Merkel cell activators, synaptic vesicle enhancers, neurotransmitter release enhancers, methods for evaluating the neurotransmitter release activity of Merkel cells, and screening methods for Merkel cell neurotransmitter release enhancers.

Abstract

The purpose of the present invention is to provide skin care focusing on the sense of touch. It was found that sandalore and an analog thereof have an action of activating Merkel cells. Provided is a Merkel cell activator containing sandalore and an analog thereof. It was also found that an activated Merkel cell contains a large number of synaptic vesicles. Provided is a synaptic vesicle enhancer containing sandalore and an analog thereof. It was also found that the neurotransmitter release activity of Merkel cells can be assessed by measuring, under a fluorescence microscope, changes in fluorescent luminance value before and after depolarization stimulation to Merkel cells that have taken in a fluorescent tracer. Provided is a method for screening for a neurotransmitter release accelerator for Merkel cells.

Description

Technical Field

[0001] The present invention relates to a Merkel cell activator and a synaptic vesicle enhancer in Merkel cells. The present invention also relates to a method for evaluating the neurotransmitter release activity of Merkel cells, a screening method for a neurotransmitter release promoter using such an evaluation method, and a neurotransmitter release promoter screened by such a screening method.

Background Art

[0002] Receptors related to touch are excited by mechanical stimuli, and signals are thereby sent to the brain via sensory nerves. Examples of such mechanical stimulus receptors include Merkel cells present in the deepest part of the epidermis, Meissner corpuscles present in the outermost layer of the dermis, Pacinian corpuscles in the deep dermis, Ruffini endings, and the like. The terminals of sensory nerves are synaptically connected to Merkel cells. When excited by mechanical stimuli, neurotransmitters are secreted at the synapses, and information is transmitted to the sensory nerves. It has been suggested that the Piezo2 channel of Merkel cells is involved in the perception of mechanical stimuli (Non-Patent Document 1: Nature (2014) volume 509, pages 622-626). Further, it has been reported that a decrease in Merkel cells causes alloknesis in which itching is induced by stimuli that usually do not cause itching, and that itching can be suppressed by activating Merkel cells (Non-Patent Document 5). Furthermore, it has been reported that PIEZO2-deficient individuals cannot induce sensitization or pain responses even at skin inflammation sites, and that the PIEZO2 channel is a therapeutic target for allodynia in which pain is caused by non-invasive stimuli that usually do not cause pain (Non-Patent Document 6).

[0003] On the other hand, it has been reported that the olfactory receptor OR2AT4 is expressed in keratinocytes, the main constituent cells of the epidermis, and that sandalol, its agonist, acts on OR2AT4 to induce intracellular signaling and promote wound healing (Non-patent Literature 2: J Investigative Dermatology (2014) vol. 134, p. 2823-2832). Furthermore, it has been reported that OR2AT4 is expressed in the sheath cells of hair follicles, and that application of sandalol induces hair elongation (Patent Literature 1: JP 2019-519502, Non-patent Literature 3: Nat Commun. (2018) Sep 18;9(1):3624). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2019-519502 [Non-patent literature]

[0005] [Non-Patent Document 1] Nature (2014) volume 509, p. 622-626 [Non-Patent Document 2] Journal of Investigative Dermatology (2014) 134:2823-2832 [Non-Patent Document 3] Nat Commun. (2018) Sep 18; 9(1):3624 [Non-Patent Document 4] Neuron (2018)19:100(6):1401-1413 [Non-Patent Document 5] Science. 2018 May 04; 360(6388): 530-533. doi:10.1126 / science.aar570 [Non-Patent Document 6] Sci Transl Med. (2018)10(462)eaat9892. doi: 10.1126 / scitranslmed.aat9892 [Overview of the project] [Problems that the invention aims to solve]

[0006] The inventors of this invention are diligently conducting research on skincare that focuses on the sense of touch, with the aim of obtaining a drug that activates Merkel cells.

[0007] Furthermore, conventionally, the activity of Merkel cells has been evaluated using changes in the amount of a specific protein expressed by Merkel cells as an indicator. However, the amount of this specific protein changes over a relatively long period, from several hours to several days, making it impossible to evaluate immediate activity. Moreover, such protein amounts only indirectly measure the activity of Merkel cells. Therefore, the present invention aims to measure the direct and immediate activity of Merkel cells during depolarization. [Means for solving the problem]

[0008] The inventors focused on OR2AT4, an olfactory receptor expressed in the skin, and investigated its presence in cell types other than keratinocytes. Surprisingly, they found that OR2AT4 is expressed in Merkel cells. Furthermore, they discovered that applying sandalol, an OR2AT4 agonist, to Merkel cells activated them, leading to the present invention.

