Active agents that modulate the activity of ion channels for use in modulating skin pigmentation - Patents.com

Abstract

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 TRPM5 or interfere with the expression of said ion channel.

Description

[Technical field]

[0001] The present invention is directed to active agents for use in regulating skin pigmentation, in particular for use in treating skin pigmentation conditions and disorders, which modulate the activity of an ion channel (including but not limited to activating, enhancing, inactivating or blocking the ion channel, or attenuating cellular responses induced by the ion channel, or interfering with the expression of the ion channel).

[0002] Furthermore, the present invention is directed to a composition for cosmetic or pharmaceutical use in the treatment of skin pigmentation, comprising 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).

[0003] Additionally, the present invention is directed to non-therapeutic methods of regulating skin pigmentation, in which 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) is administered to a subject. [Background technology]

[0004] Melanin is the skin's natural protection against ultraviolet radiation. It is the primary determinant of skin and hair color and is generated, produced and distributed in the skin through a process called melanogenesis. Melanin is first synthesized within intracellular organelles (melanosomes) in melanocytes and then delivered to neighboring keratinocytes. Strictly speaking, pigmentation encompasses several steps, including melanin synthesis itself as well as the transport and processing of melanin (mainly to and within epidermal keratinocytes), which underpins skin and hair color. Exposure to ultraviolet (UV) radiation is strongly associated with skin pigmentation. UV-induced DNA damage stimulates melanin production via upregulation and activation of tyrosinase, the enzyme that catalyzes the rate-limiting step of melanin synthesis.

[0005] Hyperpigmentation due to melanin overproduction is responsible for a variety of skin pigmentation conditions / disorders, including (but not limited to) lentigines (aging spots), solar lentigines (dark spots due to chronic sun exposure), melanosis (larger dark spots), melasma (skin discoloration caused by hormones), freckles (egg spots), (hyper)pigmented birthmarks such as nevus of Ota, Mongolian spots, cafe au lait spots, pigmented nevi or moles, 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 hyperpigmented stretch marks [striae distensae]), and dark spots of any kind. Treatment of these conditions / disorders, as well as treatment of individuals who claim to have a "darker than desired" skin color (and therefore intend to change to a lighter color), includes skin lightening products and techniques.

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

[0007] In addition, certain phenolic compounds (e.g., hydroquinone and derivatives) have also been claimed to be effective in treating hyperpigmentation because they have been shown to inhibit melanocyte tyrosinase, a key enzyme in melanin production. However, their widespread use is hindered by their widespread chronic side effects (e.g., contact dermatitis, sclera and nail hyperpigmentation, impaired wound healing) and there are significant safety concerns because hydroquinone has been shown to cause DNA damage and may therefore increase the risk of carcinogenesis.

[0008] On the other hand, generalized or localized suppression or depletion of skin melanin pigment is a feature of many hypopigmented skin conditions / disorders, including (but not limited to) vitiligo (a hypopigmented condition characterized by patches of depigmentation 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, postpartum hypopigmented stretch marks, post-bleaching hypopigmentation), and any type of white or light macules. Treatment of these conditions, as well as treatments for individuals who claim to have a "whiter or lighter than desired" skin color (and therefore intend to change it to a darker color), include skin darkening and / or "UV-free tanning" products and techniques.

[0009] Although not life-threatening complications, the above mentioned skin pigmentation conditions / disorders can result in severe psychological distress to the affected individuals.

[0010] Besides classical UV irradiation to induce photoprotective tanning of the skin, and topically applied "artificial tanning" substances that mimic natural pigmentation (e.g., dihydroxyacetone, melanins), the most extensively tested agents are analogs of melanocyte-stimulating hormone (MSH). For example, afamelanotide / melanotan, an α-MSH mimetic peptide, was shown to induce skin pigmentation in humans. However, afamelanotide / melanotan has raised great concern because it was found to induce the development / rash of melanocytic nevi, thereby increasing the risk of developing malignant melanoma. Summary of the Invention [Problem to be solved by the invention]

[0011] In light of the above, there is a continuing need for active agents for actively controlling and / or treating skin pigmentation in a subject without undesirable side effects. [Means for solving the problem]

[0012] The present inventors have discovered that the transient receptor potential ion channel TRPM5 is expressed in the basal layer of the epidermis, where melanin-producing melanocytes are located.

