WO2016013654A1

WO2016013654A1 - Allergen activity inhibitor and use thereof

Info

Publication number: WO2016013654A1
Application number: PCT/JP2015/071101
Authority: WO
Inventors: 邦宏開發; 田中　伸幸
Original assignee: 株式会社プロテクティア
Priority date: 2014-07-24
Filing date: 2015-07-24
Publication date: 2016-01-28
Also published as: JP7419416B2; JPWO2016013654A1; JP6731340B2; JP7035109B2; JP2020127418A; JP2022071097A

Abstract

Provided is a new effective substance with which safety can be maintained and allergen activity can be inhibited. According to the present invention, a drug including an epigallocatechin gallate derivative represented by chemical formula (1) is prepared. This can be used as an activity inhibitor for inhibiting allergen activity. In chemical formula (1): R¹-R⁶ are hydrogen atoms, or straight-chain or branched, saturated or unsaturated acyl groups, and may be the same or different; the acyl groups may be further substituted with one or more substituent groups; and R¹-R⁶ cannot all be hydrogen atoms at the same time.

Description

Inhibitors of allergen activity and uses thereof

The present invention relates to an allergen activity inhibitor that suppresses allergen activity.

An allergic reaction is generally an excessive immune reaction caused by a foreign substance as an allergen. In recent years, in particular, the number of patients who cause allergic symptoms due to allergens such as mites and pollen has increased significantly. For this reason, the search for an effective substance that suppresses allergic reaction is regarded as very important.

¡Allergic reactions are thought to occur, for example, by the following mechanism. First, proteins (allergens) derived from mite carcasses, feces and pollen adhere to the mucous membranes of human nose and throat and are recognized as foreign substances by lymphocytes. When the lymphocyte recognizes the allergen as a foreign substance, it produces an IgE antibody, and the produced IgE antibody is presented by binding to a receptor on the surface of mast cells. As a result, when the same protein subsequently adheres to the mucous membrane, the protein acts as an allergen on the IgE antibody on the mast cell, and a chemical substance such as histamine is released from the mast cell. These chemical substances cause an allergic reaction and cause allergic symptoms such as sneezing, runny nose / nose clogging, itchy eyes, redness of eyes and tears.

In recent years, catechins (Patent Document 1), methylated catechins and the like have been reported as inhibitors of the allergic reaction. The mechanism of action of these inhibitors is to suppress the expression of IgE receptor FcεRI and the like, which suppresses some reaction steps in a series of allergic reactions (Non-patent Document 1).

However, as a result of confirmation by the inventors of the present application, it has been found that catechins and methylated catechins as described above cannot sufficiently suppress the occurrence of allergic reactions due to allergens.

JP-A-6-279273

Therefore, an object of the present invention is to provide a new effective substance that can maintain safety and suppress allergen activity.

In order to achieve the above object, the allergen activity inhibitor of the present invention comprises a derivative of epigallocatechin gallate (EGCG) represented by the following chemical formula (1), an isomer thereof, or a salt thereof. And

In the chemical formula (1),
R ¹ to R ⁶ , R ¹² and R ¹⁴ are each a hydrogen atom, halogen, sodium, potassium, or a linear or branched saturated or unsaturated acyl group, which may be the same or different, and the acyl group May be further substituted with one or more substituents, at least one of the R ¹ to R ⁶ , R ¹² and R ¹⁴ is the acyl group, and R ⁷ to R ¹¹ , R ¹³ , R ¹⁵ And R ¹⁶ is a hydrogen atom, halogen, sodium or potassium, and may be the same or different.

The method for suppressing allergen activity of the present invention is characterized in that the allergen activity inhibitor of the present invention is brought into contact with the allergen.

The allergic reaction suppression method of the present invention is characterized in that the allergen activity inhibitor of the present invention is administered to a subject.

According to the present invention, allergic reactions can be prevented safely and effectively. For this reason, it can be said that the present invention is useful in the medical field, the food hygiene field, and the like.

In Example 1 of this invention, it is a graph which shows the allergen activity inhibitory effect of the EGCG derivative with respect to a mite allergen. In Example 1 of this invention, it is a graph which shows the allergen activity inhibitory effect of the EGCG derivative with respect to a spear allergen. In Example 1 of this invention, it is a graph which shows the allergen activity inhibitory effect of the methylation EGCG with respect to a spear allergen.

<Inhibitor of allergen activity>
As described above, the allergen activity inhibitor of the present invention (hereinafter also referred to as “activity inhibitor”) is a derivative of epigallocatechin gallate represented by the following chemical formula (1), or an isomer thereof, or an isomer thereof. It contains a salt.

In the chemical formula (1), “A to D” are notations of each ring in epigallocatechin gallate. In the present invention, epigallocatechin gallate is hereinafter referred to as “EGCG”, and a derivative of EGCG is referred to as “EGCG derivative”.

In the present invention, “suppression of allergen activity” may be, for example, inactivation of allergen activity in addition to suppression of allergen intrinsic activity.

In the present invention, EGCG derivatives include, for example, isomers such as salts, tautomers, stereoisomers, optical isomers, geometric isomers, and isomer mixtures of the compound represented by the chemical formula (1). It is. The salt is not particularly limited, and examples thereof include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, acidic or basic amino acid salts, and the like. The isomer can also be purified by a conventionally known separation method such as various types of chromatography. In the present invention, the EGCG derivative includes, for example, a compound that is produced by subjecting the compound represented by the chemical formula (1) to metabolism such as oxidation, reduction, hydrolysis, and conjugation.