[0009] Therefore, the present invention relates to the following: [A1-1] The following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A Merkel cell activator containing the compound represented by [formula]. [A1-2] The following formula for use in the treatment or prevention of at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia via activation of Merkel cells: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A compound represented by the formula. [A1-3] The following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 are the same or different and are independently selected from hydrogen and methyl, or R5 and R6 together form a double bond; or R5 and R6 together form a cyclopropyl group; R7 is methyl or ethyl; R8 is hydrogen or methyl] A cosmetic method via activation of Merkel cells, comprising administering a compound represented by: [A1-4] To a subject requiring activation of Merkel cells, the following formula: [Chemical formula] [wherein, R1 and R2 together form a double bond, or R1 and R2 together form a cyclopropyl group; R3 and R4 are the same or different and are independently selected from hydrogen and methyl, or R3 and R4 together form a double bond; R5 and R6 are the same or different and are independently selected from hydrogen and methyl, or R5 and R6 together form a double bond; or R5 and R6 together form a cyclopropyl group; R7 is methyl or ethyl; R8 is hydrogen or methyl] A method for activating Merkel cells, comprising administering a compound represented by: [A1-5] For the manufacture of a Merkel cell activator, the following formula: [Chemical formula] [wherein, R1 and R2 together form a double bond, or R1 and R2 together form a cyclopropyl group; R3 and R4 are the same or different and are independently selected from hydrogen and methyl, or R3 and R4 together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. The use of the compound represented by [the specified compound]. [A2] The activator, compound, cosmetic method, activation method, or use described in items A1-1 to A1-5, wherein the compound is sandalol or bramanol. [A3] Activators, compounds, cosmetic methods, activation methods, or uses described in items A1-1 to A1-5 or A2 that enhance the sense of touch. [A3-2] An activator, compound, cosmetic method, activation method, or use described in any one of items A1-1 to A1-5, A2, or A3, for the treatment, prevention, or reduction of at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia. [A4-1] The following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A synaptic vesicle enhancer in Merkel cells, containing the compound represented by [formula]. [A4-2] The following formula for use in the treatment, prevention, or reduction of at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia, via enhancement of synaptic vesicles in Merkel cells: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A compound represented by the formula. [A4-3] The following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A cosmetic method involving the enhancement of synaptic vesicles in Merkel cells, comprising administering a compound represented by [formula]. [A4-4] For targets requiring enhancement of synaptic vesicles in Merkel cells, use the following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A method for enhancing synaptic vesicles in Merkel cells, comprising administering a compound represented by [formula]. [A4-5] The following formula for the production of synaptic vesicle enhancers in Merkel cells: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. The use of the compound represented by [the specified compound]. [A5] The enhancer, compound, cosmetic method, enhancement method, or use described in items A4-1 to A4-5, wherein the compound is sandalol or bramanol. [A6] The enhancer, compound, cosmetic method, enhancement method, or use described in items A4-1 to A4-5 or A5, wherein the enhancer improves the efficiency of neurotransmission between Merkel cells and nerve cells. [A7] Enhancers, compounds, cosmetic methods, enhancement methods, or uses described in any one of items A4-1 to A4-5, A5, or A6, that enhance the sense of touch. [A8] Enhancers, compounds, cosmetic methods, enhancement methods, or uses described in any one of items A4-1 to A4-5 or A5 to A7 that enhance collagen production. [A9] Enhancers, compounds, cosmetic methods, enhancement methods, or uses described in any one of items A4-1 to A4-5 or A5 to A8 for the treatment, prevention, or reduction of at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia.

[0010] Furthermore, the inventors focused on the tactile sensation that can be perceived in skincare and other applications, and diligently researched the mechanism by which Merkel cells release neurotransmitters upon excitation. They discovered that the neurotransmitter-releasing activity of Merkel cells can be evaluated by measuring the change in fluorescence intensity before and after depolarization stimulation under a fluorescence microscope using Merkel cells into which a fluorescent tracer has been incorporated, leading to the present invention.

[0011] Therefore, the present invention relates to the following: [B1] A method for evaluating the neurotransmitter release activity of Merkel cells in skin samples, Skin samples are cultured in a culture medium containing a fluorescent tracer of a neurotransmitter; Apply a depolarizing stimulus; Under a fluorescence microscope, we measure the change in fluorescence intensity of Merkel cells before and after depolarization stimulation. The evaluation method, including the above. [B2] The evaluation method described in item B1, wherein the fluorescent tracer of the neurotransmitter is a fluorescent tracer of a monoamine neurotransmitter. [B3] The evaluation method described in item B1 or B2, wherein the skin sample is a human skin sample. [B4] A method for screening neurotransmitter release enhancers in Merkel cells in skin samples, Skin samples are cultured in a culture medium containing a fluorescent tracer of a neurotransmitter; Add a candidate substance for neurotransmitter release enhancer; Under a fluorescence microscope, the change in fluorescence intensity of Merkel cells before and after the addition of the additive is measured. The screening method, including the above. [B5] The screening method described in item B4, wherein the fluorescent tracer for the neurotransmitter is a fluorescent tracer for monoamine neurotransmitters. [B6] The screening method according to item B4 or B5, wherein the skin sample is a human skin sample. [B7-1] The following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A neurotransmitter release enhancer in Merkel cells, comprising the compound represented by [the formula shown]. [B7-2] The following formula for use in the treatment or prevention of at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia, via the enhancement of neurotransmitter release in Merkel cells: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A compound represented by the formula. [B7-3] The following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A cosmetic method involving the administration of a compound represented by [formula], via the enhancement of neurotransmitter release in Merkel cells. [B7-4] For targets requiring neurotransmitter release in Merkel cells, use the following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. A method for promoting neurotransmitter release in Merkel cells, comprising administering a compound represented by [formula]. [B7-5] The following formula for the production of neurotransmitter release enhancers in Merkel cells: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. The use of the compound represented by [the specified compound]. [B8] The neurotransmitter release enhancer, compound, cosmetic method, method of enhancing, or use described in items B7-1 to B7-5, wherein the compound is sandalol or bramanol. [B9] Neurotransmitter release enhancers, compounds, cosmetic methods, methods of promoting, or uses described in items B7-1 to B7-5 or B8 that enhance the sense of touch. [B10] A neurotransmitter release enhancer, compound, cosmetic method, method of promoting, or use described in any one of items B7-1 to B7-5, B8, or B9, which enhances collagen production. [B11] A neurotransmitter release enhancer, compound, cosmetic method, enhancement method, or use described in any one of items B7-1 to B7-5, B8, B9, or B10 for the treatment, prevention, or reduction of at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia. [Effects of the Invention]

[0012] By applying Sandalol and / or its analogues, it becomes possible to activate Merkel cells. Through the activation of Merkel cells, it becomes possible to provide skincare that focuses on the sense of touch.

