JP7156907B2 - autophagy inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to an autophagy inhibitor that suppresses autophagy activity in keratinocytes.

Various factors have been reported as factors that determine the color of skin and hair. Among them, the quantity and quality of melanin present in the epidermis are believed to greatly contribute to the color of skin and hair. In other words, the formation of skin and hair color occurs when melanin produced in organelles called melanosomes in melanin-producing cells (melanocytes) is transferred to keratinocytes in the epidermis and hair follicles, and spread throughout the epidermis and hair. It is said that it is greatly affected by the spread.
Therefore, if the amount of melanin in keratinocytes increases due to the enhancement of melanin production, the suppression of melanin degradation, or the like, the skin turns brown and the hair turns black.

In addition, the present applicant recently found that autophagy is involved in melanin dynamics in keratinocytes, and that the amount of melanin in keratinocytes correlates with autophagy activity (Patent Document 1). According to this, it is thought that by suppressing the activity of autophagy, the decomposition of melanin in keratinocytes is suppressed, making it possible to darken the skin and hair color.

In the skin, one of the causes of dark spots and freckles is known to be that the melanocytes present in the skin are activated and melanin production becomes active due to stimulation due to UV exposure of the skin, hormonal abnormalities, genetic factors, etc. Therefore, conventionally, whitening agents have been developed that suppress melanin production or reduce melanin produced. However, on the other hand, in Europe and the United States, many people desire brown skin. Artificial tanning by bed is also popular. In addition, due to efforts to raise awareness of the harmfulness of ultraviolet rays, self-tanning agents containing dihydroxyacetone as a main component are currently on the market.

Excessive exposure to such ultraviolet rays causes great damage to the skin and may lead to the occurrence of skin cancer. Self-tanning agents also cause the skin to turn brown due to the Maillard reaction of the stratum corneum. However, problems have been pointed out in terms of effects, such as improvement in shade, stability of shade, and UV protection.

In addition, gray hair is one of the physiological aging phenomena in which melanin is reduced due to changes in melanin-producing cells in the hair matrix, but the mechanism of its development has not yet been elucidated. As a method for changing white hair to black hair, many reports have been made on ingredients that prevent or improve gray hair, but none of them have been obtained sufficient in terms of efficacy and safety. At present, hair dyeing with hair dyes is the main focus.

Therefore, if a component that directly acts on keratinocytes to increase or accumulate the amount of melanin is found, it would promote the biological defense ability originally possessed by melanin to prevent damage to the skin, as well as prevent or improve browning of the skin and gray hair. can be realized.

Cholesterol, on the other hand, is essential for mammalian cells and is a major constituent of cell membranes. Cholesterol is believed to play an important role in cell signaling, membrane fluidity, lipid sorting, membrane traffic, membrane permeability and the like. In addition, impairments in cholesterol synthesis and catabolism lead to numerous pathological conditions such as atherosclerosis, angina pectoris, cardiovascular disorders, metabolic syndrome, diabetes, and Smith-Lemli-Opitz syndrome. It is known.

However, it is completely unknown that cholesterol production is related to autophagy activity and melanin accumulation in keratinocytes.

WO2013/162012

Moebius FF, Fitzky BU, Lee JN, Paik YK, Glossmann H (1998) Molecular cloning and expression of the human Δ7-sterol reductase. Proc. Natl. Acad.

The present invention relates to providing a skin or hair darkening agent that inhibits autophagy activity in keratinocytes and enhances melanin accumulation in skin or hair.

