Active agents that modulate the activity of ion channels for use in modulating skin pigmentation

Abstract

Active agents that modulate the activity of ion channels for use in modulating skin pigmentation In order to provide novel active agents for actively controlling and / or treating skin pigmentation in a subject without undesirable side effects, the present invention discloses active agents that activate, enhance, inactivate, block or attenuate the cellular response of the transient receptor potential ion channel TRPM8 or interfere with the expression of said ion channel.

Description

[Technical Field]

[0001] The present invention is directed to active agents for use in modulating skin pigmentation, particularly in treating skin pigmentation conditions and disorders, wherein said active agents modulate the activity of an ion channel, including but not limited to activating, enhancing, inactivating or blocking an ion channel, or attenuating a cellular response induced by an ion channel, or interfering with the expression of said ion channel. [Background technology]

[0002] Furthermore, the present invention relates to a composition for use as a cosmetic or pharmaceutical in the treatment of skin pigmentation, said composition comprising at least one active agent that modulates the activity of an ion channel, including but not limited to activating, enhancing, inactivating or blocking an ion channel, or attenuating a cellular response induced by an ion channel, or interfering with the expression of said ion channel.

[0003] Furthermore, the present invention relates to non-therapeutic methods of modulating skin pigmentation, comprising administering to a subject an effective amount of at least one active agent that modulates the activity of an ion channel, including, but not limited to, activating, enhancing, inactivating or blocking the ion channel, or attenuating a cellular response induced by the ion channel, or interfering with the expression of said ion channel.

[0004] Melanin is the skin's natural protector against ultraviolet radiation. It is the primary determinant of skin and hair color. It is produced and distributed in the skin through a process called melanogenesis. Melanin is first synthesized within intracellular organelles (melanosomes) in melanocytes and then distributed to neighboring keratinocytes. Strictly speaking, pigmentation involves several steps, including not only melanin synthesis itself but also melanin transport and processing (mainly to and within epidermal keratinocytes), which underlie skin and hair color. Ultraviolet (UV) irradiation is strongly associated with skin pigmentation. UV-induced DNA damage stimulates melanin production through the upregulation and activation of tyrosinase, the enzyme that catalyzes the rate-limiting step in melanin synthesis.

[0005] Hyperpigmentation due to overproduction of melanin includes, but is not limited to, lentigines (age spots), solar lentigines (dark spots due to chronic sun exposure), melasma (larger dark spots), chloasma (hormonal skin discoloration), freckles, hyperpigmented neonatal spots such as nevus of Ota, Mongolian spots, cafe au lait spots, and pigmented nevi (mole), hyperpigmentation as a result of skin injury (e.g., post-inflammatory hyperpigmentation, post-infectious hyperpigmentation, post-blister hyperpigmentation, post-burn hyperpigmentation, post-traumatic hyperpigmentation, postpartum hyperpigmentation, and striae atrophia), and dark spots of any kind. Skin lightening products and techniques are used to treat these conditions / conditions, as well as those who claim to have a "darker than desired" skin color (and therefore intend to lighten it).

[0006] Currently, there are several active agents on the market that are said to be useful in treating hyperpigmentation. Some of these active agents belong to the vitamin group (for example, vitamin C, α-tocopherol or vitamin E, niacinamide). The drawback of these vitamins is that they do not specifically target melanocytes (melanocytes) and the melanin production process, or only slightly, and it is claimed that their supposed anti-pigmentation effect is mainly due to their antioxidant effect on the entire skin.

[0007] Additionally, certain phenolic compounds (e.g., hydroquinone and its derivatives) have been shown to inhibit melanocyte tyrosinase, the key enzyme in melanin production, and have therefore been claimed to be effective in treating hyperpigmentation. However, their widespread chronic side effects (e.g., contact dermatitis, hyperpigmentation of the sclera and nails, and impaired wound healing) have prevented their widespread use. Furthermore, hydroquinone has been shown to cause DNA damage, raising serious safety concerns as it may increase the risk of cancer.

[0008] Meanwhile, systemic or localized suppression or reduction of skin melanin pigment is a feature of many hypopigmentary skin diseases / disorders, including, but not limited to, vitiligo (a hypopigmentary disorder characterized by patches of hyperpigmentation throughout the skin), hypopigmentation as a result of skin injury (e.g., post-inflammatory hypopigmentation, post-infectious hypopigmentation, post-blister hypopigmentation, post-burn hypopigmentation, post-traumatic hypopigmentation, post-partum hypopigmented stretch marks [striations atrophy], and post-bleaching hypopigmentation). Treatment of these conditions, as well as for people who claim to have a "whiter or lighter" skin color (and therefore intend to darken it), involves skin-darkening and / or "UV-free tanning" products and techniques.

