JP2021095358A - AGR2 expression promoter - Google Patents

Abstract

To provide an AGR2 expression promoter.SOLUTION: An AGR2 expression promoter contains chlorhexidine or a salt thereof as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to an AGR2 expression promoter that promotes AGR2 expression.

AGR2 (Antior gradient homolog 2) is a protein belonging to the Protein disulfide isomerase (PDI) family. AGR2 has one domain for thioredoxin (CXXS) and is considered to bind to the SS binding site of a protein and participate in protein folding and maturation (Non-Patent Documents 1 and 2).

AGR2 binds to mucin in the intestine and is involved in the maturation and release of mucin and is essential for the protection of the intestinal mucosa (Non-Patent Document 3). It has been reported that AGR2 is highly expressed and mucin (MUC5) is excessively secreted in the airways of asthma model mice (Non-Patent Document 4). Recently, it has been found that the expression level of AGR2 correlates with the dry state of the skin, and the expression level of AGR2 in skin cells decreases in dry skin (Patent Document 1).

On the other hand, in the field of ophthalmology, the release of secretory mucin improves dry eye (Non-Patent Document 6), and in the stomach, the gastric mucosa is stimulated by gastric acid by promoting the release of mucin in the stomach. It is thought that it can protect the stomach and prevent gastritis and gastric ulcer. In addition, the airway fluid containing mucin plays a role in preventing microorganisms and dust from foreign countries, and when mucin secretion decreases, the defense function decreases and the possibility of infection with various diseases increases. Therefore, promoting the release of mucin in the respiratory tract is useful for infection protection. Therefore, promotion of AGR2 expression is considered to be useful for prevention, treatment or amelioration of such diseases caused by decreased secretion of mucin.

On the other hand, chlorhexidine is a medicinal antiseptic, and salts such as gluconate and hydrochloride are widely used for disinfecting fingers and skin, disinfecting the skin of surgical sites (surgical fields), and the like. It is also added to medicated mouthwashes to reduce or prevent periodontal diseases such as gingival inflammation, and is also added to deodorant products for the purpose of suppressing indigenous skin bacteria. However, nothing is known about chlorhexidine being involved in AGR2 expression, mucin release, and skin moisturizing function.

On the other hand, betulinic acid is one of the lupine triterpene compounds present in bark, senburi, chowji, grape bark, chaga (chaga mushroom), etc. of chaga mushrooms. It has been found that it has an expression promoting action (Patent Document 2).

Japanese Patent No. 6462437 Japanese Unexamined Patent Publication No. 2019-104684

Mol Phylogenet Evol, 36 (3), 734-740 (2005). J Immunol Methods, 378 (1-2), 20-32 (2012). Proc Natl Acad Sci, 106 (17), 6950-6955 (2009). Am J Respir Cell Mol Biol., 47 (2), 178-185 (2012). J Huazhong Univ Sci Technolog Med Sci., 33 (1), 33-6 (2013). Kyoto Prefectural University of Medicine 122 (8), 549-558 (2013).

The present invention relates to providing an AGR2 expression promoter.

As a result of various studies, the present inventors have excellent AGR2 expression promoting action on chlorhexidines used as bactericides, for promoting AGR2 expression, improving skin moisturizing function, preventing or improving rough skin, and promoting mucin release. Found to be available in.

That is, the present invention relates to the following 1) to 4).
1) An AGR2 expression promoter containing chlorhexidine or a salt thereof as an active ingredient.
2) A skin moisturizing function improving agent containing chlorhexidine or a salt thereof as an active ingredient.
3) A preventive or ameliorating agent for rough skin containing chlorhexidine or a salt thereof as an active ingredient.
4) A mucin release promoter containing chlorhexidine or a salt thereof as an active ingredient.

According to the present invention, by promoting the expression of AGR2, improvement of skin moisturizing function, prevention or improvement of rough skin, or prevention or treatment of diseases caused by decreased mucin secretion, for example, prevention of dry eye or Pharmaceuticals, cosmetics and foods are provided that are useful for improvement, prevention of gastric inflammation and ulcers, and prevention of respiratory tract infections.

In the present invention, "chlorhexidine" is a compound represented by the following formula (1), and is also referred to as 1,1'-hexamethylenebis [5- (4-chlorophenyl) biguanide].