The present inventors have searched for a substance that increases the amount of melanin in skin or hair, and found that by suppressing the production of cholesterol in keratinocytes, the activity of autophagy in keratinocytes is suppressed, and the decomposition of melanin in these cells is suppressed. It has been found that darkening of the skin or hair color is induced by the treatment.
That is, the present invention relates to the following 1) to 6).
1) trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl] ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase An autophagy inhibitor in keratinocytes containing, as an active ingredient, a cholesterol production inhibitor selected from inhibitory siRNAs.
2) trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl] ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase A melanin degradation inhibitor in keratinocytes containing, as an active ingredient, a cholesterol production inhibitor selected from inhibitory siRNAs.
3) trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl] ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase A skin or hair darkening agent containing, as an active ingredient, a cholesterol production inhibitor selected from inhibitory siRNAs.
4) trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl] ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase An agent for preventing or improving gray hair containing, as an active ingredient, a cholesterol production inhibitor selected from inhibitory siRNAs.
5) trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl] ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase An external preparation for skin containing a cholesterol production inhibitor selected from inhibitory siRNAs.
6) A method for evaluating or searching for an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, a skin or hair darkening agent, or an agent for preventing or improving gray hair, comprising the following steps (a) to (c).
(a) Step of contacting keratinocytes with a test substance and culturing (b) Step of measuring at least one of the following indicators (i) to (iv) in keratinocytes
(i) total cholesterol
(ii) activity of cholesterol synthase
(iii) protein content of cholesterol synthase
(iv) Expression level of cholesterol synthase gene (c) A test substance that reduces any of the above indicators (i) to (iv) is an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, skin or hair color Step of selecting or evaluating as a blackening agent, or a gray hair preventing or improving agent

According to the present invention, it is possible to suppress the decomposition of melanin in the skin or hair, and to prevent or improve the browning of the skin and gray hair.

Skin darkening effect of AY9944. (A) Dose-dependent skin darkening effect of AY9944, (B) Skin darkening effect of AY9944 (100 μM). Effect of AY9944 on keratinocyte autophagy activity. Effect of AY9944 on keratinocyte cholesterol synthesis. (A) Effect of AY9944 on keratinocyte protein content, (B) Effect of AY9944 on keratinocyte intracellular total cholesterol content, (C) AY9944 effect on keratinocyte intracellular total cholesterol content (after correction for protein content) )

In the present invention, a cholesterol production inhibitor means a substance that inhibits cholesterol production by controlling the cholesterol biosynthetic system, and specifically includes cholesterol synthase inhibitors. Inhibition of cholesterol synthase includes inhibiting the activity of any enzyme in the cholesterol biosynthesis system, reducing the protein amount of the enzyme, and reducing the expression of the gene encoding the enzyme.
Here, the "substance" is not particularly limited in its type, and may be a natural product or a synthetic product, and may be a single substance, a composition, or a mixture.

Cholesterol synthetic enzymes include 7-dehydrocholesterol reductase (Δ7-dehydrocholesterol reductase; DHCR7), hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase), mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenylpyrroline acid isomerase, dimethylanyl-trans-transferase, geranyl-trans-transferase, farnesyl-PP farnesyltransferase, squalene monooxygenase, lanosterol synthase, methyl hydroxylase, zymosterol dehydrogenase, desmosterol reductase and the like, preferably 7-dehydrogenase Cholesterol reductase or hydroxymethylglutaryl-CoA reductase, more preferably 7-dehydrocholesterol reductase.

Examples of cholesterol production inhibitors include substances that inhibit any of the cholesterol synthase. For example, the DHCR7 inhibitor trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]- 1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors mevastatin, lovastatin, pravastatin, etc., 7-dehydrocholesterol reductase or HMG-CoA reductase and siRNA that suppresses the expression of

The cholesterol production inhibitor is preferably trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof. The compound means trans-N1,N4-bis[(2-chlorophenyl)methyl]-1,4-cyclohexanedimethanamine represented by the following formula.

Salts of trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane include inorganic acid salts such as hydrochlorides, sulfates, nitrates, phosphates, carbonates, hydrogencarbonates and perchlorates. organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, and ascorbate; Of these, the hydrochloride is preferred, and the dihydrochloride is more preferred. Trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride, designated AY9944, is known as a selective and potent inhibitor of 7-dehydrocholesterol reductase.
Trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane is available as AY9944 from general reagent manufacturers such as Abcam.

As shown in Examples below, when human skin tissue is cultured in a medium supplemented with AY9944, darkening of the skin color is induced in a dose-dependent manner. AY9944 also enhances LC3-II accumulation induced in the presence of the autophagy inhibitor chloroquine diphosphate. This is considered to be due to the fact that AY9944 together with chloroquine inhibits the fusion process of autophagosomes and lysosomes, resulting in accumulation of autophagosomes without degradation. In other words, the darkening of the skin color due to substances that inhibit cholesterol production is thought to be due to the accumulation and increase in the amount of melanin due to suppression of autophagy activity in keratinocytes and suppression of melanin degradation in keratinocytes.

