JP2019104684A - AGR2 expression promoter - Google Patents

Abstract

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

Description

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

  AGR2 (Anterior gradient homolog 2) is a protein belonging to the Protein disulfide isomerase (PDI) family. AGR2 has one thioredoxin domain (CXXS), and is considered to be involved in protein folding and maturation by binding to the S-S binding site of the protein (Non-patent Documents 1 and 2). And, AGR2 binds to mucin in the intestine and is involved in maturation and release of mucin and is essential for mucosal protection of the intestine (non-patent document 3), and AGR2 is expressed in the airway and is induced in asthma And hypersecretion of mucin (Non-patent Document 4), it has been reported that the airway of asthma model mice has high expression of AGR2 and hypersecretion of mucin (MUC5) (Non-patent document 5). Therefore, it is considered that the release of mucin is promoted if the expression of AGR2 is promoted.

  On the other hand, in the ophthalmology area, the release of secretory mucin improves dry eye (Non-patent document 6), and in the stomach, stimulation of gastric acid by promoting gastric mucus (mucin) release stimulates gastric acid to stimulate gastric mucosa. It is believed that it protects and prevents gastritis and gastric ulcers. In addition, mucous fluid in the airway plays a role in preventing foreign microorganisms and dust, and when mucin secretion is reduced, the protective function is reduced and the possibility of being infected with various diseases increases. Therefore, promoting mucin release in the respiratory tract is useful for protection against infection. Therefore, promotion of AGR2 expression is considered to be useful for the prevention, treatment or amelioration of such diseases that develop due to the decreased secretion of mucin.

  Betulinic acid is a kind of lupine-based triterpene compound which is present in birch bark, serpent, clove, grape peel and chaga (Kabanoanatake). Betulinic acid has anti-cancer, anti-inflammatory, antibacterial, anti-wrinkling and antiviral effects (non-patent document 7), collagen synthesis promoting effect (patent document 1), skin wrinkles and stains improving action It is reported that there are various pharmacological actions such as (Patent Document 2).

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

JP-A-8-208424 JP-A-9-87156

MoI 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 University Medical College journal 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 promoter.

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

  According to the present invention, by promoting the expression of AGR2, the prevention or treatment of a disease caused due to the decrease in mucin secretion, for example, the prevention or amelioration of dry eye, the prevention of gastritis and gastric ulcer, the prevention of respiratory tract infection Useful for medicines and foods.

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

  As salts of betulinic acid, pharmaceutically acceptable salts such as alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, 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, Chaga, etc. (Chinese Patent Application Publication No. 101485703), or it can be chemically synthesized (Japanese Patent Publication 2003- 519674). Moreover, it is also possible to use a commercial item.

As shown in the Examples below, betulinic acid has an action to promote the expression of AGR2 gene and AGR2 protein in human normal epidermal cells. AGR2 binds to mucin in the intestine and is involved in the maturation and release of mucin and is essential for mucosal protection of the intestine (above-mentioned non-patent document 3), and AGR2 is expressed in the airway and is induced in asthma It is reported that hypersecretion of mucin (the above non-patent document 4), the expression of AGR2 is high in the airways of asthma model mice, and hypersecretion of mucin (MUC5) (the above non-patent document 5) If the expression of AGR2 is promoted, it is considered that mucin release is promoted.
Therefore, betulinic acid or a salt thereof can be an AGR2 expression promoter, a mucin release promoter (hereinafter also referred to as "AGR2 expression promoter, etc.", and in order to promote the expression of AGR2, to promote mucin release. It can be used and can also be used to produce an AGR2 expression promoter, mucin release promoter.
Here, "use" may be administration or ingestion to human or non-human animals, and may be therapeutic use or non-therapeutic use. The term "non-therapeutic" refers to a concept that does not include medical practice, that is, a concept that does not include a method for surgery, treatment or diagnosis of a human being, more specifically, a doctor or a person who has received instructions from a doctor. It is a concept that does not include a method of performing surgery, treatment or diagnosis.

