Active agents modulating the activity of ion channels for use in inhibiting skin aging - Patents.com

Abstract

In order to provide novel active agents for treating skin aging in a subject without undesirable side effects, the present invention discloses active agents that activate, enhance or positively modulate the cellular response of the transient receptor potential ion channel TRPM5 or increase the expression of said ion channel.

Description

[Technical field]

[0001] The present invention is directed to active agents for use in inhibiting skin aging, in particular for use in treating premature or pathological skin aging conditions, which modulate the activity of an ion channel (including but not limited to, activating, enhancing or positively modulating cellular responses induced by the ion channel) or increase the expression of said ion channel.

[0002] Furthermore, the present invention is directed to a composition for cosmetic or pharmaceutical use in the treatment of premature or pathological skin aging conditions, comprising at least one active agent that modulates the activity of an ion channel (including, but not limited to, activation, enhancement or positive modulation of a cellular response induced by the ion channel) or increases the expression of said ion channel.

[0003] Furthermore, the present invention is directed to a non-therapeutic method of inhibiting skin aging, in which an effective amount of at least one active agent that modulates the activity of an ion channel (including, but not limited to, activation, enhancement or positive modulation of a cellular response induced by the ion channel) or increases the expression of said ion channel is administered to a subject. [Background technology]

[0004] The skin, the largest organ of the human body, establishes a barrier that insulates and protects the body from the environment. Like all other organs, the skin also exhibits a continuous, lifelong and time-dependent aging process controlled by numerous intrinsic and extrinsic factors. Intrinsic aging, which is mainly seen in areas not exposed to sunlight (e.g., the inside of the arms), is mainly due to intrinsic genetic factors and age-dependent perturbations of tissue and cell metabolism. However, extrinsic aging is induced and enhanced by a plethora of environmental factors, such as chemical exposure (e.g., air pollution, improper use of skin agents), smoking, poor or impaired nutrition, etc. Most importantly, the major driver of extrinsic skin aging is chronic lifelong exposure to solar ultraviolet (UV) radiation, i.e., photoaging.

[0005] Skin aging is characterized by multiple phenomenological and morphological changes, such as thin, dry, fragile, sensitive skin, fine or coarse wrinkles, progressive atrophy affecting both the epidermis and the dermis, loss of elasticity and laxity, reduced skin hydration, coarse texture, impaired and delayed wound regeneration, etc. These changes are determined by specific changes in the expression and / or activity of various molecules and signaling pathways induced by endogenous or exogenous pro-aging factors.

[0006] Among the increasing number of "aging markers", skin aging at the cellular and molecular level is characterized, for example, by epidermal thinning, reduced proliferation of epidermal keratinocytes, a decrease in the size of the epidermal stem cell pool resulting in impaired regenerative capacity, degradation (and therefore reduced content) of fibrous and non-fibrous extracellular matrix components (including, but not limited to, collagen, elastin, fibrillin and oligosaccharides), and changes in the expression / activity of molecules of cell metabolism, survival, stress resistance and inflammation. Moreover, these "aging markers" are routinely evaluated in preclinical and clinical situations when the efficacy and applicability of new anti-aging active substances are tested.

[0007] Importantly, skin aging, especially in premature or pathologically accelerated cases, not only constitutes a clear cosmetic problem, but also leads to the premature onset and / or aggravation of a plethora of skin diseases that require immediate and precise dermatological treatment. These include, but are not limited to, rosacea (redness, papules, bumps, and fine, superficial, dilated blood vessels that affect especially the face), asteatotic eczema (characterized by dry, itchy, cracked skin), nummular eczema (characterized by nummular, itchy, reddish patches), seborrheic dermatitis, skin infections, actinic keratosis (precancerous skin lesions associated with chronic ultraviolet radiation), and benign (non-cancerous) and malignant (basal cell carcinoma, squamous cell carcinoma) skin tumors. Thus, the proper management of skin aging, especially premature and pathologically accelerated skin aging, usually by cosmetic active substances and formulations / preparations and medical devices, has clear dermatological benefits.

[0008] Currently, there are several active substances on the market that claim to be useful for managing skin aging.Some of said active substances belong to the group of antioxidants, and include certain vitamins (e.g. vitamin C, α-tocopherol or vitamin E, niacinamide), terpenes and polyphenols.Because skin aging is overwhelmingly a more complex phenomenon than can be simplified to the level of diminished antioxidant properties (induced by endogenous or exogenous factors), the disadvantage of said antioxidants is that they do not specifically target or only slightly target the cellular and molecular processes of skin aging, and therefore their anti-aging effect is limited.