[0013] "Ion channels" are pore-forming proteins located in biological membranes. Through the protein pores, ions can pass through the membrane down their electrochemical gradient. By opening and closing the pore, ion channels can gate and control the flow of ions across the membrane, thereby modulating the intracellular concentration of the ion. Changes in intracellular ion concentrations affect numerous cellular responses and processes, including, but not limited to, growth, differentiation, survival, death, mediator release, immune mechanisms, and the like.

[0014] "Transient receptor potential channels" (TRP channels) are a group of ion channels located primarily on the plasma membrane, and there are about 30 TRP channels, including TRPC, TRPV, TRPM, TRPN, TRPA, TRPP, and TRPML. The ion channels are relatively nonselectively permeable to cations, including sodium, calcium, and magnesium.

[0015] "TRPM5" is the official gene symbol for "transient receptor potential cation channel subfamily M member 5" and identifies the protein encoded by the TRPM5 gene in humans (NCBI Gene ID: 29850; HGNC: 14323; NCBI mRNA sequence: NM_014555.3; NCBI protein sequence: NP_055370.1; Status on June 7, 2020.).

[0016] The present inventors have found that the pheromone 2,5-dimethylpyrazine (hereinafter referred to as "DMP"), an agonist of the transient receptor potential ion channel TRPM5, opening the channel, promotes human skin (epidermal) pigmentation ex vivo. Indeed, when applied topically to human ex vivo skin organotypic cultures (a suitable model for assessing multiple features of epidermal melanin production and melanin transfer), DMP upregulated epidermal melanin content, the most important marker of melanin production, as demonstrated by two independent histochemical techniques.

[0017] Furthermore, as revealed by quantitative histomorphometry, topical DMP treatment also increased the levels of a pre-melanosomal protein known to be involved in the melanosome transport compartment and contribute to the functional integrity and assembly of melanosomes and thus participate in de novo melanin synthesis, namely gp100 (also known as Pmel17 or Silver locus protein).Furthermore, topical DMP treatment also increased the intraepidermal activity of tyrosinase, the rate-limiting enzyme in melanin production, as revealed by in situ enzymatic assays.

[0018] Conversely, inhibition of TRPM5 with the selective inhibitor triphenylphosphine oxide (TPPO) consistently prevented the propigmentation effects of DMP.

[0019] In addition to the active agents described above, the present inventors have identified several further active agents which act on TRPM5 and are therefore suitable for use in the treatment of skin pigmentation.

[0020] In conclusion, the present invention features an active agent for use in the treatment of skin pigmentation, which activates, enhances, inactivates or blocks TRPM5, or attenuates cellular responses induced by the transient receptor potential ion channel TRPM5, or interferes with the expression of said ion channel.

[0021] According to an alternative of the present invention, the active agent that activates, enhances, inactivates or blocks the ion channel or attenuates the cellular response induced by the transient receptor potential ion channel TRPM5 is either an agonist or an antagonist of the ion channel.

[0022] As referred to herein, an "agonist" of the transient receptor potential ion channel TRPM5 is a substance that binds to and activates or enhances the ion channel, thereby increasing a cellular response associated with TRPM5.

[0023] As referred to herein, an "antagonist" of the transient receptor potential ion channel TRPM5 is a substance that binds to said ion channel and blocks TRPM5, thereby attenuating the cellular response to an agonist rather than activating or enhancing it as an agonist does.

[0024] An "inverse agonist" of the transient receptor potential ion channel TRPM5 is a substance that binds to said ion channel and then induces a cellular response opposite to that seen when an agonist is applied.

[0025] In the present invention, the term "cellular response" should be understood as the change in ion concentration in cells that is the result of agonist / antagonist / inverse agonist binding to the transient receptor potential ion channel TRPM5.However, this term also includes the case where agonist / antagonist / inverse agonist binding induces cellular response without changing intracellular ion concentration.

[0026] Thus, agonists of the present invention of the transient receptor potential ion channel TRPM5 activate or enhance the ion channel to produce a cellular response as do endogenous agonists, whereas antagonists of the present invention of the ion channel block or attenuate the ion channel to produce a cellular response that is normally activated / enhanced by the endogenous agonist, and inverse agonists of the present invention modulate the ion channel to produce the opposite cellular response to that normally activated / enhanced by the endogenous agonist.