In the present invention, the "salt of chlorhexidine" is not particularly limited as long as it is a pharmaceutically acceptable salt, and specifically, monocarboxylate (for example, acetate, trifluoroacetate, butyrate, palmitin). Hydrochlorides, stearate, etc.), polyvalent carboxylates (eg, fumarate, maleate, succinate, malonate, etc.), oxycarboxylates (eg, gluconate, lactate, tartrate, etc.) Organic acid salts such as salts, citrates, etc.), organic sulfonates (eg, methane sulfonates, toluene sulfonates, tosylates, etc.); hydrochlorides, sulfates, nitrates, hydrobromide, etc. Inorganic acid salts such as phosphates; salts with organic amines (eg, methylamine, triethylamine, triethanolamine, morpholine, piperazin, pyrrolidine, tripyridine, picolin, etc.); salts with inorganic bases such as ammonium salts; alkali metals (Sodium, potassium, etc.), alkaline earth metals (calcium, magnesium, etc.), salts with metals such as aluminum, etc. can be mentioned. Of these, organic acid salts and inorganic acid salts are preferable, oxycarboxylates, monocarboxylates and inorganic acid salts are more preferable, gluconates and hydrochlorides are more preferable, and hydrochloric acid is more preferable. It is a salt, more preferably a dihydrochloride. These salts of chlorhexidine may be used alone or in any combination of two or more.

Chlorhexidine or a salt thereof may be synthesized by a known method, or may be obtained as a commercially available product.

In the present invention, it is preferable to use betulinic acid or a salt thereof in combination with chlorhexidine or a salt thereof from the viewpoint of enhancing the expression promoting effect of AGR2.

"Betulinic acid" is a 3β-hydroxylpa-20 (29) -ene-28-acid represented by the following formula (2).

As the salt of bethric acid, a pharmaceutically acceptable salt, for example, an alkali metal salt such as sodium and potassium, an alkaline earth metal salt such as magnesium and calcium, and an organic amine salt such as monoethanolamine, diethanolamine and triethanolamine, Examples thereof include basic amino acid salts such as arginine, lysine, histidine and ornithine.

Betulinic acid can be obtained by separating and purifying it from plants containing it, such as birch bark and chaga (Chiga Patent Application Publication No. 101485703), and can also be chemically synthesized (Special Table 2003-). 591674 (Ab.). It is also possible to use a commercially available product.

When chlorhexidine or a salt thereof is used in combination with bethulinic acid or a salt thereof, even if each effective amount is formulated into one dosage form at an appropriate compounding ratio, a drug containing each effective amount may be used. The kit may be prepared so that it can be used separately at the same time or at intervals, but simultaneous ingestion is preferable.

In addition, when chlorhexidine or a salt thereof is combined with betulinic acid or a salt thereof to form a compounding agent, the compounding ratio can be appropriately selected depending on the material, use or type of preparation, but generally chlorhexidine or a salt thereof. : Betulinic acid or a salt thereof has a mass ratio of chlorhexidine free form to betulinic acid free form of 1: 0.1 to 10, preferably 1: 0.2 to 5, and more preferably 1: 0.5 to 2. is there.

As shown in Examples below, the salt of chlorhexidine has an effect of promoting the expression of the AGR2 gene in normal human epidermal cells. It was also found that the combined use of a salt of chlorhexidine and betulinic acid synergistically enhances the AGR2 expression promoting effect.

Since the expression level of AGR2 in skin cells is reduced by dry skin (Patent Document 3), promotion of AGR2 expression is effective in improving the skin moisturizing function, maintaining the barrier function, and preventing or improving rough skin. Is considered to be. In addition, AGR2 binds to mucin in the intestine and is involved in the maturation and release of mucin and is essential for the protection of the intestinal mucosa (Non-Patent Document 3). Also, AGR2 is expressed in the airway and expressed in asthma. It has been reported that induced mucin is over-secreted (Non-Patent Document 4), and that AGR2 expression is high in the intestine of asthma model mice and mucin (MUC5) is over-secreted (Non-Patent Document 5). Therefore, it is considered that if the expression of AGR2 is promoted, the release of mucin is promoted.

Therefore, chlorhexidine or a salt thereof, and a combination of chlorhexidine or a salt thereof and bethuric acid or a salt thereof are used as an AGR2 expression promoter, a skin moisturizing function improver, a rough skin preventive or improver, or a mucin release promoter (hereinafter, "AGR2 expression"). It can also be used as an "accelerator, etc."), to promote the expression of AGR2, to improve the skin moisturizing function, to prevent or improve rough skin, or to promote the release of mucin, and also to promote AGR2. It can be used to produce an expression promoter, a skin moisturizing function improver, a rough skin preventive or improver, or a mucin release promoter.

