KR20230051238A - autophagy activator - Google Patents

Abstract

A composition for activating autophagy, comprising an autophagy activator containing an ascorbic acid derivative or a salt thereof as an active ingredient, and the autophagy activator and a pharmaceutically acceptable carrier.

Description

autophagy activator

The present invention relates to an autophagy activator and a composition for activating autophagy.

This application claims priority based on Japanese Patent Application No. 2020-156457 for which it applied to Japan on September 17, 2020, and uses the content here.

Autophagy responds to extracellular or intracellular stresses and signals such as starvation, growth factor deficiency, and pathogen infection, and decomposes aged or damaged intracellular materials and organella, reproducing energy and removing damaged materials. It is an important mechanism for maintaining normal cell homeostasis. From past studies, it has been reported that autophagy activity in cells rapidly decreases as aging progresses (Non-Patent Document 1). In addition, when autophagy is inhibited, old mitochondria, misfolded proteins, and the like are excessively accumulated in cells, and oxidative stress in cells increases, leading to cell death, resulting in aging of the cells.

Therefore, cell homeostasis can be improved by rapidly removing unnecessary substances in cells by activating autophagy that decomposes aged substances and organella in cells and recycles the degradation products.

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 decrease with age in the human brain. In addition, in neurodegenerative diseases such as Alzheimer's disease, it is thought that the activity of autophagy is reduced. Therefore, it is known that the activation of autophagy contributes to the treatment and prevention of neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and Parkinson's disease (Non-Patent Documents 2 and 3). Particularly in Alzheimer's disease, since the function of autophagy is inhibited, an aggregation protein called amyloid β accumulates in vivo, and it is known that this is related to the onset (Non-Patent Document 4). In addition, SENDA disease (SENDA: static encephalopathy of childhood with neurodegeneration in adulthood), a neurodegenerative disease accompanied by iron deposition in the substantia nigra and globus pallidum and atrophy of the cerebrum, inflammatory bowel disease that causes serious inflammation or ulcers in the digestive tract It is known that mutations in autophagy-related genes or genes involved in selective autophagy also affect diseases such as Crohn's disease and cancer (Non-Patent Document 5).

The process of autophagy has been studied in both yeast and mammals, and up to 36 proteins are used. Among them, from the formation of the autophagosome to the differentiation of its contents, it is controlled by the Atg protein encoded in the autophagy-related gene (ATG), including the Atg12-Atg5 linkage system and the LC3-phosphatidylethanolamine (PE) linkage system. It can be classified into six groups, each of which acts in stages in each process.

As the autophagy activator, a compound that activates autophagy by increasing the LC3-related factor, which is a marker of the active state of autophagy, and a compound that promotes autophagy flux (including fusion of autophagosome to lysosome) Active ingredients This has been reported (Patent Documents 1 to 4).

International Publication No. 2018/173653 Japanese Unexamined Patent Publication No. 2018-80204 Japanese Patent Publication No. 2018-510902 Japanese Patent Publication No. 2019-529514

Yogendra S. Rajawat et al., Aging: Central role for autophagy and the lysosomal degradative system. Aging Research Reviews 8 (2009) 199-213. Aaron Barnett et al., Autophagy in Aging and Alzheimer's Disease: Pathologic or Protective?. J Alzheimers Dis. 2011; 25(3): 385-394. 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. 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. Kageyama et al., 「Autophagy and Disease」, Domain Convergence Review, 2014, 3, e006

As described above, it is known that the function of autophagy is reduced in various diseases such as Alzheimer's disease, and a drug capable of effectively activating autophagy is required. However, it can be said that the effects of conventionally known drugs are still insufficient.

Accordingly, an object of the present invention is to provide an autophagy activator capable of effectively activating autophagy and a composition for activating autophagy containing the autophagy activator.

The present invention includes the following aspects.

(1) An autophagy activator comprising an ascorbic acid derivative or a salt thereof as an active ingredient.

(2) the ascorbic acid derivative or salt thereof is at least one ascorbic acid derivative or salt thereof selected from the group consisting of ascorbyl phosphate, fatty acid esters of ascorbyl phosphate, ethyl ascorbic acid and ascorbic acid glucoside; The autophagy activator according to (1).

(3) The autophagy activator according to (2), wherein the ascorbic acid derivative or salt thereof is a fatty acid ester of ascorbyl phosphate or a salt thereof.

(4) The autophagy activator according to (3), wherein the fatty acid ester of ascorbyl phosphate or a salt thereof is palmitate ester of ascorbyl phosphate or a salt thereof.

(5) The autophagy according to any one of (1) to (4), wherein the salt of the ascorbic acid derivative is contained as an active ingredient, and the salt of the ascorbic acid derivative is a magnesium salt or a sodium salt of the ascorbic acid derivative. activator.

(6) The autophagy activator according to any one of (1) to (5), which promotes expression of the LC3 gene.

(7) The autophagy activator according to any one of (1) to (6), which promotes the expression of the LC3 gene, the ATG5 gene, the ATG7 gene, and the Beclin 1 gene.

(8) The autophagy activator according to any one of (1) to (7), for use in preventing or treating Alzheimer's disease.

(9) A composition for activating autophagy, comprising the autophagy activator according to any one of (1) to (8) and a pharmaceutically acceptable carrier.

(10) The composition for activating autophagy according to (9), wherein the total amount of the ascorbic acid derivative or salt thereof is 0.05 to 15% by mass relative to the total amount of the composition for activating autophagy.

According to the present invention, an autophagy activator capable of effectively activating autophagy by promoting the expression of at least one gene selected from the group consisting of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene, and the autophagy A composition for activating autophagy containing a phage activator may be provided.

(autophagy activator)

The autophagy activator of the present embodiment contains an ascorbic acid derivative or a salt thereof as an active ingredient.

Here, "autophagy" is a mechanism that regenerates energy and removes damaged substances by decomposing old or damaged intracellular substances and organella.

The autophagy activator of the present 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 autophagosomes, thereby activating autophagy.

<Ascorbic acid derivative or salt thereof>

Examples of the ascorbic acid derivative in the autophagy activator of the present embodiment include an ascorbic acid derivative obtained by derivatizing at least one hydroxyl group of ascorbic acid.

As the ascorbic acid derivative, more specifically, ascorbyl phosphate obtained by phosphate esterifying any of the hydroxyl groups of ascorbic acid (also referred to as ascorbic acid phosphate ester); fatty acid esters of ascorbyl phosphate obtained by phosphoesterifying one of the hydroxyl groups of ascorbic acid and esterifying the other hydroxyl group with a fatty acid; ethyl ascorbic acid obtained by ethoxylating any of the hydroxyl groups of ascorbic acid; ascorbic acid glucoside obtained by glucosidating any of the hydroxyl groups of ascorbic acid; acylated ascorbic acid obtained by acylating any of the hydroxyl groups of ascorbic acid; acylated ascorbyl phosphate in which one of the hydroxyl groups of ascorbic acid is acylated and the other hydroxyl group is phosphate esterified; glyceryl ascorbic acid obtained by substituting any of the hydroxyl groups of ascorbic acid with glycerin; phosphoric acid diesters of ascorbic acid and tocopherol (specifically, dl-α-tocopherol 2-L-ascorbic acid phosphate diester, etc.), etc., in which ascorbic acid and tocopherol are bonded through ester bonds through phosphoric acid.