[0013] Furthermore, it becomes possible to evaluate the neurotransmitter release activity when Merkel cells are depolarized. Using this evaluation method, it becomes possible to screen for neurotransmitter release activity enhancers. In addition, sandalol and its related compounds were screened as neurotransmitter release activity enhancers. [Brief explanation of the drawing]

[0014] [Figure 1A-B] Figure 1 shows the results of co-staining skin samples with CK8 and OR2AT4, markers for Merkel cells, and observing them under a fluorescence microscope. (A) shows a scalp sample, and (B) shows an abdominal skin sample, both co-stained with CK8 and OR2AT4. [Figure 1C-D] (C) The left side of the graph shows the number of OR2AT4 and CK8 double-positive cells in a given area of ​​a scalp sample. The right side of the graph shows the proportion of OR2AT4 and CK8 double-positive cells to CK8-positive cells in a scalp sample. (D) The left side of the graph shows the number of OR2AT4 and CK8 double-positive cells in a given area of ​​an abdominal sample. The right side of the graph shows the proportion of OR2AT4 and CK8 double-positive cells to CK8-positive cells in an abdominal sample. [Figure 2A-B]Figure 2 shows the results of observing skin samples co-stained with Merkel cell markers CK18 and OR2AT4 under a fluorescence microscope. (A) shows a scalp sample, and (B) shows an abdominal skin sample, both co-stained with CK18 and OR2AT4. [Figure 2C-D] (C) The left side of the graph shows the number of OR2AT4 and CK18 double-positive cells in a given area of ​​a scalp sample. The right side of the graph shows the proportion of OR2AT4 and CK18 double-positive cells to CK18-positive cells in a scalp sample. (D) The left side of the graph shows the number of OR2AT4 and CK18 double-positive cells in a given area of ​​an abdominal sample. The right side of the graph shows the proportion of OR2AT4 and CK18 double-positive cells to CK18-positive cells in an abdominal sample. [Figure 3A-B] Figure 3 shows the results of observing skin samples co-stained with CK20 and OR2AT4, markers for Merkel cells, under a fluorescence microscope. (A) shows a scalp sample, and (B) shows an abdominal skin sample, both co-stained with CK20 and OR2AT4. [Figure 3C-D] (C) The left side of the graph shows the number of OR2AT4 and CK20 double-positive cells in a given area of ​​a scalp sample. The right side of the graph shows the proportion of OR2AT4 and CK20 double-positive cells to CK20-positive cells in a scalp sample. (D) The left side of the graph shows the number of OR2AT4 and CK20 double-positive cells in a given area of ​​an abdominal sample. The right side of the graph shows the proportion of OR2AT4 and CK20 double-positive cells to CK20-positive cells in an abdominal sample. [Figure 4A-B] Figure 4 shows fluorescence microscopy images of CK20-positive cells expressing piccolo in samples treated with Sandalol (Figure 4A: control (vehicle), Figure 4B: Sandalol). [Figure 4C] Figure 4C is a graph showing the number of CK20-positive cells expressing piccolo in a predetermined region, measured in photographs taken under a fluorescence microscope. [Figure 5]Figure 5A shows iPS cell-derived sensory neural progenitor cells. Figure 5B shows the relative levels of type I collagen mRNA expression when iPS cell-derived sensory neural progenitor cell culture supernatant (conditioned medium) was added to fibroblasts, compared to when unconditioned medium was added to fibroblasts. Error bars represent the mean ± SD, and ** indicates a statistically significant difference by an unpaired t-test (**P<0.01). [Figure 6] Figure 6A shows the mRNA expression level of type I collagen when the supernatant of the culture medium of iPS cell-derived sensory neural progenitor cells stimulated with the test substance (lavender oil: LO 0.005%) was added to fibroblasts. Figure 6B shows the mRNA expression level of type I collagen when the above test substance was added directly to fibroblasts at the same concentration. In each figure, collagen production is shown as a relative value with the control (no test substance added: control) set to 100%. Error bars represent the mean ± SD, * indicates a statistically significant difference by an unpaired t-test (*P<0.05), and ns indicates no statistically significant difference. [Figure 7] Figure 7 shows the mRNA expression level of type I collagen when a higher concentration (4 times) of the test substance (lavender oil: LO 0.02%) than in Figure 6B was directly added to fibroblasts. Collagen production is shown as a relative value with the control (no test substance added: control) set to 100%. Error bars indicate the mean ± SD, ** indicates a statistically significant difference by an unpaired t-test (**p<0.01), and ns indicates no statistically significant difference. [Figure 8] Figure 8 shows photographs of Merkel cells that have taken up a fluorescent tracer (FFN206) at 0, 200, 400, and 600 seconds, illustrating the fluorescence changes. Figure 8A shows the fluorescence changes when Sandalol stimulation was applied from 300 seconds to 600 seconds, while Figure 8B shows the fluorescence changes when BSS solution was perfused from 300 seconds to 600 seconds, as a control. [Figure 9] Figure 9 is a graph showing the change in fluorescence intensity of Merkel cells during 10 minutes of fluorescence imaging. At 5 minutes (300 seconds), sandalol was introduced, and the fluorescence intensity decreased (black circles). As a control, perfusion with BSS solution was performed at 5 minutes (300 seconds) (white squares). [Modes for carrying out the invention]