Here, "use" can be administration or ingestion to a human or non-human animal, and may be therapeutic or non-therapeutic use. The term "non-therapeutic" means a concept that does not include medical practice, that is, a concept that does not include a method of surgery, treatment, or diagnosis of a human being, and more specifically, a doctor or a person who has been instructed by a doctor to treat a human being. It is a concept that does not include a method of performing surgery, treatment, or diagnosis.

In the present invention, promotion of AGR2 expression includes promotion of expression at the gene level and promotion of expression at the protein level. Promotion of expression at the gene level includes promotion of expression of mRNA, preferably promotion of transcription into mRNA, and promotion of expression at the protein level includes promotion in translation. Here, "AGR2 (Antior gradient homlog 2)" is a protein registered as "AGR2" in the NCBI database (Protein) [http://www.ncbi.nlm.nih.gov/protein/]. Means.

In the present invention, the "skin moisturizing function" means the water retention function of the skin, and the improvement or improvement of the skin moisturizing function specifically means an increase in the water retention amount of the skin and a transdermal water evaporation amount. Can be mentioned as a decrease.

In the present invention, "rough skin" is a state in which the moisturizing power of the skin is reduced and the skin is deprived of water, and desquamation and cracks are observed on the skin surface, or a state in which the skin surface roughness is increased (rough skin). ) Means the skin, also called "dry skin".

Further, in the present invention, "promotion of mucin release" means promoting the secretion of mucin in epithelial cells and goblet cells. In the present invention, the mucin is preferably a secretory mucin such as MUC2, MUC5AC, MUC5B, MUC6, and MUC7.

As described above, in the field of ophthalmology, promotion of the release of secretory mucin is useful for improving dry eye (Non-Patent Document 6), and in the gastrointestinal tract, it has a mucosal protective effect to prevent or improve gastritis and gastric ulcer. It is useful for. In addition, promoting the release of mucin in the respiratory tract is useful in protecting against bacterial and viral infections. Therefore, the AGR2 expression-promoting agent or mucin release-promoting agent of the present invention is considered to be useful for the prevention, treatment or amelioration of such diseases caused by decreased secretion of mucin.

As used herein, the term "prevention" means preventing or delaying the onset of a disease or symptom in an individual, or reducing the risk of developing a disease or symptom in an individual.

Further, "improvement" means improvement of a disease, symptom or condition, prevention or delay of deterioration of the disease, symptom or condition, or reversal, prevention or delay of progression of the disease or symptom.

Also, "treatment" includes improving symptoms in addition to complete cure of the disease.

The AGR2 expression promoter and the like of the present invention are themselves pharmaceuticals and quasi-drugs for promoting the expression of AGR2, improving the skin moisturizing function, preventing or improving rough skin, or promoting the release of mucin. It may be a product, a cosmetic product or a food product, or may be a material or a preparation used in combination with the drug, a quasi drug, a cosmetic product or a food product.

The foods are based on the concept of promoting the expression of AGR2, improving the skin moisturizing function, preventing or improving rough skin, or promoting the release of mutin, and if necessary, foods labeled to that effect, such as functional foods and foods for the sick. , Foods for specified health use, foods with functional claims, supplements are included. Since these foods are foods for which functional labeling is permitted, they can be distinguished from general foods.

The administration form of the above-mentioned pharmaceutical products (including quasi-drugs) containing chlorhexidine or a salt thereof, and chlorhexidine or a salt thereof and bethulinic acid or a salt thereof is arbitrary, and may be administered orally or parenterally. Good. Dosage forms for oral administration include solid formulations such as tablets, granules, powders and capsules, and liquid formulations such as elixir, syrup and suspension. Examples of the dosage form for parenteral administration include external skin, transdermal, transmucosal, nasal, enteral, injection, suppository, oral preparation, eye drop inhalation, and patch. Of these, the preferred formulation form is a parenteral administration formulation, more preferably an external preparation for skin or a transmucosal preparation, specifically, a form such as an ointment, an emulsion, a cream, an emulsion, a lotion, a gel, an aerosol, or a powder. Can be mentioned.

Preferred examples of cosmetics containing chlorhexidine or a salt thereof, and further chlorhexidine or a salt thereof and bethuric acid or a salt thereof are a face or body cleaning agent, a head (hair or scalp) cleaning agent (for example, shampoo, etc.). Examples include rinses, conditioners, etc.), face, body, or head cosmetics (eg, lotions, gels, creams, packs, etc.), makeup cosmetics, and the like.