[0027] In embodiments where the active agent is an agonist of the transient receptor potential ion channel TRPM5, the active agent is used to treat hypopigmentation conditions. In embodiments where the active agent is an antagonist / inverse agonist of the transient receptor potential ion channel TRPM5, the active agent is used in the treatment of hyperpigmentation conditions.

[0028] In a particular embodiment of the invention, the ion channel agonist / antagonist / inverse agonist used is a TRPM5 agonist selected from the group consisting of dimethylpyrazine, dimethylethylpyrazine, tetramethylpyrazine, 2-heptanone, eugenol, SID2848719 (CAS number 702636-90-6, SMILES, NC(=O)C1(CCN(CC1)S(=O)(=O)c1ccc2OCCCOc2c1)N1CCCCC1), rutamarin, bergapten, xanthotoxin, isopimpinellin, carbachol, 3-deoxyglucosone, glucagon-like peptide 1, (E)-N-(3,4 dimethoxybenzylidene)-2-naphthalen-1-yl)acetohydrazide, or a combination thereof.

[0029] In another embodiment of the invention, the ion channel agonist / antagonist / inverse agonist used is a TRPM5 antagonist / inverse agonist selected from triphenylphosphine oxide, econazole, miconazole, chlorpromazine, or a combination thereof.

[0030] It cannot be excluded that there exist prior art skin pigmentation treatment compositions (although the inventors are not aware of any such examples) that contain one of the specific active agents of the present invention as an ingredient, but do not attribute any effect on TRPM5 to said ingredient. In this case, such accidental antecedent active agents are to be excluded from the scope of the present invention by a corresponding disclaimer. Except for the exclusion of said accidental antecedent specific active agents, said disclaimer has no further effect on the scope of the claims.

[0031] Non-limiting examples of active agents that have inadvertently preceded the prior art include: - chemical compounds which are not attributable to any medical, pharmacological or biological activity by the corresponding prior art; - chemical compounds which, in comparison with the specific inventive active agents disclosed herein, are ascribed by the corresponding prior art to a different medical, pharmacological or biological activity, and / or - chemical compounds which, compared with a particular inventive active agent disclosed herein, are attributed by the corresponding prior art to the same medical, pharmacological or biological activity, but which are not directed against TRPM5, but against another target (e.g. receptor, enzyme, hormone, metabolite).

[0032] Alternatively, the TRPM5 agonist is an aptamer that binds to the TRPM5 ion channel and activates or enhances the ion channel to generate a cellular response, or the TRPM5 antagonist / inverse agonist is an aptamer that binds to the TRPM5 ion channel and inactivates, blocks or attenuates the ion channel to generate a cellular response.

[0033] The term "aptamer" as used herein refers to a DNA, RNA or XNA oligonucleotide or peptide molecule that binds to a specific target molecule, such as an ion channel molecule.

[0034] According to one alternative of the invention, the active agent interfering with the expression of the ion channel TRPM5 is either an miRNA, an siRNA or a ribozyme targeting the TRPM5 gene or targeting the mRNA corresponding to the TRPM5 gene.

[0035] The term "miRNA" refers to microRNAs, small non-coding RNA molecules that contain 21-23 nucleotides and 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 truncation, destabilization and / or inefficient translation of the mRNA.

[0036] The term "siRNA" refers to small interfering RNA, a small non-coding RNA molecule of 20-25 base pairs in length. siRNAs interfere with the expression of specific genes with which they have complementary nucleotide sequences by degrading the mRNA after transcription, thereby preventing translation.

[0037] In 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 TRPM5. As used herein, the phrase "selectively reduces or inhibits" refers to an miRNA, siRNA or ribozyme that affects the expression of TRPM5.

[0038] In certain embodiments of the present invention, the miRNA or siRNA interferes with gene expression of the ion channel TRPM5 by hybridizing to the gene transcript, i.e., TRPM5 mRNA, under stringent conditions, where 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., <60°C for 2 hours) and / or low salt content (e.g., 0.1xSSC).

[0039] According to the present invention, one of the above active agents is used in the treatment of skin pigmentation. In certain embodiments of the present invention, the treatment is performed locally, i.e. in, on or at the skin to be treated. In some embodiments of the present invention, the treatment is performed topically, where the term "topical" refers to a preparation that is applied to a specific location on the skin. In certain embodiments, topical application is on the skin, 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 preparation that is applied beyond the stratum corneum to deeper skin layers, 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 a preparation that is applied by penetrating the skin through skin appendage structures (e.g. hair follicles, sebaceous glands and sweat glands) to deeper skin layers.

