JP6606370B2 - Antiallergic agent and mediator release inhibitor - Google Patents

Description

  The present invention relates to an antiallergic agent and a mediator release inhibitor containing an acylated derivative of L-ascorbic acid, its isomer, or a salt thereof as an active ingredient.

In recent years, the number of patients with type I allergies such as hay fever has increased. Type I allergy is caused by the release of mediators such as histamine by degranulation of mast cells.
Therefore, Patent Document 1 describes an antihistamine containing fexofenadine hydrochloride having histamine H1 receptor antagonistic activity.
Patent Document 2 describes an antihistaminergic enhancer containing clemastine fumarate or chlorpheniramine d-maleate as an antihistamine.

JP 2013-75892 A JP 2009-185058 A

Here, the histamine receptor is known to have a wakefulness action and the like. As a result, taking the above antihistamine that antagonizes the histamine receptor may cause drowsiness, malaise, dry mouth, and the like as side effects, so care must be taken.
Therefore, a safe antiallergic agent with fewer side effects has been demanded.

  In view of the circumstances as described above, an object of the present invention is to provide a highly safe antiallergic agent and mediator release inhibitor.

In order to achieve the above object, an antiallergic agent according to one embodiment of the present invention includes an acylated derivative of L-ascorbic acid in which the hydroxyl group at the 6-position is acylated with a linear acyl group, Or a pharmaceutically acceptable salt of the acylated derivative or isomer thereof as an active ingredient.
The antiallergic agent has a high antiallergic action because it is acylated with a linear acyl group. The antiallergic agent does not have side effects such as drowsiness, malaise and thirst because it has an antiallergic effect not by antagonizing the histamine receptor but by mediator release inhibitory action. Furthermore, the antiallergic agent is very safe because it can be metabolized in the body to become ascorbic acid.

The acyl group of the antiallergic agent may have 10 or more carbon atoms.
Furthermore, the acyl group of the antiallergic agent may have 12 or more carbon atoms.
The antiallergic agent has a higher antiallergic effect as the carbon number of the acyl group is higher.

（式中、Ｒ１はアルキル基を表し、Ｒ２は水酸基又は置換基を表す。）
で表されていてもよい。 The acylated derivative of L-ascorbic acid is represented by the following general formula (1):

(In the formula, R1 represents an alkyl group, and R2 represents a hydroxyl group or a substituent.)
It may be represented by

The acylated derivative may have a glycosyl group bonded to the 2-position.
Thereby, the stability of the acylated derivative of the L-ascorbic acid, its isomer, or a pharmaceutically acceptable salt thereof can be enhanced.

  In addition, the mediator release inhibitor according to another aspect of the present invention is an acylated derivative of L-ascorbic acid in which the hydroxyl group at the 6-position is acylated with a linear acyl group, an isomer thereof, or A pharmaceutically acceptable salt of the acylated derivative or isomer thereof is used as an active ingredient.

  The external preparation according to still another aspect of the present invention is an acylated derivative of L-ascorbic acid, an isomer thereof, or the above acylated derivative or an isomer thereof, which is acylated with a linear acyl group. It has an anti-allergic action as an active ingredient.

  As described above, according to the present invention, a highly safe antiallergic agent and mediator release inhibitor can be provided.

It is a graph which shows the result of Test Example 1 of the present invention. It is a graph which shows the result of Test Example 2 of the present invention. It is a graph which shows the result of Test Example 3 of the present invention.

  Hereinafter, the present invention will be described in detail.

本発明のAA誘導体は、６位のヒドロキシル基が、直鎖状のアシル基によってアシル化されていることを特徴とする。本発明者らは、このようなAA誘導体が、高い抗アレルギー作用及びメディエーター遊離阻害作用を有することを見出し、本発明に想到した。
また、本発明のAA誘導体は、下記の一般式（１）で表されるものであってもよい。

一般式（１）中、Ｒ１はアルキル基（Ｃ_ｎＨ_２n＋１）を表し、Ｒ２は水酸基又は置換基を表す。 <Acylated derivative of L-ascorbic acid>
The antiallergic agent and mediator release inhibitor of the present invention include an acylated derivative (hereinafter also referred to as AA derivative) of L-ascorbic acid (hereinafter also referred to as AA), an isomer thereof, or the acylated derivative or an isomer thereof. The salt is an active ingredient. The structural formula of L-ascorbic acid is represented by the following general formula (2).

The AA derivative of the present invention is characterized in that the hydroxyl group at the 6-position is acylated with a linear acyl group. The present inventors have found that such AA derivatives have a high antiallergic action and mediator release inhibitory action, and have arrived at the present invention.
Moreover, the AA derivative of the present invention may be represented by the following general formula (1).

In the general formula (1), R1 represents an alkyl group _{(C n H 2n + 1)} , R2 represents a hydroxyl group or a substituent.