For each of such pharmaceutical (including quasi-drugs) and cosmetic preparations, chlorhexidine or a salt thereof, and a combination of chlorhexidine or a salt thereof and bethuric acid or a salt thereof are permitted pharmaceutically or cosmetically as necessary. It can be produced according to a conventional method in combination with a carrier, other medicinal ingredients, cosmetic ingredients, and the like. Examples of the pharmaceutically acceptable carrier for cosmetics include various oils, surfactants, gelling agents, buffers, preservatives, antioxidants, solvents, dispersants, chelating agents, thickeners, and the like. Examples thereof include an ultraviolet absorber, an emulsion stabilizer, a pH adjuster, a pigment, and a fragrance. Other medicinal or cosmetic ingredients include, for example, plant extracts, bactericides, moisturizers, anti-inflammatory agents, antibacterial agents, keratolytic agents, refreshing agents, antiseborrheic agents, detergents, makeup ingredients and the like. Be done.

The forms of the above foods include soft drinks, tea-based drinks, coffee drinks, fruit juice drinks, carbonated drinks, jelly, wafers, biscuits, bread, noodles, sausages and other foods and drinks, nutritional foods, and other foods. Examples of the nutritional supplement composition in the same form (tablets, capsules, syrup, etc.) as the above-mentioned orally administered preparation.

Various forms of foods include chlorhexidine or salts thereof, as well as chlorhexidine or salts thereof in combination with bethric acid or salts thereof, or other food materials, solvents, softeners, oils, emulsifiers, preservatives, acidulants. , Sweeteners, bitterness agents, fragrances, stabilizers, colorants, antioxidants, moisturizers, thickeners, other active ingredients and the like can be appropriately combined and prepared.

The content of chlorhexidine or a salt thereof in the above-mentioned pharmaceutical products (including quasi-drugs) and cosmetics is, for example, a chlorhexidine free form when used as a parenteral preparation such as a skin external preparation or a transmucosal preparation. In terms of conversion, the total amount is preferably 0.00001% by mass or more, more preferably 0.0001% by mass or more, further preferably 0.001% by mass or more, and preferably 40% by mass or less, more preferably. It is 20% by mass or less, more preferably 10% by mass or less. Further, it is preferably 0.00001 to 40% by mass, more preferably 0.0001 to 20% by mass, and further preferably 0.001 to 10% by mass.

The content of betulinic acid or a salt thereof in the above preparation is preferably 0.00001% by mass or more, more preferably 0.0001% by mass or more, still more preferably 0. It is 001% by mass or more, preferably 20% by mass or less, more preferably 10% by mass or less, and further preferably 1% by mass or less. Further, it is preferably 0.00001 to 20% by mass, more preferably 0.0001 to 10% by mass, and further preferably 0.001 to 1% by mass.

The dose or amount of the above-mentioned pharmaceutical products (including quasi-drugs) or cosmetics can be determined as appropriate, but when used as a parenteral preparation such as a skin external preparation or a transmucosal preparation, it is usually an adult (60 kg). ), The chlorhexidine of the present invention or a salt thereof, in terms of chlorhexidine free form, is preferably 0.01 mg or more, more preferably 0.1 mg or more, and preferably 100 mg or less, more preferably. It is 10 mg or less. The amount of betulinic acid or a salt thereof is preferably 0.001 mg or more, more preferably 0.01 mg or more, and preferably 1000 mg or less, more preferably 100 mg or less in terms of betulinic acid free form.

The above-mentioned preparation is administered or ingested according to an arbitrary plan, and the administration or ingestion period is not particularly limited, but it is preferable to administer or ingest repeatedly and continuously, and it is more preferable to administer or ingest continuously for 7 days or more. , It is more preferable to administer or ingest continuously for 28 days or more.

The target of administration or use is, for example, a person who desires improvement of skin moisturizing function, a person who desires prevention or improvement of rough skin, a person who desires dry eye or its risk, a person who desires prevention or improvement of gastritis or gastric ulcer, bacteria or viruses. Examples include humans who wish to prevent respiratory tract infections caused by humans.

Regarding the above-described embodiment, the following aspects are disclosed in the present invention.
<1> An AGR2 expression promoter containing chlorhexidine or a salt thereof as an active ingredient.
<2> A skin moisturizing function improving agent containing chlorhexidine or a salt thereof as an active ingredient.
<3> A preventive or ameliorating agent for rough skin containing chlorhexidine or a salt thereof as an active ingredient.
<4> A mucin release promoter containing chlorhexidine or a salt thereof as an active ingredient.
<5> The agent according to <4>, wherein the mucin is a secretory mucin.
<6> The agent according to any one of <1> to <5>, wherein the active ingredient is a combination of betulinic acid or a salt thereof.