[0040] As used herein, the term "treatment" refers to any action that results in a change in a physical condition. In particular, "treatment of skin pigmentation" refers to the treatment of early skin pigmentation conditions, such as lentigines (aging spots), solar lentigines (dark spots due to chronic sun exposure), melanosis (larger dark spots), melasma (skin discoloration caused by hormones), freckles (egg spots), (hyper)pigmented birthmarks, such as nevus of Ota, Mongolian spots, cafe au lait spots, pigmented nevi or pigmented moles), hyperpigmentation as a result of skin injury (e.g., post-inflammatory hyperpigmentation, post-infectious hyperpigmentation, post-blister hyperpigmentation, post-burn hyperpigmentation, post-traumatic hyperpigmentation, post-partum hyperpigmented stretch marks [striae distensae]), any kind of dark spot, "darker than desired" skin color, vitiligo (a hypopigmentation condition characterized by patches of depigmentation across 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 kind of white or light spot, and any change in "whiter or lighter than desired" skin color. In other words, the present invention is directed to any kind of skin pigmentation modulation.

[0041] In a particular embodiment of the present invention, the above active agent is used as a cosmetic agent in the treatment of skin pigmentation.In particular, the cosmetic use is carried out non-therapeutically, but to achieve a change in an initial skin pigmentation condition, such as an undesirable skin color (darker or lighter / whiter than desired), where said initial condition is not caused by a disease or disorder.

[0042] In those embodiments in which the above-mentioned active agents are used cosmetically, the active agents 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 upon application to the skin.

[0043] In another particular embodiment of the present invention, the above-mentioned active agent is used as a medicament in the topical treatment of a skin pigmentation disorder, wherein the term "disorder" refers to any functional abnormality or disturbance of normal health and the term "medicine" refers to a substance useful in the cure, treatment or prevention of the condition of the disorder.

[0044] In those embodiments in which the above-mentioned active agents are used as pharmaceuticals, the active agents used should be pharma- ceutically acceptable, where "pharmaceutically acceptable" means that the active agent should not be toxic or harmful or have other adverse side effects upon application to the skin.

[0045] In some embodiments, at least one of the active substances of 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 selected from the group consisting of carriers, excipients, adjuvants, diluents and disintegrants.

[0046] In certain embodiments of such compositions, 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.

[0047] In the composition of the present invention, the concentration of the active agent is usually in the range of 10 to 10,000 μM. In some embodiments, the lower limit of the concentration of the active agent is 30 μM or even 100 μM. In some embodiments, the upper limit is 3,000 μM or 1,000 μM. This gives preferred ranges such as 30 to 10,000 μM, 30 to 3,000 μM, 10 to 3,000 μM, 100 to 3,000 μM, etc.

[0048] Based on the total weight of the composition of the present invention, the concentration of the active agent may vary in certain embodiments within the range of 1-30% 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 5-30% by weight, 5-25% by weight, 10-25% by weight, 10-30% by weight, etc.

[0049] In certain embodiments, such compositions further comprise at least one other active agent that is effective in the treatment of skin pigmentation.

[0050] In such an embodiment, the other active agent may be selected from one of the prior art skin pigmentation agents, such as those mentioned in the introduction.

[0051] Generally, the composition of the present invention can be used in any preparation / formulation suitable for treating skin pigmentation.In a particular embodiment of the present invention, the composition is formulated in the form of an ointment, lotion, cream, gel, spray, plaster or sustained release plaster.In another particular embodiment of the present invention, the composition is formulated in the form of a solution.The solution may be applied by a microneedle device or prior to ultrasonication, electrical stimulation, etc.

[0052] As mentioned above, the inventive use of the above active agents can also be carried out non-therapeutic, where non-therapeutic refers to treatments that are not directed to curing, treating or preventing a disorder condition (see above).

[0053] Thus, the present invention is further directed to non-therapeutic methods of regulating skin pigmentation, in which an effective amount of at least one of the above-mentioned active agents is administered to a subject.

[0054] The non-therapeutic methods also include embodiments in which the above-mentioned active agents are administered simultaneously, sequentially or separately to the subject to be treated together with at least one other active agent useful for the treatment of skin pigmentation (see above).

[0055] The following examples illustrate some of the features of specific embodiments of the present invention, however, 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.