In R ¹ to R ⁶ , the main chain length of the acyl group is not particularly limited, and includes, for example, carbonyl carbon and 6 to 18 atoms, preferably 12 to 18, more preferably 12, 16 or 18, particularly preferably 12. The main chain length of the acyl group means the number of atoms of the longest chain in the acyl group, and includes, for example, a nitrogen atom, a sulfur atom, a phosphorus atom, an oxygen atom, a boron atom in addition to the carbon atom. But you can. The main chain length of the acyl group may be saturated or unsaturated, for example.

In R ¹ to R ⁶ , the number of atoms of the acyl group is not particularly limited. The number of atoms of the acyl group (for example, the number of carbon atoms) is, for example, 2 to 20, including carbonyl carbon, preferably 4 to 20, more preferably 8 to 18, and 12 to 18. For example, 8, 12, 16 or 18 is preferable. In addition, when the said acyl group is further substituted by the said substituent, it is preferable that the said carbon atom number is a number which does not contain the carbon atom number of the said substituent, for example. The unsaturated acyl group may be cis or trans, for example.

The acyl group is not particularly limited. For example, formyl group (C1), acetyl group (C2), propionyl group (C3), butyryl group (C4), isobutyryl group (C4), valeryl group (C5), isovaleryl Group (C5), pivaloyl group (C5), hexanoyl group (C6), 2-ethylhexyl group (C8), octanoyl group (C8), geranoyl group (3,7-dimethylocta-2,6-dienoyl group) (C10) ), Trans-8-methyl-6-nonenoyl group (C10), undecanoyl group (C11), lauroyl group (dodecanoyl group) (C12), tridecanoyl group (C13), 12- (dimethylamino) lauroyl group (12- ( Dimethylamino) dodecanoyl group) (C14), farnesoyl group (3,7,11-trimethyldodeca-2) 6,10-trienoyl group (C15), palmitoyl group (hexadecanoyl group) (C16), heptadecanoyl group (C17), stearoyl group (octadecanoyl group) (C18), linoleyl group (C18), linolenyl group (C18) ), Nonadecanoyl group (C19), eicosanoyl group (icosanoyl group) (C20), etc. “C” in parentheses of the listed acyl groups represents the number of carbon atoms including the carbonyl carbon.

Among the acyl groups, for example, an acyl group represented by the following chemical formula is particularly preferable. The position of the unsaturated bond in the unsaturated acyl group among the following acyl groups is not limited thereto. As a specific example, the unsaturated bond (double bond) of the trans-8-methyl-nonenoyl group (C10) is not limited to the 6-position shown below, and may be, for example, either the 2-5-position or the 7-position. May be.

The type of the acyl group is not particularly limited, and may be any of an unsaturated acyl group and a saturated acyl group as described above. The number of unsaturated bonds in the acyl group is not particularly limited, and is, for example, 1, 2, or 3.

In the chemical formula (1), for example, only ^one of R ¹ to R ⁶ , R ¹² and R ¹⁴ , or R ¹ to R ⁶ may be the acyl group, or any two of them. The above may be the acyl group. When two or more places are the said acyl groups, the said acyl group in each site | part may be the same and may differ, for example. In the chemical formula (1), R other than the acyl group is not particularly limited, and for example, a hydrogen atom is preferable.

In the chemical formula (1), the site of the acyl group is not particularly limited among R ¹ to R ⁶ , R ¹² and R ¹⁴ or among R ¹ to R ⁶ . As a specific example, for example, at least one of R ¹ and R ² of the B ring and R ⁵ and R ⁶ of the D ring preferably has the acyl group, and in particular, R ¹ , R ² , R ⁵ and R It is preferable that any one of ⁶ has the acyl group. In this case, other R is not particularly limited, and is preferably a hydrogen atom, for example.

Further, in Formula (1), it is preferable that at least one position of ^R 1, ^{R 2} and ^{R 3} of B ring is the acyl group, more preferably, ^R 1, ^{R 2} and ^{R 3} in the B ring It is preferable that only one of them is an acyl group. In Formula (1), it is preferable that at least one position of ^R 4, ^{R 5} and ^{R 6} in the D ring is the acyl group, and more preferably, the D ring ^R 4, of ^{R 5} and ^{R 6} It is preferable that only one place is an acyl group.

In the chemical formula (1), the B ring rotates around a single bond between the B ring and the C ring, for example. Therefore, a derivative having an acyl group at R ¹ is substantially the same as a derivative having an acyl group at R ³ , for example. In the chemical formula (1), the D ring rotates around a single bond between the D ring and the ester, for example. For this reason, the derivative having an acyl group at R ⁶ is substantially the same as the derivative having an acyl group at R ⁴ , for example.

In the chemical formula (1), “R ⁷ to R ¹¹ , R ¹³ , R ¹⁵ and R ¹⁶ ” or “R ⁷ to R ¹⁶ ” represents a hydrogen atom, halogen, sodium (Na) or Potassium (K), which may be the same or different. For example, as shown in the following chemical formula (2), a hydrogen atom is preferable. In the following chemical formula (2), for example, any of R ¹ to R ⁶ may be the acyl group. As a specific example, for example, it is preferable that only one or two or more of R ¹ to R ⁶ are the acyl groups described above, and more preferably R ¹ , R ² , R ⁵ and R ^6. Among these, it is preferable that only one or two or more of the above-mentioned acyl groups.

