TWI844795B - Autophagy activators - Google Patents

Abstract

An autophagy activator containing tocopherol phosphate or its salt as an active ingredient. An autophagy activating composition containing the aforementioned autophagy activator and a pharmaceutically acceptable carrier.

Description

Autophagy activators

The present invention relates to an autophagy activator and an autophagy activating composition. This application claims priority based on Japanese Patent Application No. 2020-156458 filed in Japan on September 17, 2020, and the contents thereof are hereby incorporated by reference.

Autophagy is a mechanism that responds to extracellular or intracellular stress and messages such as hunger, growth factor deficiency, and pathogen infection, and regenerates energy and removes damaged substances by decomposing aging or damaged intracellular substances and organelles. It is important for maintaining normal cell homeostasis. Previous studies have reported that the more advanced aging is, the more rapidly the autophagic activity in cells decreases (non-patent document 1). In addition, when autophagy is inhibited, aging mitochondria or misfolded proteins will accumulate excessively in cells, inducing cell death due to increased oxidative stress in cells, ultimately leading to cell aging.

Therefore, by activating autophagy, which decomposes aged substances and organelles in cells and recycles the decomposition products, it is possible to improve cell homeostasis by quickly removing unnecessary substances in cells.

On the other hand, as an effect of aging on autophagy, it is known that the expression levels of ATG5, ATG7, and Beclin 1 genes in the human brain decrease with age. In addition, it is generally believed that the activity of autophagy decreases in neurodegenerative diseases such as Alzheimer’s disease. Therefore, it is known that the activation of autophagy contributes to the treatment and prevention of neurodegenerative diseases such as Huntington’s disease and Parkinson’s disease, including Alzheimer’s disease (Non-patent Documents 2, 3). Specifically, in Alzheimer’s disease, the function of autophagy is blocked, and aggregated proteins called starch-like β accumulate in the body, which is generally considered to be related to the onset of the disease (Non-patent Document 4). In addition, it is generally believed that mutations in autophagy-related genes or genes involved in selective autophagy also have an impact on SENDA disease (static encephalopathy of childhood with neurodegeneration in adulthood), a neurodegenerative disease accompanied by iron deposition in the substantia nigra and globus alba and brain atrophy, Crohn's disease, an inflammatory bowel disease causing severe inflammation or ulcers in the digestive tract, and cancer (Non-patent document 6).

The process of autophagy has been studied in both yeast and mammals, and up to 36 proteins have been used. Among them, the formation of autophagosomes and the differentiation of their contents are controlled by Atg proteins encoded by autophagy-related genes (ATG), which can be classified into 6 groups including Atg12-ATG5 binding system and LC3-Phosphatidyl Ethanolamine (PE) binding system, each of which plays a role in each stage of the process.

On the other hand, autophagy is suppressed to a low level under stable conditions and activated under stress such as starvation. mTOR (mammalian target of rapamycin) is known to function as a major inhibitor of autophagy from yeast to mammals, but under starvation conditions, mTOR is inactivated, inducing autophagy (Non-patent document 5).

As autophagy activators, compounds that increase LC3-related factors that are markers of the active state of autophagy and activate autophagy, as well as compounds that promote autophagic flux (including fusion of autophagosomes to lysosomes) have been reported (patent documents 1 to 4). [Prior art document] [Patent document]

[Patent Document 1] International Publication No. 2018/173653 [Patent Document 2] Japanese Patent Publication No. 2018-80204 [Patent Document 3] Japanese Patent Publication No. 2018-510902 [Patent Document 4] Japanese Patent Publication No. 2019-529514 [Non-patent Document]

[Non-patent document 1]Yogendra S. Rajawat et al., Aging: Central role for autophagy and the lysosomal degradative system. Ageing Research Reviews 8 (2009) 199-213. [Non-patent document 2]Aaron Barnett et al., Autophagy in Aging and Alzheimer's Disease: Pathologic or Protective?. J Alzheimers Dis. 2011; 25(3): 385-394. [Non-patent document 3]Marta M. Lipinski et al., Genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in Alzheimer's disease. Proc Natl Acad Sci USA. 2010, 107, 14164-14169. [Non-patent document 4]Xiangqing Li et al., Cubeben induces autophagy via PI3K-AKT-mTOR pathway to protect primary neurons against amyloid beta in Alzheimer's disease. Cytotechnology (2019) 71: 679-686. [Non-patent document 5] Zhu Yu et al., "The correlation between autophagy and aging", Journal of Qiya, 2018, Vol. 45, No. 1, 1-7 [Non-patent document 6] Yingshan et al., "Autophagy and disease", Convergence Review, 2014, 3, e006

[The problem that the invention wants to solve]

As mentioned above, it is known that in various diseases such as Alzheimer's disease, the autophagy function is reduced, and drugs that can effectively activate autophagy are sought. However, the effects of the drugs known in the past are still insufficient.

Therefore, the purpose of the present invention is to provide an autophagy activator that can effectively activate autophagy and an autophagy activating composition containing the aforementioned autophagy activator. [Means for solving the problem]

The present invention includes the following aspects. [1] An autophagy activator, which contains tocopherol phosphate or a salt thereof as an active ingredient. [2] The autophagy activator as described in [1], wherein the tocopherol phosphate or a salt thereof is at least one tocopherol phosphate or a salt thereof selected from the group consisting of α-tocopherol phosphate and γ-tocopherol phosphate.

[3] The autophagy activator described in [1] or [2], which contains the salt of tocopherol phosphate as an active ingredient, wherein the salt of tocopherol phosphate is sodium salt of tocopherol phosphate. [4] The autophagy activator described in any one of [1] to [3], which promotes the expression of LC3 gene. [5] The autophagy activator described in any one of [1] to [4], which promotes the expression of ATG5 gene.

[6] An autophagy activator as described in any one of [1] to [5], which promotes the expression of the ATG7 gene. [7] An autophagy activator as described in any one of [1] to [6], which promotes the expression of the Beclin 1 gene. [8] An autophagy activator as described in any one of [1] to [7], which inhibits the expression of the mTOR gene.

[9] An autophagy activator as described in any one of [1] to [8], which is used for preventing or treating Alzheimer's disease. [10] An autophagy activating composition, which contains an autophagy activator as described in any one of [1] to [9] and a pharmaceutically acceptable carrier. [11] An autophagy activating composition as described in [10], wherein the total content of the tocopherol phosphate or its salt is 0.01 to 10% by weight relative to the total amount of the autophagy activating composition. [Effect of the invention]

The present invention provides an autophagy activator that can effectively activate autophagy and an autophagy activating composition containing the autophagy activator.