On the other hand, when AY9944 was added to keratinocytes and cultured, it was confirmed that the intracellular total cholesterol level decreased.

Therefore, by administering a cholesterol production inhibitor to animals, preferably humans, it is possible to suppress autophagy in keratinocytes, suppress melanin degradation, prevent or improve skin or hair darkening, and gray hair.

That is, the cholesterol production inhibitor is an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, a skin or hair darkening agent, and an agent for preventing or improving gray hair. It can be used for control, for skin or hair darkening, and for prevention or amelioration of gray hair.

Here, the term "suppression of autophagy in keratinocytes" means suppression of autophagy activity in keratinocytes, particularly by suppressing the activity of the steps of phagophore generation, phagophore elongation or growth in the autophagy pathway. Means to increase the amount of melanin.
Autophagy activity can also be measured and evaluated by a flux assay that measures the turnover of such proteins with or without the addition of lysosomal inhibitors, in addition to the amount of accumulated LC3-II and p62 proteins. can.

In addition, “darkening of skin or hair” means darkening of the color of skin or hair having melanin-producing ability by increasing the amount of melanin.

As used herein, the use of cholesterol production-inhibiting substances for humans may be therapeutic use or non-therapeutic use including cosmetic use.
"Non-therapeutic" means not including medical procedures, i.e., methods of surgery, treatment or diagnosis of humans, and more specifically, medical practitioners or persons under the direction of a physician It is a concept that does not include a method of performing surgery, therapy or diagnosis on a patient. Non-therapeutic uses of cholesterol synthase inhibitors include use for darkening skin or hair color for cosmetic or aesthetic purposes, such as use by estheticians, beauticians, and the like.
As used herein, the term "improvement" refers to amelioration of symptoms or conditions, prevention or delay of worsening of symptoms or conditions, or reversal, prevention or delay of progression of symptoms or conditions.
As used herein, "prevention" means preventing or delaying the onset of symptoms in an individual, or reducing the risk of developing symptoms in an individual.

In addition, cholesterol production inhibitors can be used to produce autophagy inhibitors in keratinocytes, melanin degradation inhibitors in keratinocytes, skin or hair darkening agents, or gray hair preventing or improving agents.

The autophagy inhibitor in keratinocytes, the melanin degradation inhibitor in keratinocytes, the skin or hair darkening agent, and the gray hair preventing or improving agent of the present invention are capable of suppressing melanin degradation, suppressing autophagy in keratinocytes, and darkening skin or hair color. , or it may be a drug, quasi-drug or cosmetic for the purpose of preventing or improving gray hair, or it may be a raw material or raw material for manufacturing the drug, quasi-drug or cosmetic.

The drug or quasi-drug may be administered in any form, and may be either oral administration or parenteral administration. Dosage forms for oral administration include, for example, solid preparations such as tablets, granules, powders and capsules, and liquid preparations such as elixirs, syrups and suspensions; Dosage forms include injection, external, transdermal, transmucosal, nasal, enteral, inhalation, suppository, and patch preparations. Preferably, the drug or quasi-drug is in the form of an external skin preparation, and specific examples include ointments, creams, milky lotions, lotions, gels, aerosols, patches, tapes, sprays, and the like. be done.

Forms of the above cosmetics include creams, emulsions, lotions, suspensions, gels, powders, packs, sheets, patches, sticks, cakes, hair tonics, hair liquids, hair gels, hair creams, treatments, hair sprays, aerosol mousses, Any form that can be used in cosmetics such as shampoos and rinses can be mentioned.

Pharmaceutical compositions for pharmaceuticals, quasi-drugs, or cosmetics are prepared by adding the compounds of the present invention to pharmaceutically acceptable carriers such as various oils, surfactants, gelling agents, After mixing and dispersing with preservatives, antioxidants, solvents, alcohol, water, chelating agents, thickeners, ultraviolet absorbers, emulsion stabilizers, pH adjusters, pigments, fragrances, etc., and processing them into the desired form. can be obtained by In addition to the compound of the present invention, these pharmaceutical compositions may contain plant extracts, fungicides, moisturizers, antibacterial agents, A medicinal ingredient such as a cooling agent can be appropriately blended within a range that does not interfere with the effects of the present invention.

