JP7360297B2 - autophagy activator - Google Patents

Description

The present invention relates to an autophagy activator.

Autophagy is a system that degrades intracellular organelles and proteins within cells (Non-Patent Documents 1-3). Its degradation pathways are classified into three types: "macroautophagy," "microautophagy," and "chaperone-mediated autophagy." The degradative pathway called autophagy generally refers to "macro autophagy."

Since autophagy functions constantly in the body and is responsible for intracellular clearance, it is expected that its decreased function will be the cause of various diseases. In fact, it has been found that mice with tissue-specific deletion of autophagy-related genes lead to accumulation of abnormal proteins and diseases (Non-Patent Documents 4, 5).

Representative diseases caused by dysfunction of autophagy include neurodegenerative diseases and liver diseases. Most neurodegenerative diseases are characterized by the accumulation of abnormal proteins, and the relationships between major neurodegenerative diseases and their accumulated proteins include, for example, Parkinson's disease, dementia with body mass and α-synuclein, Huntington's disease and huntingtin, and Alzheimer's disease. and amyloid β, amyotrophic lateral sclerosis, and TDP-43. Considering that autophagy is a system that degrades unnecessary substances in cells, it is understood that autophagy activity is important in neurodegenerative diseases, and in fact, by increasing autophagy activity in model animals, There are also examples of amelioration of neurodegenerative diseases (Non-patent Documents 6 and 7). Furthermore, fatty liver, which is one of the main liver diseases, is thought to be caused by the accumulation of Rubicon, a protein that suppresses autophagy, and it is thought to be important to enhance autophagy activity (non- Patent Document 8).
Therefore, autophagy activators are considered to be useful as therapeutic agents for neurodegenerative diseases and fatty liver.

On the other hand, isopropyl methylphenol has bactericidal and antibacterial properties and acts on bacteria that tend to grow in sebum, so it has traditionally been added to cosmetics, pharmaceuticals, etc. as a bactericidal agent.
However, it is completely unknown that isopropylmethylphenol has an autophagy activating effect.

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 preventive or therapeutic agent for neurodegenerative diseases and fatty liver.

In view of the above issues, the present inventors conducted studies and found that isopropyl methylphenol, which is incorporated into pharmaceuticals as a bactericidal agent, has an autophagy-activating effect. 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) Autophagy activator containing isopropylmethylphenol as an active ingredient.
2) A preventive 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, highly safe autophagy activators, preventive or therapeutic agents for neurodegenerative diseases, and preventive or therapeutic agents for fatty liver can be provided.

Evaluation of autophagic 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 in cosmetics, pharmaceuticals, quasi-drugs, etc. . 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) ), 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 the Examples below, activation of autophagy was confirmed by cell imaging using Cyto-ID (registered trademark) in human keratinocytes to which isopropylmethylphenol was added, and autophagy 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.), which is used as a marker for autophagy activity, increases when a lysosome inhibitor is added. Therefore, isopropylmethylphenol is recognized to have an autophagy activating effect.

As mentioned above, the activity of autophagy, a system that degrades unnecessary substances within cells, is thought to be important in neurodegenerative diseases, many of which are characterized by the accumulation of abnormal proteins. In fact, activating autophagy can improve neurodegenerative diseases. For example, administering rapamycin, which enhances autophagy activity, to an animal model of Huntington's disease, which is a neurodegenerative disease, can improve the motor performance characteristic of neurodegenerative diseases. It has been reported that the decline in protein aggregates is improved and the formation of protein aggregates is reduced, and that activation of autophagy by induction of starvation also reduces the accumulation of protein aggregates in the brain (non-patent document 6). , 7). Furthermore, fatty liver is said to be caused by the accumulation of Rubicon, a protein that suppresses autophagy, and activation of autophagy is thought to lead to its prevention and treatment (Non-Patent Document 8).