In R ¹ to R ⁶ of the chemical formula (1), the acyl group may be substituted with one or more substituents as described above. Specifically, for example, a hydrogen atom of the acyl group is , May be substituted with the substituent. The substituent is not particularly limited, and examples thereof include an alkyl group, an amino group, an alkylamino group, and a dialkylamino group.

Examples of the alkyl group include straight-chain or branched alkyl groups having 1 to 6 carbon atoms, preferably a methyl group. Examples of the alkyl group in the alkylamino group include straight-chain or branched alkyl groups having 1 to 6 carbon atoms, preferably a methylamino group. Examples of the alkyl group in the dialkylamino group include straight-chain or branched alkyl groups having 1 to 6 carbon atoms, and a dimethylamino group is preferable. These may be the same or different.

When the substituent or the like is a group having a chain structure, specifically, for example, an alkyl group, an alkylamino group, a dialkylamino group, an alkoxy group, a carboxyalkyl group, an alkoxycarbonylalkyl group, an alkoxyalkyl group, an alkenoxyalkyl In the case of a group or the like, it is not particularly limited, and may be linear or branched. The same applies to the case where a part of the substituent or the like includes a chain structure, for example, when a substituent in a substituted alkyl group or a substituted aryl group includes a chain structure. When an isomer exists in a substituent or the like, it is not particularly limited, and any isomer may be used. For example, when it is simply referred to as “propyl group”, it may be either n-propyl group or isopropyl group, and when it is simply referred to as “butyl group”, any of n-butyl group, isobutyl group, sec-butyl group and tert-butyl group Often, when simply referred to as a “naphthyl group”, either a 1-naphthyl group or a 2-naphthyl group may be used.

In the present invention, “halogen” refers to any halogen element. Examples of the halogen include fluorine, chlorine, bromine and iodine. In the present invention, the “alkyl group” is not particularly limited. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, hexyl group, heptyl group, octyl group. Group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, icosyl group and the like. For groups containing an alkyl group in the structure or derived from an alkyl group (alkylamino group, dialkylamino group, alkoxy group, carboxyalkyl group, alkoxycarbonylalkyl group, alkoxyalkyl group, alkenoxyalkyl group, etc.) It is the same.

When the EGCG derivative in the present invention has the acyl group only in any one of R ¹ , R ² , R ⁵ and R ^{6 in} the formula (1) or the formula (2), for example, The acyl group is preferably, for example, the following acyl group. In this case, among R ¹ , R ² , R ⁵ and R ⁶ , examples other than the acyl group include hydrogen, halogen, and alkali metals such as Na and K.

As the first form, when the acyl group is a straight-chain saturated acyl group, for example, the main chain preferably has 8 to 18 carbon atoms, preferably 12 to 18 carbon atoms. Examples include acyl groups having 12, 16 or 18 carbon atoms. In the first form, for example, the allergen is suitable for mites and pollen.

As a second form, when the acyl group is an unsaturated acyl group, for example, the main chain preferably has 12 to 18 carbon atoms, and the unsaturated bond has -C = C- at two positions. A specific example is an acyl group having 18 carbon atoms in the main chain and having —C═C— at two positions. In the second form, for example, the allergen is suitable for mites and pollen.

As the third form, when the acyl group is a branched saturated acyl group, for example, the carbon number of the main chain is preferably in the range of 6 to 20, and the carbon number of the branched chain is preferably in the range of 1 to 4. The number of branched chains is preferably 1 to 3, and specific examples include acyl groups having 6 carbon atoms in the main chain, 1 carbon atoms in the branched chain, and 2 branch chains. .

When the EGCG derivative in the present invention has the acyl group only in any ^{two of} R ¹ , R ² , R ⁵ and R ^{6 in} the formula (1) or the formula (2), for example, The acyl group is preferably, for example, the following acyl group. In this case, among R ¹ , R ² , R ⁵ and R ⁶ , examples other than the acyl group include hydrogen, halogen, and alkali metals such as Na and K.

As a fourth form, when the acyl group is a straight-chain saturated acyl group, for example, each of the two acyl groups preferably has a main chain having 8 to 18 carbon atoms. And an acyl group having 8 carbon atoms in the chain. In the fourth form, for example, the allergen is suitable for mites and pollen.

For example, one type of EGCG derivative in the present invention may be used, or two or more types may be used in combination. For example, two or more types of EGCG derivatives having acyl groups at different sites among R ¹ to R ⁶ may be used, or two or more types of EGCG derivatives having different acyl groups may be used. As specific examples, any one or more of EGCG derivatives in which R ¹ of the B ring is the acyl group, EGCG derivatives in which R ² is the acyl group, and EGCG derivatives in which R ³ is the acyl group, or It may be a mixture including all three types, among EGCG derivatives in which R ^{4 of} the D ring is the acyl group, EGCG derivatives in which R ⁵ is the acyl group, and EGCG derivatives in which R ⁶ is the acyl group, Any two or more kinds or a mixture containing all three kinds may be used. And a mixture of an EGCG derivative in which at least one of R ¹ to R ³ of the B ring is the acyl group and an EGCG derivative in which at least one of R ⁴ to R ⁶ of the D ring is the acyl group, Also good.