[0015] The present invention relates to a Merkel cell activator comprising sandalol (3-methyl-5-(2,2,3-trimethylcyclopenta-3-en-1-yl)-pentan-2-ol [CAS:65113-99-7]) or its analogues. Sandalol is represented by the following formula: [ka]

[0016] Sandalol was developed and is commercially available as a compound with a sandalwood-like scent. The scent components of sandalwood are mainly composed of santalol, and also include its stereoisomers. α- and β-isomers of santalol are known. Due to its complex structure, the synthesis of santalol is difficult, and fragrances with simpler structures that have a sandalwood-like scent have been synthesized; sandalol is one such example. Sandalol acts as an agonist of OR2AT4, one of the olfactory receptors. Examples of compounds that have OR2AT4 agonist activity include sandalol or its analogs. One example of a sandalol analog that has OR2AT4 agonist activity is bramanol (2-methyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-butan-1-ol [CAS:72089-08-8]) (Non-Patent Literature 2). Bramanol is represented by the following formula: [ka]

[0017] Examples of sandalol or its analogues that act as agonists for OR2AT4 include the following compounds: [Table 1]

[0018] Sandalol or its analogues are given, for example, by the following formula: [ka] [In the formula, R1 and R2 either form a double bond together, or R1 and R2 form a cyclopropyl group together; R3 and R4 may be the same or different, independently selected from hydrogen, methyl, or together form a double bond; R5 and R6 may be the same or different, independently selected from hydrogen, methyl, or R5 and R6 may form a double bond together; or R5 and R6 may form a cyclopropyl group together; R7 is either methyl or ethyl; R8 is either hydrogen or methyl. This relates to compounds represented by [formula]. These compounds may include optical isomers. These compounds can act as agonists of OR2AT4, similar to sandalol. Furthermore, these compounds, similar to sandalol, activate Merkel cells and promote the release of neurotransmitters from Merkel cells.

[0019] Merkel cell activation refers to the promotion of Merkel cell proliferation or maturation. Merkel cells undergo changes in expression markers depending on their maturation process. CK8, CK18, and CK20 are used as Merkel cell markers; CK8 is expressed in the undifferentiated state, while activated Merkel cells express CK18 and CK20 in the mature state (PLoS Genet (2016) Jul 14;12(7):e1006151). By applying the Merkel cell activator of the present invention, Merkel cells can be activated. Specifically, by applying the Merkel cell activator, activated Merkel cells show increased expression of proteins involved in synaptic vesicles, such as piccolo. This activation of Merkel cells promotes the release of neurotransmitters to synapses, thereby enhancing the sense of touch.

[0020] Another aspect of the present invention relates to a synaptic vesicle enhancer in Merkel cells comprising sandalol or its analogues. Application of the synaptic vesicle enhancer in Merkel cells comprising sandalol or its analogues increases the number of synaptic vesicles in Merkel cells. This enhancement of synaptic vesicles improves the efficiency of neurotransmission between Merkel cells and nerve cells, activating nerve cells involved in touch. Activation of nerve cells in the dermis increases collagen production in dermal fibroblasts. Therefore, the present invention also relates to a collagen production promoter comprising sandalol or its analogues. Yet another aspect of the present invention may relate to a composition comprising sandalol or its analogues for the treatment, prevention, or alleviation of at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia.

[0021] Another aspect of the present invention relates to a method for activating Merkel cells in vitro or in vivo, or a method for promoting the release of neurotransmitters from Merkel cells in vitro or in vivo, comprising the application of sandalol or an analog thereof. The method for activating Merkel cells in vitro or promoting the release of neurotransmitters from Merkel cells in vitro comprises the step of culturing a group of cells or tissue containing Merkel cells in a solution containing sandalol or an analog thereof. The method for activating Merkel cells in vivo or promoting the release of neurotransmitters from Merkel cells in vivo comprises the step of administering sandalol or an analog thereof to any subject. By activating Merkel cells in vivo, it is possible to treat, prevent or alleviate at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia. Therefore, the Merkel cell activator and neurotransmitter release promoter of the present invention may be a treatment, preventive, or alleviating agent for at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia. Targets for administration of sandalol or its analogues include, for example, subjects with reduced Merkel cell activity, dulled tactile sensation, reduced collagen production, or subjects suffering from or wishing to prevent or alleviate at least one symptom selected from the group consisting of itching, pruritus, allonesis, and allodynia. Sandalol or its analogues can be administered via any route, such as transdermally, orally, transmucosally, transnasally, intravenously, intra-arterially, or subcutaneously, but transdermal or transmucosal administration is preferred from the viewpoint of acting on Merkel cells. When administering sandalol or its analogues transdermally, it can be applied to any skin, such as the face, head, neck, limbs, or trunk.