[0009] Despite not being life-threatening complications, the aforementioned skin pigmentation conditions / disorders can cause significant psychological distress to affected individuals.

[0010] Besides classical UV irradiation, which induces photoprotective tanning of the skin, and topically applied "artificial tanning" agents (e.g., dihydroxyacetone, melanin) that simulate natural pigmentation, the most widely tested agents are analogs of melanocyte-stimulating hormone (MSH). For example, the α-MSH mimetic peptide, afamelanotide / melanotan, has been shown to induce skin pigmentation in humans. However, afamelanotide / melanotan has raised significant concerns because it was found to induce the development / rash of melanocytic nevi, thereby increasing the risk of developing malignant melanoma. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] HGNC:17961, NCBI Entrez Gene:79054,Ensembl:ENSG00000144481,OMIM(registered trademark):606678,UniProtKB / Swiss-Prot:Q7Z2W7;As of July 3, 2022 Summary of the Invention [Problem to be solved by the invention]

[0012] In view of the above, there is a continuing need for active agents to proactively control and / or treat skin pigmentation in patients without undesirable side effects. [Means for solving the problem]

[0013] The present inventors have found that modulation of the activity of the transient receptor potential ion channel TRPM8 significantly affects melanogenesis in human epidermis.

[0014] "Ion channels" are pore-forming proteins located in biological membranes. Ions can pass through the membrane through the protein pores, down their electrochemical gradient. By opening and closing the pore, ion channels can control the flow of ions across the membrane and regulate the intracellular ion concentration. Changes in intracellular ion concentration affect many cellular responses and processes, including, but not limited to, growth, differentiation, survival, death, mediator release, and immune mechanisms.

[0015] Transient receptor potential channels (TRP channels) are a group of ion channels that exist primarily on cell membranes, and there are approximately 30 types of TRP channels, including TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML. These ion channels are relatively nonselectively permeable to cations such as sodium, calcium, and magnesium.

[0016] "TRPM8" is the official gene symbol for "transient receptor potential cation channel subfamily M member 8" and identifies the protein encoded by the human TRPM8 gene (HGNC: 17961, NCBI Entrez Gene: 79054, Ensembl:ENSG00000144481, OMIM®: 606678, UniProtKB / Swiss-Prot:Q7Z2W7; as of July 3, 2022).

[0017] The present inventors have found that menthol, an agonist of the transient receptor potential ion channel TRPM8, promotes pigmentation in ex vivo human skin (epidermis) by opening the channel. Indeed, when applied topically to human ex vivo skin organ cultures, a suitable model for assessing multiple aspects of epidermal melanogenesis and melanin transport, menthol increased epidermal melanin content, the most important marker of melanogenesis, as verified by two independent histochemical techniques.

[0018] Furthermore, as revealed by quantitative histomorphometry, topical menthol treatment also increased the levels of gp100 (also known as Pmel17 or Silver locus protein), a pre-melanosome protein known to be involved in the melanosome transport compartment and contribute to the functional integrity and assembly of melanosomes, and therefore to de novo melanin synthesis. Furthermore, in situ enzyme assays revealed that topical menthol treatment increased the intraepidermal activity of tyrosinase, the rate-limiting enzyme in melanin production.

[0019] Conversely, inhibition of TRPM8 with the selective inhibitor N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB) consistently blocked the propigmentation effects of menthol.

[0020] In addition to the active agents described above, the present inventors have identified a number of active agents that act on TRPM8 and are therefore suitable for use in the treatment of skin pigmentation.

[0021] In conclusion, the present invention provides an active agent for use in the treatment of skin pigmentation, said active agent activating, enhancing, inactivating or blocking TRPM8 or attenuating cellular responses induced by the transient receptor potential ion channel TRPM8 or interfering with the expression of said ion channel.

[0022] According to an alternative form of the invention, the active agent that activates, enhances, inactivates, blocks or attenuates the cellular response of an ion channel induced by the transient receptor potential ion channel TRPM8 is either an agonist or an antagonist of said ion channel.

[0023] As used herein, an "agonist" of the transient receptor potential ion channel TRPM8 is a substance that binds to and activates or enhances the ion channel, thereby enhancing a cellular response associated with TRPM8.

[0024] As used herein, an "antagonist" of the transient receptor potential ion channel TRPM8 refers to a substance that binds to the ion channel and blocks TRPM8, thereby attenuating the cellular response to an agonist rather than activating or enhancing it as an agonist would.

[0025] An "inverse agonist" of the transient receptor potential ion channel TRPM8 is a substance that binds to the ion channel and induces a cellular response opposite to that observed when an agonist is applied.

[0026] With respect to the transient receptor potential ion channel TRPM8 of the present invention, the term "cellular response" is primarily understood as a change in intracellular ion concentration resulting from the binding of an agonist / antagonist / inverse agonist to the transient receptor potential ion channel TRPM8. However, this term also encompasses cases in which the binding of an agonist / antagonist / inverse agonist induces a cellular response without changing the intracellular ion concentration.