Therefore, isopropyl methylphenol can be used as an autophagy activator, a preventive or therapeutic agent for neurodegenerative diseases, or a preventive or therapeutic agent for fatty liver (hereinafter also referred to as "autophagy activator, etc."). can be used to manufacture

Furthermore, isopropylmethylphenol can be used to activate autophagy, to prevent or treat neurodegenerative diseases, and to prevent or improve fatty liver. Here, the use may be administration to humans or non-human animals, or use in specimens derived therefrom, and may be therapeutic or non-therapeutic. "Non-therapeutic" is a concept that does not include medical treatment, that is, a method that does not involve surgery, treatment, or diagnosis of humans. This concept does not include methods of performing surgery, treatment, or diagnosis.

In the present invention, "autophagy" refers to a system (macroautophagy) in which intracellular organelles and proteins are degraded through autophagosome formation, and its activation refers to the autophagy pathway. Examples include promoting the activity of any step such as phagophore generation, phagophore elongation or growth, and autophagosome formation.
In addition to measuring the accumulated amount of LC3-II, autophagy activity can also be measured and evaluated by a flux assay that measures the turnover of this protein with or without the addition of a lysosome inhibitor. .

In the present invention, "neurodegenerative disease" refers to a gradual decline and disappearance of specific nerve cell groups in the central nervous system (e.g., nerve cells involved in cognitive and motor functions) due to the accumulation of abnormal proteins. Examples include Parkinson's disease, amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease, polyglutamine disease, prion disease tauopathy, synucleinopathy, and Lewy body dementia. It will be done.
Among these, preferred 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 has been accumulated in the liver than the proportion (5%) of fat in a normal liver. Fatty liver includes alcoholic fatty liver as well as non-alcoholic fatty liver caused by obesity, diabetes, dyslipidemia, drugs, etc. In the present invention, any of these may be used, but non-alcoholic fatty liver is preferred. One example is fatty liver.

Furthermore, in the present invention, "prevention" refers to preventing, suppressing, or delaying the onset of a disease, symptom, or condition in an individual, or reducing the risk of onset of a disease, symptom, or condition in an individual. Furthermore, "treatment" includes improving the disease, symptom or condition, preventing, suppressing or delaying the worsening of the disease, symptom or condition, or reversing, preventing, suppressing or delaying the progression of the disease, symptom or condition. .

The autophagy activator of the present invention is a pharmaceutical product or quasi-drug for humans or animals that exhibits the effects of activating autophagy, preventing or treating neurodegenerative diseases, and preventing or treating fatty liver. It can also be used as a material or preparation used in combination with the drug or quasi-drug.

When using the autophagy activator of the present invention as a drug or quasi-drug, the drug can be administered in any dosage form. Examples of dosage forms include oral administration in the form of tablets, capsules, granules, powders, syrups, etc., and parenteral administration in the form of injections, suppositories, inhalants, transdermal absorption agents, external preparations, etc., but preferred are The form is for oral administration.
Pharmaceutical preparations in various dosage forms may contain the isopropyl methylphenol of the present invention alone or in combination with other pharmaceutically acceptable excipients, binders, fillers, disintegrants, surfactants, and lubricants. It can be prepared by appropriately combining agents, dispersants, buffers, preservatives, flavoring agents, fragrances, coating agents, carriers, diluents, etc.

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

In addition, the dosage of the above-mentioned drugs and quasi-drugs, etc. may vary depending on the subject's condition, weight, sex, age, or other factors, but in the case of oral administration or ingestion, the dose per adult is: The amount per day is preferably 0.0001 mg to 100 mg/kg, more preferably 0.0001 to 50 mg, and even more preferably 0.0001 mg to 25 mg/kg.