As a salt of an ascorbic acid derivative, the salt of an ascorbic acid derivative and an inorganic base, the salt of an ascorbic acid derivative and an organic base, etc. are mentioned, for example.

Examples of salts with an inorganic base include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salt; ammonium salt; Zinc salt etc. are mentioned.

Examples of salts with organic bases include alkylammonium salts and salts with basic amino acids.

As the ascorbic acid derivative or salt thereof in the autophagy activator of the present embodiment, among the above, (i) ascorbyl phosphate or a salt thereof, (ii) fatty acid ester of ascorbyl phosphate or a salt thereof, ( iii) ethyl ascorbic acid or a salt thereof, (iv) ascorbic acid glucoside or a salt thereof are preferred.

«(i) Ascorbyl phosphate or a salt thereof»

· Ascorbyl Phosphate

Ascorbyl phosphate is a compound in which a phosphate group is introduced into at least one hydroxyl group of ascorbic acid.

As the ascorbyl phosphate, compounds represented by the following formula (1) are preferably exemplified.

The compound represented by the following general formula (1) is an ascorbic acid-2-phosphate ester in which the 2-position hydroxyl group of ascorbic acid is protected with a phosphoric acid ester.

In ascorbyl phosphate, stereoisomers of the D and L forms, and the racemic DL form exist. Ascorbyl phosphate in the present embodiment may be any of these stereoisomers, but from the viewpoint of availability, an L-chain is preferable, and specifically, L-ascorbic acid-2-phosphate ester is preferable.

Salts of ascorbyl phosphate

As a salt of ascorbyl phosphate, the salt of ascorbyl phosphate and an inorganic base, the salt of ascorbyl phosphate and an organic base, etc. are mentioned, for example.

Examples of salts with an inorganic base include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salt; ammonium salt; Zinc salt etc. are mentioned.

Examples of salts with organic bases include alkylammonium salts and salts with basic amino acids.

As the salt of ascorbyl phosphate, among the above, an alkali metal salt or an alkaline earth metal salt is preferable, a sodium salt or magnesium salt is more preferable, and a magnesium salt is still more preferable.

Magnesium salts of ascorbyl phosphate are preferred from the viewpoint of high stability and less colorability.

As ascorbyl phosphate or a salt thereof in the autophagy activator of the present embodiment, among those described above, from the viewpoint of improving stability, an ascorbyl phosphate salt is preferable, and the compound represented by the above formula (1) An alkali metal salt of or an alkaline earth metal salt of a compound represented by the above formula (1) is more preferable, and a sodium salt of a compound represented by the above formula (1) or a magnesium salt of a compound represented by the above formula (1) is more preferable

Specifically, the magnesium salt of the compound represented by the formula (1) is particularly preferably a magnesium salt of L-ascorbic acid-2-phosphate.

The sodium salt of the compound represented by the above formula (1) is particularly preferably a sodium salt of L-ascorbic acid-2-phosphate ester.

Ascorbyl phosphate or a salt thereof in the autophagy activator of the present embodiment may be used alone or in combination of two or more.

Ascorbyl phosphate or a salt thereof can be produced by a known production method, for example, the method described in Japanese Patent Laid-Open No. 2-279690 and JP-A No. 6-345786.

For example, as a specific method for producing ascorbyl phosphate, it can be obtained by reacting ascorbic acid with phosphorus oxychloride and the like to phosphorylate it.

As a specific method for producing an ascorbyl phosphate salt, an ascorbyl phosphate salt can be obtained by neutralizing an ascorbyl phosphate solution with a metal oxide such as magnesium oxide or a metal hydroxide such as sodium hydroxide.

As a commercially available example of the salt of ascorbyl phosphate in the autophagy activator of the present embodiment, ascorbic acid PS (compound name; sodium salt of L-ascorbic acid-2-phosphate ester (L-ascorbic acid- Also called sodium 2-phosphate), display name; ascorbyl phosphate Na), ascorbic acid PM manufactured by Showa Denko Co., Ltd. (compound name; magnesium salt of L-ascorbic acid-2-phosphate ester (L-ascorbic acid-2- Also referred to as magnesium phosphate), display name; ascorbyl phosphate Mg), and the like.

«(ii) Fatty acid ester of ascorbyl phosphate or a salt thereof»

Fatty acid esters of ascorbyl phosphate

The fatty acid ester of ascorbyl phosphate is a compound in which a fatty acid is ester-bonded to at least one hydroxyl group of ascorbyl phosphate. The fatty acid is preferably a straight-chain or branched-chain fatty acid having 6 to 22 carbon atoms (that is, a fatty acid having 5 to 21 carbon atoms in a straight-chain or branched alkyl group bonded to a carboxyl group), and the number of carbon atoms It is more preferable that it is a 10-20 linear or branched chain fatty acid, and it is still more preferable that it is a C12-18 linear or branched chain fatty acid.

Examples of fatty acid esters of ascorbyl phosphate include compounds represented by the following general formula (2). The compound represented by the following general formula (2) is ascorbic acid-2-phosphate-6-fatty acid in which phosphoric acid is ester-bonded to the 2nd-position hydroxyl group of ascorbic acid and a fatty acid is ester-bonded to the 6-position hydroxyl group of ascorbic acid.

[Wherein, R ¹ is a straight-chain or branched-chain alkyl group having 5 to 21 carbon atoms.]

In the above general formula (2), R ¹ is a linear or branched chain alkyl group having 5 to 21 carbon atoms. Specifically, a linear or branched pentyl group, a linear or branched hexyl group, a linear or branched heptyl group, a linear or branched octyl group, a linear or branched nonyl group, a linear Or a branched decyl group, a linear or branched chain undecyl group, a linear or branched chain dodecyl group, a linear or branched chain tridecyl group, a linear or branched chain tetradecyl group, a linear or branched chain Pentadecyl group, linear or branched hexadecyl group, linear or branched heptadecyl group, linear or branched octadecyl group, linear or branched nonadecyl group, linear or branched chain of an icosyl group of , and a linear or branched henicosyl group.

In the general formula (2), among the above, R ¹ is preferably a linear or branched alkyl group having 9 to 19 carbon atoms, and is a linear or branched alkyl group having 11 to 17 carbon atoms. More preferably, it is more preferably a straight-chain or branched-chain alkyl group of 13 to 15 carbon atoms, and from the viewpoint of raw material availability, etc., it is a straight-chain alkyl group (straight-chain pentadecyl group) of 15 carbon atoms. is particularly preferred.

That is, as the compound represented by the above general formula (2), 6-O-palmitoylascorbic acid-2-phosphate ester (also referred to as ascorbic acid-2-phosphate-6-palmitic acid) is particularly preferred.