[0009] In addition, certain derivatives of vitamin A, namely retinoids, have also been claimed to be effective in managing skin aging, however their widespread use is usually hindered by their wide range of chronic adverse effects (e.g. irritation, redness, dryness, peeling, increased eczema, increased photosensitivity to UV light) and there are significant safety concerns, as retinoids have been shown to cause DNA damage and therefore may increase the risk of carcinogenesis.

[0010] In light of the above, there is a continuing need for active agents for actively controlling and / or treating skin aging in a subject without undesirable side effects.

[0011] "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, proliferation, differentiation, survival, death, mediator release, immune mechanisms, and the like.

[0012] "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.

[0013] "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.). Summary of the Invention

[0014] The inventors of the present application have discovered that the naturally occurring pheromone 2,5-dimethylpyrazine (hereinafter referred to as "DMP") (an agonist of the transient receptor potential ion channel TRPM5, opening the channel) exerts a powerful anti-aging effect.

[0015] Indeed, when applied topically or systemically to human ex vivo skin organ cultures or in vitro cultures of human epidermal keratinocytes - complementary models suitable for assessing multiple aspects of the skin aging process - DMP upregulated the expression of multiple "skin aging markers" whose levels were found to be downregulated in aging skin. Specifically, as revealed by quantitative immunohistomorphometry and, in certain cases, gene expression profiling, DMP treatment increased the expression of collagen 17A (an epidermal basement membrane and extracellular matrix component), cytokeratin 15 (a marker of epidermal stem cells and proliferation), Ki67 (a nuclear non-histone protein ubiquitously expressed across proliferating cells), phosphohistone H3 Ser10 (pH3S10) (phosphorylation of histone H3 at Ser10 tightly correlates with chromosome condensation during mitosis and cell proliferation), Lamin B1 (a regulator of cell survival, senescence and aging), peroxisome proliferator-activated receptor-coactivator-1α (PGC1α, a transcriptional coactivator that regulates genes involved in energy metabolism and mitochondrial biogenesis), and Sirtuin-1 (an NAD-dependent protein deacetylase that directly links transcriptional regulation to intracellular energetics, DNA damage, metabolism, apoptosis and autophagy).

[0016] Conversely, inhibition of TRPM5 with the selective inhibitor triphenylphosphine oxide (TPPO) consistently prevented the anti-aging effects of DMP.

[0017] In addition to the active agents mentioned above, the inventors have identified several further active agents which act on TRPM5 and are therefore suitable for use in the treatment of skin aging.

[0018] In conclusion, the present invention features an active agent for use in the treatment of skin aging, which activates, enhances, positively modulates cellular responses induced by the transient receptor potential ion channel TRPM5 or increases the expression of said ion channel.

[0019] According to one option of the invention, said active agent that activates, enhances or positively modulates the cellular response induced by the transient receptor potential ion channel TRPM5 is an agonist of said ion channel.

[0020] 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.

[0021] With respect to the transient receptor potential ion channel TRPM5 of the present invention, the term "cellular response" should be primarily understood as the change in ion concentration in a cell that is the result of agonist binding to the transient receptor potential ion channel TRPM5.However, this term also includes the case where agonist binding induces a cellular response without changing intracellular ion concentration.

[0022] Thus, the present agonists of the transient receptor potential ion channel TRPM5 activate or potentiate the ion channel to generate a cellular response similar to an endogenous agonist.

[0023] In embodiments where the active agent is an agonist of the transient receptor potential ion channel TRPM5, said active agent is used to treat skin aging and related skin conditions.

[0024] In certain embodiments of the invention, the TRPM5 agonist is 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.

[0025] It cannot be excluded that there exist prior art skin aging 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 mentioned above 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.

[0026] 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).

[0027] Alternatively, a TRPM5 agonist is an aptamer that binds to the TRPM5 ion channel and activates or enhances the ion channel to generate a cellular response.

[0028] 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.

[0029] According to the present invention, one of the above active agents is used in the treatment of skin aging. In certain embodiments of the present invention, the treatment is performed locally, 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 / preparation that is applied to a specific location on the skin. In certain embodiments, topical application is on the skin, meaning that the agonist 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 / 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 formulation / 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.