(Autophagy Activator)

In one embodiment, the present invention provides an autophagy activator containing tocopherol phosphate or a salt thereof as an active ingredient. Here, "autophagy" refers to a mechanism for regenerating energy and removing damaged substances by decomposing aged or damaged intracellular substances and organelles. The autophagy activator of this embodiment can promote the expression of the LC3 gene, which is an autophagy marker, and the ATG5 gene, ATG7 gene, and Beclin 1 gene contained in the autophagosome, thereby activating autophagy. In addition, autophagy can be activated by inhibiting the expression of the mTOR gene, which acts as an inhibitor of autophagy.

The autophagy activator of the present embodiment is not particularly limited as long as it contains tocopherol phosphate or a salt thereof as an effective ingredient. Examples of the tocopherol phosphate used in the autophagy activator of the present embodiment include compounds represented by the following general formula (1).

[In the formula, R ¹ , R ² and R ³ independently represent a hydrogen atom or a methyl group.]

Among the tocopherol phosphates, according to R ¹ , R ² , and R ³ in the above general formula (1), there are α-tocopherol phosphate (R ¹ , R ² , R ³ = CH ₃ ), β-tocopherol phosphate (R ¹ , R ³ = CH ₃ , R ² = H), γ-tocopherol phosphate (R ¹ , R ² = CH ₃ , R ³ = H), δ-tocopherol phosphate (R ¹ = CH ₃ , R ² , R ³ = H), ζ ₂ -tocopherol phosphate (R ² , R ³ = CH ₃ , R ¹ = H), η-tocopherol phosphate (R ² = CH ₃ , R ¹ , R ³ = H), and the like.

The tocopherol phosphate is not particularly limited and may be any of these tocopherol phosphates. Among these, α-tocopherol phosphate and γ-tocopherol phosphate are preferred, and α-tocopherol phosphate is more preferred.

The compound represented by the general formula (1) has an asymmetric carbon atom at the 2-position of the dihydrobenzopyran ring, and thus has d-isomers, l-isomers, and dl-isomers. Tocopherol phosphate may be any of these stereoisomers, and is preferably dl-isomers.

Among the above, as the tocopherol phosphate, dl-α-tocopherol phosphate and dl-γ-tocopherol phosphate are preferred, and dl-α-tocopherol phosphate is more preferred.

The salt of tocopherol phosphate is not particularly limited, and examples thereof include salts with inorganic bases and salts with organic bases.

Examples of salts with inorganic bases include alkaline metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salts; ammonium salts; zinc salts, etc. Examples of salts with organic bases include alkylammonium salts and salts with alkaline amino acids, etc.

Among the above, the salt of tocopherol phosphate is preferably an alkali metal salt, more preferably a sodium salt. The alkali metal salt of tocopherol phosphate, in particular the sodium salt, has the advantages of being highly soluble in water and being easy to handle due to its powdery state.

Preferred embodiments of tocopherol phosphate include alkali metal salts (e.g., sodium salts) of the compound represented by the general formula (1), alkali metal salts (e.g., sodium salts) of α-tocopherol phosphate, alkali metal salts (e.g., sodium salts) of γ-tocopherol phosphate, alkali metal salts (e.g., sodium salts) of dl-α-tocopherol phosphate, alkali metal salts (e.g., sodium salts) of dl-γ-tocopherol phosphate, and the like.

Among the alkaline metal salts of tocopherol phosphate, sodium salt of α-tocopherol phosphate and sodium salt of γ-tocopherol phosphate are preferred, and sodium salt of α-tocopherol phosphate is more preferred.

Sodium salt of dl-α-tocopherol phosphate is commercially available from Showa Denko under the product name of TPNa (registered trademark) (label name: sodium tocopherol phosphate). The above-mentioned TPNa is exemplified as a preferred example of tocopherol phosphate.

The autophagy activator of the present embodiment can be used alone or in combination of two or more selected from tocopherol phosphate and its salts. The autophagy activator of the present embodiment preferably comprises a salt of tocopherol phosphate, and more preferably, an alkali metal salt (e.g., sodium salt) of tocopherol phosphate is used alone.

Tocopherol phosphate or its salt can be produced by a known production method. Tocopherol phosphate or its salt can be produced by a method described in, for example, Japanese Patent Publication No. 59-44375, International Publication No. 97/14705, etc. For example, by allowing a phosphorylating agent such as phosphorus oxychloride to act on tocopherol dissolved in a solvent, and then appropriately purifying it after the reaction is completed, tocopherol phosphate can be obtained. Furthermore, by neutralizing the obtained tocopherol phosphate with a metal oxide such as magnesium oxide, a metal hydroxide such as sodium hydroxide, or ammonium hydroxide or alkylammonium hydroxide, a salt of tocopherol phosphate can be obtained. The production method of tocopherol phosphate or its salt is not limited to the above method.

Hereinafter, tocopherol phosphate and its salts may be collectively described as "tocopherol phosphate, etc."

The autophagy activator of this embodiment can be administered to a patient for the purpose of treating neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc. In addition, the autophagy activator of this embodiment can also be mixed into a pharmaceutical or cosmetic product for the purpose of activating autophagy. In addition, it can also be mixed into the autophagy activating composition described below for use.

The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the LC3 gene. The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the ATG5 gene. The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the ATG7 gene. The autophagy activator of the present embodiment can effectively activate autophagy by promoting the expression of the Beclin 1 gene. The autophagy activator of the present embodiment can effectively activate autophagy by inhibiting the expression of the mTOR gene. The autophagy activator of the present embodiment can effectively activate autophagy, and thus can be used to prevent or treat Alzheimer's disease.

It is known that amyloid β causes a decrease in autophagy in nerve cells. In addition, amyloid β is known to induce a decrease in autophagy and the resulting cell death of nerve cells called apoptosis. The autophagy activator of the present embodiment can promote the expression of the LC3 gene in the presence of amyloid β. The autophagy activator of the present embodiment can promote the expression of the ATG5 gene in the presence of amyloid β. The autophagy activator of the present embodiment can promote the expression of the ATG7 gene in the presence of amyloid β. The autophagy activator of the present embodiment can inhibit apoptosis in the presence of amyloid β. The autophagy activator of this embodiment, in particular, can promote the expression of at least one gene selected from the group consisting of LC3 gene, ATG5 gene and ATG7 gene in the presence of starch beta in nerve cells. In addition, the autophagy activator of this embodiment, in particular, can inhibit apoptosis in the presence of starch beta in nerve cells.

The so-called promotion of LC3 gene expression in the presence of amyloid β means that by administering the autophagy activator of the present embodiment in the presence of amyloid β, the expression amount of LC3 gene is increased compared to the case where the aforementioned autophagy activator is not administered. The same is true for the ATG5 gene and the ATG7 gene. The so-called inhibition of cell apoptosis in the presence of amyloid β means that by administering the autophagy activator of the present embodiment in the presence of amyloid β, cell apoptosis is inhibited compared to the case where the aforementioned autophagy activator is not administered.