[0027] Thus, agonists of the present invention of the transient receptor potential ion channel TRPM8 activate or enhance the ion channel to produce a cellular response as would an endogenous agonist, whereas antagonists of the present invention of the ion channel block or attenuate the ion channel, producing a cellular response that would normally be activated / enhanced by the endogenous agonist, and inverse agonists of the present invention modulate the ion channel, producing the opposite cellular response that would normally be activated / enhanced by the endogenous agonist.

[0028] In those embodiments, when the active agent is an agonist of the transient receptor potential ion channel TRPM8, the active agent is used in the treatment of hypopigmentation conditions. In those embodiments, when the active agent is an antagonist / inverse agonist of the transient receptor potential ion channel TRPM8, the active agent is used in the treatment of hyperpigmentation conditions.

[0029] In certain embodiments of the invention, the ion channel agonist / antagonist / inverse agonist used is (+)-menthol, (-)-menthol, icilin (-)-menthyl lactate (frescolat ML), menthone 1,2-glycerol ketal (frescolat MGA), N-ethyl-5-methyl-2-propan-2-ylcyclohexane-1-carboxamide (WS-3), ethyl N-[(1R,2S,5R)-5-methyl-2-propan-2-ylcyclohexanecarbonyl]carbamate, (WS-5), (1R,2S,5R)-N-(4-methoxyphenyl)-5-methyl-2-(propan-2-yl)cyclohexane-1-carboxamide (WS-13), N,2,3-trimethyl-2-propan-2-ylbutanamide (WS -23) A TRPM8 agonist selected from the group consisting of (-)-menthoxypropane-1,2-diol, p-menthane-3,8-diol (PMD38), (1R,2S,5R)-5-methyl-2-propan-2-yl-N-[4-(pyrimidin-2-ylsulfamoyl)phenyl]cyclohexane-1-carboxamide (CPS125), isopulegol, eucalyptol, geraniol, linalool, hydroxycitronellal, or a combination thereof.

[0030] In another embodiment of the present invention, the ion channel agonist / antagonist / inverse agonist used is N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(thiophen-2-ylmethyl)benzamide (AMTB), N-(2-aminoethyl)-N-[[3-methoxy-4-(phenylmethoxy)phenyl]methyl]thiophene-2-carboxamide (M8-B), erythmetrep, KPR-5714, [(1R)-1-Phenylethyl]N-(2-aminoethyl)-N-[(3-methoxy-4-phenylmethoxyphenyl)methyl]carbamate (PBMC), 3-[[(1R)-1-(4-fluorophenyl)ethyl]-(quinoline-3-carbonyl)amino]methyl]benzoic acid (PF-05105679), N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC), N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC), (5Z,8Z,11Z,14Z)-N-[2-(3,4-dihydroxyphenyl)ethyl]icosa-5,8,11,14-tetraen-1-yl]benzoic acid (ACAA), 2-aminoethoxydiphenylborate (2-APB), 5-benzyloxytryptamine, linoleic acid, clotrimazole, anandamide, 2-[3-(4-pentylphenyl)prop-2-enoylamino]benzoic acid (ACAA), (5Z,8Z,11Z,14Z)-N-[2-(3,4-dihydroxyphenyl)ethyl]icosa-5,8,11,14-tetraen-1-yl]benzoic acid (ACAA), and a TRPM8 antagonist / inverse agonist selected from NADA, baicalein, chrysin, hispidulin, oroxylin A, scutellarein, genistein, isoliquiritigenin, glycicoumarin, licochalcone A, acacetin, apigenin, butein, capsazepine, cannabidiol, cannabigerol, Δ9-tetrahydrocannabinol, or a combination thereof.

[0031] Even if the inventors have not exemplified such examples, it cannot be excluded that there exist prior art compositions for treating skin pigmentation that contain one of the specific active agents of the present invention as an ingredient, but that ingredient has no effect on TRPM8. In this case, such an unexpectedly anticipated active agent shall be excluded from the scope of the present invention by the corresponding clause. Except for the exclusion of the unexpectedly anticipated specific active agent, the clause shall not further affect the scope of the claims.

[0032] Non-limiting examples of active agents that would be foreseen in the prior art include: Chemical compounds that have no medical, pharmacological or biological activity recognized by the corresponding prior art; chemical compounds to which the corresponding prior art attributes different medical, pharmacological or biological activity compared to the specific inventive active agents disclosed herein; and / or In comparison with the specific inventive active agents disclosed herein, there are chemical compounds to which the corresponding prior art attributes the same medical, pharmacological or biological activity, but which are not directed against TRPM8 but against another target (e.g., receptor, enzyme, hormone, metabolite).