In the fatty acid ester of ascorbyl phosphate, there are stereoisomers of the D-form and the L-form, and the racemic DL-form. The fatty acid ester of ascorbyl phosphate in the present embodiment may be any of these stereoisomers, but from the viewpoint of easy availability, the L-chain is preferable, and specifically, the fatty acid of L-ascorbic acid-2-phosphate ester Esters are preferred.

Fatty acid ester salt of ascorbyl phosphate

Examples of the fatty acid ester salt of ascorbyl phosphate include a salt of an ascorbyl phosphate fatty acid ester and an inorganic base, and a salt of an ascorbyl phosphate fatty acid ester and an organic base.

As a salt with an inorganic base, For example, alkali metal salts, such as a sodium salt and a potassium salt; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salt; ammonium salt; Zinc salt etc. are mentioned.

As a salt with an organic base, the salt with an alkyl ammonium salt and a basic amino acid etc. are mentioned, for example.

As the fatty acid ester salt of ascorbyl phosphate, among the above, an alkali metal salt or an alkaline earth metal salt is preferable, a sodium salt or magnesium salt is more preferable, and a sodium salt is still more preferable.

The fatty acid ester sodium salt of ascorbyl phosphate is preferred from the viewpoints of stability and ease of blending into formulations.

Among the fatty acid esters of ascorbyl phosphate or salts thereof in the autophagy activator of the present embodiment, among those described above, from the viewpoint of stability and ease of mixing with formulations, ascorbyl phosphate fatty acid ester salts are preferred. , The alkali metal salt of the compound represented by the general formula (2), or the alkali earth metal salt of the compound represented by the general formula (2) is more preferable, and the sodium salt of the compound represented by the general formula (2), or The magnesium salt of the compound represented by the above general formula (2) is more preferred, and the sodium salt of the compound represented by the above general formula (2), specifically, L-ascorbic acid-2-phosphate-6-palmitic acid Sodium salts are particularly preferred.

The fatty acid esters of ascorbyl phosphate or salts thereof in the autophagy activator of the present embodiment may be used alone or in combination of two or more.

The fatty acid ester of ascorbyl phosphate or a salt thereof in the autophagy activator of the present embodiment can be produced by a known production method, such as the method described in Patent No. 6265550.

For example, as a specific method for producing a fatty acid ester of ascorbyl phosphate, after producing ascorbyl phosphate by a method similar to the method for producing ascorbyl phosphate described above, the ascorbyl phosphate, a fatty acid or the like It can be obtained by subjecting an ester to a condensation reaction.

Further, as a specific method for producing the fatty acid ester salt of ascorbyl phosphate, the fatty acid ester of ascorbyl phosphate is neutralized with a metal oxide such as magnesium oxide or a metal hydroxide such as sodium hydroxide, etc. ester salts can be obtained.

As a commercially available example of the fatty acid ester salt of ascorbyl phosphate in the autophagy activator of the present embodiment, Showa Denko Co., Ltd.'s Afreshie (registered trademark) (APPS) (compound name: L-ascorbic acid-2-phosphate- sodium salt of 6-palmitic acid (also referred to as sodium salt of L-6-O-palmitoyl ascorbic acid-2-phosphate ester), display name; palmitic acid ascorbyl phosphate 3Na), and the like.

«(iii) Ethyl ascorbic acid or a salt thereof»

·Ethyl ascorbic acid

Ethyl ascorbic acid is a compound in which an ethyl group is introduced into at least one hydroxyl group of ascorbic acid.

As ethyl ascorbic acid, the compound represented by following formula (3) is mentioned suitably.

The compound represented by the following formula (3) is 3-O-ethyl ascorbic acid in which a hydrogen atom, which is a hydroxyl group in the third position of ascorbic acid, is substituted with an ethyl group.

Ethyl ascorbic acid has stereoisomers of D and L forms, and a racemic DL form. Ethyl ascorbic acid may be any of these stereoisomers, but from the viewpoint of availability, it is preferably L-chain, specifically L-3-O-ethyl ascorbic acid (also referred to as 3-O-ethyl-L-ascorbic acid) this is preferable

Salt of ethyl ascorbic acid

As a salt of ethyl ascorbic acid, the salt of ethyl ascorbic acid and an inorganic base, the salt of ethyl ascorbic acid and an organic base, etc. are mentioned, for example.

As a salt with an inorganic base, For example, alkali metal salts, such as a sodium salt and a potassium salt; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salt; ammonium salt; Zinc salt etc. are mentioned.

As a salt with an organic base, the salt with an alkyl ammonium salt and a basic amino acid etc. are mentioned, for example.

Ethyl ascorbic acid or a salt thereof in the autophage activator of the present embodiment is preferably ethyl ascorbic acid, and more preferably L-3-O-ethyl ascorbic acid, from the viewpoint of easy availability among the above.

Ethyl ascorbic acid or a salt thereof in the autophagy activator of the present embodiment may be used alone or in combination of two or more.

Ethyl ascorbic acid or a salt thereof can be produced by a known production method.

For example, as a method for producing ethyl ascorbic acid, ascorbic acid is alkylated with an alkyl halide in the presence of sodium methoxide in dimethyl sulfoxide (DMSO); It can manufacture by the method described in Unexamined-Japanese-Patent No. 8-134055, Unexamined-Japanese-Patent No. 1-228977, etc.

As a specific method for producing a salt of ethyl ascorbic acid, a salt of ethyl ascorbic acid can be obtained by neutralizing a solution of ethyl ascorbic acid with a metal oxide such as magnesium oxide or a metal hydroxide such as sodium hydroxide.

Commercially available examples of ethyl ascorbic acid in the autophagy activator of the present embodiment include 3-O-ethyl-L-ascorbic acid (trade name: 3-O-ethylascorbic acid) manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. can

«(iv) ascorbic acid glucoside or a salt thereof»

· Ascorbic acid glucoside

Ascorbic acid glucoside is a compound in which at least one hydroxyl group of ascorbic acid is glucosidated. The glucosidic linkage is preferably an α-glucosidic linkage.

As the ascorbic acid glucoside, compounds represented by the following general formula (4) are preferably exemplified.

The compound represented by the following formula (4) is ascorbic acid 2-glucoside in which glucose is bonded to the 2nd hydroxyl group of ascorbic acid.

Ascorbic acid has stereoisomers of the D and L forms, and the racemic DL form. The ascorbic acid in ascorbic acid glucoside may be any of these stereoisomers, but from the viewpoint of availability, an L-chain is preferred, and the ascorbic acid glucoside is specifically preferably L-ascorbic acid-2-glucoside. Glucose in ascorbic acid glucoside may be either D or L, but from the viewpoint of availability, D is preferred.

Salts of ascorbic acid glucoside

As a salt of ascorbic acid glucoside, the salt of ascorbic acid glucoside and an inorganic base, the salt of ascorbic acid glucoside and an organic base, etc. are mentioned, for example.