<7> Use of chlorhexidine or a salt thereof for producing an AGR2 expression promoter.
<8> Use of chlorhexidine or a salt thereof for producing a skin moisturizing function improving agent.
<9> Use of chlorhexidine or a salt thereof for producing a preventive or ameliorating agent for rough skin.
<10> Use of chlorhexidine or a salt thereof for producing a mucin release promoter.
<11> The use according to <10>, wherein the mucin is a secretory mucin.
<12> The use according to any one of <7> to <11>, which comprises a combination of betulinic acid or a salt thereof.

<13> Chlorhexidine or a salt thereof for use in promoting AGR2 expression.
<14> Chlorhexidine or a salt thereof for use in improving the skin moisturizing function.
<15> Chlorhexidine or a salt thereof for use in the prevention or improvement of rough skin.
<16> Chlorhexidine or a salt thereof for use in promoting mucin release.
<17> The chlorhexidine or a salt thereof according to <16>, wherein the mucin is a secretory mucin.
<18> The chlorhexidine or a salt thereof according to any one of <13> to <17>, which is used in combination with betulinic acid or a salt thereof.

<19> A method for promoting AGR2 expression, which comprises administering or using chlorhexidine or a salt thereof to a subject.
<20> A method for improving a skin moisturizing function, which comprises administering or using chlorhexidine or a salt thereof to a subject.
<21> A method for preventing or improving rough skin, which comprises administering or using chlorhexidine or a salt thereof to a subject.
<22> A method for promoting mucin release, which comprises administering or ingesting chlorhexidine or a salt thereof to a subject.
<23> The method according to <22>, wherein the mucin is a secretory mucin.
<24> The method according to any one of <19> to <23>, wherein bethulinic acid or a salt thereof is used in combination.

In <25> <1> to <12>, the pharmaceutical form of the agent is preferably an external preparation for skin.

Example 1 Analysis of AGR2 gene expression-promoting effect Human normal epidermal cells (Thermo Fisher Scientific) were prepared by adding Humania-KG growth additive set (Kurabou) to EpiLife® medium (Thermo Fisher Scientific). The cells were cultured under 37 ° C. and 5% CO _{2 conditions.} The cells were seeded on a 12-well plate at 1 × 10 ⁵ cells per well, cultured for 24 hours, and then replaced with the medium excluding EGF and BPE in the growth additive kit, and chlorhexidine dihydrochloride (Tokyo Chemical Industry, Ltd., DMSO dilution) and bethuric acid (Tokyo Chemical Industry, EtOH dilution) were prepared to the final concentrations shown in Table 1 and added to the medium. After culturing for 24 hours, total RNA was extracted from the cells using Maxwell 16 LEV simplicity RNA Kit (Promega). The concentration of total RNA was measured using Nanodrop (Thermo Fisher Scientific), and a reverse transcription reaction was carried out using 500 ng of total RNA. A High capacity RNA-to- cDNA kit (Thermo Fisher Scientific) was used for the reverse transcription reaction.

From the obtained cDNA, the gene expression of AGR2 was quantified by Real-Time PCR. For quantification, Taqman (registered trademark) Probe (AGR2: Hs00180702_m1, Thermo Fisher Scientific) was used, and Quantum Studio 3 Real Time PCR System (Thermo Fisher Scientific) was used for quantification. In a 20 μL reaction system, the amplification conditions were a denaturation reaction at 95 ° C./15 seconds and an annealing / extension reaction at 60 ° C./1 minute. The gene expression level was corrected by the expression level of RPLP0 (Hs9999999902_m1, Thermo Fisher Scientific), and was shown as a relative value when the expression level of AGR2 in the sample-free (solvent only) control was 1. As a result, as shown in Table 1, the gene expression level of AGR2 in human normal epidermal cells was increased by the addition of chlorhexidine dihydrochloride and betulinic acid.

Claims (7)

An AGR2 expression promoter containing chlorhexidine or a salt thereof as an active ingredient. A skin moisturizing function improving agent containing chlorhexidine or a salt thereof as an active ingredient. A preventive or ameliorating agent for rough skin containing chlorhexidine or a salt thereof as an active ingredient. A mucin release promoter containing chlorhexidine or a salt thereof as an active ingredient. The agent according to claim 4, wherein the mucin is a secretory mucin. The agent according to any one of claims 1 to 5, wherein the active ingredient is a combination of betulinic acid or a salt thereof. The agent according to any one of claims 1 to 6, wherein the preparation form is an external preparation for skin.