  In the present invention, promoting expression of AGR2 includes promoting expression at the gene level and promoting expression at the protein level. Stimulation of expression at the gene level includes promotion of expression of mRNA, preferably transcription of mRNA, and promotion of expression at the protein level includes promotion of translation. Here, “AGR2 (Anterior 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, “promoting mucin release” means promoting mucin secretion in epithelial cells and goblet cells. In the present invention, mucin is preferably a secreted mucin such as MUC2, MUC5AC, MUC5B, MUC6 or MUC7.
As described above, in the ophthalmology area, promotion of release of secretory mucin is useful for the improvement of dry eye (the non-patent document 6 mentioned above), and in the digestive tract, it has a mucous membrane protective action to prevent or ameliorate gastritis and gastric ulcer Useful for Also, promoting mucin release in the respiratory tract is useful for protection against bacterial and viral infections. Therefore, the agent for promoting AGR2 expression and the agent for promoting mucin release 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, "prevention" refers to preventing or delaying the onset of a disease or condition in an individual, or reducing the risk of developing an individual's disease or condition.
Also, "improvement" refers to the improvement of disease, condition or condition, prevention or delay of deterioration of disease, condition or condition, or reversal, prevention or delay of progression of disease or condition.
Also, "treatment" includes amelioration of symptoms in addition to complete cure of the disease.

The agent for promoting AGR2 expression and the like of the present invention per se may be a drug, quasi-drug, supplement or food for promoting the release of mucin in order to promote the expression of AGR2, or It may be a material or a preparation used by being mixed with a medicine, quasi-drug or food.
The said food has a concept of promoting AGR2 expression and mucin release promotion, and displays as necessary food (AGR2 expression promoting food, mucin release promoting food), for example, functional food, for sick people Food, food for specified health use, food with functional indication, supplements are included. These foods can be distinguished from common foods because they are foods that are permitted to be labeled.

  Examples of dosage forms of the above-mentioned pharmaceuticals (including quasi-drugs) include oral administration with tablets, capsules, granules, powders, syrups etc., or parenteral administration with injections, suppositories, inhalants etc. Be In addition, such various dosage forms can be prepared by using betulinic acid or a salt thereof of the present invention alone or in combination with other pharmaceutically acceptable excipients, binders, fillers, disintegrants, surfactants. A lubricant, a dispersing agent, a buffer, a preservative, a flavoring agent, a flavor, a film, a carrier, a diluent, a medicinal component other than betulinic acid or a salt thereof, and the like can be appropriately combined and prepared. Of these dosage forms, the preferred form is oral administration.

  Examples of the form of the above-mentioned food include soft drinks, tea drinks, coffee drinks, fruit drinks, carbonated drinks, jellies, wafers, biscuits, breads, noodles, various foods such as sausages, various foods such as nutritional foods, and the like. And the nutritional composition in the same form (tablet, capsule, syrup, etc.) as the orally administered preparation described above.

  The food of various forms may be betulinic acid or a salt thereof of the present invention alone or in other food materials, solvents, softeners, oils, emulsifiers, preservatives, acidulants, sweeteners, bittering agents, aromatherapy, Stabilizers, colorants, antioxidants, humectants, thickeners, active ingredients other than chlorogenic acids and the like can be appropriately combined and prepared.

  The content of the betulinic acid or a salt thereof of the present invention in the above-mentioned pharmaceuticals (including quasi-drugs) and foods varies depending on the form of use, but usually it is preferably 0. It is 00001% by mass or more, more preferably 0.0001% by mass or more, further preferably 0.001% by mass or more, preferably 20% by mass or less, more preferably 10% by mass or less, still more preferably 1% by mass It is below. Also, it is preferably 0.00001 to 20% by mass, more preferably 0.0001 to 10% by mass, and still more preferably 0.001 to 1% by mass.

  Although the dosage or intake of the above-mentioned pharmaceuticals (including quasi-drugs) and food may be determined appropriately, it is usually betulinic acid or a salt thereof of the present invention per day for an adult (60 kg). The acid free form 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, such an amount is preferably administered or taken at one time.

  The above preparation is administered or ingested according to any schedule, and the administration or intake period is not particularly limited, but repeated or continuous administration or intake is preferable, and continuous administration or intake for 7 days or more is more preferable It is further preferable to administer or ingest continuously for 28 days or more.

  The subjects to be administered or consumed include, for example, humans who are suffering from dry eye or their fear, humans who desire to prevent or ameliorate gastritis and gastric ulcers, and humans who desire to prevent respiratory tract infections due to bacteria or viruses.

