JP2021066678A - Autophagy activator - Google Patents

Abstract

To provide an autophagy activator useful as an agent for the prevention or treatment of neurodegenerative disease and fatty liver.SOLUTION: An autophagy activator contains isopropylmethylphenol as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to an autophagy activator.

Autophagy is a system that decomposes organelles and proteins in cells (Non-Patent Documents 1-3). The degradation pathways are classified into three categories: "macro autophagy", "micro autophagy", and "chaperone-mediated autophagy". The decomposition path called autophagy generally refers to "macro autophagy."

Since autophagy works constantly in the living body and is responsible for intracellular clearance, it is expected that its functional decline will cause various diseases. In fact, it has been found that mice lacking autophagy-related genes in a tissue-specific manner lead to accumulation of abnormal proteins and diseases (Non-Patent Documents 4 and 5).

Neurodegenerative diseases and liver diseases are typical diseases caused by autophagy dysfunction. Many neurodegenerative diseases are characterized by the accumulation of abnormal proteins, and the relationship between the major neurodegenerative diseases and the accumulated proteins is, for example, Parkinson's disease and body dementia and α-synuclein, Huntington's disease and Huntington's disease, and Alzheimer's disease. And amyloid β, muscle atrophic lateral sclerosis and TDP-43 and the like. Considering that autophagy is a system that decomposes unnecessary substances in cells, it is understood that the activity of autophagy is important in neurodegenerative diseases, and by actually enhancing the activity of autophagy in model animals, There are also examples of improving neurodegenerative diseases (Non-Patent Documents 6 and 7). In addition, fatty liver, which is one of the major liver diseases, is also caused by the accumulation of Rubicon, a protein that suppresses autophagy, and it is considered important to enhance the activity of autophagy (non-). Patent Document 8).
Therefore, the autophagy activator is considered to be useful as a therapeutic agent for neurodegenerative diseases and a therapeutic agent for fatty liver.

On the other hand, isopropylmethylphenol has bactericidal activity and antibacterial activity, and acts on bacteria that easily grow on sebum, so that it has been conventionally blended as a bactericidal agent in cosmetics, pharmaceuticals, and the like.
However, it is not known at all that isopropylmethylphenol has an autophagy-activating effect.

Japanese Patent No. 6075891

Levine and Kriemer (2008) Cell 132: 27-42 Mizushima et al (2008) Nature 451: 1069-1975 Rubinsztein (2006) Nature 443: 780-786 Komatsu et al (2007) Cell 131.6: 1149-1163 Hara et al (2006) Nature 441.7095: 885 Ravikumar (2004) Nature genetics 36.6: 585 Ehrnhoefer (2018) Acta neuropathologica communications 6.1:16 Tanaka (2016) Hepatology 64.6: 1994-2014

The present invention relates to providing an autophagy activator useful as a prophylactic or therapeutic agent for neurodegenerative diseases and fatty liver.

As a result of studies in view of the above problems, the present inventors unexpectedly have an autophagy-activating effect on isopropylmethylphenol contained in pharmaceuticals and the like as a bactericidal agent, which is associated with neurodegenerative diseases and fatty liver. It has been found that it can be used for prevention or treatment.

That is, the present invention relates to the following 1) to 3).
1) An autophagy activator containing isopropylmethylphenol as an active ingredient.
2) A prophylactic or therapeutic agent for neurodegenerative diseases containing isopropylmethylphenol as an active ingredient. 3) A prophylactic or therapeutic agent for fatty liver containing isopropylmethylphenol as an active ingredient.

According to the present invention, it is possible to provide a highly safe autophagy activator, a prophylactic or therapeutic agent for neurodegenerative diseases, and a prophylactic or therapeutic agent for fatty liver.

Evaluation of autophagy activity of isopropylmethylphenol by Cyto-ID. Evaluation of autophagy activity of isopropylmethylphenol by Western blotting of LC3-II.