Regarding the embodiments described above, the following aspects are disclosed in the present invention.
<1> Autophagy activator containing isopropylmethylphenol as an active ingredient.
<2> A preventive 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 preventive or therapeutic agent for neurodegenerative diseases.
<6> Use of isopropylmethylphenol for producing a preventive 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 preventing or treating fatty liver.
<10> A method for activating autophagy in which isopropylmethylphenol is administered to a subject in need thereof.
<11> A method for preventing or treating a neurodegenerative disease, comprising administering isopropylmethylphenol to a subject in need thereof.
<12> A method for preventing or treating fatty liver, which comprises administering isopropylmethylphenol to a subject in need thereof.
<13> In the above <2>, <5>, <8>, and <11>, the neurodegenerative disease is a neurodegenerative disease caused by accumulation of abnormal proteins, preferably Parkinson's disease or amyotrophic disease. The disease is one selected from neurosclerosis, Alzheimer's disease, and Huntington's disease.
<14> In the above <3>, <6>, <9>, and <12>, the fatty liver is preferably non-alcoholic fatty liver.
<15> In the above <1> to <14>, the isopropylmethylphenol 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 a concentration of 10mM.

<Cell culture>
Cultured human epidermal cells (Human Epidermal Keratinocytes) were purchased from Kurabo Co., Ltd., and those derived from newborn foreskin were used. For culturing, a medium containing EpiLife Medium with 60 μM Calcium (hereinafter referred to as EpiLife; Life Technologies) and a growth additive kit (Humedia-KG; Kurabo Industries) was used, and the cells were cultured at 37° C. and 5% CO ₂ .

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

Example 2: Evaluation of autophagy activation ability by Western blotting Keratinocytes were seeded in a 6-well plate at 1.5×10 ⁵ cells/2 ml/well. After preculture, the medium was replaced with a medium containing isopropyl methylphenol at a final concentration of 10 μM, 50 μM, and 100 μM, and after 6 hours, the lysosome inhibitor hydroxychloroquine (HCQ; SIGMA) was added at a final concentration of 10 μM. directly added to the culture medium. After 24 hours, the cells were washed with PBS and collected in RIPA Buffer (SIGMA) supplemented with protease inhibitor (Protease/Phosphatase Inhibitor cocktail, 100X; Cell Signaling Technology). The medium used was EpiLife from Humedia-KG, which was supplemented with ingredients other than Bovine Pituitary Extract (BPE) and Human Epidermal Growth Factor (hEGF). After cell collection, protein quantification was performed using a BCA Protein Assay Kit (Thermo Fisher Scientific) to maintain a constant protein concentration. Thereafter, the expression of LC3-II was detected by SDS-PAGE and Western blotting (according to the method described in Patent Document 1). Autophagy activity was evaluated from the amount of LC3-II accumulated when autolysosome formation was inhibited by adding HCQ, a lysosome inhibitor. In addition. LC3 antibody (Anti-LC3B antibody produced in rabbit; SIGMA) and β-actin antibody (Beta-Actin Rabbit mAb; Cell Signaling Technology) were used as primary antibodies, H RP-labeled anti-rabbit antibody (A'mersham ECL Rabbit IgG, HRP-Linked F(ab')2 fragment (from donkey; GE Healthcare) was used. The LC3 antibody was diluted 1,000 times, the β-actin antibody was diluted 5,000 times, and the anti-rabbit antibody was diluted 5,000 times. For detection, SuperSignal West Pico PLUS Chemiluminescent Substrate (Themo Scientific) was used. The results are shown in Figure 2.
From Figure 2, the amount of LC3-II accumulated upon addition of HCQ increases in a concentration-dependent manner with the addition of isopropylmethylphenol (50μM, 100μM), suggesting that isopropylmethylphenol has an autophagy-activating effect. It will be done.

Claims (5)

An autophagy activator containing 4-isopropyl-3-methylphenol as an active ingredient. A prophylactic or therapeutic agent for neurodegenerative diseases containing 4-isopropyl-3-methylphenol as an active ingredient. The preventive or therapeutic agent according to claim 2, wherein the neurodegenerative disease is 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 4-isopropyl-3-methylphenol as an active ingredient. The prophylactic or therapeutic agent according to claim 4, wherein the fatty liver is non-alcoholic fatty liver.