LC3 (microtubule associated protein 1 light chain 3 alpha: NCBI Gene ID: 84557) is attached with phosphatidylethanolamine upstream of the autophagy signaling pathway and converted into LC3-II, which is attracted to the autophagosome membrane and binds to the autophagosome membrane. LC3 is used as a marker for autophagosomes. Examples of the base sequence of the human LC3 gene include NM_032514.4 and NM_181509.3 registered in the NCBI Reference Sequence database.

ATG5 (autophagy related 5: NCBI Gene ID: 9474) binds to ATG12 and functions as an E1-like activating enzyme in the ubiquitin-like conjugate system. The base sequences of the human ATG5 gene include NM_001286106.1, NM_001286107.1, NM_001286108.1, NM_001286111.1, and NM_004849.4 registered in the NCBI Reference Sequence database.

ATG7 (autophagy related 7: NCBI Gene ID: 10533) functions as an E1-like activating enzyme that activates LC3 and ATG12 in an ATP-dependent manner. Examples of the base sequence of the human ATG7 gene include NM_001136031.3, NM_001144912.2, NM_001349232.2, NM_001349233.2, and NM_001349234.2 registered in the NCBI Reference Sequence database.

Beclin 1 (NCBI Gene ID: 8678) forms a class III PI3K complex together with ATG14L and Vps34mp150, and functions as a positive control factor for autophagosome formation. The base sequences of the human Beclin 1 gene include, for example, NM_001313998.2, NM_001313999.1, NM_001314000.1, and NM_003766.4 registered in the NCBI Reference Sequence database.

mTOR (mechanistic target of rapamycin kinase: NCBI Gene ID: 2475) is a phosphatidylinositol kinase-related kinase that mediates cellular responses to stresses such as DNA damage and nutrient deficiency. mTOR functions as an inhibitor of autophagy. The base sequence of the human mTOR gene can be listed, for example, as NM_004958.4 registered in the NCBI Reference Sequence database.

The autophagy activator of this embodiment can be administered to patients with a higher risk of developing neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc., to prevent neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc. In addition, the autophagy activator of this embodiment can also be administered to patients who have already developed neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, etc., to inhibit the progression or deterioration of neurodegenerative diseases.

The autophagy activator of this embodiment can be administered to a patient by the same method as the autophagy activating composition described below, and can be administered orally or parenterally, and can be administered intravenously, intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally. It can also be administered intrarectally in the form of a suppository, or can be administered to the skin in the form of a topical preparation.

(Autophagy Activating Composition) In one embodiment, the present invention provides an autophagy activating composition, which contains the above-mentioned autophagy activator and a pharmaceutically acceptable carrier.

The autophagy activating composition of the present embodiment can be produced by mixing the above-mentioned autophagy activator, a pharmaceutically acceptable carrier and other components as appropriate and preparing the mixture according to conventional methods (for example, the method described in the Japanese Pharmacopoeia).

In this manual, the so-called "pharmaceutically acceptable carrier" means a carrier that does not hinder the physiological activity of the active ingredient and does not show substantial toxicity to the subject to which it is administered. In addition, the so-called "does not show substantial toxicity" means that the ingredient does not show toxicity to the subject to which it is administered at the dosage normally used. There are no special restrictions on pharmaceutically acceptable carriers, and examples thereof include excipients, adhesives, disintegrants, lubricants, emulsifiers, stabilizers, diluents, solvents for injections, oily bases, moisturizers, touch enhancers, surfactants, polymers/thickening/gelling agents, solvents, sprays, and anti- Oxidants, reducing agents, oxidants, chelating agents, acids, bases, powders, inorganic salts, water, metal-containing compounds, unsaturated monomers, polyols, polymer additives, auxiliary agents, wetting agents, thickeners, adhesion-imparting substances, oily raw materials, liquid bases, fat-soluble substances, polymer carboxylates, etc.

Specific examples of such ingredients include those described in International Publication No. 2016/076310. Specific examples of polymers/thickening/gelling agents include methacryloyloxyethylphosphatidylcholine, butyl methacrylate, or polymers thereof. Pharmaceutically acceptable carriers may be used alone or in combination of two or more.

In addition, other ingredients are not particularly limited, and include preservatives, antibacterial agents, ultraviolet absorbers, whitening agents, anti-inflammatory agents, anti-inflammatory agents, hair growth agents, blood circulation promoters, stimulants, hormones, anti-wrinkle agents, anti-aging agents, firming agents, cooling agents, warming agents, wound healing promoters, irritation relievers, analgesics, cell activators, plant/animal/microorganism extracts, seed oils, anti-itching agents, keratin peeling/dissolving agents, and antiperspirants. , coolants, astringents, enzymes, nucleic acids, fragrances, pigments, colorants, dyes, pigments, anti-inflammatory analgesics, antifungal agents, antihistamines, hypnotic sedatives, tranquilizers, antihypertensives, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptics, coronary vasodilators, herbal medicines, antipruritics, keratin softening and exfoliating agents, ultraviolet light blocking agents, preservatives and bactericidal agents, antioxidant substances, pH adjusters, additives, metal soaps, etc. As specific examples of such ingredients, for example, those described in International Publication No. 2016/076310 can be cited. Furthermore, as specific examples of plant/animal/microorganism extracts, Lapsana communis flowers/leaves/stems, tea leaves, etc. can be cited. As specific examples of seed oils, Moringa seed oil can be cited. As specific examples of spices, perilla aldehyde can be cited. Other ingredients can be used alone or in combination of two or more.

The composition for activating autophagy of the present embodiment may contain a therapeutically effective amount of the aforementioned autophagy activator. The so-called "therapeutically effective amount" means the amount of the agent that is effective for treating or preventing the patient's disease. The therapeutically effective amount may vary depending on the state of the disease, age, gender, and weight of the subject to be administered. In the composition for activating autophagy of the present embodiment, the therapeutically effective amount of the aforementioned autophagy activator may be an amount of tocopherol phosphate, etc. that can activate autophagy. In addition, the therapeutically effective amount of the aforementioned autophagy activator may be an amount of tocopherol phosphate, etc. that can promote the expression of at least one gene selected from the group consisting of LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene. Alternatively, it may be an amount of tocopherol phosphate, etc. that can inhibit the expression of mTOR gene, which is an autophagy inhibitor. In addition, the therapeutically effective amount of the above-mentioned autophagy activator can be an amount of tocopherol phosphate, etc. that can inhibit cell apoptosis in the presence of starch beta. The therapeutically effective amount of the above-mentioned autophagy activator in the autophagy activating composition of this embodiment is the total content of tocopherol phosphate or its salt relative to the total amount of the autophagy activating composition, which can be, for example, 0.01-10 mass%, or 0.1-10 mass%, or 0.1-5 mass%, or 0.1-3 mass%, or 0.1-2 mass%, or 0.3-2 mass%, or 0.6-1.5 mass%.