[0033] Alternatively, a TRPM8 agonist is an aptamer that binds to the TRPM8 ion channel and activates or enhances the ion channel to produce a cellular response, or a TRPM8 antagonist / inverse agonist is an aptamer that binds to the TRPM8 ion channel and inactivates, blocks or attenuates the ion channel to produce a cellular response.

[0034] As used herein, the term "aptamer" refers to a DNA, RNA, XNA oligonucleotide or peptide molecule that binds to a specific target molecule, such as an ion channel molecule.

[0035] According to another aspect of the present invention, the active agent that inhibits the expression of the ion channel TRPM8 is either an miRNA, an siRNA, or a ribozyme targeted to the TRPM8 gene or to the mRNA corresponding to the TRPM8 gene.

[0036] The term "miRNA" refers to microRNAs, small non-coding RNA molecules containing 21–23 nucleotides, which function in RNA knockdown and post-transcriptional regulation of gene expression. miRNAs function through base pairing with complementary sequences within mRNA molecules. As a result, these mRNA molecules are knocked down by cleavage, destabilization, and / or reduced mRNA translation efficiency.

[0037] The term "siRNA" refers to small interfering RNA, a small non-coding RNA molecule 20-25 base pairs in length that disrupts the expression of specific genes with complementary base sequences by degrading mRNA after transcription and inhibiting translation.

[0038] According to the present invention, a gene is "targeted" by an miRNA, siRNA, or ribozyme if the miRNA, siRNA, or ribozyme molecule selectively reduces or inhibits the expression of TRPM8. As used herein, the phrase "selectively reduces or inhibits" refers to an miRNA, siRNA, or ribozyme that affects the expression of TRPM8.

[0039] In a specific embodiment of the present invention, the miRNA or siRNA disrupts gene expression of the ion channel TRPM8 by hybridizing to the gene transcript, i.e., TRPM8 mRNA, under stringent conditions. Here, hybridizing "under stringent conditions" means annealing to the target mRNA region under standard conditions that tend to disfavor hybridization, such as high temperature (e.g., below 60°C for 2 hours) and / or low salt content (e.g., 0.1xSSC).

[0040] According to the present invention, one of the above active agents is used to treat skin pigmentation. In a specific embodiment of the present invention, the treatment is performed topically, i.e., in, on, or in the skin area to be treated. In some embodiments of the present invention, the treatment is performed topically, where the term "topical" refers to a formulation applied to a specific location on the skin. In a specific embodiment, topical application is epicutaneous application, meaning that the agonist or antagonist is applied directly to the skin. In other embodiments of the present invention, the treatment is performed transdermally, where the term "transdermal" refers to a formulation applied across the stratum corneum to the deeper layers of the skin, for example, by injection with a standard needle or microneedle. In other embodiments of the present invention, the treatment is performed transappendageally, where the term "transappendageal" refers to an applied formulation that penetrates into the deeper layers of the skin through skin appendage structures (such as hair follicles, sebaceous glands, sweat glands, etc.).

[0041] The term "treatment" as used herein refers to any action that results in a change in physical condition. In particular, "treatment of skin pigmentation" refers to any change in the initial skin pigmentation state, such as lentigines (age spots), solar lentigines (dark spots due to chronic sun exposure), melasma (age spots, larger dark spots), chloasma (hormonal skin discoloration), freckles (age spots), hyperpigmented birthmarks, hyperpigmented neonatal spots, e.g., nevus of Ota, Mongolian spots, cafe au lait spots, pigmented nevi (mole), hyperpigmentation as a result of skin injury (e.g., post-inflammatory hyperpigmentation), The present invention is directed to the modulation of skin pigmentation in a variety of ways, including: hyperpigmentation, post-infectious hyperpigmentation, post-blister hyperpigmentation, post-burn hyperpigmentation, post-traumatic hyperpigmentation, post-partum hyperpigmented stretch marks (striae distensae), any type of dark spot, "darker than desired" skin color, vitiligo (a hypopigmentation disorder characterized by patches of pigmentation throughout the skin), hypopigmentation as a result of skin injury (e.g., post-inflammatory hypopigmentation, post-infectious hypopigmentation, post-blister hypopigmentation, post-burn hypopigmentation, post-traumatic hypopigmentation, post-partum hypopigmented stretch marks (striae distensae), post-bleaching hypopigmentation), any type of white or pale blemish, and "whiter or lighter than desired" skin color. In other words, the present invention is directed to the modulation of skin pigmentation in a variety of ways.

[0042] In a particular embodiment of the present invention, the active agent is used as a cosmetic in the treatment of skin pigmentation.In particular, the cosmetic use is carried out to achieve a change in an initial skin pigmentation condition that occurs non-therapeutically, such as an undesirable skin color (darker or lighter / whiter than desired), where the initial condition is not caused by a disease or disorder.