[0030] As used herein, the term "treatment" refers to any action that results in a change in physical condition. In particular, "treatment of skin aging" refers to any change in initial skin condition, including (but not limited to) thin, dry, fragile, sensitive skin, fine or coarse wrinkles, progressive atrophy affecting both the epidermis and dermis, loss of elasticity and laxity, reduced skin moisture, coarse texture, impaired and delayed wound regeneration. Additionally, "treatment of skin aging" also refers to the prevention or treatment of skin diseases associated with aging, including, but not limited to, rosacea (redness, papules, bumps, and fine, superficial, dilated blood vessels especially affecting the face), asteatotic eczema (characterized by dry, itchy, cracked skin), nummular eczema (characterized by nummular, itchy, reddish patches), seborrheic dermatitis, skin infections, actinic keratosis (precancerous skin lesions associated with chronic ultraviolet radiation), and benign (noncancerous) and malignant (basal cell carcinoma, squamous cell carcinoma) skin tumors.

[0031] In a particular embodiment of the invention, the above active agents are used as cosmetics in the treatment of skin aging. In particular, the cosmetic use is carried out non-therapeutic, but to maintain an initial skin condition, such as a younger or less aged skin, where said initial condition is not caused by a disease or disorder.

[0032] 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.

[0033] In another particular embodiment of the present invention, the above-mentioned active agent is used as a medicine in the topical treatment of a disorder related to skin aging, where 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 disorder condition.

[0034] In those embodiments in which the above-mentioned active agents are used as pharmaceuticals, the active agents used should be pharma- ceutically acceptable, where "pharma-ceutically acceptable" means that the active agent should not be toxic or harmful or have other adverse side effects upon application to the skin.

[0035] 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 aging, said composition further comprising at least one auxiliary selected from the group consisting of carriers, excipients, adjuvants, diluents and disintegrants.

[0036] 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.

[0037] In the composition of the present invention, the concentration of the active agent is usually in the range of 10 nM 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 nM to 3,000 μM, 100 to 3,000 μM, etc.

[0038] 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 0.1-30% 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.

[0039] In certain embodiments, such compositions further comprise at least one other active agent that is effective in the treatment of skin aging.

[0040] In such embodiments, the other active agent may be selected from one of the prior art skin anti-aging agents, such as those mentioned in the introduction.

[0041] Generally, the composition of the present invention can be used in any preparation / formulation suitable for treating skin aging.In a particular embodiment of the present invention, the composition is formulated in the form of ointment, lotion, cream, gel, spray, plaster or sustained release plaster.In another particular embodiment of the present invention, the composition is formulated / prepared in the form of solution.The solution may be applied by microneedle device or prior to ultrasonication, electrical stimulation, etc.

[0042] 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).

[0043] Thus, the present invention is further directed to non-therapeutic methods of regulating skin aging, in which an effective amount of at least one of the above-mentioned active agents is administered to a subject.

[0044] 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 aging (see above).

[0045] 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.

[0046] In describing the examples, reference is made to the following drawings: [Brief description of the drawings]

[0047] [Figure 1] FIG. 1 contains two microscopic images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases epidermal expression of collagen 17A (an epidermal basement membrane and extracellular matrix component whose levels have been shown to decrease with skin aging) in human skin organ cultures. [Diagram 2] FIG. 2 contains two microscopic images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases epidermal expression of cytokeratin 15 (a marker of epidermal stem cells and proliferation, the levels of which have been shown to decrease with skin aging) in human skin organotypic cultures. [Diagram 3] FIG. 3 contains two microscopy images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases epidermal expression of LaminB1 (a regulator of cell survival, senescence and aging, the levels of which have been shown to decrease with skin aging) in human skin organotypic cultures. [Figure 4] FIG. 4 contains two microscopy images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases epidermal expression of peroxisome proliferator-activated receptor-coactivator-1α (PGC1α), a transcriptional coactivator that regulates genes involved in energy metabolism and mitochondrial biogenesis and whose levels have been shown to decrease with skin aging, in human skin organ cultures. [Diagram 5] FIG. 5 contains two microscopy images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases epidermal expression of Sirtuin-1 (an NAD-dependent protein deacetylase that directly links transcriptional regulation to intracellular energetics, DNA damage, metabolism, apoptosis and autophagy and whose levels have been shown to decrease with skin aging) in human skin organ cultures. [Figure 6] FIG. 6 contains two microscopic images and a graph showing that 2,5-dimethylpyrazine (DMP) increases the expression of collagen 17A in human epidermal keratinocyte cultures. [Figure 7] FIG. 7 contains two microscopic images and a graph showing that 2,5-dimethylpyrazine (DMP) increases the expression of cytokeratin 15 in human epidermal keratinocyte cultures. [Figure 8] FIG. 8 contains two graphs showing that 2,5-dimethylpyrazine (DMP) increases gene expression of collagen 17A and cytokeratin 15 in human epidermal keratinocyte cultures. [Figure 9] FIG. 9 contains two microscopy images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases the expression of Ki67, a nuclear non-histone protein that is ubiquitously expressed across proliferating cells and whose levels have been shown to decrease with skin aging, in human epidermal keratinocyte cultures. [Figure 10] FIG. 10 contains two microscopy images and a bar graph showing that 2,5-dimethylpyrazine (DMP) increases the expression of phosphohistone H3 Ser10 (pH3S10; phosphorylation of histone H3 at Ser10 has been shown to closely correlate with chromosome condensation during mitosis and cell proliferation and to decrease with skin aging) in human epidermal keratinocyte cultures. [Figure 11] FIG. 11 includes three microscopic images and a graph showing that triphenylphosphine oxide (TPPO) blocks the effect of 2,5-dimethylpyrazine (DMP) to increase epidermal expression of collagen 17A in human skin organotypic cultures. [Figure 12] FIG. 12 includes three microscopic images and a graph showing that triphenylphosphine oxide (TPPO) blocks the effect of 2,5-dimethylpyrazine (DMP) to increase epidermal expression of Ki67 in human skin organotypic cultures. [Figure 13]FIG. 13 includes three microscopic images and a graph showing that triphenylphosphine oxide (TPPO) blocks the effect of 2,5-dimethylpyrazine (DMP) to increase collagen 17A expression in human epidermal keratinocyte cultures. [Figure 14] FIG. 14 includes three microscopic images and a graph showing that triphenylphosphine oxide (TPPO) blocks the effect of 2,5-dimethylpyrazine (DMP) to increase the expression of cytokeratin 15 in human epidermal keratinocyte cultures. EXAMPLES