The content of the above-mentioned tocopherol phosphate ester in the composition for activating autophagy means the content of the compound when one tocopherol phosphate ester is used alone, and means the total content of these compounds when two or more tocopherol phosphate esters are used in combination.

The composition for activating autophagy of this embodiment may be a pharmaceutical composition or a cosmetic.

(Pharmaceutical composition) In one embodiment, the present invention provides a pharmaceutical composition for activating autophagy, which contains the above-mentioned autophagy activator and a pharmaceutically acceptable carrier.

In the pharmaceutical composition of the present embodiment, there is no particular limitation on the pharmaceutically acceptable carrier, and in addition to the above-mentioned carriers, carriers generally used in pharmaceuticals can be used. For example, general raw materials described in the Japanese Pharmacopoeia, Japanese Pharmacopoeia Non-Pharmacopoeia Pharmaceutical Specifications, Pharmaceutical Additives Specifications 2013 (Yakuji Nichipōsha, 2013), Pharmaceutical Additives Dictionary 2016 (Japan Pharmaceutical Additives Association, Yakuji Nichipōsha, 2016), Handbook of Pharmaceutical Excipients, 7th edition (Pharmaceutical Press, 2012), etc. can be used. The pharmaceutically acceptable carrier may be used alone or in combination of two or more.

The pharmaceutical composition of the present embodiment may contain other ingredients in addition to the aforementioned autophagy activator and pharmaceutically acceptable carrier. As other ingredients, there are no particular restrictions, and general pharmaceutical additives can be used. In addition, as other ingredients, active ingredients other than the aforementioned autophagy activator can also be used. As pharmaceutical additives and active ingredients used as other ingredients, in addition to those listed above, general raw materials such as those recorded in the Japanese Pharmacopoeia, Japanese Pharmacopoeia Non-Pharmacopoeia Pharmaceutical Specifications, Pharmaceutical Additives Specifications 2013 (Yakuji Nichipōsha, 2013), Pharmaceutical Additives Dictionary 2016 (Compiled by the Japanese Pharmaceutical Additives Association, Yakuji Nichipōsha, 2016), Handbook of Pharmaceutical Excipients, 7th edition (Pharmaceutical Press, 2012), etc. can also be used. Other ingredients can be used alone or in combination of two or more.

The dosage form of the pharmaceutical composition of this embodiment is not particularly limited, and can be prepared into dosage forms generally used as pharmaceutical preparations. Examples include oral dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, and parenteral dosage forms such as injections, suppositories, and skin external preparations. Pharmaceutical compositions of these dosage forms can be prepared according to conventional methods (e.g., methods described in the Japanese Pharmacopoeia).

The administration method of the pharmaceutical composition of the present embodiment is not particularly limited, and can be administered by methods generally used for the administration of pharmaceuticals. For example, it can be administered orally in the form of tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., or it can be administered intravenously, intraarterially, intramuscularly, intradermally, subcutaneously, or intraperitoneally in the form of injections, infusion preparations, etc., alone or mixed with common infusions such as glucose solution and Ringer's solution, or it can be administered intrarectally in the form of suppositories, or it can be administered to the skin in the form of topical preparations for skin application.

The dosage of the pharmaceutical composition of this embodiment can be set to a therapeutically effective amount. The therapeutically effective amount can be appropriately determined according to the patient's symptoms, weight, age, and gender, as well as the dosage form and administration method of the pharmaceutical composition. For example, the dosage of the pharmaceutical composition of this embodiment can be exemplified as follows: in the case of oral administration, 0.01 to 500 mg per administration unit form of tocopherol phosphate, etc., in the case of injections, 0.02 to 250 mg per administration unit form of tocopherol phosphate, etc., in the case of suppositories, 0.01 to 500 mg per administration unit form of tocopherol phosphate, etc. In the case of a skin external preparation, for example, the dosage per administration unit of tocopherol phosphate is 0.15 to 500 mg, 0.15 to 300 mg, 0.15 to 200 mg, or 0.2 to 100 mg.

The administration interval of the pharmaceutical composition of this embodiment can be appropriately determined according to the patient's symptoms, weight, age, gender, etc., as well as the dosage form and administration method of the pharmaceutical composition, etc. For example, it can be set to once a day or about 2 to 3 times a day.

The pharmaceutical composition of this embodiment can be used to treat or prevent diseases caused by decreased autophagy activity. Examples of such diseases include neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and SENDA disease; inflammatory bowel diseases such as Crohn's disease; and cancer.

The pharmaceutical composition of this embodiment can be administered to patients suffering from neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, SENDA disease, inflammatory bowel diseases such as Crohn's disease, or cancer, to inhibit the progression of neurodegenerative diseases, inflammatory bowel diseases, or cancer. In addition, the pharmaceutical composition of this embodiment can be administered to patients suffering from neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, SENDA disease, inflammatory bowel diseases such as Crohn's disease, or cancer, to treat neurodegenerative diseases, inflammatory bowel diseases, or cancer. In addition, the pharmaceutical composition of this embodiment can be used to treat diseases caused by starch beta. In addition, the pharmaceutical composition of this embodiment can be used to treat diseases caused by reduced expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene. In addition, the pharmaceutical composition of this embodiment can be used to treat diseases caused by increased expression of mTOR. Among the above, the pharmaceutical composition of this embodiment can be particularly suitable for treating Alzheimer's disease.

The pharmaceutical composition of this embodiment can also be administered to patients at high risk of developing neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and SENDA disease, for the prevention of neurodegenerative diseases. In addition, the pharmaceutical composition of this embodiment can also be administered to patients at high risk of developing inflammatory bowel diseases such as Crohn's disease, for the prevention of inflammatory bowel diseases. In addition, the pharmaceutical composition of this embodiment can also be administered to patients at high risk of developing cancer, for the prevention of cancer.

(Cosmetics) In one embodiment, the present invention provides a cosmetic for activating autophagy, which contains the above-mentioned autophagy activator and a pharmaceutically acceptable carrier.

In the cosmetic of this embodiment, there is no particular limitation on the pharmaceutically acceptable carrier, and in addition to the above-mentioned carriers, carriers generally used in cosmetics can also be used. For example, general raw materials described in the Notes to the Second Edition of the Cosmetic Raw Materials Standard (edited by the Japan Kondo Association, Yakuji Nichipō, 1984), Specifications for Ingredients Outside the Cosmetic Raw Materials Standard (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Supplement to the Specifications for Ingredients Outside the Cosmetic Raw Materials Standard (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Category Permit Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Raw Materials Dictionary (Nikko Chemicals, Japan, 1993), International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol.1~4, by CTFA, etc. can be used. The pharmaceutically acceptable carriers may be used alone or in combination of two or more.