[0022] The Merkel cell activator, synaptic vesicle enhancer, and / or neurotransmitter release enhancer of the present invention may each be incorporated into cosmetics, pharmaceuticals, or quasi-drugs. These agents may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated into topical skin preparations. Topical skin preparations are not particularly limited as long as they are applicable to the skin, and any dosage form can be used, such as solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated into topical skin preparations, bases and excipients commonly used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used. When incorporated into cosmetics, it can be used in facial or body cosmetics such as lotions, emulsions, serums, creams, packs, essences, and gels, as well as makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. By using cosmetics containing the ingredients of the present invention, Merkel cells are activated, leading to the activation of nerve cells and the resulting promotion of collagen production. Due to the promotion of collagen production, it may also be used in cosmetics aimed at improving wrinkles and sagging.

[0023] The Merkel cell activator, synaptic vesicle enhancer, and / or neurotransmitter release enhancer of the present invention can be arbitrarily selected from the viewpoint of exhibiting a desired effect, such as a Merkel cell activation effect, a synaptic enhancement effect, or an effect of promoting the release of neurotransmitters from Merkel cells, or from the viewpoint of promoting collagen production. From the viewpoint of formulation as a topical skin preparation, sandalol or its analogs can be incorporated at a concentration of 0.0005% to 0.5%. From the viewpoint of fully exhibiting the effect, it is preferably incorporated at a concentration of 0.001% or more, and more preferably at a concentration of 0.005% or more. From the viewpoint of avoiding a strong odor, it is preferably incorporated at a concentration of 0.1% or less, and more preferably at a concentration of 0.05% or less.

[0024] This invention relates to a method for evaluating the neurotransmitter release activity of Merkel cells in skin samples. Specifically, this evaluation method consists of the following steps: Skin samples are cultured in a culture medium containing a fluorescent tracer of a neurotransmitter; Apply a depolarizing stimulus; Under a fluorescence microscope, we measure the change in fluorescence intensity of Merkel cells before and after depolarization stimulation. This includes a step of culturing skin samples in a culture medium containing a fluorescent tracer, followed by a washing step or a medium replacement step. Such a method can be performed under perfusion conditions. This method makes it possible to directly and immediately evaluate the neurotransmitter-releasing activity of Merkel cells.

[0025] Merkel cells are cells located in the deepest layer of the epidermis and function as receptors for mechanical stimuli. When Merkel cells are excited and depolarized by mechanical stimuli, neurotransmitters are secreted into the synaptic cleft, and signals are transmitted to sensory nerves via the synapse. It has been suggested that the Piezo2 channel of Merkel cells is involved in the sensing of mechanical stimuli. Merkel cells undergo changes in expression markers depending on their maturation process. To identify Merkel cells in a skin sample, fluorescent dyes that are specifically taken up by Merkel cells, such as quinacrine or FMdye, may be used as markers. Alternatively, proteins expressed in Merkel cells may be detected as markers, and for example, at least one cell surface marker selected from the group consisting of CK8, CK18, and CK20 may be used.

[0026] A skin sample can be any sample containing Merkel cells, but may also contain cells other than Merkel cells. The skin sample may be derived from any animal, but from the viewpoint of cosmetic and pharmaceutical development, human-derived samples are preferred. Cells other than Merkel cells may include cells found in the skin, such as epidermal cells and dermal cells, or cells of the nervous system, such as nerve cells. The skin sample may be a skin sample obtained from a subject, or it may be a cultured sample. More preferably, a cultured skin model constructed in three dimensions by culturing epidermal cells or dermal cells may be used.

[0027] Neurotransmitter release activity is determined by the amount of neurotransmitters released from Merkel cells. When Merkel cells receive a depolarizing stimulus, they transmit the stimulus by releasing neurotransmitters into the synaptic cleft. Some of the neurotransmitters released into the synaptic cleft are received by nerve receptors, causing nerve excitation. In addition, some of the neurotransmitters released into the synaptic cleft are reuptaken via receptors expressed on Merkel cells. Examples of neurotransmitters released by Merkel cells include monoamine neurotransmitters. In this invention, neurotransmitter release activity is determined by introducing a fluorescent tracer of a neurotransmitter into Merkel cells and measuring the change in the intracellular fluorescent tracer during depolarization. For example, the fluorescent tracer of a neurotransmitter is dissolved in a culture medium at a concentration of 1 to 50 μM and cultured for 30 minutes to 12 hours to be taken up by Merkel cells. A fluorescent tracer refers to an analog of a neurotransmitter that can be observed by fluorescence. From the perspective of observing uptake into and release from Merkel cells, examples include FFN206 (4-(2-aminoethyl)-7-(methylamino)-2H-1-benzopyran-2-one dihydrochloride) (Non-patent Literature 4: Neuron (2018) 19:100(6):1401-1413) and FFN511 ((9-(2-aminoethyl)-2,3,6,7-tetrahydro-1H,5H,11H-[1]-benzopyrano[6,7,8-ij]quinoridine-11-one)).

[0028] For intracellular fluorescent tracers during depolarization, the sample is observed under a fluorescence microscope, and the fluorescence intensity values ​​before and after depolarization stimulation are measured. Fluorescence intensity may be measured over time. When measured over time, fluorescence fades, so the fluorescence intensity values ​​may be corrected to account for this fading. After depolarization stimulation, the fluorescent tracer is released into the synaptic cleft along with neurotransmitters, resulting in a decrease in intracellular fluorescence intensity. Fluorescence intensity may be measured throughout the entire cell, or it may be determined in a specific region near the synapse, such as the synaptic vesicle region.