The isopropylmethylphenol used in the present invention is a phenol having an isopropyl group and a methyl group as substituents, and is not particularly limited as long as it can be used for cosmetics, pharmaceuticals, non-pharmaceutical products and the like. .. Specifically, 4-isopropyl-3-methylphenol (also known as p-thymol), 2-isopropyl-5-methylphenol (also known as m-thymol), 3-isopropyl-6-methylphenol (also known as carvacrol). ), Etc., and 4-isopropyl-3-methylphenol is preferably used in the present invention. Such isopropylmethylphenol may be chemically synthesized by a known method (for example, DE 102007035515), or a commercially available product may be used.

As shown in Examples below, in human keratinocytes supplemented with isopropylmethylphenol, activation of autophagy was confirmed by cell imaging using Cyto-ID (registered trademark), and it was localized on the autophagosome membrane. , The amount of LC3-II (Mizushima N, Yoshimori T (2007). How to interpret LC3 immunoblotting. Autophagy 3: 542-545.) Used as a marker of autophagy activity increases with the addition of lysosome inhibitors. Therefore, it is recognized that isopropylmethylphenol has an autophagy-activating effect.

As already mentioned, the activity of autophagy, which is a system that decomposes unnecessary substances in cells, is considered to be important in neurodegenerative diseases, many of which are characterized by the accumulation of abnormal proteins. It is possible to improve neurodegenerative diseases by actually activating autophagy. For example, when an animal model of Huntington's disease, which is a neurodegenerative disease, is administered with rapamycin that enhances autophagy activity, the motor ability characteristic of neurodegenerative diseases It has been reported that the decrease in protein aggregates is improved, the formation of protein aggregates is reduced, and the accumulation of protein aggregates in the brain is also reduced in the activation of autophagy by inducing starvation (Non-Patent Document 6). , 7). In addition, fatty liver is caused by the accumulation of Rubicon, which is a protein that suppresses autophagy, and it is considered that activation of autophagy leads to its prevention and treatment (Non-Patent Document 8).

Therefore, isopropylmethylphenol becomes an autophagy activator, a prophylactic or therapeutic agent for neurodegenerative diseases, or a prophylactic or therapeutic agent for fatty liver (hereinafter, these are also referred to as "autophagy activators and the like"), and these Can be used to manufacture.

In addition, isopropylmethylphenol can be used for autophagy activation, prevention or treatment of neurodegenerative diseases, and prevention or improvement of fatty liver. Here, the use may be administration to a human or non-human animal, or use in a sample derived from them, and may be therapeutic or non-therapeutic use. The term "non-therapeutic" means a concept that does not include medical practice, that is, a concept that does not include a method of surgery, treatment, or diagnosis of a human being, more specifically, a doctor or a person who has been instructed by a doctor to treat a human being. It is a concept that does not include a method of performing surgery, treatment or diagnosis.

In the present invention, "autophagy" means a system (macroautophagy) in which organelles and proteins are degraded in cells via autophagosome formation, and its activation is defined in the autophagy pathway. It includes promoting the activity of any step such as fagophore development, fagophore elongation or growth, autophagosome formation and the like.
In addition to measuring the amount of LC3-II accumulated, the activity of autophagy can also be measured and evaluated by a flux assay that measures the turnover of such proteins with or without the addition of a lysosomal inhibitor. ..

In the present invention, "neurodegenerative disease" means that a specific group of nerve cells in the central nervous system (for example, nerve cells involved in cognitive function and motor function) gradually declines and disappears due to accumulation of abnormal proteins. It means a neurological disease that goes on, for example, Parkinson's disease, muscular atrophic lateral sclerosis, Alzheimer's dementia, Huntington's disease, polyglutamine's disease, prion's disease tauopathy, sineculinopathies, Lewy body dementias, etc. Be done.
Of these, preferred examples of neurodegenerative diseases include Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, and Huntington's disease.

In the present invention, "fatty liver" means that a larger amount of fat is accumulated in the liver than the proportion of fat in the normal liver (5%). In addition to alcoholic fatty liver, fatty liver includes non-alcoholic fatty liver caused by obesity, diabetes, dyslipidemia, drugs, etc. In the present invention, any of them may be used, but non-alcoholic fatty liver is preferable. Sexual fatty liver can be mentioned.