The cosmetic of this embodiment may contain other ingredients in addition to the autophagy activator and the pharmaceutically acceptable carrier. There is no particular limitation on the other ingredients, and general cosmetic additives may be used. In addition, active ingredients other than the above-mentioned autophagy activator may also be used as other ingredients. In addition to the above, cosmetic additives and active ingredients that are deemed to be other ingredients may include those listed in the Notes to the Second Edition of the Cosmetic Ingredient Standards (edited by the Japan KOKU Association, Yakuji Nichipō, 1984), Specifications for Ingredients Outside the Cosmetic Ingredient Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Supplement to the Specifications for Ingredients Outside the Cosmetic Ingredient Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Category Permit Standards (supervised by the Inspection Division of the Pharmaceutical Bureau of the Ministry of Health, Labor and Welfare of Japan, Yakuji Nichipō, 1993), Cosmetic Ingredient Dictionary (Nikko Chemicals, Japan, 1993), International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol.1~4, by CTFA and other general raw materials. Other ingredients may be used alone or in combination of two or more.

The form of the cosmetics of this embodiment is not particularly limited, and can be made into forms commonly used as cosmetics. Examples include hair cosmetics such as shampoo, conditioner, and hair spray; basic cosmetics such as facial cleansers, cleaners, toners, emulsions, skin lotions, creams, gels, sunscreens, facial masks, face packs, and beauty serums; makeup cosmetics such as foundations, primers, lipsticks, lip glosses, and blushers; and body cosmetics such as body washes, body powders, and deodorant cosmetics. These cosmetics can be manufactured according to conventional methods.

In addition, the dosage form of the cosmetic of this embodiment is not particularly limited, and examples include emulsion types such as oil in water (O/W), water in oil (W/O), W/O/W, and O/W/O, emulsified polymer types, oily, solid, liquid, paste, stick, volatile oil type, powder, jelly, gel, ointment, cream, sheet, film, mist, spray, aerosol, multilayer, bubble, and fragments.

The amount of the cosmetic used in this embodiment is not particularly limited and can be set to an amount effective for activating autophagy. For example, the amount of the cosmetic used in this embodiment can be 0.15 to 500 mg, 0.15 to 300 mg, 0.15 to 200 mg, or 0.2 to 100 mg per use, for example, in terms of the amount of tocopherol phosphate, etc.

The usage interval of the cosmetic of this embodiment is not particularly limited, and can be set to, for example, once a day or 2 to 3 times a day.

The cosmetic of this embodiment can be used to alleviate symptoms caused by reduced autophagic activity. Alternatively, it can also be used by subjects with a high risk of developing symptoms caused by reduced autophagic activity for daily skin care or makeup in order to prevent the onset of such symptoms.

[Other embodiments] In one embodiment, the present invention provides a method for activating autophagy, which comprises the step of administering tocopherol phosphate or a salt thereof to a subject.

In one embodiment, the present invention provides a method for promoting the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene, comprising the step of administering tocopherol phosphate or a salt thereof to a subject.

In one embodiment, the present invention provides a method for inhibiting the expression of mTOR gene, comprising the step of administering tocopherol phosphate or a salt thereof to a subject.

In one embodiment, the present invention provides a method for inhibiting apoptosis in the presence of amyloid β, comprising the step of administering tocopherol phosphate or a salt thereof to a subject.

In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof for activating autophagy.

In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof, which is used to promote the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene.

In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof, which is used to inhibit the expression of the mTOR gene.

In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof for inhibiting apoptosis in the presence of starch beta.

In one embodiment, the present invention provides a tocopherol phosphate or a salt thereof for preventing or treating Alzheimer's disease, Huntington's disease, Parkinson's disease, SENDA disease, Crohn's disease or cancer.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing an autophagy activator.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for producing an agent for promoting the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for producing an inhibitor of mTOR gene expression.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for producing an agent for promoting the expression of LC3 gene, ATG5 gene or ATG7 gene in the presence of starch beta.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing an inhibitor of cell apoptosis in the presence of starch beta.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for activating autophagy.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for promoting the expression of LC3 gene, ATG5 gene, ATG7 gene or Beclin 1 gene.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for inhibiting mTOR gene expression.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for promoting the expression of LC3 gene, ATG5 gene or ATG7 gene in the presence of starch beta.

In one embodiment, the present invention provides a use of tocopherol phosphate or a salt thereof for preparing a composition for inhibiting cell apoptosis in the presence of starch beta. [Example]

The present invention is described in more detail below based on embodiments, but the present invention is not limited to these embodiments.

[Tocopherols] In the following examples and formulations, the following sodium salts of tocopherol phosphates were used. α-TPNa: dl-α-tocopherol sodium phosphate (label name: tocopherol sodium phosphate, product name; TPNa (registered trademark), manufactured by Showa Denko Co., Ltd.) γ-TPNa: dl-γ-tocopherol sodium phosphate (manufactured by Showa Denko Co., Ltd.) α-TPNa solution: obtained by dissolving α-TPNa in a 0.05% (V/V) ethanol aqueous solution. γ-TPNa solution: obtained by dissolving γ-TPNa in a 0.05% (V/V) ethanol aqueous solution. In the following comparative examples, the following tocopherols were used. Tocopherol acetate: manufactured by Eisai Co., Ltd. α-Tocopherol: manufactured by Sigma-Aldrich γ-Tocopherol: manufactured by Sigma-Aldrich Tocopherol acetate solution: obtained by dissolving tocopherol acetate in a 0.05% (V/V) ethanol aqueous solution. α-Tocopherol solution: obtained by dissolving α-tocopherol in a 0.05% (V/V) ethanol aqueous solution. γ-Tocopherol solution: obtained by dissolving γ-tocopherol in a 0.05% (V/V) ethanol aqueous solution.

<Evaluation of the effect of promoting the expression of LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene in human aged fibroblasts> In order to produce artificially aged cells, fibroblasts were used for the experiment. Aged fibroblasts were produced by the following procedure.

(Preparation of aged fibroblasts) Human normal fibroblasts (NB1RGB; RIKEN BRC Cell Bank) were cultured in D-MEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (MP Biomedicals) until they became confluent. Then, they were treated with 250 μM hydrogen peroxide for 2 hours and cultured in new D-MEM medium supplemented with 10% fetal bovine serum for 24 hours. This process of hydrogen peroxide treatment and cell culture was repeated three times to obtain aged fibroblasts.

(Evaluation of the effect of promoting gene expression) The prepared aged fibroblasts were prepared at an inoculation density of 10,000 cells/ ^cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 1, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa became 1 μM. In Example 2, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa became 10 μM. In Example 3, a γ-TPNa solution was added to the medium in such a manner that the final concentration of γ-TPNa became 10 μM. In Comparative Example 2, a tocopherol acetate solution was added to the culture medium in such a manner that the final concentration of tocopherol acetate became 10 μM. In Comparative Example 3, an α-tocopherol solution was added to the culture medium in such a manner that the final concentration of α-tocopherol became 10 μM. In Comparative Example 4, a γ-tocopherol solution was added to the culture medium in such a manner that the final concentration of γ-tocopherol became 10 μM. In addition, in Comparative Example 1, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium. Then, each culture medium was cultured at 37°C and 5% _CO2 for 24 hours.