[0029] Depolarization stimulation may be mechanical or pharmacological. Mechanical stimulation can be applied to Merkel cells by applying a probe. Mechanical stimulation may be applied until cell deformation is observed. Pharmacological stimulation is applied by applying a drug that causes depolarization. Such drugs include sandalol or its analogues. More specifically, depolarization stimulation can be applied by introducing a culture medium containing a depolarizing drug under perfusion conditions.

[0030] Another aspect of the present invention may relate to a screening method for Merkel cell neurotransmitter release enhancers utilizing a method for evaluating the neurotransmitter release activity of Merkel cells. Specifically, such a screening method involves the following steps: Skin samples are cultured in a culture medium containing a fluorescent tracer of a neurotransmitter; Add a candidate substance for neurotransmitter release enhancer; Under a fluorescence microscope, the change in fluorescence intensity of Merkel cells before and after the addition of the additive is measured. This includes the following. This screening method allows for the screening of neurotransmitter release enhancers that promote neurotransmitter release in Merkel cells. After culturing a skin sample in a medium containing a fluorescent tracer, a washing step or a medium replacement step may be performed. This method can be carried out under perfusion conditions. If the fluorescence intensity value decreases significantly, the candidate substance can be selected as a neurotransmitter release enhancer for Merkel cells.

[0031] Candidate substances for neurotransmitter release enhancers can be any substance belonging to any library, such as a cosmetic material library, an extract library, a pharmaceutical library, or a compound library. Neurotransmitter release enhancers can induce depolarization of Merkel cells and can also be called depolarization stimulants or Merkel cell activators.

[0032] Neurotransmitter release enhancers induce depolarization of Merkel cells and promote the release of neurotransmitters from Merkel cells. By promoting neurotransmitter release, the efficiency of neurotransmission between Merkel cells and nerve cells is improved, and nerve cells involved in touch are activated. From another perspective, by using neurotransmitter release enhancers at lower concentrations, Merkel cells can be made more susceptible to depolarization, i.e., sensitized, and can therefore be called Merkel cell sensitization enhancers. As a result, Merkel cells are activated even with weaker mechanical stimuli. This improves the efficiency of neurotransmission between Merkel cells and nerve cells, and activates nerve cells involved in touch. Activation of nerve cells in the dermis increases collagen production in dermal fibroblasts. Therefore, the present invention also relates to collagen production enhancers, including neurotransmitter release enhancers.

[0033] In another aspect of the present invention, the invention may also relate to neurotransmitter release promoters selected by the screening method described above. Specifically, sandalol and its analogues have been screened as neurotransmitter release promoters. Therefore, the present invention may also relate to neurotransmitter release promoters or collagen production promoters comprising at least one selected from the group consisting of sandalol and its analogues. [Examples]

[0034] Example 1: Distribution of Merkel cells in scalp and abdominal skin samples skin sample Human scalp from the temporal and occipital regions, or abdominal skin, was obtained from healthy subjects (20-60 years old: 13 subjects in total) who had undergone routine cosmetic or reconstructive surgery. Informed consent and ethical approval (Muenster University) were obtained before use. Human scalp was obtained from 3 men and 3 women, and abdominal skin was obtained from 6 women and 1 man.

[0035] Fluorescent immunohistochemical staining Scalp and abdominal skin samples were cut into 4 mm sections, embedded in OCT, and frozen in liquid nitrogen. The OCT-embedded samples were sectioned using a Leica cryostat. For primary antibody staining, the frozen tissue sections were fixed with 4% paraformaldehyde, pre-incubated with 10% goat serum (for OR2AT4) or 5% goat serum + 0.3% Triton X-100, and incubated overnight at 4°C in the corresponding primary antibody solution. The tissues were incubated at room temperature for 45 minutes in the secondary antibody solution. Nuclei were stained using DAPI (1 μg / ml). As primary antibodies, anti-OR2AT4 antibody (distributor: Eurogentech, product number: custom made, 1910432), anti-CK8 antibody (distributor: Progen, product number: GP-CK8), and anti-CK18 antibody (distributor: Progen, product number: GP-CK18) were used. Subsequently, as secondary antibodies, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (distributor: Invitrogen, product number: A-11008), Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (distributor: Invitrogen, product number: A-11030), and Fluorescein (FITC) - conjugated AffiniPure Goat Anti Guinea Pig IgG (H+L) (distributor: Jackson ImmunoResearch, product number: 106-095-003) were used, each in combination with the primary antibody used. DAPI (distributor: Sigma-Aldrich, product number: 10236276001) was used for nuclear staining.

[0036] The results of co-staining for OR2AT4 and CK8 are shown (Figure 1A: scalp, Figure 1B: abdominal skin). The number of OR2AT4 and CK8 double-positive cells in designated areas was counted (Figure 1C left, Figure 1D left). The ratio of OR2AT4 and CK8 double-positive cells to the total number of CK8-positive cells is shown (Figure 1C right, Figure 1D right). In the epidermis of the scalp and abdominal skin, approximately 50-60% of CK8-positive immature Merkel cells expressed OR2AT4.

[0037] The results of co-staining for OR2AT4 and CK18 are shown (Figure 2A: scalp, Figure 2B: abdominal skin). The number of OR2AT4 and CK18 double-positive cells in designated areas was counted (Figure 2C left, Figure 2D left). The ratio of OR2AT4 and CK18 double-positive cells to the total number of CK18-positive cells is shown (Figure 2C right, Figure 2D right). In the epidermis of the scalp and abdominal skin, approximately 50% of CK18-positive intermediate-mature Merkel cells expressed OR2AT4.