[0048] example 1. Ex vivo Skin Organ Culture Model for Assessing Skin Aging Human abdominal, thoracic, 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 penicillin-streptomycin, insulin, hydrocortisone and L-glutamine, as described above.

[0049] To model the direct application of actives to the skin surface (topical application), 2,5-dimethylpyrazine (DMP), a TRPM5 activator, was 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) 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 epidermal surface of skin biopsies. To model the effect of active substances reaching the skin via the bloodstream (systemic application), DMP (12.5 μM) was applied daily (up to 3 days) to the culture medium.

[0050] 2. In vitro culture of normal human epidermal keratinocytes for assessing skin aging Primary adult normal human epidermal keratinocytes (NHEK) were cultured in serum-free Keratinocyte Growth Medium 2 (KGM2) for up to six passages and then cultured at 15–20x10 3 cells / cm 2 The cells were seeded onto glass slides at a density of 1000 μg / mL. The next day, the KGM2 culture medium was replaced and supplemented with the compounds shown below, their combinations or ethanol as a vehicle control.

[0051] For application of active substances to the medium (systemic application), for the TRPM5 activator 2,5-dimethylpyrazine (DMP), DMP stocks were dissolved in KGM2 at a final concentration of 10, 12.5 or 100 nM at 37° C. For the TRPM5 inhibitor triphenylphosphine oxide (TPPO), stocks were prepared in ethanol and dissolved in KGM2 at a final concentration of 50 μM at 37° C. After 24 h, NHEKs were washed with PBS and then fixed in 4% formaldehyde for 10 min at room temperature (immunofluorescence labeling) or dissolved in buffer RLT containing 1% β-mercaptoethanol (for quantitative RT-qPCR).

[0052] 3. Activation of TRPM5 using 2,5-dimethylpyrazine (DMP) For detection of collagen 17A (an epidermal basement membrane and extracellular matrix component whose levels have been shown to decrease with skin aging), formalin-fixed human skin cryosections were incubated overnight at 4°C with anti-collagen 17A antibody diluted 1:100 in antibody diluent. After successive rinses with PBS, samples were incubated for 45 min at room temperature with Alexa fluorophore-conjugated secondary antibody diluted 1:500 in antibody diluent containing 2% normal goat serum. After successive rinses with PBS, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) (SouthernBiotech) was used to mount the samples on slides.

[0053] Images were then obtained using a digital microscope (Keyence). Quantitative immunohistomorphometric analysis was performed by evaluating relative fluorescence or color intensity in standardized reference areas using ImageJ (NIH; Bethesda, MD, USA). Briefly, 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. Evaluation was performed in multiple different microscopic fields per section. 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).