Further, in the present invention, "prevention" means preventing, suppressing or delaying the onset of a disease, symptom or condition in an individual, or reducing the risk of developing a disease, symptom or condition in an individual. In addition, "treatment" includes improvement of disease, symptom or condition, prevention, suppression or delay of deterioration of disease, symptom or condition, or reversal, prevention, suppression or delay of progression of disease, symptom or condition. ..

The autophagy activator and the like of the present invention are pharmaceuticals and non-pharmaceutical products for humans or animals that exert the effects of activation of autophagy, prevention or treatment of neurodegenerative diseases, prevention or treatment of fatty liver. In addition, it can be a material or preparation used by blending with the drug or non-pharmaceutical product.

When the autophagy activator or the like of the present invention is used as a drug or a quasi drug, the drug can be administered in any administration form. Examples of the administration form include oral administration with tablets, capsules, granules, powders, syrups and the like, or parenteral administration with injections, suppositories, inhalants, transdermal absorbents, external preparations and the like. The form is oral administration.
Such various dosage forms of pharmaceutical preparations contain the isopropylmethylphenol of the present invention alone or other pharmaceutically acceptable excipients, binders, bulking agents, disintegrants, surfactants, and lubricants. It can be prepared by appropriately combining agents, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents and the like.

When the autophagy activator or the like of the present invention is used as a drug or a quasi-drug, the content of isopropylmethylphenol in the preparation is usually 0.01 to 5% by weight, preferably 0.01 to 1. By weight%.

In addition, the dose of the above-mentioned drugs and quasi-drugs may vary depending on the condition, weight, sex, age or other factors of the subject, but in the case of oral administration or ingestion, as isopropylmethylphenol per adult. The daily dose is preferably 0.0001 mg to 100 mg / kg, more preferably 0.0001 to 50 mg, and further preferably 0.0001 mg to 25 mg / kg.

Regarding the above-described embodiment, the following aspects are disclosed in the present invention.
<1> An autophagy activator containing isopropylmethylphenol as an active ingredient.
<2> A prophylactic or therapeutic agent for neurodegenerative diseases containing isopropylmethylphenol as an active ingredient.
<3> A prophylactic or therapeutic agent for fatty liver containing isopropylmethylphenol as an active ingredient.
<4> Use of isopropylmethylphenol for producing an autophagy activator.
<5> Use of isopropylmethylphenol for producing a prophylactic or therapeutic agent for neurodegenerative diseases.
<6> Use of isopropylmethylphenol for producing a prophylactic or therapeutic agent for fatty liver.
<7> Isopropylmethylphenol for use in autophagy activation.
<8> Isopropylmethylphenol for use in the prevention or treatment of neurodegenerative diseases. <9> Isopropylmethylphenol for use in the prevention or treatment of fatty liver.
<10> A method for activating autophagy, in which isopropylmethylphenol is administered to a subject who needs it.
<11> A method for preventing or treating a neurodegenerative disease in which isopropylmethylphenol is administered to a subject who needs it.
<12> A method for preventing or treating fatty liver in which isopropylmethylphenol is administered to a subject who needs it.
<13> In the above <2>, <5>, <8>, and <11>, the neurodegenerative disease is a neurodegenerative disease caused by the accumulation of abnormal proteins, preferably Parkinson's disease and amyotrophic lateral lateral side. It is selected from sclerosis, Alzheimer's disease, and Huntington's disease.
<14> In the above <3>, <6>, <9>, and <12>, fatty liver is preferably non-alcoholic fatty liver.
<15> In the above <1> to <14>, the isopropyl methylphenol is preferably 4-isopropyl-3-methylphenol.

<Preparation of test substance>
4-Isopropyl-3-methylphenol (Wako Pure Chemical Industries, Ltd.) was used as isopropylmethylphenol and dissolved in dimethyl sulfoxide (DMSO) to 10 mM.