[0038] The results of co-staining for OR2AT4 and CK20 are shown (Figure 3A: scalp, Figure 3B: abdominal skin), the number of cells double-positive for OR2AT4 and CK20 in a specified area was counted (Figure 3C left, Figure 3D left), and the ratio of OR2AT4 and CK8 double-positive cells to the number of CK20-positive cells is shown (Figure 3C right, Figure 3D right). In the epidermis of the scalp and abdominal skin, approximately 70-85% of CK20-positive mature Merkel cells expressed OR2AT4.

[0039] Quantitative immunohistomorphometry Staining intensity was evaluated using quantitative (immunohistomorphometry)31,32 with NIH ImageJ software (NIH, Bethesda, MD, USA) in a clearly defined reference region.

[0040] Example 2: Changes in piccolo expression after application of Sandalol to scalp samples skin sample Human scalp samples from the temporal and occipital regions were obtained from healthy female subjects (47-60 years old) who had undergone conventional cosmetic surgery. These samples were used after obtaining informed consent and ethical approval (Muenster University). Fluorescent immunohistochemical staining Scalp samples containing hair follicles were obtained from the occipital region (Donors 1, 2, 3, and 4) or temporal region (Donor 5) of female subjects: a 60-year-old woman (Donor 1), a 55-year-old man (Donor 2), a 47-year-old man (Donor 3), a 40-year-old woman (Donor 4), and a 40-year-old woman (Donor 5) using a 6mm punch. The obtained samples were placed in DMEM / F12 medium supplemented with 25 ng / ml NGF and cultured at 37°C under a 5% CO2 humidified atmosphere (Day 0). On Day 1, the medium was replaced with a medium containing sandalol (500 μM). A medium containing 0.1% DMSO was used as a control. On Day 3, the samples were collected, embedded in OCT, and frozen in liquid nitrogen. The OCT-embedded samples were sectioned using a Leica cryostat. For primary antibody staining, frozen tissue sections were fixed with 4% paraformaldehyde, pre-incubated with 5% goat serum + 0.3% Triton X-100, and incubated overnight at 4°C in the corresponding primary antibody solution. Secondary antibody solution was incubated at room temperature for 45 minutes. Nuclei were stained using DAPI (1 μg / ml). As primary antibodies, anti-CK20 antibody (manufacturer: Progen, product number: 61054) and anti-piccolo antibody (manufacturer: Novus Biologicals (Tocris), product number: NBP1-90250) were used. Subsequently, as secondary antibodies, Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (manufacturer: Invitrogen, product number: A-11008) and Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (manufacturer: Invitrogen, product number: A-11030) were used, depending on the primary antibody used. DAPI (manufacturer: Sigma-Aldrich, product number: 10236276001) was used for nuclear staining. Fluorescence microscope images are shown (Figure 4A: Vehicle (control), Figure 4B: Sandalol). The number of CK20-positive cells expressing Piccolo in a designated region was counted in images taken under a fluorescence microscope and graphed (Figure 4C). Sandalol increased the number of Merkel cells expressing Piccolo.

[0041] Example 3: Sensory nerve cells promote type I collagen production in fibroblasts. (1) Culture of sensory nerve cells Human iPS cell-derived sensory neural progenitor cells (i-SNs, ax0055, Axol Bioscience, Cambridge, United Kingdom) were cultured according to the manufacturer's instructions (Figure 5A). Specifically, these cells were plated onto 24-well plates coated with Sure-Bond XF (ax0053) and cultured in Sensory Neuron Maintenance Medium (SNMM, ax0060) supplemented with 25 ng / mL GDNF, 25 ng / mL BDNF, 10 ng / mL NGF, and 10 ng / mL NT-3. The cells were cultured for 5 weeks, with half of the culture medium being replaced every 3-4 days, to differentiate into sensory neurons. After 5 weeks of culture, the culture medium was collected and the supernatant was obtained. (2) Type I collagen production in fibroblasts stimulated by sensory nerve cells (2-1) Stimulation by sensory nerve cells Fibroblasts were obtained by isolating neonatal foreskin fibroblasts after informed consent and growing them for 2 days at 37°C in a humid atmosphere of 95% air and 5% CO2 in Dulbecco's Modified Eagle Medium (Life Technologies Japan Ltd., Tokyo, Japan) containing 10% fetal bovine serum. The supernatant (300 μl) of the culture medium obtained by the method in (1) above was mixed with the fibroblast culture medium in a 1:1 ratio and cultured for 4 hours, and collagen production was measured by the method in (2-2) below. As a control, instead of the culture medium supernatant, the medium for iPS cell-derived sensory nerve progenitor cells described above was mixed with the fibroblast culture medium in a 1:1 ratio and used. (2-2) Measurement of type I collagen production by quantitative real-time PCR Total RNA was isolated from fibroblasts cultured for 4 hours in or without serum starvation using the RNeasy Mini Kit (250) (QIAGEN #74106). Type I collagen mRNA expression levels were measured by quantitative real-time PCR. Specifically, this was performed using the TaqMan RNA-to-C 1Step Kit (Applied Biosystems #4392938), a TaqMan probe, and a type I collagen α1 primer (Hs00164004, Taqman, Applied Biosystems). β-actin (Hs01060665) was used as an internal control, and expression levels were normalized relative to β-actin.

[0042] The results are shown in Figure 5B. When the supernatant of nerve cell culture medium was added (conditioned medium), the mRNA expression level of type I collagen in fibroblasts was significantly increased compared to the control group without the addition (control). This suggests that components derived from nerve cells promote collagen production in fibroblasts.