[0043] In these embodiments, when the active agent is used as a cosmetic, the active agent used should be cosmetically acceptable, where "cosmetically acceptable" means that the active agent should not be toxic or harmful or have other adverse side effects when applied to the skin.

[0044] In another specific embodiment of the present invention, the active agent is used as a pharmaceutical in the topical treatment of skin pigmentation disorders, where the term "disorder" means any functional abnormality or disturbance of a normal healthy state, and the term "pharmaceutical" means a substance useful in curing, treating, or preventing the condition of the disorder.

[0045] In these embodiments, when the active agent is used as a pharmaceutical, the active agent used should be pharmaceutically acceptable, where "pharmaceutically acceptable" means that the active agent should not be toxic or harmful or have other adverse side effects when applied to the skin.

[0046] In some embodiments, at least one of the actives in the present invention is used as a component of a composition for cosmetic or pharmaceutical use in the topical treatment of skin pigmentation, said composition further comprising at least one auxiliary agent selected from the group consisting of carriers, excipients, adjuvants, diluents, and disintegrants.

[0047] In specific embodiments of such compositions, the adjuvant is selected from the group consisting of liposomes, nanoparticles, carboxymethylcellulose, hydroxyethylcellulose, mineral oil, petrolatum, glycerin, polysorbate 80, hydroxyethyl starch, dextran, and polyethylene glycol.

[0048] In the compositions of the present invention, the concentration of the active agent is typically in the range of 10 to 10,000 μM. In some embodiments, the lower limit of the active agent concentration is 30 μM or 100 μM. In some embodiments, the upper limit is 3,000 μM or 1,000 μM. As a result, preferred ranges include, for example, 30 to 10,000 μM, 30 to 3,000 μM, 10 to 3,000 μM, and 100 to 3,000 μM.

[0049] In reference to the total weight of the composition of the present invention, the concentration of the active agent can vary in specific embodiments within a range of 1 to 30% by weight. In some embodiments, the lower limit is 5% by weight, or even 10% by weight. In some embodiments, the upper limit is 25% by weight or 20% by weight. This results in preferred ranges such as 5 to 30% by weight, 5 to 25% by weight, 10 to 25% by weight, and 10 to 30% by weight.

[0050] In certain embodiments, such compositions further comprise at least one other active agent effective in the treatment of skin pigmentation.

[0051] In such embodiments, the other active agent may be selected from one of the prior art skin pigmentation agents as mentioned in the introduction.

[0052] Generally, the composition of the present invention can be used in any formulation suitable for treating skin pigmentation. In a specific embodiment of the present invention, the composition is formulated in the form of an ointment, lotion, cream, gel, spray, patch or sustained-release patch. In another specific embodiment of the present invention, the composition is formulated in the form of a solution. The solution can be applied by a microneedle device or before sonication, electrical stimulation, etc.

[0053] As mentioned above, the use of the above-mentioned active agents in the present invention also occurs in non-therapeutic uses, where non-therapeutic refers to treatments that are not intended to cure, treat or prevent a disorder condition (see above).

[0054] Accordingly, the present invention is further directed to non-therapeutic methods of modulating skin pigmentation, in which an effective amount of at least one of the above active agents is administered to a subject.

[0055] Non-therapeutic methods also include embodiments in which the above-mentioned active agent is administered to the subject being treated simultaneously, sequentially, or separately with at least one other active agent useful in the treatment of skin pigmentation (see above).

[0056] The following examples illustrate some of the features of specific embodiments of the present invention, but those skilled in the art will understand that these embodiments are merely illustrative and are not intended to limit the inventive concept to the precise features or combinations of features of the example embodiments.

[0057] For a description of the example, please refer to the following figures: [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1 contains two microscopic images and a bar graph showing that menthol increases epidermal melanin content in human skin organ cultures. [Figure 2] FIG. 2 contains two microscopic images and a graph showing that menthol increases the expression of the melanosome-melanogenic marker gp100 in human skin organ cultures. [Figure 3] FIG. 3 contains two microscopic images and a graph showing that menthol increases tyrosinase activity, a marker of melanin production, in human skin organ cultures. [Figure 4] FIG. 4 contains three microscopic images and a graph showing that N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB) inhibits the effect of menthol on increasing epidermal melanin content in human skin organ cultures. [Figure 5] Figure 5 contains three microscopic images and a graph showing that N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB) inhibits the effect of menthol to increase the expression of the melanosome-melanogenic marker gp100 in human skin organ cultures. [Figure 6]Figure 6 contains three microscopic images and a graph showing that N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB) blocks the effect of menthol to increase tyrosinase activity, a marker of melanin production, in human skin organ cultures. [Example]

[0059] example 1. Ex Vitro Skin Organ Culture Model for Assessing Skin Pigmentation Human abdominal, temporal, and occipital skin samples were obtained from healthy adult human subjects during elective plastic surgery for cosmetic reasons, under ethical approval and with written patient consent. Human full-thickness skin samples were prepared as 4-6 mm punches, placed on filter paper, and cultured at the air-liquid interface under serum-free conditions in William's medium E supplemented only with insulin, hydrocortisone, and L-glutamine.