As a salt with an inorganic base, For example, alkali metal salts, such as a sodium salt and a potassium salt; alkaline earth metal salts such as calcium salts and magnesium salts; aluminum salt; ammonium salt; Zinc salt etc. are mentioned.

As a salt with an organic base, the salt with an alkyl ammonium salt and a basic amino acid etc. are mentioned, for example.

As the ascorbic acid glucoside or salt thereof in the autophagy activator of the present embodiment, among the above, ascorbic acid glucoside is preferable from the viewpoint of availability, and L-ascorbic acid 2-glucoside is more desirable.

Ascorbic acid glucoside or a salt thereof in the autophagy activator of the present embodiment may be used alone or in combination of two or more.

Ascorbic acid glucoside or a salt thereof can be produced by, for example, the method described in Japanese Unexamined Patent Publication No. 03-139288.

For example, as a specific method for producing ascorbic acid glucoside, it can be produced by subjecting one molecule of glucose to α-glucoside linkage to the 2nd hydroxyl group of ascorbic acid through an enzymatic reaction.

Further, as a specific method for producing a salt of ascorbic acid glucoside, a salt of ascorbic acid glucoside can be obtained by neutralizing an ascorbic acid glucoside solution with a metal oxide such as magnesium oxide or a metal hydroxide such as sodium hydroxide.

As a commercially available example of ascorbic acid glucoside in the autophagy activator of the present embodiment, ascorbic acid 2-glucoside (compound name: L-ascorbic acid 2-glucoside, display name: ascorbyl glucoside) manufactured by Hayashibara Co., Ltd. etc. can be mentioned.

The ascorbic acid derivative or salt thereof in the autophagy activator of the present embodiment may be used alone or in combination of two or more.

As the ascorbic acid derivative or salt thereof in the autophagy activator of the present embodiment, among the above, from the viewpoint of being able to activate autophagy more, (i) ascorbyl phosphate or a salt thereof or (ii) phosphoric acid It is preferably an ascorbyl fatty acid ester or a salt thereof, and (ii) an ascorbyl phosphate fatty acid ester or a salt thereof is more preferable.

The autophagy activator of the present embodiment can be used by administering itself to a patient for the purpose of treating neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. In addition, the autophagy activator of the present embodiment can be used in combination with pharmaceuticals or cosmetics for the purpose of activating autophagy. In addition, it may be used in combination with a composition for activating autophagy described later.

The autophagy activator of the present embodiment can effectively activate autophagy by promoting 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.

Since the autophagy activator of the present embodiment can effectively activate autophagy, it can be used for preventing or treating Alzheimer's disease.

It is known that amyloid β causes a decrease in autophagy in nerve cells. In addition, it is known that amyloid β induces a decrease in autophagy and thereby induces cell death called apoptosis of nerve cells.

The autophagy activator of the present embodiment can promote LC3 gene expression in the presence of amyloid β.

The autophagy activator of the present embodiment can promote ATG5 gene expression in the presence of amyloid β.

The autophagy activator of the present embodiment can promote ATG7 gene expression in the presence of amyloid β.

The autophagy activator of the present embodiment can promote Beclin 1 gene expression in the presence of amyloid β.

The autophagy activator of the present embodiment can suppress apoptosis in the presence of amyloid β.

The autophagy activator of the present embodiment can promote expression of at least one gene selected from the group consisting of LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene in the presence of amyloid β, particularly in nerve cells. . In addition, the autophagy activator of the present embodiment can suppress apoptosis in the presence of amyloid β, particularly in nerve cells.

The promotion of LC3 gene expression in the presence of amyloid β means that, in the presence of amyloid β, by administering the autophagy activator of the present embodiment, the expression level of the LC3 gene is increased compared to the case where the autophagy activator is not administered. means to do The same applies to the ATG5 gene, ATG7 gene and Beclin 1 gene.

Suppression of apoptosis in the presence of amyloid β means that apoptosis is suppressed by administering the autophagy activator of the present embodiment in the presence of amyloid β, compared to the case where the autophagy activator is not administered.

LC3 (microtubule associated protein 1 light chain 3 alpha: NCBI Gene ID: 84557) is converted to LC3-II, which is attracted to the autophagosome membrane by adding phosphatidylethanolamine upstream of the autophagy signal transduction, to the autophagosome membrane. combine LC3 is used as an autophagosome marker. Examples of the nucleotide 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 a ubiquitin-like conjugated system. Examples of the nucleotide sequence 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 nucleotide 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 cres III PI3K complex with ATG14L and Vps34mp150, and functions as a defining control factor for autophagosome formation. Examples of the nucleotide sequence of the human Beclin 1 gene include NM_001313998.2, NM_001313999.1, NM_001314000.1 and NM_003766.4 registered in the NCBI Reference Sequence database.

The autophagy activator of the present embodiment may be administered to patients with a high risk of developing neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, and Parkinson's disease, and used to prevent neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. do. In addition, the autophagy activator of the present embodiment may be administered to a patient who has developed a neurodegenerative disease such as Alzheimer's disease, Huntington's disease, or Parkinson's disease, and may be used to suppress progression or aggravation of the neurodegenerative disease.

The autophagy activator of the present embodiment can be administered to a patient by the same method as the composition for activating autophagy described later, and may be administered orally or parenterally, intravenously, intraarterially, or intramuscularly. It may be administered internally, intradermally, subcutaneously, intraperitoneally, etc., intrarectally as a suppository, or administered to the skin as an external preparation for the skin.

(Composition for activating autophagy)

The composition for activating autophagy of the present embodiment contains an autophagy activator containing the above-described ascorbic acid derivative or a salt thereof, and a pharmaceutically acceptable carrier.

The composition for activating autophagy of the present embodiment is prepared by mixing the above-described autophagy activator, a pharmaceutically acceptable carrier, and other components in accordance with a conventional method (for example, the method described in the Japanese Pharmacopoeia). It can be prepared by formulating.

In the present specification, "pharmaceutically acceptable carrier" means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the subject of administration.

In addition, "no substantial toxicity" means that the component does not exhibit toxicity to the subject of administration at a dose normally used.

The pharmaceutically acceptable carrier is not particularly limited, and excipients, binders, disintegrants, lubricants, stabilizers, diluents, solvents for injections, moisturizers, texture improvers, surfactants, polymers, thickeners and gelling agents, solvents, propellants , antioxidant, reducing agent, oxidizing agent, chelating agent, acid, alkali, powder, inorganic salt, water, metal-containing compound, unsaturated monomer, polyhydric alcohol, polymer additive, wetting agent, thickener, tackifier, oil-based raw material, liquid matrix, oil-soluble substances, polymeric carboxylates, and the like.

As a specific example of these components, the thing of international publication 2016/076310, etc. are mentioned, for example. Further, specific examples of the polymer/thickener/gelling agent include methchloroyloxyethylphosphorylcholine, butyl methacrylate or polymers thereof.

The pharmaceutically acceptable carriers in the composition for activating autophagy of the present embodiment may be used alone or in combination of two or more.