The activity inhibitor of the present invention can be used against allergens as described above. The allergen causes, for example, an allergic reaction in humans and non-human animals, and is not particularly limited. Specific examples of the allergen include house dust, pollen, insect-derived protein and plant-derived protein. House dust includes, for example, animal skin (including dandruff); mites, worm bodies, carcasses and feces thereof; mold and bacteria. Examples of the animal include humans and non-human animals such as dogs and cats. Examples of the mites include leopard mites (dust mites) such as the leopard mite, mites such as the mite mite, tick mites such as the tick mite, house mites such as the house mite, and the like. Examples of the pollen include pollen of plants such as cypress, such as cedar, asteraceae, such as ragweed and mugwort, pine, such as pine, and gramineous, such as rice. Examples of the insect-derived protein include proteins such as cockroaches. Examples of the plant-derived protein include proteins such as soybean, egg and milk.

The activity inhibitor of the present invention can be used, for example, for preventing the onset of allergic reactions and treating allergic reactions. Specifically, for example, by bringing the allergen and the activity inhibitor into contact with each other, the activity of the allergen can be suppressed, and as a result, the onset of an allergic reaction can be suppressed.

The activity inhibitor of the present invention may contain the EGCG derivative, and its form is not limited at all. Examples of the form include liquids such as solutions and dispersions, solids, powders, and the like. The dosage form is not particularly limited, and can be appropriately set according to the method of use. Examples thereof include liquids, capsules, tablets, granules (fine granules), powders, and the like.

Examples of the usage method include a method of administering the activity inhibitor to a subject that may cause an allergic reaction. The subject is, for example, an animal that may cause an allergic reaction due to an allergen. The administration method is not particularly limited, and examples thereof include parenteral administration and oral administration. Examples of the parenteral administration include transdermal administration, intraperitoneal administration, intravenous administration such as intravenous injection, intramuscular administration, subcutaneous administration such as subcutaneous injection, rectal administration, etc., preferably transdermal administration. . The transdermal administration is not particularly limited, and includes, for example, mucous membranes in addition to skin. The activity-suppressing agent of the present invention is, for example, in accordance with these administration forms, for example, a coating containing the EGCG derivative, a nasal spray, a nasal coating, a nasal irrigant, a mouth rinse, a mouthwash, a sublingual agent, an internal drug, It can be administered as a disinfectant such as a finger, and can be administered as an EGCG derivative or a solution or dispersion containing the same using a nebulizer, aspirator, injection, or the like. Moreover, it can administer using a nebulizer, a suction device etc. as said EGCG derivative | guide_body or powder containing the same, for example.

Examples of the animals include humans and non-human animals. Examples of the non-human animals include non-human mammals such as pigs, ferrets, rats, mice, and cows, and birds such as ducks and chickens.

The activity inhibitor of the present invention may be brought into contact with the subject before the subject such as the animal and the allergen are contacted, or after the subject and the allergen are brought into contact with each other. You may make it contact with a subject. Since the onset of an allergic reaction in the subject can be more effectively suppressed by suppressing the allergen activity, the subject and the activity inhibitor are brought into contact before the subject and the allergen are brought into contact as in the former. It is preferable to make it contact.

Other examples of the method of use include a method of treating a subject in which allergen may be present with the activity inhibitor, and the subject may be in an environment in which allergen may be present, for example. Examples thereof are described below. In this case, the activity inhibitor of the present invention may be in the form of a cleaning agent such as a hand-washing agent, a wiping agent, or a laundry containing the EGCG derivative. In this way, the activity inhibitor of the present invention is used to suppress the activity of existing allergens, for example, hands and desks, and to prevent the development of allergic reactions. Is also possible. The activity inhibitor of the present invention may be carried on, for example, clothing; bedding such as futons and pillows; furniture such as tatami mats, carpets, desks, chairs, and beds; and masks. In addition, for example, a filter in an air conditioner or an air cleaner may be treated with the activity inhibitor of the present invention. In this case, for example, the activity inhibitor of the present invention may be carried on the filter, or the filter may be washed with the activity inhibitor of the present invention.

Further, the subject may be, for example, the living body of the animal itself, cells and tissues collected from the living body, or a culture thereof. When the subject is, for example, the cells or tissues, the method for administering the activity inhibitor is not particularly limited, and examples thereof include addition to a medium.

In the activity inhibitor of the present invention, the content of the EGCG derivative is not particularly limited, and can be appropriately determined according to, for example, the purpose of use or the method of use. When the activity inhibitor of the present invention is a mouthwash, for example, it is preferable to contain 10 to 100 μmol of the EGCG derivative per time. When the activity inhibitor of the present invention is a nasal spray, for example, it is preferable to contain 10 to 100 μmol of the EGCG derivative per time.

The activity inhibitor of the present invention may further contain an additive, a base and the like as appropriate depending on, for example, the dosage form and method of use. Examples of the additive include excipients, binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flavoring agents, emulsifiers, surfactants, solubilizing agents, suspending agents, and isotonic agents. , Buffers, preservatives, antioxidants, stabilizers, absorption promoters and the like. These addition ratios are not particularly limited, and can be added within a range not impairing the effect of the EGCG derivative.

The activity inhibitor of the present invention may further contain other agents having anti-allergen activity. Specific examples thereof include organic anti-allergen agents such as tannic acid and inorganic anti-allergen agents such as zeolite and silver. Etc. Moreover, these may be contained in the activity inhibitor of this invention and may be used together with the activity inhibitor of this invention.