<Substituent>
In the present invention, the substituent of R2 in the general formula (1) is a substituent bonded to the 2-position of the AA derivative, preferably a glycosyl group, a phosphate group, a sulfate group, an alkyl group, an alkenyl group, It shall mean a phenyl group or the like.
Specific examples of the glycosyl group include α-D-monoglucopyranosyl group, β-D-monoglucopyranosyl group and other glucopyranosyl groups, and β-D-monogalactopyranosyl group and others. Examples thereof include a galactopyranosyl group.
Specific examples of the phosphoric acid group include a monophosphoryloxy group, a pyrophosphoryl group, a triphosphoryl group, and a polyphosphoryl group.
When the substituent is bonded to the 2-position of the AA derivative of the present invention, the high reducibility of AA can be reduced. Thereby, the stability of the AA derivative against light, heat, oxygen, metal ions, etc. can be enhanced.

一般式（３）中、Ｒ１はアルキル基（Ｃ_ｎＨ_２n＋１）を表す。 <Substituent: glycosyl group>
Furthermore, as described above, the AA derivative of the present invention may have a glycosyl group bonded to the 2-position as the substituent. Thereby, the stability of the AA derivative against light, heat, oxygen and the like can be further increased, and the AA derivative can be easily produced. Furthermore, since the above substituents are also sugars when metabolized in vivo, safety against the living body can be ensured.
Further, the glycosyl group may be an α-D-monoglucopyranosyl group as represented by the following general formula (3).

In the general formula (3), R1 represents an alkyl group _{(C n H 2n + 1)} .

<Acylation / Linear acyl group>
In the present invention, acylation refers to introducing an acyl group into the 6-position hydroxyl group of ascorbic acid. When the AA derivative of the present invention contains a linear acyl group, it can exhibit a high antiallergic action and mediator release inhibitory action.
The linear acyl group may be an acyl group composed of a saturated fatty acid, as described using the general formula (1). Thereby, the stability of the AA derivative can be further improved.
The acyl group may preferably have 10 or more carbon atoms, more preferably 12 or more carbon atoms. As described later, the AA derivative of the present invention has higher antiallergic action and mediator release inhibitory action as the carbon number of the acyl group is larger.
Further, the upper limit of the carbon number of the acyl group is not particularly limited, and is appropriately set in view of the physical properties of the AA derivative, the base used for formulation, and the like. For example, an AA derivative having 16 or less carbon atoms has a relatively high water solubility and can be dissolved in an aqueous base or the like. For example, since an AA derivative having 18 or more carbon atoms has high lipophilicity, an oily base or an emulsified base can be used.
Specific examples of the acyl groups of the present invention, (R1 is _C 9 _{H 19} in the general formula (1)) decanoyl group of 10 carbon atoms, R1 in dodecanoyl group having a carbon number of 12 (the general formula (1) Is C ₁₁ H ₂₃ ), a tetradecanoyl (myristyl) group having 14 carbon atoms (R1 in the general formula (1) is C ₁₃ H ₂₇ ), a hexadecanoyl (palmityl) group having 16 carbon atoms (the general formula (1) R1 in can be mentioned _C 15 _{H 31).}

<Isomer of acylated derivative of L-ascorbic acid>
The isomer of the AA derivative of the present invention is a compound in which the hydroxyl group at the 6-position of the isomer of L-ascorbic acid is acylated with a linear acyl group. Examples of isomers of L-ascorbic acid include D-ascorbic acid, L-araboascorbic acid, and D-araboascorbic acid (also referred to as erythorbic acid and isoascorbic acid). Of these, erythorbic acid is preferably used in view of the flowability and the like.
These isomers are known to have the same function as L-ascorbic acid (see Suzuki et al., J. Nutr. Sci. Vitaminol., 41, 17-24, 1995, etc.). Can also be used in the same manner as L-ascorbic acid.

<Acylated derivative of L-ascorbic acid or salt of isomer thereof>
The antiallergic agent and mediator release inhibitor of the present invention may contain, as an active ingredient, an acylated derivative of L-ascorbic acid or a pharmaceutically acceptable salt thereof.
As the acylated derivative of L-ascorbic acid of the present invention or a salt of an isomer thereof, one having particularly low toxicity is preferable. Examples of inorganic bases that generate these salts include alkali metals (for example, sodium, potassium, etc.), ammonium, alkaline earth metals (for example, calcium, magnesium, strontium, barium, etc.), aluminum salts, etc. Examples thereof include one or a mixture of two or more selected from trimethylamine, triethylamine, pyridine, picoline, N, N-dibenzylethylenediamine, ethanolamine, diethanolamine, trishydroxymethylaminomethane, dicyclohexylamine and the like. Among these, those selected from one or a mixture of two or more selected from sodium, potassium, magnesium, calcium, and aluminum are particularly easy to apply.
The AA derivative or isomer salt thereof may be, for example, a structure in which the 3-position of L-ascorbic acid is an anion or a structure in which the substituent at the 2-position is an anion. Good.