In addition, other components are not particularly limited, and preservatives, antibacterial agents, ultraviolet absorbers, whitening agents, vitamins other than ascorbic acid derivatives or salts thereof and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, hair growth agents, blood circulation promoters, stimulants, hormones , Anti-wrinkle agent, Anti-aging agent, Tightening agent, Cooling agent, Warming agent, Wound healing accelerator, Irritation reliever, Pain reliever, Cell activator, Plant/animal/microbial extract, Seed oil, Antiseptic agent, Keratin exfoliation/dissolving agent, Perspiration inhibitor, Refreshers, astringents, enzymes, nucleic acids, fragrances, pigments, colorants, dyes, pigments, anti-inflammatory drugs, antifungals, antihistamines, hypnotic sedatives, tranquilizers, antihypertensives, antihypertensives, antibiotics, anesthetics, antibacterial substances, antiepileptics, coronary vascular expanders, herbal medicines, anti-itch agents, keratin softening exfoliators, sunscreens, disinfectants, antioxidants, pH adjusters, additives, metal soaps, and the like. As a specific example of these components, the thing of international publication 2016/076310, etc. are mentioned, for example. Further, specific examples of plant/animal/microorganism extracts include flowers/leaves/stems of Lapsana communis, tea leaves, and the like. As a specific example of seed oil, moringa seed oil is mentioned. As a specific example of the perfume, perylaldehyde is exemplified.

Other components may be used individually by 1 type, and may use 2 or more types together.

The composition for activating autophagy of the present embodiment may contain a therapeutically effective amount of the autophagy activator. "Therapeutically effective amount" means an amount of a drug effective for the treatment or prevention of a patient's disease. A therapeutically effective amount may vary depending on the disease state, age, sex, and weight of the subject to be administered.

In the composition for activating autophagy of the present embodiment, the therapeutically effective amount of the autophagy activator may be an amount capable of activating autophagy of an ascorbic acid derivative or a salt thereof. In addition, the therapeutically effective amount of the autophagy activator described above is such that the ascorbic acid derivative or salt thereof promotes the expression of at least one gene selected from the group consisting of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene. amount that can be In addition, the therapeutically effective amount of the autophagy activator may be an amount capable of inhibiting apoptosis in the presence of amyloid β.

The therapeutically effective amount (total content of ascorbic acid derivatives or salts thereof) of the autophagy activator in the composition for activating autophagy of the present embodiment is, for example, 0.05 to 15% by mass, based on the total amount of the composition for activating autophagy. It may be, for example, 0.1 to 10 mass% may be sufficient, for example, 0.5 to 5 mass% may be sufficient.

In addition, the total content of an ascorbic acid derivative or salt thereof means the content of the compound when one type of ascorbic acid derivative or salt thereof is used alone, and when two or more types of ascorbic acid derivatives or salts thereof are used in combination In this case, the total content of these compounds is meant.

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

(Pharmaceutical composition)

In one embodiment, the present invention provides a pharmaceutical composition for activating autophagy, containing the above-described autophagy activator and a pharmaceutically acceptable carrier.

In the pharmaceutical composition of the present embodiment, the pharmaceutically acceptable carrier is not particularly limited, and carriers generally used for pharmaceuticals can be used in addition to those listed above. For example, Japanese Pharmacopoeia, Japanese Non-Pharmacopeia Drug Specifications, Pharmaceutical Excipients Specifications 2013 (Yakuji Nippo Co., Ltd., 2013), Pharmaceutical Excipients Dictionary 2016 (Japanese Pharmaceutical Excipients Association Edition, Yakuji Nippos Co., 2016), Handbook of Pharmaceutical Common raw materials described in Excipients, 7th edition (Pharmaceutical Press, 2012) can be used.

A pharmaceutically acceptable carrier may be used individually by 1 type, or may use 2 or more types together.

The pharmaceutical composition of the present embodiment may contain other components in addition to the autophagy activator and pharmaceutically acceptable carrier. As the other component, it is not particularly limited, and general pharmaceutical additives can be used. In addition, as other components, active ingredients other than the aforementioned autophagy activator may be used. As pharmaceutical additives and active ingredients as other ingredients, in addition to those listed above, for example, Japanese Pharmacopoeia, Japanese Pharmacopoeia Non-Pharmacopoeia Drug Standards, Pharmaceutical Additives Standards 2013 (Yakuji Nippo Co., Ltd., 2013), Pharmaceutical Excipients Dictionary 2016 (Japanese Pharmaceuticals) Excipient Association Edition, Yakuji Nippos, 2016), Handbook of Pharmaceutical Excipients, 7th edition (Pharmaceutical Press, 2012), etc. can use general raw materials described. Other components may be used individually by 1 type, and may use 2 or more types together.

The dosage form of the pharmaceutical composition of the present embodiment is not particularly limited, and a dosage form generally used as a pharmaceutical preparation can be used. For example, orally administered dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, and emulsions; and parenterally administered formulations such as injections, suppositories, and skin external preparations. Pharmaceutical compositions of these dosage forms can be formulated according to a conventional method (for example, the method 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 a method generally used as a pharmaceutical administration method. For example, it may be administered orally as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions, etc., alone as injections, infusion preparations, etc., or general infusions such as glucose solution and Ringer's solution. and may be administered intravenously, intraarterially, intramuscularly, intradermally, subcutaneously, intraperitoneally, etc., intrarectally as a suppository, or administered to the skin as an external skin preparation.

The dose of the pharmaceutical composition of the present embodiment can be a therapeutically effective amount. A therapeutically effective amount may be appropriately determined depending on the patient's symptoms, body weight, age, sex, etc., and the formulation and administration method of the pharmaceutical composition.

For example, the dosage of the pharmaceutical composition of the present embodiment is 0.01 to 500 mg per dosage unit form as the total content of the ascorbic acid derivative or salt thereof in the case of oral administration, and of the ascorbic acid derivative or salt thereof in the case of injection. 0.02 to 250 mg per dosage unit form as a total content, 0.01 to 500 mg per dosage unit form as the total content of ascorbic acid derivatives or salts thereof in the case of suppositories, and as the total content of ascorbic acid derivatives or salts thereof in the case of external skin preparations 0.01 to 500 mg per dosage unit form; and the like.

The administration interval of the pharmaceutical composition of the present embodiment may be appropriately determined depending on the condition, body weight, age, sex, etc. of the patient, and the dosage form and administration method of the pharmaceutical composition. For example, it can be set to once a day or about 2 to 3 times.

The pharmaceutical composition of the present embodiment can be used for the treatment or prevention of diseases caused by a decrease in 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; Cancer etc. are mentioned.