The production method of the EGCG derivative in the present invention is not particularly limited. As the method, for example, a conventionally known method such as an organic synthesis method, a chemical synthesis method using an enzyme or the like can be adopted. The chemical synthesis method using the enzyme is not particularly limited, and examples thereof include a method using lipase disclosed in WO2007 / 105280. That is, this is a method of acylating EGCG by carrying out an enzymatic reaction with lipase using EGCG and an acyl group donor as substrates in an organic solvent. According to this method, for example, EGCG can be selectively acylated. In addition, although the method of using a lipase is illustrated below as an example, this invention is not restrict | limited at all to the manufacturing method of an EGCG derivative | guide_body.

Examples of the lipase include IUB No. 3.1.1.3. Lipase can be used. Specific examples, Aspergillus sp lipase derived from such Aspergillus niger; Candida rugosa, Candida cylindracea , Candida sp lipase derived from such Candida antarctica; Pseudomonas fluorescens, Pseudomonas cepacia , Pseudomonas sp lipase derived from such Pseudomonas stutzeri; Alcaligenes sp lipase; Burkholderia cepacia Lipases derived from the genus Burkholderia, etc .; lipases derived from porcine pancreas and the like. These can be prepared by a conventionally known method. For example, Lipase AS “AMANO”, Lipase AYS “AMANO”, Lipase PS “AMANO”, Lipase AK “AMANO” 20, Lipase AH “AMANO” (all products Name: Amano Enzyme), Lipase MY, Lipase OF, Lipase PL, Lipase PLC, Lipase PLG, Lipase QLM, Lipase QLC, Lipase QLG, Lipase SL, Lipase TL Commercial products such as PPL, L4777 Lipase acrylic resin from Candida Antarctica, and L3126 Lipase from porcine pancreas (all trade names: Sigma Aldrich) can also be used. In addition, the physicochemical properties of each commercially available product are as described in each product description, and enzymes exhibiting similar physicochemical properties can be used in the same manner.

Further, it may be a lipase having the physicochemical properties and enzymological properties of any one of (1) to (8) as shown below.
(1) Molecular weight 35,000, isoelectric point 4.10 (for example, derived from Aspergillus niger )
(2) Inactivation by treatment with molecular weight 64,000, isoelectric point 4.30, 80 ° C. for 10 minutes (eg, derived from Candida rugosa )
(3) Optimal pH 8, optimal temperature 60 ° C, particularly stable in the range of pH 4-10, particularly stable below 70 ° C (eg derived from Pseudomonas fluorescens )
(4) Particularly stable in a solution state at a molecular weight of 60,000, optimum pH 6-7 , pH stability 3-8 , optimum temperature 40-50 ° C., 37 ° C. or less (eg derived from Candida cylindracea, derived from Candida rugosa )
(5) Particularly stable at a molecular weight of 30,000, an isoelectric point of 4.5, an optimum pH of 8 to 9.5, a pH stability of 7 to 10, an optimum temperature of 50 ° C. and 40 ° C. or less (eg, derived from the genus Alcaligenes )
(6) Particularly stable at a molecular weight of 31,000, an isoelectric point of 4.9, an optimal pH of 7-9, a pH stability of 6-10, an optimal temperature of 65-70 ° C., and below 50 ° C. (eg derived from the genus Alcaligenes )
(7) Particularly stable at a molecular weight of 31,000, an isoelectric point of 5.2, an optimal pH of 7-9, a pH stability of 6-10, an optimal temperature of 65-70 ° C., and below 60 ° C. (for example, derived from Pseudomonas cepacia , Burkholderia cepacia origin)
(8) Particularly stable at a molecular weight of 27,000, an isoelectric point of 6.6, an optimum pH of 7 to 8, a pH stability of 6 to 9, an optimum temperature of 50 ° C. and 40 ° C. or less (eg derived from Pseudomonas stutzeri )

The organic solvent is not particularly limited, and for example, acetonitrile, acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and the like can be used. Further, for example, an organic solvent having a hydrophobicity parameter (log P value) in the range of −0.35 to 0.28 may be used. Examples of such an organic solvent include acetonitrile (log P value: −0.45 to 0.19), acetone (log P value: -0.16 to 0.19), DMF (log P value: -1.01 to 0.28), DMSO (log P value: -1.35 to 0.28). can give. In addition to these, conventionally known solvents satisfying the above parameters can be used. Since logP is a value specific to the solvent, those skilled in the art can select a solvent that satisfies the parameters. In addition, logP is the one in which the target substance is added to a mixed solution of octanol and water, and the concentration ratio of the target substance in the octanol layer and the aqueous layer when equilibrium is reached is displayed in the common logarithm. As described above, it is a general parameter indicating the hydrophobicity of a substance.

In the present invention, examples of the acyl group (R—CO—) donor include carboxylic acid vinyl ester (R—CO—O—CH═CH ₂ ). Examples of the acyl group include linear or branched saturated or unsaturated acyl groups as described above.

When DMF is used for the enzyme reaction solution, the addition ratio of EGCG is not particularly limited, but is, for example, 0.2 to 100 mmol / L, preferably 0.5 to 50 mmol / L, more preferably 0.5. ~ 20 mmol / L. The addition ratio of the acyl group donor is not particularly limited, and can be appropriately determined according to, for example, the addition ratio of EGCG in the reaction solution. As a specific example, the addition ratio (molar ratio) of EGCG and acyl group donor is, for example, 1: 1 to 1:10, preferably 1: 1 to 1: 5, more preferably 1: 1 to 1. : 3. Further, the addition ratio of the lipase in the reaction solution can be appropriately determined according to, for example, the addition ratio of EGCG or an acyl group donor, the specific activity of the lipase, etc., but is not particularly limited. For example, for EGCG 1 mmol / L, for example, 500 to 50,000 U / L, preferably 500 to 5,000 U / L, more preferably 1,000 to 2,500 U / L.