The content of the cholesterol production-inhibiting substance in the pharmaceutical composition is generally preferably 0.0001% by mass or more, more preferably 0.0005% by mass or more, still more preferably 0.001% by mass or more, and preferably 10% by mass or less, more preferably 1% by mass or less, still more preferably 0.1% by mass or less, preferably 0.0001 to 10% by mass, more preferably 0.005 to 1% by mass, still more preferably It is 0.01 to 0.1% by mass.

The dose of the cholesterol production-suppressing substance in the pharmaceutical composition can be an amount that can achieve the effects of the present invention. The dosage may vary according to the subject's condition, body weight, sex, age or other factors, but is preferably 0.001 mg or more, more preferably 0.01 mg or more per day per adult (60 kg), It is more preferably 0.1 mg or more, and preferably 10,000 mg or less, more preferably 1,000 mg or less, and still more preferably 100 mg or less. Also, it is preferably 0.001 to 10,000 mg, more preferably 0.01 to 1,000 mg, and still more preferably 0.1 to 100 mg.
In the case of administration by application of an external preparation such as cosmetics, the dose per 100 cm ² is preferably 0.001 mg or more, more preferably 0.005 mg or more, still more preferably 0.01 mg or more, and It is preferably 1,000 mg or less, more preferably 500 mg or less, and even more preferably 100 mg or less. Further, it is preferably 0.001 to 1,000 mg, more preferably 0.005 to 500 mg, still more preferably 0.01 to 100 mg.

In addition, the formulation can be ingested and administered according to any intake and administration schedule, but it is preferable to divide it once to several times a day and administer it continuously for several weeks to several months.
Subjects to which the above cosmetics, pharmaceuticals, or quasi-drugs are applied are not particularly limited as long as they are in need, but are preferably people who desire prevention or improvement of browning of the skin and graying of hair. Usually, it is preferable to apply directly to the site where the above effects are expected. Areas where the above effects are expected to appear include, in the case of browning of the skin, areas where the UV protection ability is desired to be improved, and areas where the skin is desired to be browned from an aesthetic point of view, for example, where the skin is exposed to ultraviolet rays and the eyes on a daily basis. Examples include the skin of the face, limbs, etc., which are frequently exposed, and the skin to which a self-tanning agent is administered. In addition, in the case of prevention or improvement of gray hair, hairy skin is mentioned, and hairy head skin is particularly preferable.

The autophagy inhibitor in keratinocytes of the present invention, the melanin degradation inhibitor in keratinocytes, the skin or hair darkening agent, or the method for evaluating or searching for the agent for preventing or improving gray hair is shown below in (a) to (c). It can be done by a process.
(a) Step of contacting keratinocytes with a test substance and culturing (b) Step of measuring at least one of the following indicators (i) to (iv) in keratinocytes
(i) total cholesterol
(ii) activity of cholesterol synthase
(iii) protein content of cholesterol synthase
(iv) Expression level of cholesterol synthase gene (c) A test substance that reduces any of the above indicators (i) to (iv) is an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, skin or hair color Step of selecting or evaluating as a blackening agent, or a gray hair preventing or improving agent

Here, as the "keratinocytes", any normal self-proliferating epidermal keratinocytes can be used, and human-derived cells can be used. Alternatively, a piece of human skin surgically collected or a 3D skin model may be used as it is. When using human-derived keratinocytes, keratinocytes can be collected from human skin and processed according to a conventional method. HaCaT cells, etc.) can also be purchased and used. Normal epidermal keratinocytes include, for example, normal human epidermal keratinocytes, neonatal (HEKn).

The contact between the keratinocytes and the test substance in step (a) is performed while culturing the keratinocytes under conditions in which the keratinocytes can grow. For example, Epilife medium (Thermo Fisher Scientific) containing a growth additive (Humedia-KG), Epilife medium and CnT-Prime medium (CELLnTEC) containing no hEGF among the above growth additives, and antibiotics as appropriate can be cultured in commercially available media such as media supplemented with , serum, growth factors and the like. Cultivation is usually carried out at 30° C. to 40° C. in the presence of 1 to 10 vol % carbon dioxide, preferably at 37° C. in the presence of 5 vol % carbon dioxide.