<Cell culture>
Human cultured epidermal cells (Human Epidermal Keratinocyte) were purchased from Kurabou and derived from neonatal foreskin. For culturing, a medium in which a growth additive kit (Humedia-KG; Kurabo Industries) was added to EpiLife Medium, with 60 μM Calcium (hereinafter referred to as EpiLife; Life Technology) was used, and the cells were cultured under the conditions of _{37 ° C. and 5% CO 2.}

Example 1; Evaluation of autophagy activation ability using CYTO-ID Keratinocytes were ^{seeded on 96-well plates at a cell density of 2 × 10 4} cells / 100 μl / well. After culturing for 24 hours, 10 μM of Chloroquine (Enzo Life Sciences), which is a lysosome inhibitor, was added, and then isopropylmethylphenol was added to 1 μM and 10 μM media. 20 hours after sample addition, Cyto-ID manufactured by Enzo Life Sciences. The autophagy activity was measured by measuring the fluorescence intensity (fluorescence wavelength: 480 nm, excitation wavelength: 530 nm) with a fluorescent plate reader using an autophagy detection dye (registered trademark). The results are shown in FIG. The autophagy activity was expressed as a relative value when the autophagy activity value of the control was 1.
From FIG. 1, it was shown that isopropylmethylphenol activates autophagy.

Example 2; Evaluation of autophagy activation ability by Western blotting Keratinocytes were ^{seeded on a 6-well plate at 1.5 × 10 5} cells / 2 ml / well. After pre-culture, the isopropylmethylphenol is replaced with a medium added to the final concentrations of 10 μM, 50 μM, and 100 μM, and after 6 hours, the lysosome inhibitor hydroxychloroquine (HCQ; SIGMA) has a final concentration of 10 μM. Was added directly to the medium. After 24 hours, the cells were washed with PBS and cells were collected with RIPA Buffer (SIGMA) supplemented with a protease inhibitor (Protease / Phosphatase Inhibitor cocktile, 100X; Cell Signaling Technology). As the medium, EpiLife to which bovine pituitary extract (BPE; Bovine Pituitary Extract) and human epidermal growth factor (hEGF; Human Epidermal Growth Factor) other than Human Epidermal Growth Factor (BPE) was added was used. After cell recovery, protein quantification was performed using BCA Protein Assay Kit (Thermo Fisher Scientific) to keep the protein concentration constant. Then, the expression of LC3-II was detected by SDS-PAGE and Western blotting (according to the method described in Patent Document 1). The autophagy activity was evaluated from the accumulated amount of LC3-II when HCQ, which is a lysosome inhibitor, was added and the formation of autolysosomes was inhibited. In addition. LC3 antibody (Anti-LC3B antibody producted in rabbit; SIGMA) and β-actin antibody (Beta-Actin Rabbit mAb; Cell Signaling Technology) as the primary antibody, and Cell Signaling Technology (Cell Signaling Technology) as the secondary antibody. Rabbit IgG, HRP-Linked F (ab') 2 antibody (from monoclonal); GE Healthcare) was used. The LC3 antibody was diluted 1,000-fold, the β-actin antibody was diluted 5,000-fold, and the anti-rabbit antibody was diluted 5,000-fold. For detection, SuperSignal West Pico PLUS Chemiluminescence Substrate (Themo Scientific) was used. The results are shown in FIG.
From FIG. 2, since the amount of LC3-II accumulated when HCQ was added increased in a concentration-dependent manner with the addition of isopropylmethylphenol (50 μM, 100 μM), it is considered that isopropylmethylphenol has an autophagy activating effect. Be done.

Claims (6)

An autophagy activator containing isopropylmethylphenol as an active ingredient. A prophylactic or therapeutic agent for neurodegenerative diseases containing isopropylmethylphenol as an active ingredient. The prophylactic or therapeutic agent according to claim 2, wherein the neurodegenerative disease is any one selected from Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, and Huntington's disease. A prophylactic or therapeutic agent for fatty liver containing isopropylmethylphenol as an active ingredient. The prophylactic or therapeutic agent according to claim 4, wherein the fatty liver is a non-alcoholic fatty liver. The autophagy activator according to claim 1, the prophylactic or therapeutic agent for a neurodegenerative disease according to claim 2 or 3, or the agent according to claim 4 or 5, wherein the isopropylmethylphenol is 4-isopropyl-3-methylphenol. A prophylactic or therapeutic agent for fatty liver.

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