[0056] In describing the examples, reference is made to the following drawings: [Brief description of the drawings]

[0057] FIG. 1 includes two microscopic images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases epidermal melanin content in human skin organotypic cultures.

[0058] FIG. 2 contains two microscopic images and a graph showing that 2,5-dimethylpyrazine (DMP) increases epidermal melanin content in human skin organotypic cultures.

[0059] FIG. 3 contains two microscopic images and a graph showing that 2,5-dimethylpyrazine (DMP) increases the expression of gp100, a melanosome-melanin production marker, in human skin organotypic cultures.

[0060] FIG. 4 contains two microscopic images and a graph showing that 2,5-dimethylpyrazine (DMP) increases tyrosinase activity, a marker of melanogenesis, in human skin organotypic cultures.

[0061] FIG. 5 includes three microscopic images and a graph showing that triphenylphosphine oxide (TPPO) blocks the effect of 2,5-dimethylpyrazine (DMP) to increase epidermal melanin content in human skin organotypic cultures.

[0062] FIG. 6 contains three microscopic images and a graph showing that triphenylphosphine oxide (TPPO) blocks the effect of 2,5-dimethylpyrazine (DMP) to increase expression of gp100, a melanosomal-melanogenic marker, in human skin organotypic cultures.

[0063] FIG. 7 contains three microscopic images and a graph showing that triphenylphosphine oxide (TPPO) blocks the effect of 2,5-dimethylpyrazine (DMP) to increase tyrosinase activity, a marker of melanin production, in human skin organotypic cultures. EXAMPLES

[0064] example 1. Ex vivo Skin Organ Culture Model for Assessing Skin Pigmentation Human abdominal, temporal and occipital skin samples were obtained from healthy adult subjects during elective plastic surgery performed for cosmetic reasons, with written patient consent and ethical approval. 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 E medium supplemented only with insulin, hydrocortisone and L-glutamine, as described above.

[0065] For topical application of the TRPM5 activator 2,5-dimethylpyrazine (DMP) (Sigma), stocks were prepared in ethanol and dissolved in a 50% (v / v) solution of isopropanol in PBS containing 1 g / mL polyethylene glycol 6000 at a final concentration of 12.5 mM at 37° C. For topical application of triphenylphosphine oxide (TPPO) (Sigma) and TRPM5 inhibitors, stocks were prepared in ethanol and dissolved in a 50% (v / v) solution of isopropanol in PBS containing 1 g / mL polyethylene glycol 6000 at a final concentration of 1.5 mM at 37° C. Drops of 4 μL of this viscous solution containing DMP (12.5 mM), TPPO (1.5 mM), their combination, or ethanol as a vehicle control were applied daily (up to 3 days) to the epidermis of skin biopsies.

[0066] 2. Activation of TRPM5 using 2,5-dimethylpyrazine (DMP) Histochemical visualization of melanin in skin sections was performed using the Warthin-Starry staining kit (Abcam) or Fontana-Masson histochemistry. For the Warthin-Starry histochemistry, formalin-fixed human skin / HF cryosections were rinsed in distilled water and incubated with AgNO3 (0.5% in acidified water) for 30 s at 50 °C. The samples were then incubated with developer (0.42% AgNO3, 5% gelatin and 0.15% hydroquinone in acidified water) and incubated for 2–5 min at 50 °C. The reaction was stopped by successive rinses in hot (60 °C) tap water for 2 min. Incubation with Na2S2O3 (5% in distilled water) for 1 min, followed by successive washes in tap water for 3 min, can also be used to remove unbound AgNO3 and to tone the black color of melanin to a brownish hue. A simple counterstain with hematoxylin was used to visualize the nuclei. After dehydration, the samples were (登録商標) was embedded in.

[0067] Images were then obtained using a digital microscope (Keyence). Quantitative histomorphometric analysis was performed by evaluating the relative intensity and area of ​​melanin staining in standardized reference areas using ImageJ (NIH; Bethesda, MD, USA). Briefly, for each parameter evaluated, multiple non-consecutive skin sections (at least 280 μm apart from each other) were stained per punch biopsy. Two skin punch biopsies were analyzed per experimental group unless otherwise stated. Melanin content was then calculated based on the product of the percentage of area occupied by melanin and the average grey shade of melanin, as described in detail elsewhere. Evaluation was performed in multiple different microscopic fields per section. For all parameters, the entire epidermis (or only the basal layer) was used for the measurements. Analysis was performed in the center of the skin biopsy, at least 500 μm away from the edges (as these sites show significant wound healing processes such as re-epithelialization).