<Method for producing acylated derivative of L-ascorbic acid>
Various methods can be used for producing the AA derivative of the present invention. For example, Yamamoto et al., “Synthesis and characterization of a series of novel monoacylated ascorbic acid derivatives, 6-O-acyl-2-O-α- Reference can be made to the methods described in “D-glucopyranosyl-L-ascorbic acids, as skin antioxidants” J. Med. Chem., 45, 462-468, 2002 and Japanese Patent No. 4981198.
For example, as the above production method, the hydroxyl group at the 2-position may be glycosylated, and then the hydroxyl group at the 6-position may be acylated using an acylating agent or the like.
As the glycosylated 2-glycosyl-L-ascorbic acid, for example, a commercially available product can be used. Alternatively, 2-glycosyl-L-ascorbic acid can also be obtained by a known method (JP-A-3-139288, JP-A-3-135992, JP-A-3-183492, JP-A-6-126). 263790 publication etc.).
For example, 2-phosphate-L-ascorbic acid can be obtained by a known method, or commercially available 2-phosphate-L-ascorbic acid or a salt thereof (2-phosphate-L-ascorbic acid). Sodium, etc.) can be used.
Alternatively, as the above production method, the 6-position hydroxyl group of L-ascorbic acid can be acylated and then the 2-position hydroxyl group can be glycosylated.
The AA derivative of the present invention obtained by these methods can be purified by applying a usual method for purifying a fatty acid ester of L-ascorbic acid. Examples of the purification method include salting out, dialysis, filtration, concentration, fractional precipitation, liquid separation extraction, gel chromatography, ion exchange chromatography, high performance liquid chromatography, gas chromatography, affinity chromatography, gel electrophoresis, isoelectric focusing, Examples include crystallization. These may be applied in combination as appropriate.

<Method for producing acylated derivative of L-ascorbic acid: acylation>
Examples of the acylating agent used in the production of the AA derivative of the present invention include acids or acid halides, acid anhydrides or acid esters, and more specifically, decanoic acid, dodecanoic acid (lauric acid), Examples thereof include carboxylic acids such as myristic acid and palmitic acid, acid halides, acid anhydrides, and carboxylic acid esters.
The acylation in the AA derivative of the present invention can be typically carried out by a non-aqueous chemical reaction that blocks water from entering the reaction system. In this case, for example, in an organic solvent such as pyridine, dimethyl sulfoxide, and dimethylformamide, if necessary, a catalyst such as p-toluenesulfonic acid is allowed to coexist, and carboxylic acid anhydride is added to 2-glycosyl-L-ascorbic acid or the like. Can be reacted.
Alternatively, acylation in the AA derivative of the present invention can also be performed by an enzymatic reaction. In this case, glycosyl-L-ascorbic acid or the like and an acylating agent are applied as substrates, and an organic solvent corresponding to these substrates and enzymes can be appropriately used.

<Antiallergic agent>
The antiallergic agent of the present invention contains the acylated AA derivative as an active ingredient and can be applied to allergies, particularly type I allergies. Specifically, the antiallergic agent can suppress symptoms such as rhinitis, conjunctivitis, bronchial asthma, food allergy, anaphylaxis, urticaria, or atopic dermatitis. The allergic allergen to which the antiallergic agent is applied is not particularly limited, and can be, for example, pollen, mites, animal dander, mold, chemical substances, protein in foods, and the like.

<Mediator release inhibitor>
The mediator release inhibitor of the present invention contains the acylated AA derivative as an active ingredient, and can inhibit the release of inflammatory mediators such as histamine that cause type I allergy. As described above, type I allergy is caused by the release of mediators such as histamine by the degranulation reaction of mast cells. As will be described later, the AA derivative can suppress the degranulation reaction of mast cells and can exert the mediator release inhibitory action.

<Antiallergic agent and mediator release inhibitor: Form>
The antiallergic agent and mediator release inhibitor of the present invention can take various forms such as pharmaceuticals, quasi drugs, and cosmetics. The case where it is used as a medicine will be described later.
The antiallergic agent and mediator release inhibitor of the present invention are applied to creams, emulsions, lotions, packs, bath preparations, etc. for sensitive skin or allergic skin when used as medicinal cosmetics for cosmetics and quasi drugs. Can do. In this case, the antiallergic agent and mediator release inhibitor are oily or aqueous bases, vitamins, emollients, whitening agents, moisturizers, antioxidants, buffers, UV absorbers, chelating agents, pH adjusters, Preservatives, thickeners, alcohols, cooling agents, colorants, fragrances and the like can be appropriately blended as necessary.