The pharmaceutical composition of this embodiment is suitable for, for example, neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and SENDA disease; inflammatory bowel diseases such as Crohn's disease; Alternatively, it can be administered to cancer patients to inhibit progression of neurodegenerative diseases, inflammatory bowel diseases, or cancers. Moreover, the pharmaceutical composition of this embodiment is neurodegenerative diseases, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and SENDA disease; inflammatory bowel diseases such as Crohn's disease; Alternatively, it can be administered to cancer patients and used to treat neurodegenerative diseases, inflammatory bowel diseases, or cancer. In addition, the pharmaceutical composition of the present embodiment can be used to treat a disease caused by amyloid β. In addition, the pharmaceutical composition of the present embodiment can be used to treat a disease caused by a decrease in the expression level of the LC3 gene, the ATG5 gene, the ATG7 gene, or the Beclin 1 gene.

Among the above, the pharmaceutical composition of the present embodiment can be particularly suitably used for treating Alzheimer's disease.

The pharmaceutical composition of the present embodiment can be administered to patients with a high risk of onset of neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and SENDA disease, and can also be used to prevent neurodegenerative diseases. In addition, the pharmaceutical composition of the present embodiment can be administered to a patient with a high risk of developing inflammatory bowel disease such as Crohn's disease, and can also be used to prevent inflammatory bowel disease. In addition, the pharmaceutical composition of the present embodiment can be administered to a patient with a high risk of developing cancer and used to prevent cancer.

(cosmetics)

In one embodiment, the present invention provides a cosmetic for activating autophagy containing the aforementioned autophagy activator and a pharmaceutically acceptable carrier.

In the cosmetic composition of the present embodiment, the pharmaceutically acceptable carrier is not particularly limited, and carriers commonly used in cosmetics other than those listed above can be used. For example, Commentary on the 2nd Edition of Standards for Cosmetics Ingredients (Japan Compendium Association, Yakuji Nippo, 1984), Standards for Ingredients Outside of Standards for Cosmetics (Ministry of Health and Welfare Pharmaceutical Affairs Bureau, Yakuji Nippo, 1993), Cosmetics Supplementation of ingredient specifications other than raw material standards (Ministry of Health and Welfare Pharmaceutical Affairs Bureau, Yakuji Nippo, 1993), cosmetics category approval criteria (Ministry of Health, Welfare and Pharmaceutical Affairs Bureau, Yakuji, Nippo, 1993), Cosmetic Ingredient Dictionary (Nikko) Chemicals Co., 1991), International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol. 1 to 4, by CTFA, etc. can be used.

A pharmaceutically acceptable carrier may be used individually by 1 type, or may use 2 or more types together.

In addition to the autophagy activator and the pharmaceutically acceptable carrier, the cosmetic of the present embodiment may contain other components. As the other component, it is not particularly limited, and general cosmetic additives can be used. In addition, as other components, active ingredients other than the aforementioned autophagy activator may be used. As cosmetic additives and active ingredients as other ingredients, in addition to those listed above, for example, Cosmetic Ingredient Standards 2nd Edition Commentary (Japanese Compendium Association edition, Yakuji Nippo, Inc., 1984), Standards for Ingredients Outside of Standards for Cosmetic Ingredients ( Ministry of Health and Welfare Pharmaceutical Affairs Bureau Examination Division Supervision, Yakuji Nippo Co., Ltd., 1993), Cosmetic Ingredient Standard Ingredient Standard Supervision (Ministry of Health and Welfare Pharmaceutical Affairs Bureau Examination Division Supervision, Yakuji Nippo Corporation, 1993), Cosmetic Type Approval Standards (Ministry of Health and Welfare Pharmaceutical Affairs Bureau Examination Division Supervision, Ya Kuji Nippo Co., Ltd., 1993), Cosmetic Ingredient Dictionary (Nikko Chemicals Co., Ltd., 1991), International Cosmetic Ingredient Dictionary and Handbook 2002 Ninth Edition Vol. 1 to 4, by CTFA, etc. can be used. Other components may be used individually by 1 type, and may use 2 or more types together.

The form of the cosmetic of the present embodiment is not particularly limited, and can be a form generally used as a cosmetic. For example, cosmetics for hair, such as a shampoo, conditioner, and a hair conditioner; basic cosmetics such as face wash, cleansing agent, lotion, lotion, lotion, cream, gel, sunscreen agent, pack, mask, beauty essence; makeup cosmetics such as foundations, makeup foundations, lipsticks, lip glosses, and cheek touches; and body cosmetics such as body washing, body powder, and deodorizing cosmetics. These cosmetics can be manufactured according to a conventional method.

In addition, the dosage form of the cosmetic of the present embodiment is not particularly limited, and examples thereof include oil-in-water (O/W) type, water-in-oil (W/O) type, W/O/W type, and O/W/O type. Emulsion type, emulsified polymer type, oil, solid, liquid, smoke, stick, volatile type, powder, jelly, gel, paste, cream, sheet, film, mist, spray Mold, aerosol form, multilayer form, bubble form, flake form, etc. are mentioned.

The usage amount of the cosmetic of the present embodiment is not particularly limited, but can be an amount effective for activating autophagy.

For example, the amount of cosmetics used in the present embodiment is 0.01 to 500 mg per use, for example, 0.15 to 300 mg, for example, 0.15 to 200 mg, as the total content of the ascorbic acid derivative or salt thereof. It may be, for example, 0.2 to 100 mg.

The use interval of the cosmetic composition of the present embodiment is not particularly limited, but may be, for example, once a day or about 2 to 3 times a day.

The cosmetic of the present embodiment can be used to alleviate symptoms caused by a decrease in autophagy activity. Alternatively, in order to prevent the onset of symptoms due to a decrease in autophagy activity, it may be used for daily skin care or makeup by subjects at high risk of developing these symptoms.

(other embodiments)

In one embodiment, the present invention provides a method for activating autophagy, comprising administering an ascorbic acid derivative or a salt thereof to a subject.

In one embodiment, the present invention provides a method for stimulating expression of LC3 gene, ATG5 gene, ATG7 gene, or Beclin 1 gene, comprising administering an ascorbic acid derivative or a salt thereof to a target.

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

In one embodiment, the present invention provides an ascorbic acid derivative or salt thereof for activating autophagy.

In one embodiment, the present invention provides an ascorbic acid derivative or salt thereof for promoting expression of the LC3 gene, the ATG5 gene, the ATG7 gene, or the Beclin 1 gene.

In one embodiment, the present invention provides an ascorbic acid derivative or salt thereof for inhibiting apoptosis in the presence of amyloid β.

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

In one embodiment, the present invention provides use of an ascorbic acid derivative or a salt thereof to prepare an stimulator of expression of the LC3 gene, the ATG5 gene, the ATG7 gene, or the Beclin 1 gene.

In one embodiment, the present invention provides use of an ascorbic acid derivative or a salt thereof to prepare an stimulator of LC3 gene, ATG5 gene, ATG7 gene, or Beclin 1 gene expression in the presence of amyloid β.

In one embodiment, the present invention provides the use of an ascorbic acid derivative or salt thereof for preparing an inhibitor of apoptosis in the presence of amyloid β.

In one embodiment, the present invention provides the use of an ascorbic acid derivative or a salt thereof to prepare a composition for activating autophagy.