[0054] Figure 1 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of collagen 17A in skin sections. It can be seen that topical application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) significantly increases the expression of collagen 17A in the basal layer of the epidermis after 3 days in human skin organotypic cultures compared to vehicle. Since the expression level of collagen 17A decreases with skin aging, this suggests that DMP exerts an anti-aging effect.

[0055] For detection of cytokeratin 15 (a marker of epidermal stem cells and proliferation, whose levels have been shown to decrease with skin aging), formalin-fixed human skin cryosections were incubated overnight at 4°C with anti-cytokeratin 15 antibody diluted 1:100 in antibody diluent. After successive rinses with PBS, samples were incubated for 45 min at room temperature with Alexa fluorophore-conjugated secondary antibody diluted 1:500 in antibody diluent containing 2% normal goat serum. After successive rinses with PBS, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) (SouthernBiotech) was used to mount the sections on slides. Image acquisition and quantitative immunohistomorphometric analysis were performed as described above.

[0056] Figure 2 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of cytokeratin 15 in skin sections. It can be seen that topical application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) significantly increases the expression of cytokeratin 15 in the basal layer of the epidermis after 3 days in human skin organotypic cultures compared to vehicle. As the expression level of cytokeratin 15 decreases with skin aging, this suggests that DMP exerts an anti-aging effect.

[0057] For detection of Lamin B1 (a regulator of cell survival, senescence and ageing, whose reduced levels have been shown to decrease with skin ageing), formalin-fixed human skin cryosections were permeabilized and blocked with PBS containing 5% BSA, 0.1% Triton and 0.1% saponin for 10 min at room temperature. Samples were then incubated overnight at 4°C with anti-Lamin B1 antibody diluted 1:100 in PBS containing 1% BSA, 0.05% Triton and 0.1% saponin. After sequential rinsing with PBS, samples were incubated for 45 min at room temperature with Alexa fluorophore-conjugated antibody diluted 1:500 in PBS containing 1% BSA, 0.05% Triton and 0.1% saponin. After sequential rinsing with PBS, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) (SouthernBiotech) was used to mount the sections on slides. Image acquisition and quantitative immunohistomorphometric analysis were performed as described above.

[0058] Figure 3 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of LaminB1 in skin sections. It can be seen that systemic application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 μM) significantly increases LaminB1 expression in the epidermis (mainly in the basal layer) after 3 days in human skin organotypic cultures compared to vehicle. Since the expression level of LaminB1 decreases with skin aging, this suggests that DMP exerts an anti-aging effect.

[0059] For detection of peroxisome proliferator-activated receptor-coactivator-1α (PGC1α, a transcriptional coactivator that regulates genes involved in energy metabolism and mitochondrial biogenesis and whose levels have been shown to decrease with skin aging), formalin-fixed human skin cryosections were permeabilized and blocked for 5 min at room temperature with Tris-buffered saline (TBS) containing 0.1% TritonX-100. Samples were rinsed with TBS, blocked for 20 min in TBS containing 10% normal goat serum (NGS), and then incubated overnight at 4°C with anti-PGC1α antibody diluted 1:100 in antibody diluent. After successive rinses with TBS, samples were incubated with fluorophore-conjugated secondary antibodies diluted 1:200 in antibody diluent containing 2% NGS. After successive rinses with PBS, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) (SouthernBiotech) was used to mount the sections on slides. Image acquisition and quantitative immunohistomorphometric analysis were performed as described above.

[0060] Figure 4 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of PGC1α in skin sections. It can be seen that systemic application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 μM) significantly increases the expression of PGC1α in the epidermis after 3 days in human skin organotypic cultures compared to vehicle. Since the expression level of PGC1α decreases with skin aging, this suggests that DMP exerts an anti-aging effect.

[0061] For detection of Sirtuin-1 (an NAD-dependent protein deacetylase that directly links transcriptional regulation to intracellular energetics, DNA damage, metabolism, apoptosis and autophagy and whose levels have been shown to decrease with skin aging), acetone-fixed human skin cryosections were washed with Tris-buffered saline (TBS). Endogenous peroxidase activity was blocked by incubation in TBS containing 3% H2O2 for 15 min at room temperature. Endogenous biotin was blocked using an avidin / biotin blocking kit according to the manufacturer's instructions. Samples were rinsed with TBS and incubated overnight at 4 °C with anti-Sirtuin antibody diluted 1:400 in antibody diluent. After successive rinses with TBS, samples were incubated with biotinylated anti-rabbit secondary antibody diluted 1:200 in antibody diluent. After successive rinses with TBS, samples were incubated with avidin-biotin peroxidase for 30 min at 37 °C. After successive rinses with TBS, samples were incubated with AEC peroxidase substrate according to the manufacturer's instructions. Samples were rinsed with TBS for 5 min and embedded in Faramount. Image acquisition and quantitative immunohistomorphometric analysis were performed as described above.