In addition, the number of keratinocytes to be contacted with the test substance is usually about 10 ⁴ to about 10 ⁶ cells/well, and 5×10 ⁴ to 5×10 ⁵ cells/well when a 6-well plate is used, for example. is preferred. The treatment time with the test substance is generally 12 to 96 hours, preferably 48 to 72 hours.

In step (b), one or a plurality of indicators selected from indicators (i) to (iv) are measured according to the following.
(i) Total Cholesterol Level Measurement of total cholesterol level is not particularly limited, and can be carried out according to the following procedure using a known measuring method (cholesterol oxidase method). That is, cholesterol ester present in total cholesterol is decomposed into free cholesterol and fatty acid by cholesterol esterase, and the obtained free cholesterol and originally existing free cholesterol are oxidized to Δ4-cholestenone by cholesterol oxidase, The hydrogen peroxide produced as a by-product at that time is used as a coloring reagent (eg, 4-aminoantipyrine/3,5-dimethoxy-N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline sodium (DAOS), or N- Acetyl-3,7-dihydroxyphenoxazine, etc.), peroxidase (POD) is allowed to act, and absorbance or fluorescence intensity is measured. For example, a commercially available kit such as Cholesterol/Cholesteryl Ester Quantitation Assay kit (Abcam) may be used.

(ii) Activity of Cholesterol Synthetase The activity of cholesterol synthase is not particularly limited, and is measured by a known measurement method, i. is captured as an absorbance change and measured by a spectrophotometer. For example, when measuring the activity of 7-dehydrocholesterol reductase, it is known that it can be calculated from the conversion rate of ergosterol to brassicasterol in 1 hour (Zou L, Porter TD (2015) Rapid suppression of 7-dehydrocholesterol reductase activity in keratinocytes by vitamin DJ Steroid. Biochem. Mol. Biol. 148, 64-71.). That is, 1 hour after the cells were treated with ergosterol, the cells and the culture supernatant were collected, lysed with alkali, and the sterol components were extracted using hexane. The amount of brassicasterol present in the sterol component can be estimated by using Gas Chromatography-mass Spectrometry (GC-MS), and it is recommended to use stigmasterol as an internal standard. In addition, when measuring the activity of hydroxymethylglutaryl-CoA reductase, a commercially available kit such as HMG-CoA Reductase Activity Assay kit (Abcam) may be used.

(iii), (iv) Protein level and gene expression level of cholesterol synthase Measurement of the protein expression level and gene expression level of cholesterol synthase is not particularly limited, and any analysis method commonly used in the field may be used. For gene expression analysis, for example, dot blotting, Northern blotting, RNase protection assay, reporter assay using luciferase, RT-PCR, DNA microarray, etc., for protein expression analysis, Western blotting, Immunostaining methods, ELISA, binding assays and the like can be used.

In step (c), an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, a skin or hair darkening agent, or a gray hair preventing or improving agent is added based on the indicators (i) to (iv) measured according to the above. select or evaluate.
Specifically, in step (a), when the test substance is brought into contact with the keratinocytes, a cell group to which the test substance is added (addition group) and a cell group to which the test substance is not added or a control substance is added (control group) are prepared. , as an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, a skin or hair darkening agent, or a gray hair preventing or improving agent if the index in the addition group is reduced compared to the index in the control group can be evaluated or selected. If a plurality of indices are selected from (i) to (iv) as indices, it is sufficient that any one of the selected indices decreases.
The following aspects are further disclosed in this invention regarding embodiment mentioned above.