As Reference Example 1, the human normal fibroblasts were prepared at a seeding density of 10,000 cells/ ^cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Only a 0.05% (V/V) ethanol aqueous solution was added to the medium. Then, the cells were cultured for 24 hours at 37°C and 5% _CO2 .

Then, RNA was extracted from aged fibroblasts or normal human fibroblasts of each case using Nucleospin (registered trademark) RNA Kit (manufactured by Takara Bio), and cDNA was synthesized from the obtained RNA. Then, using this cDNA as a template, quantitative real-time PCR was performed to quantify the expression level of each gene using primers specific to the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene (manufactured by Takara Bio).

The expression level of the housekeeping gene GAPDH (primer; manufactured by Takara Bio) which is an internal standard gene and does not change in expression due to the addition of compounds was quantified, and the expression level of each gene was standardized by using its value. For the gene expression levels in the above examples, the relative gene expression level was calculated when the expression level of each gene in Comparative Example 1 was set to 1.00. The results are shown in Table 1.

[Table 1] Autophagy activator or ethanol aqueous solution Cells Relative gene expression LC3 ATG5 ATG7 Beclin 1 Reference Example 1 0.05% (V/V) ethanol aqueous solution Normal fibroblasts 8.13 1.12 1.29 0.93 Comparison Example 1 0.05% (V/V) ethanol aqueous solution Aging fibroblasts 1.00 1.00 1.00 1.00 Embodiment 1 1μM α-TPNa 1658.65 2.04 1.10 1.08 Embodiment 2 10μM α-TPNa 1044.58 1.58 1.23 1.28 Embodiment 3 10 μM γ-TPNa 1274.13 1.55 1.76 1.01 Comparison Example 2 10 μM Tocopherol acetate 0.06 1.68 0.68 1.01 Comparison Example 3 10 μM α-tocopherol 0.12 1.54 0.34 0.89 Comparison Example 4 10 μM γ-tocopherol 2.51 0.99 1.13 0.26

As shown in Table 1, in Reference Example 1 and Comparative Example 1, for human normal fibroblasts and aged fibroblasts cultured with only 0.05% (V/V) ethanol aqueous solution, the expression levels of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene in the aged fibroblasts of Comparative Example 1 were reduced or almost unchanged. In addition, for the aged fibroblasts cultured with α-TPNa in Examples 1 and 2 and the aged fibroblasts cultured with γ-TPNa in Example 3, the expression-promoting effect of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene was confirmed compared with Comparative Example 1. Furthermore, compared with the aged fibroblasts cultured with tocopherol acetate added in Comparative Example 2 used in the past, a higher performance-enhancing effect was also confirmed.

Compared with the human normal fibroblasts of Reference Example 1, the expression of LC3 gene was reduced in the aged fibroblasts of Comparative Example 1, while the expression of LC3 gene was increased in the aged fibroblasts cultured with the addition of tocopherol phosphate in Examples 1 to 3 compared with the normal fibroblasts of Reference Example 1, and an excellent LC3 gene expression promoting effect was confirmed. On the other hand, no LC3 gene expression promoting effect was observed in the aged fibroblasts cultured with the addition of tocopherol acetate or α-tocopherol in Comparative Examples 2 to 3, and the LC3 gene expression promoting effect was weak in the aged fibroblasts cultured with the addition of γ-tocopherol in Comparative Example 4.

In addition, compared with the human normal fibroblasts of Reference Example 1, the expression levels of ATG5 and ATG7 genes in the aged fibroblasts of Comparative Example 1 were reduced, while the expressions of ATG5 and ATG7 genes in the aged fibroblasts cultured with the addition of tocopherol phosphate in Examples 1 to 3 were promoted compared with the aged fibroblasts of Comparative Example 1. Furthermore, the expression promotion effect of Beclin 1 gene was also confirmed in the aged fibroblasts cultured with the addition of tocopherol phosphate in Examples 1 to 3. That is, in the aged fibroblasts cultured with the addition of tocopherol phosphate, which is an autophagy activator in Examples 1 to 3, the expression-promoting effect of the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene was confirmed.

<Evaluation of the inhibitory effect on the expression of the mTOR gene in human aged fibroblasts> The aged fibroblasts prepared as described above were prepared at a seeding density of 10,000 cells/ ^cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 4, an α-TPNa solution was added to the culture medium in such a manner that the final concentration of α-TPNa became 1 μM. In Example 5, an α-TPNa solution was added to the culture medium in such a manner that the final concentration of α-TPNa became 10 μM. In Example 6, a γ-TPNa solution was added to the culture medium in such a manner that the final concentration of γ-TPNa became 10 μM. In Comparative Example 6, a tocopherol acetate solution was added to the culture medium in such a manner that the final concentration of tocopherol acetate was 10 μM. In Comparative Example 7, an α-tocopherol solution was added to the culture medium in such a manner that the final concentration of α-tocopherol was 10 μM. In Comparative Example 8, a γ-tocopherol solution was added to the culture medium in such a manner that the final concentration of γ-tocopherol was 10 μM. In addition, in Comparative Example 5, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium. Then, each culture medium was cultured at 37°C and 5% CO ₂ for 24 hours.

As Reference Example 2, the human normal fibroblasts were prepared at a seeding density of 10,000 cells/ ^cm2 and cultured for 24 hours in a D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Only a 0.05% (V/V) ethanol aqueous solution was added to the medium. Then, the cells were cultured at 37°C and 5% _CO2 for 24 hours.

Then, RNA was extracted from aged fibroblasts or normal human fibroblasts of each case using Nucleospin (registered trademark) RNA Kit (manufactured by Takara Bio), and cDNA was synthesized from the obtained RNA. Then, the expression level of the mTOR gene was quantified by quantitative real-time PCR using mTOR gene-specific primers (manufactured by Takara Bio) using this cDNA as a template.

The expression level of the housekeeping gene GAPDH (primer; manufactured by Takara Bio) which is an internal standard gene and does not change in expression due to the addition of compounds was quantified, and the expression level of each gene was standardized by using its value. For the gene expression levels in the above examples, the relative gene expression level was calculated when the expression level of the mTOR gene in Comparative Example 5 was set to 1.00. The results are shown in Table 2.

