such as methyl paraoxybenzoate, propyl paraoxybenzoate, phenoxyethanol, timole, and antiseptic such as sodium hydrogen sulfite, ascorbic acid, tocopherol, dibutylhydroxytoluene, sodium edetate hydrate, benzotriazole and the like. Oxidants, glycerin monostearate, sorbitan monostearate, polyoxyethylene hydrogenated castor oil 60, polysorbate 60 and other surfactants, citric acid hydrate, sodium citrate hydrate, lactic acid, diisopropanolamine, acetic acid, There are pH adjusters such as sodium acetate hydrate, moisturizers, thickeners, inorganic fillers, colorants, fragrances, ultraviolet absorbers, cell activators, and various skin nutritional components. Other base materials and the like can be appropriately added to prepare external liquids, external solids, sprays, ointments, creams, gels, patches, and others.

(III-4) Feed The immunomodulatory composition according to the present invention can be prepared in the form of feed. The present invention provides a feed for immunomodulation (for immunosuppression release, immunoactivation).

The target for feeding the feed of the present invention is a mammal including livestock, pet animals, experimental (test) animals and the like. In particular, mammals whose immune function has deteriorated due to aging, illness, treatment, etc., and mammals whose immune function tends to decrease constitutionally or environmentally are preferable as targets for administering the feed of the present invention.

Next, the present invention will be specifically described with reference to examples, but these examples do not limit the present invention in any way.

Example 1 Preparation of Compound I A ginkou extract was prepared using agarwood (grade 4) derived from A. filaria Merrill of the family Thymelaeaceae, and compound I was isolated from the thymelaeaceae extract. (See FIG. 1). As the above-mentioned Jinkou, "Jinkou chopped crude drug" sold by Takasago Yakuhin Co., Ltd. was used. The following preparation operations were performed under room temperature conditions unless otherwise specified.

(1) Preparation of agarwood extract (AcOEt fraction) (see Fig. 1)
(I) 50 g of chopped crude drug (Takasago Pharmaceutical Co., Ltd.) was immersed in 1000 mL of 100% methanol overnight for extraction. After extraction, filtration was performed with filter paper, 1000 mL of 100% methanol was added to the residue again, and the residue was immersed overnight under the same conditions for extraction (total of 3 extraction treatments). The extracts obtained in the three extraction treatments were combined and concentrated to dryness with an evaporator to obtain 3.7 g of agarwood MeOH extract.

(Ii) The Jinkou MeOH extract obtained above was dispensed and extracted with a mixed solution of ethyl acetate (AcOEt) (400 mL) and water (500 mL) using a 1 L volume separatory funnel. The AcOEt layer, which had become two layers after being allowed to stand, was recovered, and ethyl acetate (400 mL) was added to the aqueous layer again for partition extraction (a total of three partition extraction treatments). The AcOEt layer was allowed to stand again to recover the two layers of AcOEt, and the previously recovered AcOEt layers were put together and filtered using a filter paper to remove insoluble matter (insoluble fraction). The recovered AcOEt layer was concentrated and dried by an evaporator to obtain an AcOEt fraction (2.7 g). Hereinafter, compound I was isolated using this as an agarwood extract and this as a material.

(2) Normal phase column chromatography (separation 1)
2.7 g of the Jinkou extract (AcOEt fraction) prepared above is dissolved in a sufficient amount of methanol (sample), _{adsorbed on a normal phase carrier (SiO 2} ), and subjected to normal phase open column chromatography having the following separation conditions. Served at the top of the graffiti. In addition, a solution of ethyl acetate: n-Hexane = 9: 1 (volume ratio) was previously flowed through an open column packed with the following carrier for equilibration.

[Separation condition 1]
Column: GL Science (inner diameter 45 mm, length 500 mm)
Filling carrier: Silica Gel 60 (manufactured by Nacalai Tesque) (inner diameter 45 mm, length 300 mm)
Elution solvent: AcOEt: n-Hexane = 9: 1 (volume ratio) 2000 mL

After supplying the sample to an open column packed with the above carrier and equilibrated with a solution of AcOEt: n-Hexane = 9: 1, 2000 mL of the same solution was flowed as an elution solvent, and 100 mL of the eluate from the column was separated. , 16 fractions (Fr.1 to Fr.16). The yield in each fraction is 1198 mg for Fr.1-9 (total amount), 82 mg for Fr.10-12 (total amount), 37 mg for Fr.13-14 (total amount), and 801 mg for Fr.15-16 (total amount). there were.