<Antiallergic agents and mediator release inhibitors: Pharmaceuticals>
The antiallergic agent and mediator release inhibitor of the present invention can take various dosage forms depending on the application, for example, as a pharmaceutical product. Examples of the dosage form include liquids, ointments, eye drops, suspensions, patches, injections, powders, tablets, granules, powders, capsules, suppositories and the like. When the antiallergic agent and mediator release inhibitor of the present invention are used as pharmaceuticals, either parenteral or oral administration is possible, but parenteral administration is more preferable.
In particular, the acylated AA derivative or a salt thereof can be used as an active ingredient of an external medicine having an antiallergic action. The topical medicine referred to here is a medicine excluding internal medicines and injections, and includes, for example, a topical skin medicine (skin medicine), eye drops, nasal drops, inhalants and the like. By using the acylated AA derivative as an external medicine, it is difficult to be metabolized in the tissue to which the AA derivative has been administered. Therefore, as will be described later, the antiallergic action can be exerted using the AA derivative itself as an action body.
The antiallergic agent and mediator release inhibitor of the present invention can be formulated into an AA derivative or salt thereof as an active ingredient according to a known pharmaceutical manufacturing method (16th revised Japanese Pharmacopoeia (Heisei 16) March 24, 2011 See Ministry of Health, Labor and Welfare Notification No. 65)).
In this case, the antiallergic agent and the mediator release inhibitor can contain a pharmaceutically acceptable base, other medicinal components, additives and the like in addition to the above active ingredients.
The base may be any of an oily base, an emulsified base and an aqueous base.
As other medicinal ingredients, for example, analgesic / anti-inflammatory agents, bactericidal / disinfectants, vitamins, emollients, whitening agents, moisturizers and the like can be used as needed.
As additives, buffering agents, tonicity agents, ultraviolet absorbers, chelating agents, pH adjusters, preservatives, excipients, thickeners, alcohols, cooling agents, coloring agents, flavors, and the like can be blended. .
Moreover, when the antiallergic agent and mediator release inhibitor of the present invention are used as an external medicine, the content of the AA derivative can be, for example, 0.0005 to 25 w / v%. Moreover, although administration frequency is not specifically limited, It can be set about 1 to 6 times a day.

<Operational effect of the present invention>
The antiallergic agent and mediator release inhibitor of the present invention can effectively inhibit the release of inflammatory mediators such as histamine. In addition, side effects such as sleepiness, malaise and thirst in antihistamines having a receptor antagonistic action can be avoided. Thereby, it is possible to provide a drug having a higher safety and an antiallergic action.
In addition, the AA derivatives and salts thereof of the present invention can be metabolized in the body to become AA. As a result, the safety is very high, and it can be expected that AA exhibits an activity such as an antioxidant effect.

  Hereinafter, the present invention will be further described based on examples and the like.

[Example 1: 6-sDeca-AA-2G]
As Example 1, an AA derivative in which a decanoyl group having 10 carbon atoms was bonded to the 6-position and an α-D-monoglucopyranosyl group was bonded to the 2-position was prepared. That is, this derivative is 6-decanoyl-2-O-α-D-monoglucosyl-L-ascorbic acid and is hereinafter referred to as 6-sDeca-AA-2G.
6-sDeca-AA-2G is written by Yamamoto et al., `` Synthesis and characterization of a series of novel monoacylated ascorbic acid derivatives, 6-O-acyl-2-O-α-D-glucopyranosyl-L-ascorbic acids, as skin. It was prepared as follows with reference to "antioxidants", J. Med. Chem., 45, 462-468, 2002.
First, 2-O-α-D-monoglucosyl-L-ascorbic acid (produced by Hayashibara Biochemical Laboratories Co., Ltd.) (hereinafter referred to as AA-2G) and n-caprylic anhydride are mixed with pyridine. The mixture was stirred at 60 ° C. for 30 min and the reaction was concentrated under reduced pressure. Ethyl acetate was added to the residue obtained by concentration to obtain a white precipitate. The residue was then filtered, washed with warm ethyl acetate, dried, and 6-sDeca-AA-2G recrystallized. The obtained substance was analyzed by UV spectrum, elemental analysis, nuclear magnetic resonance spectrum measurement method, etc., and confirmed to be 6-sDeca-AA-2G.

[Example 2: 6-sDode-AA-2G]
As Example 2, an AA derivative in which a dodecanoyl group having 12 carbon atoms was bonded to the 6-position and an α-D-monoglucopyranosyl group was bonded to the 2-position was prepared. That is, this derivative is 6-dodecanoyl-2-O-α-D-monoglucosyl-L-ascorbic acid, hereinafter referred to as 6-sDode-AA-2G.
6-sDode-AA-2G was produced in the same manner as in Example 1 except that lauric anhydride was used as the acid anhydride. The obtained substance was analyzed in the same manner as in Example 1 and confirmed to be 6-sDode-AA-2G.

[Example 3: 6-sMyri-AA-2G]
As Example 3, an AA derivative in which a myristyl (tetradecanoyl) group having 14 carbon atoms was bonded to the 6-position and an α-D-monoglucopyranosyl group was bonded to the 2-position was prepared. That is, this derivative is 2-O-α-D-monoglucosyl-6-myristyl (tetradecanoyl) -L-ascorbic acid, hereinafter referred to as 6-sMyri-AA-2G.
6-sMyri-AA-2G was produced in the same manner as in Example 1 except that myristic acid anhydride was used as the acid anhydride. The obtained substance was analyzed in the same manner as in Example 1 and confirmed to be 6-sMyri-AA-2G.