<1> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl ]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, a 7-dehydrocholesterol reductase inhibitor, a statin HMG-CoA reductase inhibitor, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase An autophagy inhibitor in keratinocytes containing, as an active ingredient, a cholesterol production inhibitor selected from siRNAs that inhibit autophagy.
<2> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl ]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, a 7-dehydrocholesterol reductase inhibitor, a statin HMG-CoA reductase inhibitor, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase A melanin degradation inhibitor in keratinocytes containing, as an active ingredient, a cholesterol production inhibitor selected from siRNAs that inhibit
<3> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl ]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, a 7-dehydrocholesterol reductase inhibitor, a statin HMG-CoA reductase inhibitor, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase A skin or hair darkening agent containing, as an active ingredient, a cholesterol production-inhibiting substance selected from siRNAs that inhibit
<4> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl ]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, a 7-dehydrocholesterol reductase inhibitor, a statin HMG-CoA reductase inhibitor, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase An agent for preventing or improving gray hair containing, as an active ingredient, a cholesterol production-inhibiting substance selected from siRNAs that inhibit
<5> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl ]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, a 7-dehydrocholesterol reductase inhibitor, a statin HMG-CoA reductase inhibitor, and expression of 7-dehydrocholesterol reductase or HMG-CoA reductase A skin external preparation containing a cholesterol production-inhibiting substance selected from siRNAs that inhibit
<6> A method for evaluating or searching for an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, a skin or hair darkening agent, or a gray hair preventing or improving agent, comprising the following steps (a) to (c).
(a) Step of contacting keratinocytes with a test substance and culturing (b) Step of measuring at least one of the following indicators (i) to (iv) in keratinocytes
(i) total cholesterol
(ii) activity of cholesterol synthase
(iii) protein content of cholesterol synthase
(iv) Expression level of cholesterol synthase gene (c) A test substance that reduces any of the above indicators (i) to (iv) is an autophagy inhibitor in keratinocytes, a melanin degradation inhibitor in keratinocytes, skin or hair color Step of selecting or evaluating as a blackening agent, or a gray hair preventing or improving agent

<7> Trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4), for producing an autophagy inhibitor in keratinocytes -Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and Use of a cholesterol production inhibitor selected from siRNA that inhibits the expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<8> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4-[3-(4 -Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and Use of a cholesterol production inhibitor selected from siRNA that inhibits the expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<9> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-( 4-Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitor selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitor, and siRNA that suppresses the expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<10> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4-[3-(4- Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7 - Use of cholesterol production inhibitors selected from siRNAs that inhibit the expression of dehydrocholesterol reductase or HMG-CoA reductase.

<11> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4-[3-(4- Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7 - A cholesterol production inhibitor selected from siRNA that inhibits the expression of dehydrocholesterol reductase or HMG-CoA reductase.
<12> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4-[3-(4- Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7 - A cholesterol production inhibitor selected from siRNA that inhibits the expression of dehydrocholesterol reductase or HMG-CoA reductase.
<13> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4), for use in skin or hair darkening -Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and A substance suppressing cholesterol production selected from siRNA that suppresses the expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<14> Trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl )-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7- A substance suppressing cholesterol production selected from siRNAs that suppress the expression of dehydrocholesterol reductase or HMG-CoA reductase.

<15> Trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl) -2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7-dehydro Use of a cholesterol production inhibitor selected from siRNA that inhibits the expression of cholesterol reductase or HMG-CoA reductase.
<16> Trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl) for inhibiting melanin degradation in keratinocytes -2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7-dehydro Use of a cholesterol production inhibitor selected from siRNA that inhibits the expression of cholesterol reductase or HMG-CoA reductase.
<17> Trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl )-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7- Use of a cholesterol production inhibitor selected from siRNA that inhibits the expression of dehydrocholesterol reductase or HMG-CoA reductase.
<18> trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or its salt, BM15766 (4-[2-[4-[3-(4-Chlorophenyl)- 2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 7-dehydrocholesterol reductase inhibitors selected from 2-chlorobenzylamine, statin HMG-CoA reductase inhibitors, and 7-dehydrocholesterol Use of a cholesterol production inhibitor selected from siRNA that inhibits the expression of reductase or HMG-CoA reductase.
<19> In <15> to <18> above, the use is preferably non-therapeutic use for preventing or improving skin or hair darkening or gray hair for cosmetic or aesthetic purposes. .