[0062] Figure 5 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunohistochemical visualization of Sirtuin1 in skin sections. It can be seen from Figure 5 that systemic application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 μM) significantly increases the expression of Sirtuin1 in the epidermis after 3 days in human skin organotypic cultures compared to vehicle. Since the expression level of Sirtuin1 decreases with skin aging, this suggests that DMP exerts an anti-aging effect.

[0063] For detection of collagen 17A in cell culture, formalin-fixed NHEKs were cultured in 0.05% Triton (商標) The samples were permeabilized with PBS containing X-100 for 10 min, rinsed with PBS, and then blocked with PBS containing 10% normal goat serum (NGS) for 20 min. Samples were then incubated overnight at 4°C with anti-collagen 17A diluted 1:100 in antibody diluent. After successive rinses with PBS, samples were incubated with Alexa fluorophore-conjugated secondary antibodies diluted 1:500 in antibody diluent containing 2% NGS for 45 min at room temperature. After successive rinses with PBS, samples were counterstained with DAPI and Fluoromount-G (登録商標) (SouthernBiotech) was used to mount the samples on slides.

[0064] For analysis of NHKE, images were then obtained using a digital microscope (Keyence). Quantitative immunohistomorphometric analysis was performed by evaluating the number of positive cells or the relative fluorescence intensity in standardized reference areas using ImageJ (NIH; Bethesda, MD, USA). Briefly, for evaluation of collagen 17A, the relative target-specific intensity was measured in 14–20 cells per microscopic field. For each parameter, 14–20 microscopic fields were evaluated.

[0065] Figure 6 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of collagen 17A in NHEK cultures. It can be seen that application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 nM) significantly increases collagen 17A expression in NHEKs after 24 hours in culture compared to vehicle. This supports the findings on human organotypic cultured skin samples (see Figure 1) that DMP exerts an anti-aging effect.

[0066] For detection of cytokeratin 15 in cell culture, formalin-fixed NHEKs were cultured in 0.05% Triton (商標) The samples were permeabilized with PBS containing X-100 for 10 min, rinsed with PBS, and then blocked with PBS containing 10% normal goat serum (NGS) for 20 min. Samples were then incubated overnight at 4°C with anti-cytokeratin 15 diluted 1:100 in antibody diluent. After successive rinses with PBS, samples were incubated with Alexa fluorophore-conjugated secondary antibodies diluted 1:500 in antibody diluent containing 2% NGS for 45 min at room temperature. After successive rinses with PBS, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) was used to mount it on a glass slide.

[0067] For analysis of NHKE, images were then obtained using a digital microscope (Keyence). Quantitative immunohistomorphometric analysis was performed by evaluating the number of positive cells or relative fluorescence intensity in standardized reference areas using ImageJ (NIH; Bethesda, MD, USA). Briefly, for evaluation of cytokeratin 15, relative target-specific intensity was measured in 14–20 cells per microscopic field. For each parameter, 14–20 microscopic fields were evaluated.

[0068] Figure 7 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of cytokeratin 15 in NHEK cultures. It can be seen that application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 nM) significantly increases the expression of cytokeratin 15 in NHEKs after 24 hours in culture compared to vehicle. This supports the findings on human organotypic cultured skin samples (see Figure 2) that DMP exerts an anti-aging effect.

[0069] For the evaluation of collagen 17A and cytokeratin 15 specific mRNA transcripts (COL17A, KRT15) by quantitative RT-qPCR technique, RNA was isolated from cells lysed in RLT buffer containing 1% β-mercaptoethanol using the RNeasy Mini Kit according to the manufacturer's instructions. RNA purity and concentration were determined using BioDrop. For reverse transcription, 500 ng of sample RNA was transferred to a Tetro (商標) The TaqMan cDNA Synthesis Kit was used for subsequent cDNA synthesis according to the manufacturer's instructions. (商標) Real-time quantitative polymerase chain reaction (qRT-PCR) was performed in triplicate using Fast Advanced Master Mix and TaqMan Gene Expression Assays. Real-time quantitative plots and Ct values ​​were collected and stored by qPCRsoft2.1 software. Transcript abundance was normalized to that of the housekeeping gene GAPDH by the ΔΔCT method.