[Example 4: 6-sPalm-AA-2G]
As Example 4, an AA derivative in which a palmityl (hexadecanoyl) group having 16 carbon atoms was bonded to the 6-position and an α-D-monoglucopyranosyl group was bonded to the 2-position was prepared. That is, this derivative is 2-O-α-D-monoglucosyl-6-palmityl (hexadecanoyl) -L-ascorbic acid, which is hereinafter referred to as 6-sPalm-AA-2G.
6-sPalm-AA-2G was produced in the same manner as in Example 1 except that palmitic acid anhydride was used as the acid anhydride. The obtained substance was analyzed in the same manner as in Example 1 and confirmed to be 6-sPalm-AA-2G.

[Example 5: 6-sPalm-AA]
As Example 5, an AA derivative in which a palmityl group having 16 carbon atoms was bonded to the 6-position was prepared. That is, this derivative is 6-palmityl-L-ascorbic acid and is hereinafter referred to as 6-sPalm-AA.
6-sPalm-AA purchased the product name 6-O-palmitoyl-L-ascorbic acid from Tokyo Chemical Industry Co., Ltd.

[Comparative Example 1: AA]
As Comparative Example 1, AA was prepared. AA purchased the product name sodium ascorbate from Wako Pure Chemical Industries, Ltd.

[Comparative Example 2: AA-2G]
As Comparative Example 2, AA-2G (manufactured by Hayashibara Biochemical Laboratory Co., Ltd.) was prepared.

  The following Test Example 1, Test Example 2, and Test Example 3 were performed using the drugs of Examples 1 to 5 and Comparative Examples 1 and 2.

<Test Example 1: Hyaluronidase inhibition test>
Hyaluronidase is a hyaluronic acid hydrolase and is known to be activated during inflammation and involved in degranulation of mast cells. Therefore, a hyaluronidase inhibition test was performed in order to confirm the effectiveness of the drug according to the example as an antiallergic agent.

(Adjustment of reagents)
The acetate buffer was prepared to 0.1 M, pH 4.0 using acetic acid and sodium acetate solution.
The aqueous sodium borate solution was adjusted to pH 9.1 with NaOH by adding 40 ml of water to 4.95 g of boric acid, and then filled up to 100 ml with water.
Hyaluronidase solution is prepared by dissolving hyaluronidase (product name `` Hyaluronidase Type IV-S from Bovine Testis (lot; SLBF8562V) '', SIGMA-ALDRICH CO. LLC) (1148 units / mg) in acetate buffer to 615.4 units / ml It was done.
The hyaluronic acid solution was prepared by dissolving Hyaluronic acid sodium salt (product name “hyaluronic acid sodium salt from rooster Comb”, Wako Pure Chemical Industries, Ltd.) in an acetate buffer to 4.0 mg / ml.
The coloring solution was prepared by adding 100 mg of p-dimethylaminobenzaldehyde (Wako Pure Chemical Industries, Ltd.) to 875 μl of acetic acid and 125 μl of HCl, and diluted 10 times with acetic acid at the time of use.
The drugs of Examples 1 to 5 and Comparative Examples 1 and 2 were prepared with an acetate buffer containing 30% DMSO. The final concentration of DMSO during the enzyme reaction was 3%. In addition, Examples 1 to 5 and Comparative Examples 1 and 2 thus prepared are referred to as samples in this test example, respectively.
As a positive control, DSCG (Disodium chromoglycate, SIGMA-ALDRICH Co. LLC) was used. DSCG is known as a mediator release inhibitor (see Hisao et al., Chem. Pharm. Bull., 33, 642-646, 1985).

(Measurement of hyaluronidase inhibitory activity)
First, 325 μl of the prepared hyaluronidase acid solution and 50 μl of each sample were incubated at 37 ° C. for 20 minutes. Subsequently, Compound 48/80 (SIGMA-ALDRICH CO. LLC) was prepared in 2.0 mg / ml Compound 48/80 solution prepared in acetic acid buffer, CaCl ₂ solution prepared in 50 mM CaCl ₂ in acetic acid buffer, and NaCl in acetic acid buffer. 25 μl each of the prepared 3 M NaCl solution was added, and further incubated at 37 ° C. for 20 minutes. Subsequently, 50 μl of the prepared hyaluronic acid solution was added and reacted at 37 ° C. for 40 minutes (containing a final concentration of 3% DMSO during the enzyme reaction).
Thereafter, 100 μl of 0.4 N NaOH was added to each reaction solution to stop the reaction. After cooling with ice for 10 minutes, 120 μl of the prepared sodium borate solution was added to each reaction solution and heated at 100 ° C. for 3 minutes. After ice-cooling for 5 minutes, 30 μl was dispensed into a 96-well microplate containing 150 μl of the coloring reagent and allowed to react at 37 ° C. for 20 minutes.
Subsequently, hyaluronidase inhibitory activity was measured with reference to the Morgan-Elson method (see J. Biol. Chem., 217, 959-966, 1955). First, the absorbance at 585 nm of each reaction solution was measured, and the inhibition rate relative to the positive control was calculated. The inhibition rate (Inhibition) is expressed by the following mathematical formula.
Inhibition (%) = 100-(absorbance of sample / absorbance of positive control) x 100
Finally, a 50% inhibitory concentration (IC ₅₀ ), which is the concentration of the sample at which the inhibition rate is 50%, was determined.