[0060] For topical application of the TRPM8 activator menthol (Sigma), a stock (5M) was prepared in ethanol and dissolved in a 30% v / v solution of polyethylene glycol 6000 (Sigma) in ethanol to a final concentration of 1 mM at 37°C. For topical application of the TRPM8 inhibitor N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB) (Sigma), a stock (50 mM) was prepared in water and dissolved in a 30% v / v solution of polyethylene glycol 6000 (Sigma) in ethanol to a final concentration of 10 μM at 37°C. 2 μL drops of this viscous solution containing menthol (1 mM), AMTB (10 μM), their combination, or ethanol as a solvent control (vehicle) were applied daily (for up to 3 days) to the epidermis of skin biopsies.

[0061] 2. Activation of TRPM8 by menthol Histochemical visualization of melanin in skin sections was performed using the Warthin-Starry Stain Kit (Abcam) or Fontana Masson histochemistry. For Warthin-Starry histochemistry, formalin-fixed human skin / HF cryosections were rinsed in distilled water and incubated with AgNO3 (0.5% in acidified water) at 50°C for 30 seconds. The specimens were then incubated with a developer (0.42% AgNO3, 5% gelatin, 0.15% hydroquinone in acidified water) for 2–5 minutes at 50°C. The reaction was stopped by rinsing with hot (60°C) tap water for 2 minutes consecutively. Unbound AgNO3 was removed by incubation with Na2SO3 (5% in distilled water) for 1 minute, followed by rinsing with tap water for 3 minutes consecutively. The brown melanin staining can also be toned to black by a brief counterstain with hematoxylin to visualize nuclei. After dehydration, the specimens were embedded in Eukitt®.

[0062] Images were then captured using a digital microscope (Keyence). Quantitative histomorphometric analysis was performed using ImageJ (NIH; Bethesda, MD, USA) by assessing the relative intensity and area of ​​melanin staining in standardized reference areas. Briefly, for each parameter assessed, multiple non-contiguous skin sections (separated by at least 280 μm) were stained per punch biopsy. Unless otherwise indicated, two skin punch biopsies were analyzed per experimental group. Melanin content was calculated based on the product of the percentage of area occupied by melanin and the mean grayness of melanin, as described in detail elsewhere. Evaluations were performed in multiple different microscopic fields per section. For all parameters, the entire epidermis (or the basal layer only) was used for measurement. This analysis was performed at least 500 μm away from the center of the skin biopsy, as these areas are where significant wound healing processes, such as re-epithelialization, are observed.

[0063] Figure 1 shows representative images (a, b) of histochemical visualization of melanin in skin sections using the Warthin-Starry method and the results of quantitative histomorphometric analysis (c). Figure 1 demonstrates that topical application of the TRPM8 agonist menthol (1 mM) significantly increased epidermal melanin content after 3 days compared to vehicle in human skin organ cultures. Furthermore, Figure 1 shows that menthol treatment increased melanin content not only in the basal layer (i.e., the epidermal compartment where melanocytes reside) but also in the suprabasal layers, suggesting that menthol-mediated TRPM8 activation enhanced melanosome migration.

[0064] For detection of gp100 (known to be involved in the melanosome transport compartment and contribute to the functional integrity and assembly of melanosomes, thus contributing to de novo melanin synthesis), acetone-fixed human skin / HF cryosections were incubated overnight at 4°C with anti-gp100 (clone NKI / beteb; MONOSAN) diluted 1:100 in antibody diluent (Agilent Dako). After sequential rinses with PBS, samples were incubated for 45 minutes at room temperature with rhodamine-conjugated secondary antibody (Jackson ImmunoResearch) diluted 1:200 in normal goat serum (2% in PBS). After washing with PBS, samples were counterstained with DAPI and mounted on glass slides using Fluoromount-G® (SouthernBiotech). Image acquisition and quantitative histomorphometric analysis were performed similarly to that described for the Warthin-Starry method, with particular emphasis on assessing the relative intensity and area of ​​gp100-specific immunoreactivity (IR) in the basal epidermal layer (i.e., the epidermal compartment where melanocytes reside).

[0065] Figure 2 shows representative images (a, b) of immunohistochemical visualization of gp100-specific IR in skin sections and the results of quantitative histomorphometric analysis (c). Figure 2 confirms that topical application of the TRPM8 agonist menthol (1 mM) significantly increased the intensity level of gp100-specific IR in the basal layer of the epidermis after 3 days in human skin organ culture compared to vehicle, thereby further supporting the pro-pigmentation effect of TRPM8 activation.