[Table 2] Autophagy activator or ethanol aqueous solution Cells Relative gene expression mTOR Reference Example 2 0.05% (V/V) ethanol aqueous solution Normal fibroblasts 0.96 Comparison Example 5 0.05% (V/V) ethanol aqueous solution Aging fibroblasts 1.00 Embodiment 4 1μM α-TPNa 0.83 Embodiment 5 10μM α-TPNa 0.55 Embodiment 6 10 μM γ-TPNa 0.74 Comparative Example 6 10 μM Tocopherol acetate 1.06 Comparison Example 7 10 μM α-tocopherol 1.76 Comparative Example 8 10 μM γ-tocopherol 1.92

As shown in Table 2, in Reference Example 2 and Comparative Example 5, for human normal fibroblasts and aged fibroblasts cultured with only 0.05% (V/V) ethanol aqueous solution, the expression of mTOR gene was increased in the aged fibroblasts of Comparative Example 5 compared with the human normal fibroblasts of Reference Example 2. In the aged fibroblasts cultured with the addition of α-TPNa in Examples 4 and 5 and the aged fibroblasts cultured with the addition of γ-TPNa in Example 6, the expression of mTOR gene was decreased compared with the normal fibroblasts of Reference Example 2, and the mTOR gene expression inhibitory effect was confirmed.

On the other hand, in the aged fibroblasts cultured with the addition of tocopherol acetate in Comparative Example 6, the aged fibroblasts cultured with the addition of α-tocopherol in Comparative Example 7, and the aged fibroblasts cultured with the addition of γ-tocopherol in Comparative Example 8, the expression levels of the mTOR gene were all equal to or higher than those in Comparative Example 5, and no mTOR gene expression inhibitory effect was confirmed.

<Evaluation of the effect of promoting the expression of LC3 gene, ATG5 gene and ATG7 gene in human neuroblastoma> The expression levels of LC3 gene, ATG5 gene and ATG7 gene in human neuroblastoma (SH-SY5Y; obtained from ATCC) were measured by the following test method, and the effect of promoting the expression of LC3 gene, ATG5 gene and ATG7 gene induced by tocopherol phosphate was evaluated. In the following examples and comparative examples, starch β, which is known to cause a decrease in autophagy in neurons, was added to each culture medium to perform the test.

SH-SY5Y cells were prepared at a seeding density of 10,000 cells/ ^cm2 and cultured for 24 hours in D-MEM/Ham's F-12 medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 7, α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 1 μM. In Example 8, α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 10 μM. In Example 9, γ-TPNa solution was added to the medium in such a manner that the final concentration of γ-TPNa was 10 μM. In Comparative Example 10, a tocopherol acetate solution was added to the culture medium so that the final concentration of tocopherol acetate was 10 μM. In Comparative Example 11, an α-tocopherol solution was added to the culture medium so that the final concentration of α-tocopherol was 10 μM. In Comparative Example 12, a γ-tocopherol solution was added to the culture medium so that the final concentration of γ-tocopherol was 10 μM. In Comparative Example 9, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium.

Next, a starch β-like solution was prepared by dissolving a starch β-like solution (manufactured by Sigma-Aldrich) in a 0.01% (V/V) DMSO aqueous solution, and the starch β-like solution was added to each culture medium so that the final concentration of the starch β-like solution in each culture medium was 20 μM. In addition, in Reference Example 3, only a 0.05% (V/V) ethanol aqueous solution was added, and the starch β-like solution was not added. Then, each culture medium was cultured at 37°C and 5% CO ₂ for 48 hours.

Then, RNA was extracted from SH-SY5Y cells of each case using Nucleospin (registered trademark) RNA Kit (manufactured by Takara Bio), and cDNA was synthesized from the obtained RNA. Then, using this cDNA as a template, quantitative real-time PCR was performed to quantify the expression level of each gene using primers specific to the LC3 gene, ATG5 gene, and ATG7 gene (manufactured by Takara Bio).

The expression level of the housekeeping gene GAPDH (primer; manufactured by Takara Bio) which is an internal standard gene and does not change in expression due to the addition of compounds was quantified, and the expression level of each gene was standardized by using its value. For the gene expression levels in the above examples, the relative gene expression level when the expression level of each gene in Reference Example 3 was set to 1.00 was calculated. The results are shown in Table 3.

[table 3] Autophagy activator or ethanol aqueous solution Starch beta Relative gene expression LC3 ATG5 ATG7 Reference Example 3 0.05% (V/V) ethanol aqueous solution without 1.00 1.00 1.00 Comparative Example 9 0.05% (V/V) ethanol aqueous solution have 0.56 0.45 0.76 Embodiment 7 1μM α-TPNa 0.98 1.24 1.08 Embodiment 8 10μM α-TPNa 1.32 2.56 3.29 Embodiment 9 10 μM γ-TPNa 1.41 1.36 1.01 Comparative Example 10 10 μM Tocopherol acetate 0.67 0.48 0.68 Comparative Example 11 10 μM α-tocopherol 0.47 0.23 0.55 Comparative Example 12 10 μM γ-tocopherol 0.71 0.79 0.61

As shown in Table 3, in Reference Example 3 and Comparative Example 9, compared with the SH-SY5Y cells of Reference Example 3, which only added 0.05% (V/V) ethanol aqueous solution and did not add starch beta, the SH-SY5Y cells of Comparative Example 9, which were cultured by further adding starch beta, showed a decrease in the expression of the LC3 gene. In Examples 7 to 9, the SH-SY5Y cells cultured by adding tocopherol phosphate, which is an autophagy activator, showed a higher expression of the LC3 gene than the SH-SY5Y cells of Comparative Example 9, confirming the effect of promoting the expression of the LC3 gene caused by tocopherol phosphate. On the other hand, in the SH-SY5Y cells cultured in Comparative Examples 10 to 12 supplemented with tocopherol acetate, α-tocopherol, or γ-tocopherol, no significant increase in LC3 gene expression was observed compared to Comparative Example 9.

In addition, compared with the SH-SY5Y cells of Reference Example 3 to which starch beta was not added, the SH-SY5Y cells of Comparative Example 9 obtained by further adding starch beta and cultured showed reduced expression of ATG5 gene and ATG7 gene. In the SH-SY5Y cells obtained by adding tocopherol phosphate, which is an autophagy activator, in Examples 7 to 9, the expression levels of ATG5 gene and ATG7 gene increased compared with the SH-SY5Y cells of Comparative Example 9, and the expression-promoting effect of tocopherol phosphate on ATG5 gene and ATG7 gene was confirmed. On the other hand, in the SH-SY5Y cells cultured in Comparative Examples 10 to 12 supplemented with tocopherol acetate, α-tocopherol, or γ-tocopherol, no significant increase in the expression levels of the ATG5 gene and the ATG7 gene was observed compared to Comparative Example 9.