(result)
FIG. 1 is a graph showing the results of this test example, where the horizontal axis represents each sample and the vertical axis represents IC ₅₀ . In addition, the numerical value of a bar graph shows an average value, and each error bar shows a standard deviation.
As shown in the figure, the IC ₅₀ of the samples of Comparative Examples 1 and 2 (AA and AA-2G) was higher than that of the positive control. This confirmed that these samples had low hyaluronidase inhibitory activity.
On the other hand, the IC ₅₀ of the samples of Examples 1 to 5 were all lower than the positive control DSCG. Thereby, it was confirmed that the samples of Examples 1 to 5 have very high hyaluronidase inhibitory activity even when compared with the existing mediator release inhibitors.
From the above results, it was suggested that the samples of Examples 1 to 5 have an anti-inflammatory action and a mediator release inhibitory action.
Furthermore, when comparing the IC ₅₀ in the samples of Examples 1 to 4 (hereinafter also referred to as 6-sAcyl-AA-2G), the longer the carbon number of the acyl group, the lower the IC ₅₀ , and the lower the concentration, the higher the inhibition of hyaluronidase. It was confirmed to show activity.
Moreover, it is known that 6-sAcyl-AA-2G of Examples 1 to 4 is metabolized in vivo to AA-2G of Comparative Example 2 and AA of Comparative Example 1. From this, it was confirmed that the action of 6-sAcyl-AA-2G itself is greater than that of metabolites AA-2G and AA with respect to the hyaluronidase inhibitory activity of 6-sAcyl-AA-2G.
In addition, when the IC ₅₀ in the sample of Example 4 (6-sPalm-AA-2G) and Example 5 (6-sPalm-AA) was compared, the former had higher hyaluronidase inhibitory activity than the latter. Thereby, it was confirmed that the AA derivative having an acyl group bonded to the 6-position and a substituent bonded to the 2-position has higher hyaluronidase inhibitory activity.

<Test Example 2: Degranulation inhibition test (Examples 1 to 4, Comparative Examples 1 and 2)>
Subsequently, in order to confirm the action of the drugs according to Examples 1 to 4 on degranulation of mast cells causing symptoms of type I allergy, a degranulation inhibition test using rat basophilic leukemia cell RBL-2H3 was conducted. went.

(Used cells)
Rat basophilic leukemia cells (RBL-2H3) used in this test example were purchased from Human Science Cell Bank (JCRB). All the cells (RBL-2H3) used in the experiment were cultured in a DMEM medium containing 10% FBS (described later) under conditions of 37 ° C. and 5% CO ₂ and subcultured every 2 or 3 days.

(Adjustment of reagents)
The antibody solution was prepared as follows. First, Anti-DNP IgE (Nacalai Tesque Co., Ltd.) was added to Dulbecco's phosphate buffered saline (PBS (-)) and dissolved to 250 μg / ml. Thereafter, it was stored at −30 ° C. and diluted to 500 ng / ml with 10% FBS-containing DMEM medium at the time of use. FBS (Fetal bovine serum (lot. AVH77993)) was purchased from HyClone. The DMEM medium was purchased from Corning and used with 100 U / ml penicillin G and 100 μg / ml streptomycin (both from Nacalai Tesque).
MT buffer (modified tyrode buffer) is ultrapure water, 137 mM sodium chloride (Wako Pure Chemical Industries, Ltd.), 2.7 mM potassium chloride (Nacalai Tesque), 1.8 mM calcium chloride, 1 mM magnesium chloride hexahydrate Product (Wako Pure Chemical Industries, Ltd.), 5.6 mM (+)-glucose, 20 mM HEPES (Wako Pure Chemical Industries, Ltd.), and 0.1% bovine serum albumin (BSA) (Nacalai Tesque, Inc.) And sodium hydroxide (Wako Pure Chemical Industries, Ltd.). The prepared MT buffer was filter sterilized and stored at 4 ° C. In use, it was heated to 37 ° C.
The antigen solution was prepared as follows. First, DNP-HSA (SIGMA-ALDRICH CO. LLC) was added to PBS (−), dissolved to 10 mg / ml, and stored at 4 ° C. At the time of use, it was diluted to 500 ng / ml with the above MT buffer.
When using 0.1% Triton solution, Triton X-100 (SIGMA-ALDRICH CO. LLC) was diluted to 0.1% with MT buffer.
The substrate solution was prepared by adding 3.3 mM p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside (Nacalai Tesque) to 100 mM citrate buffer (pH 4.5) at the time of use.
The enzyme reaction stop solution (2 M Glycine buffer) was prepared by adding 2 M glycine solution (Glycine purchased from Wako Pure Chemical Industries, Ltd.) to sodium hydroxide to pH 10.4.
Each of the drugs of Examples 1 to 4 and Comparative Examples 1 and 2 was prepared to a predetermined concentration with MT buffer containing 0.25% DMSO. Examples 1 to 4 and Comparative Examples 1 and 2 thus prepared are referred to as samples in this test example, respectively.
As a negative control, an MT buffer containing 0.25% DMSO to which no drug was added was used (indicated as “control” in FIG. 2).
As a positive control, 75 μM oxatomide (Wako Pure Chemical Industries, Ltd.) was used. Oxatimide is known as an H1 receptor blocker and an inflammatory mediator release inhibitor.