[0070] Figure 8 shows the results of RT-qPCR analysis in NHEK cultures. It can be seen from Figure 8 that application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP) (a: 10 nM; b: 100 nM) significantly increases the expression of COL17A1 (a) and KRT15 (b) in NHEKs after 24 hours in culture compared to vehicle. This supports the immunohistomorphometric findings on human organotypic cultured skin samples and NHEKs (see Figures 1, 2, 6, 7) that DMP exerts an anti-aging effect.

[0071] For detection of Ki67 in cell culture, which is a nuclear non-histone protein ubiquitously expressed across proliferating cells, formalin-fixed NHEKs were permeabilized and incubated with 5% normal goat serum and 0.3% Triton X-rays. (商標) After rinsing with PBS, the samples were blocked with 1% BSA and 0.05% Triton X-100 for 30 min. 商標 The samples were incubated overnight at 4° C. with anti-Ki67 antibody diluted 1:800 in PBS containing X-100. After sequential rinsing with PBS, the samples were resuspended in 1% BSA and 0.05% Triton X-100. 商標 After incubation with Alexa fluorophore-conjugated antibodies diluted 1:500 in PBS containing X-100 for 45 min at room temperature, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) was used to mount it on a glass slide.

[0072] For analysis of NHKE, images were then obtained using a digital microscope (Keyence). Quantitative immunohistomorphometric analysis was performed by evaluating the number of positive cells or relative fluorescence intensity in standardized reference areas using ImageJ (NIH; Bethesda, MD, USA). Briefly, for evaluation of Ki67, the number of cells with positive nuclear staining per field was counted and normalized to the total number of DAPI-positive nuclei. For each parameter, 14–20 microscopic fields were evaluated.

[0073] Figure 9 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of Ki67 in NHEK cultures. It can be seen from Figure 9 that application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 100 nM) significantly increases the number of Ki67-positive (i.e., proliferating) NHEKs in cultures after 24 hours compared to vehicle. Since the rate of proliferation of human epidermal keratinocytes decreases with aging, this suggests that DMP exerts an anti-aging effect.

[0074] For detection of phospho-histone H3 Ser10 (pH3S10) in cell culture (phosphorylation of histone H3 at Ser10 closely correlates with chromosome condensation during mitosis and cell proliferation), formalin-fixed NHEKs were permeabilized and incubated with 5% normal goat serum and 0.3% Triton X-10. (商標) After rinsing with PBS, the samples were blocked with 1% BSA and 0.05% Triton X-100 for 30 min. 商標 The samples were incubated overnight at 4° C. with anti-pH3S10 antibody diluted 1:200 in PBS containing X-100. After sequential rinses with PBS, the samples were washed with 1% BSA and 0.05% Triton X-100. 商標 After incubation with Alexa fluorophore-conjugated antibodies diluted 1:500 in PBS containing X-100 for 45 min at room temperature, samples were counterstained with DAPI and stained with Fluoromount-G. (登録商標) was used to mount it on a glass slide.

[0075] For analysis of NHKE, images were then obtained using a digital microscope (Keyence). Quantitative immunohistomorphometric analysis was performed by evaluating the number of positive cells or relative fluorescence intensity in standardized reference areas using ImageJ (NIH; Bethesda, MD, USA). Briefly, for evaluation of pH3S10, the number of cells with positive nuclear staining per field was counted and normalized to the total number of DAPI-positive nuclei. For each parameter, 14–20 microscopic fields were evaluated.

[0076] Figure 10 shows representative images (a, b) and quantitative immunohistomorphometric analysis (c) of immunofluorescence visualization of pH3S10 in NHEK cultures. It can be seen from Figure 10 that application of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 100 nM) significantly increases the number of pH3S10 positive (i.e. dividing and proliferating) NHEKs in cultures after 24 hours compared to vehicle. Since the rate of cell division (and therefore proliferation) of human epidermal keratinocytes decreases with age, this suggests that DMP exerts an anti-aging effect.

[0077] 4. Inactivation of TRPM5 using triphenylphosphine oxide (TPPO) Figure 11 shows representative images (a, b, c) and quantitative immunohistomorphometric analysis (d) of immunofluorescence visualization of collagen 17A in skin sections. From Figure 11, it can be seen that topically applied TRPM5 antagonist triphenylphosphine oxide (TPPO, 1.5 mM) prevented the effect of topically applied TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) in upregulating collagen 17A expression in human skin organ cultures after 3 days compared to vehicle. This suggests that the anti-aging effect of DMP is mediated by TRPM5.