[0066] For in situ detection of tyrosinase activity (the enzyme that catalyzes the rate-limiting step in melanin synthesis), human skin cryosections were fixed in an acetone / methanol solution (1:1 ratio) and blocked with HO (0.3% in PBS) for 15 min. Endogenous biotin was blocked using a streptavidin / biotin blocking kit (Vector Labs). Samples were then blocked with normal goat serum (5%) and BSA (1%) in PBS for 30 min. Tyrosinase activity was detected using the TSA® Biotin System (Akoya Biosciences).

[0067] Briefly, samples were incubated with biotin tyramide (1:50 in amplification diluent) and washed sequentially with IGEPAL® CA-630 (Sigma, 0.1% in PBS) and PBS. To visualize biotin crosslinked near tyrosinase activity, samples were incubated with Strepdavidin-Cy3 (Sigma, 1:600 ​​in 5% BSA in PBS) for 1 hour. After further washing with IGEPAL® CA-630 (0.1% in PBS) and PBS, samples were counterstained with DAPI and mounted on slides with Fluoromount-G® (SouthernBiotech).

[0068] Image acquisition and quantitative histomorphometric analysis were performed similarly as described for the Warthin-Starry method, with particular emphasis on assessing the relative intensity and area of ​​tyrosinase activity-specific immunoreactivity (IR) in the basal epidermal layer (i.e., the epidermal compartment where melanocytes reside).

[0069] Figure 3 shows representative images (a, b) of histochemical visualization of tyrosinase activity in skin sections and the results of quantitative histomorphometric analysis (c). Figure 3 demonstrates that topical application of the TRPM8 agonist menthol (1 mM) significantly increased the intensity of tyrosinase activity in the basal layer of the epidermis after 3 days in human skin organ cultures compared to vehicle, further supporting the pro-pigmentation effect of TRPM8 activation.

[0070] 3. Inactivation of TRPM8 by N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB) Figure 4 shows representative images (a, b, c) of melanin histochemical visualization in skin sections using the Warthin-Starry method and the results of quantitative histomorphometric analysis (d). Figure 4 shows that the TRPM8 antagonist N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB, 10 μM) inhibited the effect of the TRPM8 agonist menthol (1 mM) on increasing epidermal melanin content after 3 days in human skin organ cultures, compared with vehicle. This suggests that the pigmentation-promoting effect of menthol is mediated by TRPM8.

[0071] Figure 5 shows representative images (a, b, c) of immunohistochemical visualization of gp100-specific IR in skin sections and the results of quantitative histomorphometric analysis (d). Figure 5 shows that the TRPM8 antagonist N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB, 10 μM) inhibited the effect of the TRPM8 agonist menthol (1 mM) on increasing gp100-specific IR in the basal layer of the epidermis after 3 days in human skin organ culture, compared to vehicle. This suggests that the pigmentation-promoting effect of menthol is mediated by TRPM8.

[0072] Figure 6 shows representative images (a, b, c) of histochemical visualization of tyrosinase activity in skin sections and the results of quantitative histomorphometric analysis (d). Figure 6 shows that the TRPM8 antagonist N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide hydrochloride (AMTB, 10 μM) inhibited the effect of the TRPM8 agonist menthol (1 mM) on increasing tyrosinase activity in the basal layer of the epidermis after 3 days in human skin organ cultures, compared to vehicle. This suggests that the pigmentation-promoting effect of menthol is mediated by TRPM8.

Claims

1. An activator for use in the treatment of skin pigmentation, wherein the activator activates, enhances, inactivates, blocks or attenuates the cellular response of the transient receptor potential ion channel TRPM8, or interferes with the expression of the ion channel.

2. The activator for use in the treatment of skin pigmentation according to claim 1, wherein the activator is an agonist of the transient receptor potential ion channel TRPM8 for use in the treatment of reducing pigmentation.

3. The activator for use in the treatment of skin pigmentation according to claim 1, wherein the activator is an antagonist / inverse agonist of the transient receptor potential ion channel TRPM8 for use in the treatment of hyperpigmentation.