(Measurement of degranulation inhibitory activity)
First, the used cells cultured until they became semi-confluent were detached with a 0.05% trypsin solution and seeded at a density of 5.0 × 10 ⁴ cells / well / 200 μl in a 96-well flat bottom multiplate (NUNC, 167008). After culturing the cells for 24 hours, remove the culture supernatant, add 100 μl of antibody solution prepared to 500 ng / ml to each well, and incubate for 2 hours at 37 ° C and 5% CO ₂ did.
After incubation, the cell layer was washed twice with MT buffer warmed to 37 ° C., 90 μl of sample solution was added to each well, and incubated for 20 minutes at 37 ° C. and 5% CO ₂ .
Subsequently, 10 μl of the antigen solution was added to each well and incubated for 1 hour, followed by ice cooling for 10 minutes to stop the reaction, and the supernatant was collected. 20 μl of this supernatant was dispensed into 96-well microplates for measurement (“WATSON (registered trademark)”, Fukae Kasei Co., Ltd.).
On the other hand, 100 μl of 0.1% Triton solution was added to the cell layer, dissolved by stirring (1,000 rpm, 3 min), and 20 μl of this cell lysate was dispensed into a 96-well microplate for measurement. .
Subsequently, 40 μl of the substrate solution was added to each well and reacted at 37 ° C. for 90 minutes. After the reaction, the enzyme reaction was stopped with 40 μl of enzyme reaction stop solution (glycine buffer), the color was developed, and the absorbance at 405 nm was measured (product name “MULTISKAN FC”, Thermo Fisher Scientific Co., Ltd.). The β-hexosaminidase release rate (degranulation rate) was calculated by the following formula.
Degranulation rate (%) = [(S-Sc) / {(S-Sc) + (CL-CLc)}] x 100
S: Absorbance of supernatant
Sc: Absorbance of supernatant added in the order of enzyme stop solution and substrate solution
CL: Absorbance of cell lysate
CLc: Absorbance of cell lysate added in the order of enzyme reaction stop solution and substrate solution In the above formula, Sc and CLc indicate the absorbance of the sample solution not subjected to enzyme reaction. For the measurement of Sc and CLc, the enzyme reaction stop solution was not added after the above substrate solution was added, but the enzyme reaction stop solution was added first to suppress the enzyme reaction and then the substrate solution was added. And cell disruption fluid was used.

(Statistical processing)
Quantitative data are expressed as mean +/- standard deviation.
About comparison of the average value between many groups, it was determined by Dunnett's test after one-way analysis of variance, and judged to be significant with a risk factor of less than 0.05.

(result)
FIG. 2 is a graph showing the results of Examples 1 to 4 and Comparative Examples 1 and 2 in this test example, where the horizontal axis represents the sample and the vertical axis represents the degranulation ratio. In addition, the numerical value of a bar graph shows an average value, and each error bar shows a standard deviation.
As shown in the figure, Examples 1 to 4 all showed a decrease in degranulation in a concentration-dependent manner, so it was confirmed that each drug according to Examples 1 to 4 has a degranulation inhibitory effect. .
Moreover, like the result of Test Example 1, Examples 1 to 4 exhibited a high degranulation inhibitory effect as the carbon number of the acyl group increased. In particular, the sample of Example 4 (6-sPalm-AA-2G) exhibited an activity equivalent to that of the positive control oxatomide.
In particular, in Examples 2 to 4, significant activity was recognized as compared with the negative control. Thereby, it was confirmed that when the number of carbon atoms of the 6-position acyl group is 12 or more, a higher degranulation inhibitory effect can be exhibited.
On the other hand, Comparative Examples 1 and 2 showed a tendency to suppress degranulation in a concentration-dependent manner, but sufficient activity was not observed as compared with Examples 1 to 5.
Furthermore, from this, it is considered that the degranulation inhibitory action according to Examples 1 to 4 is not due to the activity of metabolites but is due to the AA derivative itself of the present invention in which a fatty acid is added at the 6-position.