(3) Isolation of the compound contained in Fr.10-12 Fr.10-12 (82 mg) prepared above was dissolved in 100% methanol and subjected to HPLC under the following conditions.

[HPLC conditions]
Stationary phase (column): COSMOSIL 5C8-MS (inner diameter 20 mm, length 250 mm) (Nacalai Tesque)
Mobile phase (eluent): 80% MeOH aqueous solution (40 min) Isocratic elution flow rate: 5 mL / min
Liquid feed pump: LC-10AD (SHIMADZU)
Detector: SPD-M10A (SHIMADZU)
System controller: SCL-10A (SHIMADZU)
Software: LC-Solution (SHIMADZU)
Degasser: DGU-14A (SHIMADZU)

As a result, a chromatogram having a main peak at a retention time of 27 minutes was obtained. The peak fraction with a retention time of 27 minutes was fractionated (dry weight 7.6 mg), and the UV spectrum was measured. As shown in FIG. 2, maximum absorption was achieved near 229 nm, 248 nm, and 330 nm, respectively. It was confirmed that the compound had a wavelength.

(4) Determination of compound structure The compound isolated above shows a pseudomolecular ion peak at ^{m / z 571.1734 [M + Na] + (Calcd. 571.1733) in HR-ESI / TOF / MS, and thus has a molecular formula C.} _{It was estimated to be 34} H ₂₈ O ₇ (unsaturation: 21). ^{In the 1} H-NMR spectrum (Fig. 3), there is a proton signal for _{10 H in ortho-coupling proton signal [δ H} 7.08 (1H, d, J = 9.5), 7.27 (1H, overlap)] and 7.14-7.27. Therefore, the presence of two monosubstituted benzenes was presumed. Four more methine [δ _H 4.37 (1H, dd, J = 2.0, 9.3), 4.46 (1H, brq), 4.69 (1H, m), 6.17 (1H, brt)] signals were observed. ..

¹³ C-NMR spectrum (Fig. 4) and DEPT spectrum show 8 quaternary carbons (δ _C : 120.5, 121.0, 121.1, 148.9, 150.9, 161.7, 165.9, 167.3), 18 methines (δ _C : 29.5). , 63.0, 68.2, 73.7, 110.2, 112.7, 118.1, 121.4, 128.4-126.2), 4 methylene (δ _C : 31.9 × 2, 33.9, 34.2), 2 carbonyl groups (δ _C : 177.1, 178.1) Was observed. As a result of analyzing the HMQC spectrum (Fig. 5), the connection between carbon and hydrogen as shown in Table 1 was estimated. Further ¹ H- ¹ H COZY (Fig. 6) and HMBC were examined in detail the correlation (Figure 7), the correlation shown in Table 1 and 8 were observed. Based on these results, this compound was determined. Below Table 1, the structure of the compound and its position number are shown.

また図９に示すように、NOESYの相関で、化合物Ｉの水酸基は同じ方向にあることが確認された。このことから、化合物Ｉは下式(II)または(III)に示す立体構造を有すると考えられる。

Further, as shown in FIG. 9, it was confirmed that the hydroxyl groups of compound I were in the same direction by the correlation of NOESY. From this, it is considered that compound I has a three-dimensional structure represented by the following formula (II) or (III).

Example 2 Regulatory T cell (Treg) differentiation inhibitory effect It was confirmed by the following test that the compound I isolated in Example 1 has a regulatory T cell differentiation inhibitory effect.

(1) Test method (a) The spleen and lymph collected from mice (B6.Cg-Foxp3 tm1Mal / J: The Jackson Laboratory) were ground and cells were collected.
(B) Naive CD4 + T cells (CD4 + CD62L + T cells) were isolated from the collected cells using an automatic magnetic cell separator (MACS [registered trademark] system: Miltenyi biotec).
(C) The isolated naive CD4 + T cells were subjected to the following "iTreg conditions" and cultured for 3 days.
(D) Cultured naive CD4 + T cells are stained with an antibody (anti-CD4 antibody) and used for CD4 + T using flow cytometry (FACS: FACS Canto (BD biolegend) and EC-800 (Sony)). The ratio of Foxp3 + T cell number to cell number (Foxp3 positive ratio) was measured to determine the ratio of regulatory T cells (Treg) to CD4 + T cells. The analysis software FlowJo (FLOWJO) was used for the analysis.
(E) In the above test system, the compound depends on whether or not the number of regulatory T cells (Treg) is reduced when the compound I is added (Example) as opposed to the case where the compound I is not added (control). The Treg differentiation inhibitory effect of I was evaluated.

