JP6965137B2 - AGR2 expression promoter - Google Patents

Description

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

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 mucin is over-secreted (Non-Patent Document 4), and that AGR2 is highly expressed in the respiratory tract 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.

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 release of mucus (mucin) in the stomach is promoted, so that the gastric mucosa is stimulated by gastric acid. It is thought that it can protect the stomach and prevent gastric inflammation and gastric ulcer. In addition, the respiratory tract 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.

Betulinic acid is one of the lupine triterpene compounds present in birch bark, senburi, chowji, grape bark, chaga (Chaga mushroom) and the like. Betulinic acid has an anticancer effect, an anti-inflammatory effect, an antibacterial effect, an antifungal effect, an antiviral effect (Non-Patent Document 7), a collagen synthesis promoting effect (Patent Document 1), and an effect of improving skin wrinkles and stains. It has been reported that it has various pharmacological actions such as (Patent Document 2).

However, it is not known that betulinic acid promotes the expression of AGR2 and the release of mucin.

Japanese Unexamined Patent Publication No. 8-208424 Japanese Unexamined Patent Publication No. 9-87156

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). Nat. Prod. Rep., 23, 919-942 (2006).

The present invention relates to providing an AGR2 expression promoter.

As a result of various studies on naturally-derived components, the present inventors have found that betulinic acid has an excellent AGR2 expression-promoting action and is useful as an AGR2 expression-promoting agent and a mucin release-promoting agent.

That is, the present invention relates to the following 1) to 4).
1) An AGR2 expression promoter containing bethulinic acid or a salt thereof as an active ingredient.
2) A mucin release promoter containing bethulinic acid or a salt thereof as an active ingredient.
3) A food for promoting AGR2 expression containing bethulinic acid or a salt thereof as an active ingredient.
4) A food for promoting mucin release containing bethulinic acid or a salt thereof as an active ingredient.

According to the present invention, by promoting the expression of AGR2, prevention and treatment of diseases caused by decreased mucin secretion, for example, prevention or amelioration of dry eye, prevention of gastric inflammation and gastric ulcer, prevention of respiratory tract infections. Useful medicines and foods are provided.

In the present invention, "betulinic acid" is 3β-hydroxylpa-20 (29) -ene-28-acid represented by the following formula.

Pharmaceutically acceptable salts of bethric acid, such as alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, and organic amine salts 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.

As shown in Examples below, bethulinic acid has an action of promoting the expression of AGR2 gene and AGR2 protein in normal human epidermal cells. AGR2 binds to mucin in the intestine and is involved in mucin maturation and release, and is essential for intestinal mucosal protection (Non-Patent Document 3). AGR2 is expressed in the airway and is induced to be expressed in asthma. It has been reported that mucin is over-secreted (Non-Patent Document 4), AGR2 is highly expressed in the intestine of asthma model mice, and mucin (MUC5) is over-secreted (Non-Patent Document 5). , It is considered that the release of mucin is promoted if the expression of AGR2 is promoted.
Therefore, betulinic acid or a salt thereof can be an AGR2 expression-promoting agent or a mucin release-promoting agent (hereinafter, also referred to as "AGR2 expression-promoting agent or the like"), and in order to promote the expression of AGR2, in order to promote the release of mucin. It can be used, and can also be used to produce an AGR2 expression promoter and 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 mRNA expression, preferably transcription into mRNA, and promotion of expression at the protein level includes promotion in translation. Here, "AGR2 (Antior gradient homolog 2)" is a protein registered as "AGR2" in the NCBI database (Protein) [http://www.ncbi.nlm.nih.gov/protein/]. Means.

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 and the mucin release-promoting agent of the present invention are 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.
In addition, "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 of the present invention may themselves be pharmaceuticals, quasi-drugs, supplements or foods for promoting the release of mucin in order to promote the expression of AGR2, or the said. It may be a drug, a quasi-drug, or a material or preparation used in combination with food.
The foods are based on the concept of promoting the expression of AGR2 and promoting the release of mutin, and foods labeled to that effect (foods for promoting the expression of AGR2, foods for promoting the release of mutin), for example, functional foods and for the sick. Includes foods, foods for specified health uses, foods with functional claims, and supplements. Since these foods are foods for which functional labeling is permitted, they can be distinguished from general foods.

Examples of the administration form of the above-mentioned drugs (including quasi-drugs) include oral administration with tablets, capsules, granules, powders, syrups, etc., or parenteral administration with injections, suppositories, inhalants, etc. Be done. In addition, the preparations of such various dosage forms are the bethric acid of the present invention or a salt thereof alone or other pharmaceutically acceptable excipients, binders, bulking agents, disintegrants, surfactants. , A lubricant, a dispersant, a buffer, a preservative, a flavoring agent, a fragrance, a coating agent, a carrier, a diluent, a medicinal ingredient other than bethric acid or a salt thereof, and the like can be appropriately combined and prepared. Of these dosage forms, the preferred form is oral administration.

The forms of the above foods include soft drinks, tea-based beverages, coffee beverages, fruit juice beverages, carbonated beverages, 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 the bethric acid of the present invention or a salt thereof alone, or other food materials, solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bitterness agents, fragrances, etc. Stabilizers, colorants, antioxidants, moisturizers, thickeners, active ingredients other than chlorogenic acids and the like can be appropriately combined and prepared.