[0068] Figure 1 shows representative images (a, b) and quantitative histomorphometric analysis (c) of histochemical visualization of melanin in skin sections using the Warthin-Starley technique. It can be seen from Figure 1 that topical application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) significantly increased epidermal melanin content after 3 days in human skin organotypic cultures compared to vehicle. Furthermore, Figure 1 also shows that DMP treatment increased melanin content not only in the basal layer (i.e., the epidermal compartment where melanocytes reside) but also in suprabasal layers, suggesting increased melanosome migration upon TRPM5 activation by DMP.

[0069] For Fontana-Masson staining, ethanol / acetic acid fixed human skin / HF cryosections were rinsed with two and three washes of Tris-buffered saline and distilled water, respectively. Samples were then incubated with AgNO3 (5% in distilled water) for 40 min at 56 °C in the dark. Samples were rinsed three times with distilled water and then incubated with Na2S2O3 (5% in distilled water) for 1 min at room temperature. A simple counterstain with hematoxylin was used to visualize nuclei. After dehydration, samples were stained with Eukitt (登録商標) Image acquisition and quantitative histomorphometric analysis were performed as described for the Warthin-Starley technique.

[0070] Figure 2 shows representative images (a, b) and quantitative histomorphometric analysis (c) of histochemical visualization of melanin in skin sections using the Fontana-Masson technique, a complementary technique to the Warthin-Starley technique. From Figure 2, it can be confirmed that topical application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) significantly increases epidermal melanin content after 3 days in human skin organotypic cultures compared to vehicle, thereby further supporting the pro-pigmentation effect of TRPM5 activation. Furthermore, Figure 2 also shows that DMP treatment increases melanin content not only in the basal layer (i.e., the epidermal compartment where melanocytes reside) but also in suprabasal layers, suggesting increased melanosome migration upon TRPM5 activation by DMP.

[0071] For detection of gp100 (known to be involved in the melanosome transport compartment and contribute to the functional integrity and assembly of melanosomes and thus 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 successive rinses in PBS, samples were incubated with rhodamine-conjugated secondary antibody (Jackson ImmunoResearch) diluted 1:200 in normal goat serum (2% in PBS) for 45 min at room temperature. After rinsing in PBS, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) (SouthernBiotech) on glass slides. Image acquisition and quantitative histomorphometric analysis were performed as described for the Warthin-Starley technique, 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).

[0072] Representative images (a, b) and quantitative histomorphometric analysis (c) of immunohistochemical visualization of gp100-specific IR in skin sections are shown in Figure 3. It can be seen that topical application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) significantly increased the intensity levels of gp100-specific IR in the basal epidermal layer after 3 days in human skin organotypic cultures compared to vehicle, thereby further supporting the pro-pigmentation effect of TRPM5 activation.

[0073] 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 H2O2 (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 determined by TSA block. 商標 Detection was performed using the Biotin System (Akoya Biosciences).

[0074] Briefly, samples were incubated with biotin tyramide (1:50 in Amplification Diluent) and then subjected to IGEPAL (登録商標) Successive rinses were performed with CA-630 (Sigma, 0.1% in PBS) and PBS. For visualization of cross-linked biotin in the vicinity of tyrosinase activity, samples were incubated with streptavidin-Cy3 (Sigma, 1:600 ​​in 5% BSA in PBS) for 1 h. GEPAL (登録商標) After further rinsing with CA-630 (0.1% in PBS) and PBS, samples were counterstained with DAPI and Fluoromount-G (登録商標) (SouthernBiotech) was used to mount the samples on slides.

[0075] Image acquisition and quantitative histomorphometric analysis were performed similarly as described for the Warthin-Starley technique, 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).

[0076] Representative images (a, b) and quantitative histomorphometric analysis (c) of histochemical visualization of tyrosinase activity in skin sections are shown in Figure 4. It can be seen that topical application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) significantly increased the intensity level of tyrosinase activity in the basal epidermal layer after 3 days in human skin organotypic cultures compared to vehicle, thereby further supporting the pro-pigmentation effect of TRPM5 activation.