<20> A method for suppressing autophagy in keratinocytes, wherein trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4- 7-dehydrocholesterol reductase inhibitor selected from [3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, statin HMG-CoA A method comprising administering or ingesting an effective amount of a cholesterol production inhibitor selected from reductase inhibitors and siRNA that suppresses the expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<21> A method for inhibiting melanin degradation in keratinocytes, wherein trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4- 7-dehydrocholesterol reductase inhibitor selected from [3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, statin HMG-CoA A method comprising administering or ingesting an effective amount of a cholesterol production inhibitor selected from reductase inhibitors and siRNA that suppresses the expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<22> A method for darkening skin or hair, wherein trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4 -[3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, 7-dehydrocholesterol reductase inhibitor, statin HMG- A method comprising administering or ingesting an effective amount of a cholesterol production inhibitor selected from CoA reductase inhibitors and siRNA that inhibits expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<23> A method for preventing or improving gray hair, wherein trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof, BM15766 (4-[2-[4-[ 3-(4-Chlorophenyl)-2-propenyl]-1-piperazinyl]ethyl]benzoic acid sulfate), triparanol, 2-chlorobenzylamine, 7-dehydrocholesterol reductase inhibitor, statin HMG-CoA reductase A method comprising administering or having an effective amount of a cholesterol production inhibitor selected from inhibitors and siRNAs that suppress the expression of 7-dehydrocholesterol reductase or HMG-CoA reductase.
<24> In <20> to <23> above, the method is preferably a non-therapeutic method for preventing or improving skin or hair darkening or gray hair for cosmetic or aesthetic purposes. .

<25> In <1> to <24> above, the cholesterol production inhibitor is trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane or a salt thereof.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.

Example 1 Effects of AY9944 on Skin Color (1) Tissue Culture A normal human skin tissue derived from surgery was provided by the Department of Plastic Surgery, a medical university. trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride (AY9944) was purchased from Abcam. Human skin tissue was trimmed of subcutaneous fat, cut into small pieces of about 1 cm×1 cm with a knife, and cultured in a 6-well plate at 37° C. and 5 vol % CO ₂ . An Advanced DMEM medium containing 10% (v/v) of Fetal Bovine Serum (FBS) (both from Thermo Fisher Scientific) was used for culture.

(2) Effect of AY9944 on skin color Human skin tissue derived from the flank of a 47-year-old female was placed on a 6-well plate, and AY9944 dissolved in DMSO was added to final concentrations of 5, 10, 50, and 100 µM. Tissue culture was performed after adding to the medium as described (n=1). As a control, the same amount of DMSO was added. The appearance of the skin tissue was observed and photographed after 7 days from the start of the culture while the medium was replaced every 2 to 3 days. As a result, it was confirmed that AY9944 dose-dependently induces darkening of the skin color (Fig. 1A). In addition, human skin tissue derived from a different donor (55-year-old female flank) was treated with AY9944 to a final concentration of 100 μM, and the same procedure was performed (n=3). As a result, it was confirmed that darkening of the skin color was induced independently of the donor (Fig. 1B).

Example 2 Effect of AY9944 on Epidermal Cells (Keratinocytes) (1) Cell Culture Normal human neonatal foreskin-derived epidermal keratinocytes (Human Epidermal Keratinocytes, neonatal (HEKn) lightly; keratinocytes) were purchased from Thermo Fisher Scientific. Keratinocytes were cultured under conditions of 37° C. and 5 vol% CO ₂ in an epidermal keratinocyte growth medium (Epilife) (Thermo Fisher Scientific) supplemented with a medium growth additive (Humedia-KG) (Kurashiki Boseki).