The conditions for the enzyme reaction are not particularly limited, but the reaction temperature is, for example, in the range of 45 to 75 ° C. The reaction time can be appropriately determined depending on, for example, the amount of the substrate and the enzyme, and is not particularly limited. For example, the reaction time is 30 minutes to 24 hours (1440 minutes), preferably 1 hour (60 minutes) to 3 hours (180 minutes). ), More preferably 1.5 hours (90 minutes) to 3 hours (180 minutes).

A basic catalyst may be further added to the reaction solution. Examples of the basic catalyst include tertiary amines such as triethylamine, pyridine and the like. The addition rate of the basic catalyst in the reaction solution is not particularly limited, but is, for example, 5 to 720 mmol / L, preferably 12 to 240 mmol / L, more preferably 12 to 48 mmol / L.

The position where the acyl group is introduced in EGCG can be selected depending on, for example, the type of lipase used. Further, the number of acyl groups introduced into EGCG can be determined, for example, depending on the type of organic solvent used and the reaction time. For example, the higher the hydrophobicity of the organic solvent (the lower the hydrophilicity), the lower the number of acyl groups introduced, and the higher the hydrophilicity of the organic solvent ( The lower the hydrophobicity), the greater the number of acyl groups introduced. The number of acyl groups to be introduced can also be adjusted by using a mixture of two or more organic solvents. As a specific example, it is preferable to use acetonitrile or the like when introducing one acyl group, for example, acetone or acetonitrile or the like is used when introducing one or two acyl groups. For example, when introducing 3 to 5 acyl groups, it is preferable to use DMSO, DMF, or the like.

Furthermore, even when the same organic solvent is used, the number of acyl groups to be introduced can be adjusted by combining with control of temperature time and reaction temperature. Examples thereof are shown below, but are not limited thereto. When using DMF, for example, by setting the reaction temperature in the range of about 57 ° C. to about 70 ° C. and increasing the reaction temperature (eg, about 3-5 hours), two acyl groups are selected for EGCG. Can be preferentially obtained, while reducing the reaction temperature (eg, 57 ° C. to about 5 ° C. lower) and shortening the reaction time (eg, about 1-3 hours) Thus, one acyl group can be selectively introduced. Also, one acyl group can be selectively introduced into EGCG by using a mixed solvent in which acetone and DMF are mixed in the same amount (mass).

Also, the number of acyl groups to be introduced can be increased by adding the aforementioned basic catalyst to the reaction solution. In this case, to which site in EGCG the acyl group is further introduced depends on, for example, the regioselectivity of the lipase.

The yield of the EGCG derivative by the enzyme reaction can be relatively improved, for example, by setting the reaction temperature relatively high. The reaction temperature is usually 45 to 75 ° C. as described above, but preferably 57 to 75 ° C., more preferably 57 to 70 ° C. from the viewpoint of improving the yield. In particular, when the reaction temperature is 57 to 70 ° C., the yield of the EGCG acylated derivative can be about 35 to 45%. In addition, the said yield means the ratio (conversion efficiency) of EGCG acylated derivatives (for example, all monoacylated derivatives) when EGCG used for reaction is 100%.

In the present invention, as described above, for example, any one kind of EGCG derivative may be used, or a mixture of two or more kinds may be used. When one kind of EGCG derivative is isolated from the mixture, it can be prepared by a conventionally known method using, for example, chromatography.

<Method for suppressing allergen activity>
As described above, the method for suppressing allergen activity of the present invention is characterized in that the activity inhibitor of the present invention is brought into contact with the allergen. The suppression method of the present invention is characterized by using the activity inhibitor of the present invention, and other configurations and conditions are not limited at all. The activity inhibitor of the present invention and the method of using the same are, for example, the same as described above, and can all be incorporated.

In the present invention, the method for contacting the allergen and the activity inhibitor is not particularly limited. As described above, for example, the allergen of the animal may be brought into contact with the activity inhibitor after being administered to the subject, or the subject in which the allergen may be present is treated with the activity inhibitor. By doing so, the allergen and the activity inhibitor may be brought into contact with each other. In the former, the subject is, for example, an animal (human or non-human animal) as described above, and in the latter, the subject is an environment in which allergens may exist, for example. As an example, the above-mentioned garments and bedding can be cited.

<Method of suppressing allergic reaction>
As described above, the method for suppressing an allergic reaction of the present invention is characterized in that the activity inhibitor of the present invention is administered to a subject. The present invention is characterized in that the activity inhibitor of the present invention is used, and other configurations and conditions are not limited at all. The activity inhibitor of the present invention and the method of using the same are, for example, the same as described above, and can all be incorporated. Moreover, the description in the allergen activity suppression method of the said this invention can also be used for the suppression method of the allergic reaction of this invention, for example.

The method for suppressing an allergic reaction of the present invention can treat, for example, a disease caused by an allergic reaction in the subject by administering the activity inhibitor of the present invention. For this reason, the method for suppressing an allergic reaction of the present invention can also be referred to as a method for treating allergic diseases, for example. In the present invention, “treatment” includes, for example, the meaning of prevention of the disease, improvement of the disease, and improvement of the prognosis of the disease.