[ITreg condition]
(A) Control test conditions (control)
2-Mercapto prepared in culture plates (Biolite) pre-applied with 3 μg / mL of anti-CD3ε antibody (145-2C11) (BioLegend) and incubated overnight at 4 ° C or 37 ° C. Naive CD4 + T using RPMI1640 medium (Nakalitesk) containing 10% FBS containing 55 μM ethanol, 2 μg / mL anti-CD28 antibody, 5 ng / mL TGF-β, and 20 ng / mL IL-2. _{The cells were cultured in a CO 2} incubator for 3 days and evaluated by flow cytometry (to step (d) above).

(B) Compound I test conditions (Example)
2-Mercapto prepared in culture plates (Biolite) pre-applied with 3 μg / mL of anti-CD3ε antibody (145-2C11) (BioLegend) and incubated overnight at 4 ° C or 37 ° C. Compound I in multiple concentrations in RPMI1640 medium (Nakalitesk) containing 55 μM ethanol, 2 μg / mL anti-CD28 antibody, and 20 ng / mL TGF-β and 20 ng / mL IL-2 containing 10% FBS. It was added, and using this, naive CD4 + T cells were _{cultured in a CO 2} incubator for 3 days and evaluated by flow cytometry (to step (d) above).

(2) Test Results As shown in FIG. 10, in the test system (Example) to which Compound I was added, Treg differentiation was suppressed in a concentration-dependent manner. From this, it was confirmed that Compound I has a regulatory T cell differentiation inhibitory effect.

Reference example Profilaggrin expression promoting action It was confirmed by the following test that compound I has a profilelaggrin expression promoting action in epidermal cells as another action.

(1) Test material (cells)
In this test example, the following epidermis model cells were used as cells.
(A) Epidermal model cells (HaCaT cells)
HaCaT cells (human epidermal keratinized cells (Deutsches Krebsforschungszentrum, Stiftung desoffentlichen Rechts (DKFZ), Heidelberg, Germany)) were used as epidermal model cells.

(Culture medium)
D-MEM / Ham's F-12 (Wako Pure Chemical Industries, Ltd.) / 10% FCS added

(Test substance)
Compound I isolated in Example 1

(2) Test method Epidermal model cells were seeded in each well of a 12-well plate at ^{a concentration of 4 × 10 5} cells / well, and cultured at _{37 ° C. under 5% CO 2 conditions.} The next day, compound I was added to the wells to 1 μg / mL or 10 μg / mL and cultured at _{37 ° C. under 5% CO 2 conditions.} The next day, epidermal model cells were collected and total RNA was extracted using RNeasy mini kit (manufactured by Qiagen Co., Ltd.). Then, reverse transcription was performed using ReverTra Ace (manufactured by Toyobo Co., Ltd.) to synthesize cDNA. Next, the profilaggrin gene was amplified by qRT-PCR using MyGo Pro (manufactured by IT-IS Life Science Co., Ltd.). β-actin was used as an internal standard to standardize the expression level of profilaggrin mRNA. The expression level of this mRNA was divided by the expression level (control value) of the mRNA confirmed in the same manner as above except that compound I was not added.

3. 3. Test Results The results obtained are shown in FIG. As can be seen from FIG. 11, in the epidermal model cells, an increase in the expression level of profilaggrin mRNA, which is a precursor of filaggrin, was observed under the condition of addition of compound I. From this result, it was shown that Compound I has an action of increasing the expression level of profilaggrin mRNA in epidermal cells, and as a result, promotes the production of filaggrin.

Claims (5)

A regulatory T cell differentiation inhibitor containing the compound represented by the following formula (I), a salt thereof, or a solvate thereof as an active ingredient:

The regulatory T cell differentiation inhibitor according to claim 1, wherein the compound represented by the above (I) is at least one of the stereoisomers represented by the following formula (II) or (III):

An immunomodulatory composition comprising the regulatory T cell differentiation inhibitor according to claim 1 or 2. The immunomodulatory composition according to claim 3, which has an immunostimulatory action. The immunomodulatory composition according to claim 3 or 4, which is a food or drink, a pharmaceutical product, a quasi drug, a cosmetic product, or a feed.

Images