<Test example 3: degranulation suppression test (Example 5)>
Subsequently, rat basophilic leukemia cells RBL-2H3 were used in the same manner as in Test Example 2 in order to confirm the action of the drug according to Example 5 on mast cell degranulation causing symptoms of type I allergy. A degranulation inhibition test was conducted. As a negative control, an MT buffer containing 0.25% DMSO to which no drug was added was used (indicated as “control” in FIG. 3). As a positive control, 75 μM oxatomide (Wako Pure Chemical Industries, Ltd.) was used.
In addition, since it is the same as that of Test Example 2 about adjustment of a use cell, reagents, measurement of degranulation inhibitory activity, and statistical processing, description is abbreviate | omitted.

(result)
FIG. 3 is a graph showing the results of Example 5 in this test example, in which the horizontal axis indicates the sample and the vertical axis indicates the degranulation ratio, as in FIG. In addition, the numerical value of a bar graph shows an average value, and each error bar shows a standard deviation.
As shown in the figure, similar to the results of Test Example 2, since Example 5 showed a decrease in degranulation in a concentration-dependent manner, it was confirmed that the drug of Example 5 has a degranulation inhibitory effect. .
Further, the drug of Example 5 showed a significant difference in the degranulation rate with respect to the negative control (control) even at a relatively low concentration of 40 μM. Accordingly, it was confirmed that 6-sPalm-AA according to Example 5 has a degranulation inhibitory effect comparable to or higher than that of 6-sPalm-AA-2G according to Example 4.

<Discussion>
From the results of Test Example 1, Test Example 2 and Test Example 3, it was confirmed that the AA derivatives of Examples 1 to 5 of the present invention have an antiallergic action and a mediator release inhibitory action. Furthermore, it was confirmed that the said effect | action increases more as carbon number of the 6-position acyl group becomes large.
From these results, while considering the physical properties and formulation technology of AA derivatives, by using AA derivatives having an acyl group with a larger number of carbon atoms, it has a strong antiallergic action and mediator release inhibitory action, and safety. High antiallergic agents and mediator release inhibitors can be provided.
Furthermore, from the results of the degranulation test of Example 5, it was confirmed that the presence of the acyl group at the 6-position was important for the degranulation inhibitory action. In other words, regardless of the substituent at the 2-position, it is suggested that the hydroxyl group at the 6-position is acylated by a linear acyl group in order to exert a degranulation inhibitory action and an antiallergic action. It was done.
It was also suggested that the action body is an AA derivative itself having an acyl group. AA derivatives having an acyl group are said to be easily metabolized to AA-2G and AA, especially in oral administration and intravenous administration, so by using as an external medicine, suppress metabolism to AA-2G and AA, It is thought that the said effect | action can be exhibited more effectively.
Here, it is known that an AA derivative having an acyl group penetrates the skin without being metabolized at least partially (Yamamoto et al., J. Med. Chem., 45, 462-468, 2002, Tai). Others, see Bioorg. Med. Chem., 14, 623-627, 2004). Therefore, by administering the AA derivative of the present invention to, for example, the skin, the AA derivative itself acts as a main body in the skin and exerts a hyaluronidase inhibitory action and a degranulation inhibitory action. Further, AA etc. in the blood and other tissues It is thought that it is metabolized and exhibits various activities. Similarly, by using the AA derivative of the present invention as an external medicine such as eye drops or nasal drops, the AA derivative directly acts on tissues such as the conjunctiva, eyeball surface, nasal mucosa, etc., and exhibits a high antiallergic effect. It is considered possible.

Claims (5)

Acylated derivatives of L- ascorbic acid represented by the following general formula (1), its isomers, or the acylated derivatives or antiallergic agent with a pharmaceutically acceptable salt thereof as an active ingredient of the isomer (except , Proanthocyanidins, and complexes of proanthocyanidins with phospholipids) .

(Wherein R ₁ represents a linear alkyl group having 9 or more carbon atoms, and R ₂ represents a hydroxyl group, glycosyl group, phosphate group, sulfate group, alkyl group, alkenyl group, or phenyl group.) The antiallergic agent according to claim 1,
R _{1 in the} general formula (1) is a linear alkyl group having 11 or more carbon atoms. The antiallergic agent according to claim 1 or 2,
R _{2 in the} general formula (1) is a glycosyl group. Mediator release inhibitor comprising as an active ingredient an acylated derivative of L-ascorbic acid represented by the following general formula (1), an isomer thereof, or a pharmaceutically acceptable salt of the acylated derivative or the isomer : However, it does not include proanthocyanidins and complexes of proanthocyanidins with phospholipids) .

(Wherein R ₁ represents a linear alkyl group having 9 or more carbon atoms, and R ₂ represents a hydroxyl group, glycosyl group, phosphate group, sulfate group, alkyl group, alkenyl group, or phenyl group.) An external preparation containing the antiallergic agent according to any one of claims 1 to 3 .

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