The allergic disease is not particularly limited, and examples thereof include hay fever, allergic rhinitis, urticaria, animal allergy, food allergy, insect allergy to cockroaches, house dust allergy, and the like.

The administration timing of the activity inhibitor to the subject is not particularly limited, and may be, for example, before contact with the allergen or after contact with the allergen.

<Use of inhibitors of allergic activity>
The present invention is the use of the activity inhibitor of the present invention for suppressing an allergic reaction and the use of the activity inhibitor of the present invention for treating allergic diseases. Moreover, this invention is use of the activity inhibitor of the said this invention for manufacture of the pharmaceutical for allergic reactions, and use of the activity inhibitor of the said this invention for manufacture of the pharmaceutical for allergic diseases.

Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

As compounds, acylated EGCG derivatives corresponding to the examples, EGCG corresponding to the comparative examples and methylated catechins were prepared.

(1) Acylated EGCG Derivatives In the following chemical formula (2), EGCG derivatives in which one or two of R ¹ to R ⁶ are the following acyl groups were used (all manufactured by Protectia). R ¹ to R ⁶ other than the acyl group were hydrogen atoms.

EGCG-C12, EGCG-C16, EGCG-C18, EGCG-C18DE and EGCG-EH are an acylated derivative of R ¹ , an acylated derivative of R ² , an acylated derivative of R ^{5 and} an acylated derivative of R ⁶ , respectively. These are the four types of mixtures. EGCG-C8 × 2 is an acylated derivative of R ¹ and R ⁵ , an acylated derivative of R ¹ and R ⁶ , an acylated derivative of R ² and R ^5, and an acyl derivative of R ² and R ⁶ 8 kinds of acylated derivatives, acylated derivatives of R ¹ and R ² , acylated derivatives of R ¹ and R ³ , acylated derivatives of R ⁴ and R ⁵ and acylated derivatives of R ⁴ and R ⁶ It is a mixture.

(2) EGCG
In the chemical formula (2), natural type EGCG (trade name Sunphenon EGCG, manufactured by Taiyo Kagaku Co., Ltd.) in which R ¹ to R ⁶ are all hydrogen atoms (non-acyl groups) was used.

(3) Methylated catechin In the above formula (2), monomethylated catechin (manufactured by Protectia) in which only R ⁵ is a methyl group (non-acyl group), and dimethylation in which R ² and R ⁵ are methyl groups Catechin (manufactured by Protectia) was used. In addition, R ¹ to R ⁶ other than the methyl group were hydrogen atoms.

[Example 1]
About the acylated EGCG derivative, the activity suppression ability with respect to various allergens was confirmed.
(1) Mite Allergen Activity Inhibition Test Acarlet mite extract (trade name Mite Extract Df, manufactured by Cosmo Bio) was added to a Dulbecco phosphate physiological buffer (D-PBS (−), Japanese A mite allergen solution was prepared by diluting to a final concentration of 100 μg / mL with Koyo Pure Chemical). In addition, a DMSO solution containing 10 mmol / L of the acylated EGCG derivative was diluted with a phosphate physiological buffer (PBS) to a final concentration of 111 μmol / L to prepare an example sample.

The mite allergen solution 50 μL and the sample 450 μL were mixed in a tube and incubated at room temperature for 2 hours. Thereafter, the mite allergen activity remaining in the mixed solution was measured using a mite allergen measurement ELISA kit (trade name mite allergen (Derf1) measurement kit, manufactured by Nitinichi Pharmaceutical Co., Ltd.). The measurement using the kit was performed according to the instruction manual.

Further, as a control, PBS or 1% DMSO solution (hereinafter referred to as 1% DMSO) was used as a control sample in place of the above-mentioned example sample, and mite allergen activity was measured in the same manner. In the comparative example, natural EGCG was used as a comparative example sample in place of the above-described example sample, and a sample was prepared in the same manner to measure mite allergen activity.

Then, assuming that the mite allergen activity of the control was 100%, the relative values (%) of the mite allergen activities of the examples and comparative examples were calculated, and this was used as the mite allergen activity remaining rate (%). In this example, the measurement was performed twice for each sample, and the average value was used as the result.

These results are shown in FIG. FIG. 1 is a graph showing the residual rate (%) of mite allergen allergen activity. In FIG. 1, the Y axis is the allergen activity remaining rate (%), C12 is EGCG-C12, C16 is EGCG-C16, C18 is EGCG-C18, C18DE is EGCG-C18DE, and EH is EGCG. -EH, C8x2 indicates the result of EGCG-C8x2. In FIG. 1, the lower the mite allergen activity remaining rate, the better the mite allergen activity suppression effect of the sample. As shown in FIG. 1, natural EGCG showed little activity-inhibiting effect on mite allergens, whereas EGCG derivatives (C12, C16, C18, C18DE, EH, C8 × 2) The activity was significantly suppressed against mite allergens. Among them, C12 and C18DE showed a very excellent activity suppressing effect on mite allergen, and in particular, C12 suppressed mite allergen activity to about 10% and was found to be extremely excellent.

(2) Inhibition test of cedar allergen activity A cedar pollen antigen (trade name purified cedar pollen antigen Cryj1, manufactured by Funakoshi Co., Ltd.) was diluted with D-PBS (−) to a final concentration of 500 ng / mL to prepare a squirrel allergen solution. Was prepared. Moreover, the Example sample of the said acylated EGCG derivative prepared by said (1) was used.