In one embodiment, the present invention provides the use of an ascorbic acid derivative or a salt thereof to prepare a composition for stimulating the expression of LC3 gene, ATG5 gene, ATG7 gene, or Beclin 1 gene.

In one embodiment, the present invention provides the use of an ascorbic acid derivative or a salt thereof to prepare a composition for stimulating the expression of LC3 gene, ATG5 gene, ATG7 gene, or Beclin 1 gene in the presence of amyloid β.

In one embodiment, the present invention provides use of an ascorbic acid derivative or a salt thereof to prepare a composition for inhibiting apoptosis in the presence of amyloid β.

Example

Hereinafter, the present invention will be described in more detail by examples, but the present invention is not limited by these examples.

[Ascorbic acid derivatives or salts thereof]

In the following Examples and Formulation Examples, the following ascorbic acid derivatives or salts thereof were used.

APM: magnesium salt of L-ascorbic acid-2-phosphate ester (labeled name: ascorbyl phosphate Mg, product name: ascorbic acid PM, manufactured by Showa Denko Co., Ltd.)

APPS: Sodium salt of L-ascorbic acid-2-phosphate-6-palmitic acid (labeled name: palmitic acid ascorbic acid 3Na, product name: APPS, manufactured by Showa Denko Co., Ltd.)

AG: L-ascorbic acid 2-glucoside (labeled name: ascorbyl glucoside, product name: ascorbic acid 2-glucoside, manufactured by Hayashibara Co., Ltd.)

AE: 3-O-ethyl-L-ascorbic acid (labeled name; 3-O-ethylascorbic acid, product name; 3-O-ethyl-L-ascorbic acid, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

<Evaluation of the expression promoting effect of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene in human senescent fibroblast cells>

In order to prepare artificially senescent cells, tests were conducted using fibroblasts. Senescent fibroblasts were produced by the following procedure.

≪Production of senescent fibroblasts≫

Normal human fibroblasts (NB1RGB; manufactured by RIKEN BRC Cell Bank) were cultured until confluent in D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Thereafter, they were treated with 250 μM hydrogen peroxide for 2 hours and cultured for 24 hours in D-MEM medium supplemented with fresh 10% fetal bovine serum. Treatment with hydrogen peroxide and cell culture were repeated three times, and the obtained fibroblasts were used as senescent fibroblasts.

≪Evaluation test of gene expression promoting effect≫

The prepared senescent fibroblasts were prepared at a seeding density of 10000 cells/cm 2 and cultured for 24 hours in D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals). Next, in Example 1, APM dissolved in purified water was added to the medium so that the final concentration of APM was 10 µM. In Example 2, APPS dissolved in purified water was added to the medium so that the final concentration of APPS was 1 μM. In Example 3, APPS dissolved in purified water was added to the medium so that the final concentration of APPS was 10 µM. In Example 4, AG dissolved in purified water was added to the medium so that the final concentration of AG was 100 µM. In Example 5, AE dissolved in purified water was added to the medium so that the final concentration of AE was 100 µM.

In Comparative Example 1, only purified water was added to the medium.

Then, each medium was cultured for 24 hours at 37°C and 5% CO ₂ .

On the other hand, as Reference Example 1, the human normal fibroblasts were prepared at a seeding density of 10,000 cells/cm 2 and cultured in D-MEM medium (manufactured by Sigma-Aldrich) supplemented with 10% fetal bovine serum (manufactured by MP Biomedicals) at 24 After incubation for a period of time, only purified water was added to the medium. Subsequently, the medium was cultured for 24 hours at 37°C and 5% CO ₂ .

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

As an internal standard gene, the expression level of GAPDH (primer; Takara Bio Co., Ltd.), a housekeeping gene that showed no change in gene expression by the addition of the compound, was quantified, and the expression level of each gene was standardized according to the value. Regarding the gene expression level in each of the above examples, the relative gene expression level when the expression level of each gene in Comparative Example 1 was set to 1.00, respectively, was determined. The results are shown in Table 1.

As shown in Table 1, in Reference Example 1 and Comparative Example 1, in normal human fibroblasts and senescent fibroblasts cultured by adding only purified water, respectively, the senescent fibroblasts had the LC3 gene, the ATG5 gene, the ATG7 gene and It was confirmed that all the expression levels of the Beclin 1 gene were decreased.

On the other hand, in senescent fibroblasts cultured with the addition of the autophagy activators of Examples 1 to 5, compared to senescent fibroblasts cultured with only purified water in Comparative Example 1, the expression level of LC3 gene was particularly high, and the expression of LC3 gene The accelerating effect was confirmed.

In particular, when the autophagy activators of Examples 1 to 3 were added, even at low concentrations of ascorbic acid derivatives (APM and APPS), the LC3 gene expression level was high, and a remarkable LC3 gene expression promoting effect was confirmed. .

Further, in Examples 2 and 3, even at a very low concentration of APPS, the expression level of any of the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene can be improved, and APPS is particularly effective in LC3 gene, ATG5 gene, and ATG7 gene. And it was confirmed that the effect of promoting the expression of the Beclin 1 gene was excellent.

<Evaluation of the effect of promoting expression of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene in human neuroblastoma>

In the presence of an autophagy activator, the expression levels of the LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene in human neuroblastoma (SH-SY5Y; obtained from ATCC) were measured by the following test method, and the autophagy activator The expression promotion effect of LC3 gene, ATG5 gene, ATG7 gene and Beclin 1 gene by was evaluated.

In the following Examples and Comparative Examples, tests were conducted by adding amyloid β, which is known to cause a decrease in autophagy in nerve cells, to each medium.

The SH-SY5Y cells were prepared at a seeding density of 10000/cm2 and cultured for 24 hours in D-MEM/Ham's F-12 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (MP Biomedicals). . Next, in Example 6, APM dissolved in purified water was added to the medium so that the final concentration of APM was 10 µM. In Example 7, APPS dissolved in purified water was added to the medium so that the final concentration of APPS was 1 µM. In Example 8, APPS dissolved in purified water was added to the medium so that the final concentration of APPS was 10 µM. In Example 9, AG dissolved in purified water was added to the medium so that the final concentration of AG was 100 µM. In Example 10, AE dissolved in purified water was added to the medium so that the final concentration of AE was 100 µM.

In Comparative Example 2, only purified water was added to the medium.

Subsequently, an amyloid β solution in which amyloid β (manufactured by Sigma-Aldrich) was dissolved in 0.01% (V/V) DMSO was prepared, and the amyloid β solution was added to each medium so that the final concentration of amyloid β in each medium was 20 µM. added to.

In Reference Example 2, only purified water was added, and no amyloid β solution was added.

Then, each medium was cultured for 48 hours at 37°C and 5% CO ₂ .

Thereafter, 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, primers specific to the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene (manufactured by Takara Bio) were respectively used by quantitative real-time PCR to quantify the expression level of each gene.