[0077] 3. Inactivation of TRPM5 using triphenylphosphine oxide (TPPO) Figure 5 shows representative images (a, b, c) and quantitative histomorphometric analysis (d) of histochemical visualization of melanin in skin sections using the Fontana-Masson technique. From Figure 5, it can be seen that the TRPM5 antagonist triphenylphosphine oxide (TPPO, 1.5 mM) prevented the effect of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) in increasing epidermal melanin content after 3 days in human skin organotypic cultures compared to vehicle. This suggests that the pro-pigmentation effect of DMP is mediated by TRPM5.

[0078] Figure 6 shows representative images (a, b, c) and quantitative histomorphometric analysis (d) of immunohistochemical visualization of gp100-specific IR in skin sections. It can be seen from Figure 6 that the TRPM5 antagonist triphenylphosphine oxide (TPPO, 1.5 mM) prevented the effect of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) in increasing gp100-specific IR in the basal epidermal layer after 3 days in human skin organotypic cultures compared to vehicle. This suggests that the pigmentation-promoting effect of DMP is mediated by TRPM5.

[0079] Figure 7 shows representative images (a, b, c) and the results of quantitative histomorphometric analysis (d) of histochemical visualization of tyrosinase activity in skin sections. It can be seen from Figure 7 that the TRPM5 antagonist triphenylphosphine oxide (TPPO, 1.5 mM) prevented the effect of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) in increasing tyrosinase activity in the basal epidermal layer after 3 days in human skin organotypic cultures compared to vehicle. This suggests that the pro-pigmentation effect of DMP is mediated by TRPM5.

Claims

1. An active agent for use in the treatment of skin pigmentation, which activates, enhances, inactivates, blocks or attenuates the cellular response of the transient receptor potential ion channel TRPM5 or interferes with the expression of said ion channel.

2. 2. An active agent for use in the treatment of skin pigmentation according to claim 1, which is an agonist of the transient receptor potential ion channel TRPM5 for use in the treatment of hypopigmentation.

3. 2. An active agent for use in the treatment of skin pigmentation according to claim 1, which is an antagonist / inverse agonist of the transient receptor potential ion channel TRPM5 for use in the treatment of hyperpigmentation.

4. a) any one of the TRPM5 activating agonists dimethylpyrazine, dimethylethylpyrazine, 2-heptanone, SID2848719 (CAS No. 702636-90-6), rutamarin, xanthotoxin, isopimpinellin, carbachol, 3-deoxyglucosone, (E)-N-(3,4-dimethoxybenzylidene)-2-naphthalen-1-yl)acetohydrazide, or a combination thereof, or an aptamer that binds to TRPM5 and activates or enhances the ion channel to generate a cellular response; or b) TRPM5 inactivation antagonists / inverse agonists triphenylphosphine oxide, econazole, miconazole, chlorpromazine, or any one or combination thereof, or an aptamer that binds to TRPM5 and inactivates, blocks, or attenuates the ion channel to generate a cellular response; or c) selected from miRNA, siRNA, or ribozymes that target the TRPM5 gene or target the mRNA corresponding to the TRPM5 gene; 10. An active agent for use in the treatment of skin pigmentation according to claim 1.

5. The treatment a) for the treatment of a hyperpigmentation disorder selected from lentigines, solar lentigines, melanosis, melasma, freckles, hyperpigmented birthmarks such as nevus of Ota, Mongolian spots, cafe au lait spots, pigmented nevi or moles, post-inflammatory hyperpigmentation, post-infectious hyperpigmentation, post-blister hyperpigmentation, post-burn hyperpigmentation, post-traumatic hyperpigmentation, post-partum hyperpigmented stretch marks [striae distensae], or b) for treating hypopigmentation disorders selected from vitiligo, post-inflammatory hypopigmentation, post-infectious hypopigmentation, post-blister hypopigmentation, post-burn hypopigmentation, post-traumatic hypopigmentation, post-partum hypopigmented stretch marks [striae distensae], and post-bleaching hypopigmentation; An active agent for use in the treatment of skin pigmentation according to any one of claims 1 to 4.

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

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

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

9. 8. The cosmetic or medical skin pigmentation composition of claim 7, further comprising at least one other active agent effective in the treatment of skin pigmentation.

10. 8. The cosmetic or medical skin pigmentation composition of claim 7, 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, wherein an effective amount of at least one active agent that activates, enhances, inactivates, blocks or attenuates the cellular response of the transient receptor potential ion channel TRPM5 or interferes with the expression of said ion channel is administered to a subject.

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