The 50 μL of the Sumier allergen solution and 450 μL of the sample were mixed in a tube and incubated at room temperature for 2 hours. Thereafter, the cedar allergen activity remaining in the mixed solution was measured using an ELISA kit for measuring cedar pollen antigen (trade name cedar pollen antigen (Cryj1) measurement kit, manufactured by Nitinichi Pharmaceutical Co., Ltd.). The measurement using the kit was performed according to the instruction manual. Controls and comparative examples were the same as (1) above.

These results are shown in FIG. FIG. 2 is a graph showing the residual ratio (%) of allergen activity of a spear allergen. In FIG. 2, the Y-axis is the allergen activity remaining rate (%), C12 is EGCG-C12, C16 is EGCG-C16, C18 is EGCG-C18, C18DE is EGCG-C18DE, and C8 × 2 is , EGCG-C8 × 2 results are shown. In FIG. 1, it means that the activity suppression effect with respect to a spear allergen by a sample is excellent, so that a spear allergen activity residual rate is low. As shown in FIG. 2, natural EGCG showed almost no activity-suppressing effect on squirrel allergens, whereas EGCG derivatives (C12, C16, C18, C18DE, C8 × 2) respectively showed no effect on squirrel allergens. The activity was significantly suppressed.

In addition, as a comparative example, using the methylated catechin, which has been reported to have an antiallergic effect, in place of the acylated EGCG derivative, the ability to suppress the activity against swarm allergen was confirmed. This comparative example sample uses the methylated catechin (monomethylated catechin or dimethylated catechin) instead of the acylated EGCG derivative, except that the final concentration is 50 μmol / L or 100 μmol / L. Prepared in the same manner as the sample. And using the said comparative example sample, the suger allergen activity was measured like the above-mentioned, and the allergen activity residual rate was calculated | required. The allergen activity remaining rate was determined as a relative value (%) with the allergen activity of 1% DMSO as 100%.

These results are shown in FIG. FIG. 3 is a graph showing the residual ratio (%) of squirrel allergen activity. In FIG. 3, the Y-axis is the allergen activity remaining rate (%), mM EGCG is monomethylated catechin, dM EGCG is dimethylated catechin, and μM (μmol / L) is the methylated catechin in the sample. Indicates the concentration. In FIG. 3, it means that the activity inhibitory effect with respect to a spear allergen is excellent, so that a spear allergen activity residual rate is low. As shown in FIG. 3, methylated catechins showed an allergen activity residual ratio exceeding 100% at any concentration and did not show an activity-inhibiting effect on allergens.

Claims

The inhibitor of allergen activity characterized by including the epigallocatechin gallate derivative represented by following Chemical formula (1), its isomer, or those salts.

In the chemical formula (1),
R 1 to R 6 , R 12 and R 14 are
Each represents a hydrogen atom, halogen, sodium, potassium, or a linear or branched saturated or unsaturated acyl group, which may be the same or different;
The acyl group may be further substituted with one or more substituents,
At least one of the R 1 to R 6 , R 12 and R 14 is the acyl group;
R 7 to R 11 , R 13 , R 15 and R 16 are a hydrogen atom, halogen, sodium or potassium, and may be the same or different.
2. The allergen activity inhibitor according to claim 1, wherein in R 1 to R 6 , the main chain length of the acyl group is 6 to 18 atoms.
The inhibitor of allergen activity according to claim 2, wherein in R 1 to R 6 , the main chain length of the acyl group is 12 to 18 atoms.
The allergen activity inhibitor according to any one of claims 1 to 3, wherein in R 1 to R 6 , the acyl group has 8 to 18 atoms.
The allergen activity inhibitor according to any one of claims 1 to 4, wherein in R 1 to R 6 , the acyl group has 12 to 18 carbon atoms.
The allergen activity inhibitor according to any one of claims 1 to 5, wherein at least one of R 1 , R 2 , R 5 and R 6 is the acyl group.
The allergen activity inhibitor according to claim 6, wherein at least one of R 1 , R 2 , R 5 and R 6 is the acyl group, and the other is a hydrogen atom.
The allergen activity inhibitor according to any one of claims 1 to 5, wherein at least two of R 1 , R 2 , R 5 and R 6 are the acyl group.
The inhibitor of allergen activity according to claim 8, wherein at least two of R 1 , R 2 , R 5 and R 6 are the acyl group, and the other is a hydrogen atom.
The allergen activity inhibitor according to any one of claims 1 to 9, wherein R 7 to R 11 , R 13 , R 15 and R 16 are hydrogen atoms.
The allergen activity inhibitor according to any one of claims 1 to 10, wherein the acyl group is at least one of a linear saturated acyl group and a linear unsaturated acyl group.
The acyl group is a butyryl group, 2-ethylhexanoyl group, octanoyl group, trans-8-methyl-6-octenoyl group, geranoyl group, lauroyl group, 12- (dimethylamino) lauroyl group, farnesoyl group, palmitoyl group, The inhibitor of allergen activity according to any one of claims 1 to 11, which is at least one acyl group selected from the group consisting of a stearoyl group, a linoleyl group, a linolenyl group, an eicosanoyl group and isomers thereof.
The inhibitor of allergen activity as described in any one of Claim 1 to 12 whose said inhibitor of allergen activity is an inhibitor with respect to a mite allergen or a spear allergen.
A method for suppressing allergen activity, comprising contacting the allergen with the allergen activity inhibitor according to any one of claims 1 to 13.
A method for suppressing an allergic reaction, comprising administering an inhibitor of allergen activity according to any one of claims 1 to 13 to a subject.