As an internal standard gene, the expression level of GAPDH (primer; Takara Bio Co., Ltd.), a housekeeping gene whose expression was not changed by addition of the compound, was quantified, and the expression level of each gene was standardized according to the value. Regarding the gene expression level in each of the above examples, the relative gene expression level when the expression level of each gene in Reference Example 2 was set to 1.00, respectively, was determined. The results are shown in Table 2.

As shown in Table 2, in Reference Example 2 and Comparative Example 2, compared to SH-SY5Y cells cultured with only purified water added, in SH-SY5Y cells cultured with the addition of amyloid β, LC3 gene and ATG5 gene , It was confirmed that the expression levels of both the ATG7 gene and the Beclin 1 gene were decreased. Moreover, among these, it was confirmed that the expression level of the LC3 gene in particular was reduced.

On the other hand, in SH-SY5Y cells cultured with the addition of an autophagy activator and an amyloid β solution in Examples 6 to 10, compared to SH-SY5Y cells cultured with the addition of purified water and an amyloid β solution in Comparative Example 2, In particular, the expression level of the LC3 gene was high, and the effect of promoting the expression of the LC3 gene by the autophagy activators of Examples 6 to 10 was confirmed.

In addition, the SH-SY5Y cells cultured with the addition of the autophagy activator and the amyloid β solution of Examples 6 to 10 showed higher levels of ATG5, ATG7 and Beclin 1 than the SH-SY5Y cells cultured with only the purified water of Reference Example 2. It was confirmed that the expression level of the gene was increased.

Among the Examples, in Examples 6 to 8, even at a low concentration of ascorbic acid derivatives (APM and APPS), the expression level of the LC3 gene was high, and a remarkable LC3 gene expression promoting effect was confirmed.

Further, in Examples 7 and 8, even at a very low concentration of APPS, the expression levels of any of the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 gene can be improved, and in particular, the LC3 gene, ATG5 gene, ATG7 gene, and Beclin 1 It was confirmed that the gene expression promotion effect was excellent.

<Evaluation of apoptosis inhibitory effect due to amyloid β in human neuroblastoma> The ratio of apoptotic cells in human neuroblastoma (SH-SY5Y; obtained from ATCC) in the presence of an autophagy activator was measured by the following test method. And, the apoptosis inhibitory action by the autophagy activator was evaluated.

In the following Examples and Comparative Examples, tests were conducted by adding amyloid β, which is known to induce a decrease in autophagy and thereby induce cell death called apoptosis of nerve cells, to each medium.

SH-SY5Y cells were prepared at a seeding density of 50000 cells/cm 2 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). Next, in Example 11, APM dissolved in purified water was added to the medium so that the final concentration of APM was 10 µM. In Example 12, APM dissolved in purified water was added to the medium so that the final concentration of APM was 100 µM. In Example 13, APPS dissolved in purified water was added to the medium so that the final concentration of APPS was 1 µM. In Example 14, APPS dissolved in purified water was added to the medium so that the final concentration of APPS was 10 µM. In Example 15, AG dissolved in purified water was added to the medium so that the final concentration of AG was 100 µM. In Example 16, AE dissolved in purified water was added to the medium so that the final concentration of AE was 100 µM.

In Comparative Example 3, only purified water was added to the culture medium.

Subsequently, an amyloid β solution was prepared by dissolving amyloid β (manufactured by Sigma-Aldrich) in 0.01% (V/V) DMSO, and the amyloid β solution was added to each medium so that the final concentration of amyloid β in each medium was 30 µM. added to.

In Reference Example 3, only purified water was added and no amyloid β solution was added.

Then, each medium was cultured for 48 hours at 37°C and 5% CO ₂ .

Thereafter, an aqueous solution of 10 μM Hoechst33342 (manufactured by Sigma-Aldrich) was added to each SH-SY5Y cell from which the medium was removed, and the cells were allowed to stand at room temperature (25° C.) for 10 minutes. After removing the solution, each SH-SY5Y cell was washed with phosphate buffer (PBS, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the number of cells showing strong Hoechst fluorescence such as apoptosis due to chromatin aggregation under a fluorescence microscope (Hoechst(+) cells) were counted. In 4 experiments of independent trials, 5000 or more cells in each field were counted, and the average value of the percentages of Hoechst (+) cells in the 4 tests was taken as the percentage of cells undergoing apoptosis. For each case, the relative value when the ratio of apoptotic cells in Reference Example 3 was set to 1.00 was calculated, and the results are shown in Table 3.

As shown in Table 3, in Reference Example 3 and Comparative Example 3, compared to the SH-SY5Y cells of Reference Example 3 cultured with the addition of purified water only, the SH-SY5Y cells of Comparative Example 3 cultured with the addition of amyloid β. , it was confirmed that the proportion of apoptotic cells increased.

On the other hand, in the SH-SY5Y cells cultured with the addition of the autophagy activator and the amyloid β solution of Examples 11 to 16, compared to the SH-SY5Y cells cultured with the addition of purified water and the amyloid β solution of Comparative Example 3, apoptosis It was confirmed that the percentage of cells decreased.

[Prescription example]

As compositions for activating autophagy, Formulation Examples 1 to 4 for external use are shown in Table 4.

According to the present invention, an autophagy activator capable of effectively activating autophagy and a composition for activating autophagy containing the autophagy activator are provided.

In the above, preferred embodiments of the present invention have been described, but the present invention is not limited to these embodiments. Addition, omission, substitution, and other changes to the configuration are possible without departing from the spirit of the present invention. The present invention is not limited by the foregoing description, but only by the scope of the appended claims.

Claims (10)

An autophagy activator comprising an ascorbic acid derivative or a salt thereof as an active ingredient. The method according to claim 1, wherein the ascorbic acid derivative or salt thereof is at least one ascorbic acid derivative selected from the group consisting of ascorbyl phosphate, fatty acid ester of ascorbyl phosphate, ethyl ascorbic acid and ascorbic acid glucoside; Its salt, an autophagy activator. The autophagy activator according to claim 2, wherein the ascorbic acid derivative or salt thereof is a fatty acid ester of ascorbyl phosphate or a salt thereof. The autophagy activator according to claim 3, wherein the fatty acid ester of ascorbyl phosphate or a salt thereof is a palmitate ester of ascorbyl phosphate or a salt thereof. The autophagy activator according to any one of claims 1 to 4, which contains a salt of the ascorbic acid derivative as an active ingredient, and the salt of the ascorbic acid derivative is a magnesium salt or a sodium salt of the ascorbic acid derivative. . The autophagy activator according to any one of claims 1 to 5, which promotes expression of the LC3 gene. The autophagy activator according to any one of claims 1 to 6, which promotes expression of the LC3 gene, the ATG5 gene, the ATG7 gene and the Beclin 1 gene. The autophagy activator according to any one of claims 1 to 7, which is used for preventing or treating Alzheimer's disease. A composition for activating autophagy, comprising the autophagy activator according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. The composition for activating autophagy according to claim 9, wherein the total content of the ascorbic acid derivative or salt thereof is 0.05 to 15% by mass based on the total amount of the composition for activating autophagy.