(2) Effect of AY9944 on Autophagic Activity of Keratinocytes Keratinocytes were seeded on a 6-well plate at a cell density of 1.5×10 ⁵ cells/well (2 mL/well) and dissolved in DMSO the next day. AY9944 was added to final concentrations of 1, 5 and 10 μM, and cultured at 37° C. and 5 vol % CO ₂ . As a control, the same amount of DMSO was added. The test medium at this time was Epilife (proliferation additive (Humedia-KG) containing hydrocortisone, insulin, gentamicin/amphotericin B, human epidermal growth factor (hEGF; Human Epidermal Growth Factor) and bovine A pituitary extract (BPE; Bovine Pituitary Extract) was used. On the following day, Chloroquine diphosphate attached to LC3B Antibody Kit for Autophagy (Thermo Fisher Scientific) was added to a final concentration of 10 μM, and cultured for 24 hours at 37° C. and 5 vol% CO ₂ . After the culture was completed, the cells were washed with PBS, recovered using RIPA buffer (Sigma-Aldrich), and disrupted by sonication. After that, the supernatant was centrifuged at 15,000 rpm for 15 minutes, and the protein content of the supernatant was quantified by the BCA (bicinchoninic acid) method using Bovine Serum Albumin (BSA) as a standard substance. Subjected to SDS-PAGE and Western blot according to. Anti-LC3B (Sigma-Aldrich, 1:1000) and anti-p62 (MBL, 1:1000) were used as primary antibodies. Anti-rabbit IgG, HRP-Linked F(ab') ₂ Fragment Sheep (GE Healthcare Life Science) was used as a secondary antibody. Thereafter, color was developed using ECL plus western blotting detection reagents (GE healthcare bioscience), and the expression level was visualized using LAS4000 (Fuji Film). β-actin expression as an internal standard was evaluated using β-actin (13E5) rabbit monoclonal antibody (Cell Signaling Technology, 1:5000). LC3-II is used as an indicator of its activity because it localizes to the autophagosomal membrane and is ultimately degraded by autophagy. Addition of Chloroquine diphosphate (Chloroquine), an autophagy inhibitor, induced the accumulation of LC3-II, which was enhanced by AY9944 in a concentration-dependent manner (Fig. 2). This is considered to mean that AY9944 inhibited the fusion process of autophagosomes and lysosomes together with Chloroquine, and as a result, autophagosomes accumulated without being decomposed. In addition, the fact that AY9944 treatment induced the accumulation of p62, a substrate selectively degraded by autophagy, also seems to support the above interpretation.

(3) Effect of AY9944 on Cholesterol Synthesis of Keratinocytes Keratinocytes were seeded in a 6-well plate at a cell density of 2×10 ⁵ cells/well (2 mL/well), and on the following day, AY9944 dissolved in DMSO was terminated. It was added at concentrations of 1, 5 and 10 μM, and cultured for 3 days at 37° C. and 5 vol % CO ₂ (n=6). As a control, the same amount of DMSO was added. In addition, as the test medium at this time, Epilife (proliferation additive (Humedia-KG) containing hydrocortisone, insulin, gentamicin/amphotericin B, human epidermal growth factor (hEGF; Human Epidermal Growth Factor) and bovine A pituitary extract (BPE; Bovine Pituitary Extract) was used. After culturing, the cells were washed with PBS, 3-well out of 6-wells were recovered using RIPA buffer (Sigma-Aldrich), and the cells were crushed by sonication. Thereafter, the mixture was centrifuged at 15,000 rpm for 15 minutes, and the protein content of the supernatant was quantified by the BCA (bicinchoninic acid) method using Bovine Serum Albumin (BSA) as a standard substance (Fig. 3A). For the remaining 3-wells, a cholesterol/cholesteryl Ester Quantitation Assay kit (Abcam) was used to measure the total intracellular cholesterol level for each well according to the manufacturer's protocol (Fig. 3B). A value obtained by dividing the total cholesterol amount by the protein amount was calculated and used as corrected data (Fig. 3C). As a result, when AY9944 was treated with 10 μM, a decrease in the amount of protein was confirmed, so it is possible that cell growth inhibition or cell death induction affected the number of cells, but at least AY9944 was treated with 1 μM or It was confirmed that when treated with 5 μM, the amount of intracellular cholesterol decreased regardless of the increase or decrease in the number of cells.

Claims (4)

An autophagy inhibitor in keratinocytes containing trans-1,4- bis (2-chlorobenzylaminomethyl)cyclohexane or a salt thereof as an active ingredient. A melanin degradation inhibitor in keratinocytes containing trans-1,4- bis (2-chlorobenzylaminomethyl)cyclohexane or a salt thereof as an active ingredient. A skin or hair darkening agent containing trans-1,4- bis (2-chlorobenzylaminomethyl)cyclohexane or a salt thereof as an active ingredient. An agent for preventing or improving gray hair containing trans-1,4- bis (2-chlorobenzylaminomethyl)cyclohexane or a salt thereof as an active ingredient.

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