<Evaluation of the inhibitory effect of amyloid beta on apoptosis in human neuroblastoma> The ratio of apoptotic cells in human neuroblastoma (SH-SY5Y; obtained from ATCC) in the presence of tocopherol phosphate was measured by the following test method, and the inhibitory effect of tocopherol phosphate-induced apoptosis was evaluated. In the following examples and comparative examples, amyloid beta, which is known to induce a decrease in autophagy and the cell death of neurons called apoptosis, was added to each culture medium to perform the test.

SH-SY5Y cells were prepared at a seeding density of 50,000 cells/ ^cm2 and cultured for 24 hours in a D-MEM/Ham's F-12 medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Then, in Example 10, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 1 μM. In Example 11, an α-TPNa solution was added to the medium in such a manner that the final concentration of α-TPNa was 10 μM. In Example 12, a γ-TPNa solution was added to the medium in such a manner that the final concentration of γ-TPNa was 10 μM. In Comparative Example 14, a tocopherol acetate solution was added to the culture medium so that the final concentration of tocopherol acetate was 10 μM. In Comparative Example 15, an α-tocopherol solution was added to the culture medium so that the final concentration of α-tocopherol was 10 μM. In Comparative Example 16, a γ-tocopherol solution was added to the culture medium so that the final concentration of γ-tocopherol was 10 μM. In Comparative Example 13, only a 0.05% (V/V) ethanol aqueous solution was added to the culture medium.

Next, a starch β-like solution was prepared by dissolving starch β-like (manufactured by Sigma-Aldrich) in a 0.01% (V/V) DMSO aqueous solution, and the starch β-like solution was added to each culture medium so that the final concentration of starch β-like in each culture medium was 30 μM. In addition, in Reference Example 4, only a 0.05% (V/V) ethanol aqueous solution was added, and the starch β-like solution was not added. Then, each culture medium was cultured at 37°C and 5% CO ₂ for 48 hours.

Then, 10 μM Hoechst 33342 (Sigma-Aldrich) aqueous solution was added to each SH-SY5Y cell from which the culture medium had been removed, and the cells were left to stand at room temperature (25°C) for 10 minutes. After the solution was removed, each SH-SY5Y cell was washed with phosphate buffered saline (PBS, Wako Pure Chemical Industries, Ltd.), and the number of cells (Hoechst(+) cells) with strong Hoechst fluorescence, which indicated cell apoptosis caused by chromatin condensation, was counted under a fluorescent microscope. In four independent experiments, more than 5,000 cells were counted in each field of view, and the average of the proportion of Hoechst(+) cells in the four experiments was regarded as the proportion of cells undergoing cell apoptosis. For each example, the relative value was calculated when the ratio of apoptotic cells in Reference Example 4 was set to 1.00, and the results are shown in Table 4.

[Table 4] Autophagy activator or ethanol aqueous solution Starch beta The percentage of apoptotic cells Reference Example 4 0.05% (V/V) ethanol aqueous solution without 1.00 Comparative Example 13 0.05% (V/V) ethanol aqueous solution have 3.78 Embodiment 10 1μM α-TPNa 2.54 Embodiment 11 10μM α-TPNa 1.22 Embodiment 12 10 μM γ-TPNa 1.46 Comparative Example 14 10 μM Tocopherol acetate 3.89 Comparative Example 15 10 μM α-tocopherol 4.09 Comparative Example 16 10 μM γ-tocopherol 3.76

As shown in Table 4, in Reference Example 4 and Comparative Example 13, compared with the SH-SY5Y cells of Reference Example 4 to which only 0.05% (V/V) aqueous ethanol solution was added and no starch beta was added, it was confirmed that the proportion of apoptotic cells increased in the SH-SY5Y cells of Comparative Example 13 to which starch beta was further added.

In the SH-SY5Y cells cultured with tocopherol acetate, α-tocopherol or γ-tocopherol in Comparative Examples 14 to 16, a decrease in the proportion of apoptotic cells was not observed compared with the SH-SY5Y cells in Comparative Example 13. On the other hand, in the SH-SY5Y cells cultured with tocopherol phosphate, which is an autophagy activator, in Examples 10 to 12, a decrease in the proportion of apoptotic cells was observed compared with the SH-SY5Y cells in Comparative Example 13.

[Formulation Examples] As compositions for activating autophagy, the formulation examples 1 and 2 of external preparations are shown in Table 5.

[table 5] Material Formula Example 1 (Mass %) Formula Example 2 (Mass %) α-TPNa 2.0 - γ-TPNa - 0.5 glycerin 4.0 4.0 PG(propylene glycol) 6.0 6.0 Ethanol 3.0 3.0 Phenoxyethanol 0.1 0.1 Potassium Phosphate 3.5 3.5 EDTA4Na 0.2 0.2 PEG(50) Hydrogenated Castor Oil 0.5 0.5 water 80.7 82.2 Total 100.0 100.0

The above is a description of the preferred embodiments of the present invention, but the present invention is not limited to these embodiments. Additions, omissions, substitutions and other changes can be made to the structure without departing from the scope of the present invention. The present invention is not limited by the above description, but only by the scope of the attached patent application. [Industrial Applicability]

The present invention provides an autophagy activator that can effectively activate autophagy and an autophagy activating composition containing the autophagy activator.

Claims (9)

A use of tocopherol phosphate or its salt for preparing an autophagy activator for treating or preventing Huntington's disease, Parkinson's disease, SENDA disease or Crohn's disease, wherein the tocopherol phosphate or its salt is at least one tocopherol phosphate or its salt selected from the group consisting of α-tocopherol phosphate and γ-tocopherol phosphate. For use as claimed in claim 1, the aforementioned autophagy activator contains the aforementioned salt of tocopherol phosphate as an active ingredient, and the aforementioned salt of tocopherol phosphate is sodium salt of tocopherol phosphate. The use of claim 1 or 2, wherein the aforementioned autophagy activator promotes the expression of the LC3 gene. The use of claim 1 or 2, wherein the aforementioned autophagy activator promotes the expression of the ATG5 gene. As for the use of claim 1 or 2, wherein the aforementioned autophagy activator promotes the expression of the ATG7 gene. The use of claim 1 or 2, wherein the aforementioned autophagy activator promotes the expression of Beclin 1 gene. For use as claimed in claim 1 or 2, wherein the aforementioned autophagy activator inhibits the expression of the mTOR gene. A use of tocopherol phosphate or its salt for preparing an autophagy activating composition for treating or preventing Huntington's disease, Parkinson's disease, SENDA disease or Crohn's disease, wherein the autophagy activating composition contains tocopherol phosphate or its salt and a pharmaceutically acceptable carrier, wherein the tocopherol phosphate or its salt is at least one tocopherol phosphate or its salt selected from the group consisting of α-tocopherol phosphate and γ-tocopherol phosphate. For use as in claim 8, wherein the total content of the tocopherol phosphate or its salt in the aforementioned autophagy activating composition is 0.01-10% by weight relative to the total amount of the autophagy activating composition.