With regard to the embodiments described above, the following aspects are disclosed in the present invention.
The AGR2 expression promoter which uses <1> betulinic acid or its salt as an active ingredient.
The mucin release promoter which uses <2> betulinic acid or its salt as an active ingredient.
The food for AGR 2 expression promotion which uses <3> betulinic acid or its salt as an active ingredient.
The food for mucin release promotion which uses <4> betulinic acid or its salt as an active ingredient.
Use of betulinic acid or a salt thereof for producing a <5> AGR2 expression promoter.
Use of betulinic acid or a salt thereof for producing a <6> 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 betulinic acid or a salt thereof to a subject.
<10> A method for promoting mucin release, which comprises administering or ingesting betulinic acid or a salt thereof to a subject.
In <11><2>,<4>,<6>,<8>, mucin is a secretory mucin.
The digestive tract mucous membrane protective agent which uses <12> betulinic acid or its salt as an active ingredient.
The dry eye prevention or improvement agent which uses <13> betulinic acid or its salt as an active ingredient.
The respiratory infection control agent which uses <14> betulinic acid or its salt as an active ingredient.

Example 1 Analysis of AGR2 Gene Expression Promoting Action Human normal epidermal cells (NHEK) were cultured under conditions of 37 ° C., 5% CO ² using EpiLife (registered trademark) (purchased from Kurabo). The cells are seeded at 1 × 10 ⁵ in a 12-well plate and cultured for 24 hours, the medium is replaced with a serum-free medium, and the test substance is prepared to a final concentration (100 nM to 1 μM) and added to the medium did. After 1-day culture, total RNA was extracted from the cells using Maxwell 16 LEV simply RNA kit (Promega). The concentration of total RNA was measured, and reverse transcription was performed using a fixed amount of total RNA. High capacity RNA-to-cDNA kit (life technologies) was used for reverse transcription reaction.

  From the resulting cDNA, quantification of AGR2 gene expression was performed by Real-Time PCR. The quantification was detected and quantified with PRISM 7500 (life technologies) using Taqman (registered trademark) Probe (AGR2: Hs00180702_m1, life technologies). The amplification conditions were a denaturation reaction at 95 ° C. for 15 seconds, an annealing reaction at 60 ° C. for 1 minute, and an elongation reaction in a 20 μL reaction system. The gene expression level was corrected based on the expression level of RPLP0 (Hs99999902_m1, life technologies), and it was shown as a relative value when the expression level of AGR2 in the control with no sample added (solvent only) was 1. The results are shown in Table 1.

Example 2 Analysis of AGR2 protein expression level promotion action Human normal epidermal cells (NHEK) were cultured under conditions of 37 ° C. and 5% CO ² using EpiLife (registered trademark) (purchased from Kurabo). The cells were seeded at 1 × 10 ⁵ in a 12-well plate, cultured for 24 hours, the medium was replaced with a serum-free medium, and the test substance was adjusted to a final concentration (10 μM) and added to the medium. After 1 day of culture, cells were used for a protein extract using a lysate [RIPA Buffer (Sigma)]. The concentration of protein mass was measured, and Sample Buffer (Themo Scientific) and 100 mM DTT were added to a sample of a fixed amount of protein mass, and heat treatment (95 ° C. for 5 minutes) was performed. Electrophoresis was performed on 4-20% Gradient gel (Bio-Rad), and transferred to a PVDF membrane with Trans-Blot Turbo (Bio-Rad). After blocking for 1 hour with a 5% skimmed milk solution, they were reacted with a primary antibody (anti-AGR2, abcam) (4 ° C., O / N). After reacting for 1 hour with a secondary antibody (anti-Rabbit IgG HRP conjugate, GE Healthcare), luminescence was performed with Clarity Western ECL Substrate (Bio-Rad). The detection used LAS-4000 (Fujifilm). After that, the antibody is removed with Restore Western Blot Stripping Buffer (Themo Scientific), and reaction is performed again with a primary antibody (anti-Actin, Santacruz), a secondary antibody (anti-Goat IgG HRP conjugate, GE Healthcare) and detection. Did. The protein expression level was corrected based on the expression level of Actin, and was shown as a relative value when the expression level of AGR2 in the control with no sample added (solvent only) was taken as 1. The results are shown in Table 2.

Claims (5)

  An AGR2 expression promoter comprising betulinic acid or a salt thereof as an active ingredient.   A mucin release promoter comprising betulinic acid or a salt thereof as an active ingredient.   A food for promoting AGR2 expression, which comprises betulinic acid or a salt thereof as an active ingredient.   A food for promoting mucin release, which comprises betulinic acid or a salt thereof as an active ingredient.   The agent according to claim 2, wherein the mucin is a secretory mucin, or the food according to claim 4.