4. The aforementioned activator, a) Any one of the following TRPM8 activating agonists: (+)-menthol, (-)-menthol, ishirin, (-)-menthyl lactate (frescolat ML), menthone 1,2-glycerol ketal (frescolat MGA), N-ethyl-5-methyl-2-propane-2-ylcyclohexane-1-carboxamide (WS-3), ethyl N-[(1R,2S,5R)-5-methyl-2-propane-2-ylcyclohexanecarbonyl]carbamate (WS-5), (1R,2S,5R)-N-(4-methoxyphenyl)-5-methyl-2-(propane-2-yl)cyclohexane-1-carboxamide (WS-13), N,2,3-trimethyl-2-propane-2-ylbutanamide (WS-23), (-)-menthoxy Propane-1,2-diol, p-menthane-3,8-diol (PMD38), (1R,2S,5R)-5-methyl-2-propane-2-yl-N-[4-(pyrimidine-2-ylsulfamoyl)phenyl]cyclohexane-1-carboxamide (CPS125), isopulegol, eucalyptol, geraniol, linalool, hydroxycitronellal, or combinations thereof, or aptamers that bind to TRPM8 and activate or enhance the ion channel to produce a cellular response, or b) One of the following TRPM8 inactivating antagonists / inverse agonists: N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(thiophen-2-ylmethyl)benzamide (AMTB), N-(2-aminoethyl)-N-[[3-methoxy-4-(phenylmethoxy)phenyl]methyl]thiophen-2-carboxamide (M8-B), elismetrep, KPR-5714, [(1R)-1-phenylethyl]N -(2-aminoethyl)-N-[(3-methoxy-4-phenylmethoxyphenyl)methyl]carbamate (PBMC), 3-[[[(1R)-1-(4-fluorophenyl)ethyl]-(quinoline-3-carbonyl)amino]methyl]benzoic acid (PF-05105679), N-(4-tert-butylphenyl)-4-(3-chloropyridine-2-yl)piperazine-1-carboxamide (BCTC), N-(4-tert-butylphenyl)-4-(3-chloropyridine-2-yl) Piperazine-1-carbothioamide (thioBCTC), 2-aminoethoxydiphenylborate (2-APB), 5-benzyloxytryptamine, linoleic acid, clotrimazole, anandamide, 2-[3-(4-pentylphenyl)prop-2-enoylamino]benzoic acid (ACAA), (5Z,8Z,11Z,14Z)-N-[2-(3,4-dihydroxyphenyl)ethyl]icosa-5,8,11,14-tetraenamide (NADA), baicalein, chrysin, hispi Hyspidulin, oroxylin A, scutellain, genistein, isoliquirtigenin, glycicoumarin, licochalcone A, acacetin, apigenin, butein, capsazepine, cannabidiol, cannabigerol, Δ9-tetrahydrocannabinol, or combinations thereof, or aptamers that bind to TRPM8 and inactivate, block, or attenuate the ion channel to produce a cellular response, or c) An activator selected from miRNA, siRNA, or ribozyme that is targeted to the TRPM8 gene or to mRNA corresponding to the TRPM8 gene. Selected from, An activator for use in the treatment of skin pigmentation as described in claim 1.

5. The treatment a) Treatment of hyperpigmentation disorders selected from among moles, solar lentigines, melasma, brown spots, freckles, hyperpigmented neonatal spots, e.g., nevus of Ota, Mongolian spot, café-au-lait spot, pigmented nevi (moles), post-inflammatory hyperpigmentation, post-infection hyperpigmentation, post-vesicular hyperpigmentation, post-burn hyperpigmentation, post-traumatic hyperpigmentation, and postpartum hyperpigmented stretch marks [linear atrophy]. b) Treatment of hyperpigmentation disorders selected from vitiligo, post-inflammatory hyperpigmentation, post-infection hyperpigmentation, post-vesicular hyperpigmentation, post-burn hyperpigmentation, post-traumatic hyperpigmentation, postpartum hyperpigmentation-induced stretch marks [linear atrophy], and post-bleaching hyperpigmentation. That is, An activator for use in the treatment of skin pigmentation according to any one of claims 1 to 4.

6. Use of the activator according to any one of claims 1 to 4 as a cosmetic for the non-therapeutic treatment of skin pigmentation.

7. A cosmetic or medical skin pigmentation composition comprising at least one activator according to any one of claims 1 to 4, and at least one auxiliary agent selected from the group consisting of carriers, excipients, adjuvants, diluents, and disintegrants.

8. A cosmetic or medical skin pigmentation composition for claim 7, wherein the auxiliary agent is selected from the group consisting of liposomes, nanoparticles, carboxymethylcellulose, hydroxyethylcellulose, mineral oil, petrolatum, glycerin, polysorbate 80, hydroxyethyl starch, dextran, and polyethylene glycol.

9. The cosmetic or medical skin pigmentation composition according to claim 7, further comprising at least one other active agent effective in treating skin pigmentation.

10. The cosmetic or medical skin pigmentation composition according to claim 7, wherein the composition is formulated in the form of an ointment, lotion, cream, gel, solution, spray, plaster or sustained-release plaster.

11. A non-therapeutic method for regulating skin pigmentation, comprising administering to a subject an effective amount of at least one activator that activates, enhances, inactivates, blocks, or attenuates the cellular response of the transient receptor potential ion channel TRPM8, or interferes with the expression of the said ion channel.

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