The content of the betulinic acid of the present invention or a salt thereof in the above-mentioned pharmaceuticals (including non-medicinal products) and foods varies depending on the mode of use thereof, but is usually 0. 0.001% by mass or more, more preferably 0.0001% by mass or more, still more preferably 0.001% by mass or more, and preferably 20% by mass or less, more preferably 10% by mass or less, still more preferably 1% by mass. It is as follows. 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 intake of the above-mentioned pharmaceutical products (including quasi-drugs) and foods can be appropriately determined, but usually, betulin as the betulinic acid of the present invention or a salt thereof per day for an adult (60 kg). In terms of acid free form, it 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 the present invention, it is preferable to administer or ingest such an amount at a time.

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.

Examples of the administration or ingestion target include humans who desire to prevent or ameliorate dry eye or its risk, gastritis and gastric ulcer, and humans who desire to prevent respiratory tract infections caused by bacteria and viruses.

Regarding the above-described embodiment, the following aspects are disclosed in the present invention.
<1> An AGR2 expression promoter containing bethulinic acid or a salt thereof as an active ingredient.
<2> A mucin release promoter containing betulinic acid or a salt thereof as an active ingredient.
<3> A food for promoting AGR2 expression containing bethulinic acid or a salt thereof as an active ingredient.
<4> A food for promoting mucin release containing bethulinic acid or a salt thereof as an active ingredient.
<5> Use of bethulinic acid or a salt thereof for producing an AGR2 expression promoter.
<6> Use of betulinic acid or a salt thereof for producing a mucin release promoter.
<7> Betulinic acid or a salt thereof for use in promoting AGR2 expression.
<8> Betulinic acid or a salt thereof for use in promoting mucin release.
<9> A method for promoting AGR2 expression, which comprises administering or ingesting bethulinic acid or a salt thereof to a subject.
<10> A method for promoting mucin release, which comprises administering or ingesting bethulinic acid or a salt thereof to a subject.
In <11>, <2>, <4>, <6>, and <8>, the mucin is a secretory mucin.
<12> A gastrointestinal mucosa protective agent containing bethulinic acid or a salt thereof as an active ingredient.
<13> A dry eye preventive or ameliorating agent containing bethulinic acid or a salt thereof as an active ingredient.
<14> A respiratory tract infection protective agent containing bethulinic acid or a salt thereof as an active ingredient.

Example 1 Analysis of AGR2 gene expression promoting effect Human normal epidermal cells (NHEK) were ^{cultured using EpiLife® (purchased from Kurabo) under 37 ° C. and 5% CO 2} conditions. The cells were seeded on a 12-well plate at 1 × 10 ⁵ cells, cultured for 24 hours, the medium was replaced with a serum-free medium, and the test substance was prepared to a final concentration (100 nM to 1 μM) and added to the medium. bottom. After culturing for 1 day, total RNA was extracted from the cells using Maxwell 16 LEV simplicity RNA kit (Promega). The concentration of total RNA was measured, and a reverse transcription reaction was carried out using a constant amount of total RNA. A high capacity RNA-to- cDNA kit (life technologies) 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, life technologies) was used, and PRISM 7500 (life technologies) was detected and quantified. In a 20 μL reaction system, the amplification conditions were 95 ° C., 15 seconds of denaturation reaction, 60 ° C., 1 minute of annealing, and extension reaction. The gene expression level was corrected by the expression level of RPLP0 (Hs9999999902_m1, life technologies), and was shown as a relative value when the expression level of AGR2 in the sample-free (solvent only) control was 1. The results are shown in Table 1.

Example 2 Analysis of AGR2 protein expression promoting action Human normal epidermal cells (NHEK) were ^{cultured using EpiLife® (purchased from Kurabo) under 37 ° C. and 5% CO 2} conditions. The cells were seeded on a 12-well plate at 1 × 10 ⁵ cells, cultured for 24 hours, the medium was replaced with a serum-free medium, and the test substance was prepared to a final concentration (10 μM) and added to the medium. After culturing for 1 day, the cells were solubilized using a solubilizing solution [RIPA Buffer (Sigma)] to prepare a protein extract. The protein mass concentration was measured, and Sample Buffer (Themo Scientific) and 100 mM DTT were added to a sample having a certain amount of protein mass, and heat treatment (95 ° C. for 5 min) was performed. Electrophoresis was performed on a 4-20% Gradient gel (Bio-Rad) and transferred to a PVDF membrane on a Trans-Blot Turbo (Bio-Rad). After locking with 5% skim milk solution for 1 hour, the reaction was carried out with a primary antibody (anti-AGR2, abcam) (4 ° C., O / N). After reacting with a secondary antibody (anti-Rabbit IgG HRP conjugate, GE Healthcare) for 1 hour, it was made to emit light with a Clarity Western ECL Substrate (Bio-Rad). For detection, LAS-4000 (Fujifilm) was used. After that, the antibody was removed with Restore Western Blot Stripping Buffer (Themo Scientific), and again the primary antibody (anti-Actin, Horseradish) was used to detect the secondary antibody (anti-Goat IgG HRP reaction), and the secondary antibody (anti-Goat IgG HRP reaction) was detected. Was done. The protein expression level was corrected by the expression level of Actin, and was shown as a relative value when the expression level of AGR2 in the sample-free (solvent only) control was 1. The results are shown in Table 2.

Claims (4)

AGR2 expression promoter containing bethulinic acid or a salt thereof as an active ingredient (except when used for the prevention or treatment of gastric ulcer or asthma) . The AGR2 expression promoter according to claim 1, which is used as a preventive or ameliorating agent for dry eye. Foods for promoting AGR2 expression containing bethulinic acid or a salt thereof as an active ingredient (except when used for the prevention or treatment of gastric ulcer or asthma) . The food for promoting AGR2 expression according to claim 3, which is used as a food for preventing or improving dry eye.