[0078] For detection of Ki67, formalin-fixed human skin cryosections were blocked with PBS containing 10% normal goat serum (NGS) for 30 min at room temperature and then incubated overnight at 4°C with anti-Ki67 antibody diluted 1:800 in PBS containing 2% NGS. After successive rinses with PBS, samples were incubated for 45 min at room temperature with secondary antibodies, namely anti-rabbit Alexa 488 fluorophore conjugate diluted 1:400 and anti-mouse rhodamine conjugate diluted 1:200 in PBS containing 2% NGS. After successive rinses with PBS, samples were counterstained with DAPI and Fluoromount-G. (登録商標) The sections were mounted on glass slides using a 100% ethanol centrifuge. Image acquisition and quantitative immunohistomorphometric analysis were performed as described above.

[0079] Figure 12 shows representative images (a, b, c) and quantitative immunohistomorphometric analysis (d) of immunofluorescence visualization of Ki67 in skin sections. From Figure 12, it can be seen that topically applied TRPM5 antagonist triphenylphosphine oxide (TPPO, 1.5 mM) prevented the effect of topically applied TRPM5 agonist 2,5-dimethylpyrazine (DMP, 12.5 mM) in upregulating Ki67 expression in human skin organ cultures after 3 days compared to vehicle. This suggests that the anti-aging effect of DMP is mediated by TRPM5.

[0080] Figure 13 shows representative images (a, b, c) and quantitative immunohistomorphometric analysis (d) of immunofluorescence visualization of collagen 17A in NHEK cultures. It can be seen from Figure 13 that the TRPM5 antagonist triphenylphosphine oxide (TPPO, 50 μM) prevented the effect of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 100 nM) in upregulating collagen 17A expression in cultures after 24 hours compared to vehicle. This suggests that the anti-aging effect of DMP is mediated by TRPM5.

[0081] Figure 14 shows representative images (a, b, c) and quantitative immunohistomorphometric analysis (d) of immunofluorescence visualization of cytokeratin 15 in NHEK cultures. It can be seen from Figure 14 that the TRPM5 antagonist triphenylphosphine oxide (TPPO, 50 μM) prevented the effect of the TRPM5 agonist 2,5-dimethylpyrazine (DMP, 100 nM) in upregulating cytokeratin 15 expression in cultures after 24 hours compared to vehicle. This suggests that the anti-aging effect of DMP is mediated by TRPM5.

Claims

1. An active agent for use in the treatment of skin aging, in particular premature or pathological skin aging, and pathological skin conditions related to or induced by skin aging, which activates, enhances or positively modulates the cellular response of the transient receptor potential ion channel TRPM5 or increases the expression of said ion channel.

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

3. 2. The active agent for use in the treatment of skin aging according to claim 1, wherein the active agent is selected from 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.

4. 4. An active agent for use in the treatment of skin aging according to any one of claims 1 to 3, wherein said treatment comprises: a) for the treatment of any form of skin ageing characterized by thin, dry, fragile, sensitive skin, fine or coarse wrinkles, gradual atrophy affecting both the epidermis and the dermis, loss of elasticity and laxity, reduced skin moisture, coarse texture, impaired and delayed wound regeneration, and / or b) for the treatment of age-related skin disorders selected from rosacea (redness, papules, bumps, and fine, superficial, dilated blood vessels particularly affecting the face), asteatotic eczema (characterized by dry, itchy, cracked skin), nummular eczema (characterized by nummular, itchy, reddish patches), seborrheic dermatitis, skin infections, actinic keratoses (precancerous skin lesions associated with chronic ultraviolet radiation), and benign (non-cancerous) and malignant (basal cell carcinoma, squamous cell carcinoma) skin tumors, The activator.

5. Use of an active agent according to any one of claims 1 to 3 as a cosmetic agent for the non-therapeutic treatment of skin ageing and related conditions.

6. A cosmetic or medical skin anti-aging composition comprising at least one active agent according to any one of claims 1 to 3 and at least one auxiliary selected from the group consisting of carriers, excipients, adjuvants, diluents and disintegrants.

7. 7. The cosmetic or medical skin anti-aging composition according to claim 6, wherein 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.

8. 7. A cosmetic or medical skin anti-aging composition according to claim 6, further comprising at least one other active agent effective in the treatment of skin aging.

9. 7. The cosmetic or medical skin anti-aging composition according to claim 6, formulated in the form of an ointment, lotion, cream, gel, solution, spray, plaster or sustained-release plaster.

10. A non-therapeutic method for controlling skin aging, wherein an effective amount of at least one active agent that activates, enhances or modulates the cellular response of the transient receptor potential ion channel TRPM5 or increases the expression of said ion channel is administered to a subject.

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