[0043] Example 4: Collagen production promotion effect via nerve activation induced by stimulation of various substances Next, to confirm that type I collagen production in fibroblasts is promoted through nerve activation, we measured type I collagen production using the culture supernatant of nerve cells stimulated with lavender oil, which has been shown to activate nerves.

[0044] (1) Test substance We used lavender oil purchased from Bayan, France (essential oil containing 30% or more linalyl acetate, obtained by steam distillation of Lavandula vera DC. flowers). In addition, we used capsaicin (Cap) (Sigma-Aldrich) and cinnamaldehyde (CNM 125 μM) (Sigma-Aldrich), which are known TRPV1 ligands, as test substances. (2) Culture of human iPS cell-derived sensory nerve progenitor cells The entire supernatant (600 μl) of neural progenitor cells cultured for more than 5 weeks using the same materials and method as in Example 4 was collected. 500 μl of medium prepared by adding the test substance described in (1) above to the medium containing fibroblasts so that the final concentration was 0.005% lavender oil was added back to the neural progenitor cells, and the mixture was left to stand in an incubator for 15 minutes. The same test was performed using 0.25 μM capsaicin (Cap 0.25 μM) and cinnamaldehyde (CNM 125 μM) as test substances. Subsequently, the supernatant containing the test substances was collected, DMEM was added, and the supernatant:DMEM was mixed in a ratio of 1:3. 1 ml of the above mixture was applied to fibroblasts cultured in a 12-well plate using the same method as in Example 4, and RNA was collected after standing for 4 hours. As a control, DMEM was prepared by adjusting the above test substance to a final concentration of 0.005% lavender oil, and this was directly mixed with fibroblasts. After standing for 4 hours, RNA was recovered. The recovered RNA was measured for type I collagen by quantitative real-time PCR, as in Example 4.

[0045] The results are shown in Figure 6. As shown in Figure 6A, the culture supernatant of iPS cell-derived sensory neural progenitor cells stimulated with lavender oil promoted type I collagen production in fibroblasts. However, as shown in Figure 6B, when the same concentration of lavender oil was added directly to fibroblasts without neural activation, no significant promotion of type I collagen production was observed.

[0046] Example 5: Effects of direct addition of high-concentration test substance The effects of direct addition were investigated using a higher concentration of the test substance than that used in Example 5. Specifically, the test substance from Example 5 was added to fibroblasts directly using the same method as the control experiment in Example 5, except that the final concentration was 0.02% lavender oil, and collagen levels were measured. The results are shown in Figure 7. As shown in Figure 7, high concentrations of the test substance promoted type I collagen production in fibroblasts. However, this effect was not observed at low concentrations (Figure 6B), suggesting that type I collagen production in fibroblasts was promoted via nerve activation by neuroactivating substances such as lavender oil.

[0047] Example 6: Release of neurotransmitters by Merkel cells in fresh human skin (ex vivo) Sample preparation Fresh skin was obtained from the abdomens of a 42-year-old healthy female subject and a 48-year-old healthy male subject who had undergone cosmetic surgery. After obtaining informed consent and ethical approval, the skin was used in the experiment within 5 days of excision. The skin was exposed to a 25 units / ml dispase-PBS solution at 37°C for 15 minutes, and the epidermis was peeled off.

[0048] Epidermis exfoliated from fresh human skin was cultured for 30 minutes in a high-potassium extracellular solution (5 mM NaCl, 140 mM KCl, 10 mM HEPES (pH 7.4), 10 mM D-glucose, 2 mM MgCl2, and 2 mM CaCl2) containing FFN206 (Tocris, Catalog No. 5043), allowing the fluorescently labeled neurotransmitter FFN206 to be specifically taken up by Merkel cells.

[0049] Epidermal cells containing Merkel cells that had taken up FFN206 under perfusion conditions were perfused, and the FFN206 signal was observed for 10 minutes. For the first 5 minutes, a Balanced Salt Solution (BSS) solution (140 mM NaCl, 5 mM KCl, 10 mM HEPES (pH 7.4), 10 mM D-glucose, 2 mM MgCl2, and 2 mM CaCl2) was perfused as a baseline. From 5 minutes (300 s) until the end of observation (600 s), a 500 μM Sandalol (Givaudan) / BSS solution was perfused (A). As a control, the BSS solution was perfused from 5 minutes (300 s) until the end of observation (600 s). Brightness values ​​were acquired using Fluoview (Olympus).

[0050] cellSens (Olympus) and MATLAB (Mathworks) were used to evaluate cell responses based on FFN206 luminance values. ROIs were created along the FFN206 signal, and the time evolution of the average luminance value within each ROI was evaluated. For each ROI, baseline luminance value changes were determined using nonlinear approximation, and luminance values ​​were calculated based on the baseline approximation results, excluding the effect of fading. Normalized luminance values ​​(%) were calculated by dividing the baseline average luminance value over 300 s by the luminance value at each time point. As an indicator of cell response evaluation, a decrease of 3 S.D. or more in the normalized luminance value from the baseline mean of each ROI after sandalol stimulation (from 300 s onward) was defined as the luminance value (Figure 8A). In contrast, no decrease in luminance value was observed in the control group after 5 minutes of BSS solution perfusion (Figure 8B).

[0051] To confirm the release of FFN206 in response to 500 μM Sandalore stimulation, 10 minutes of live-cell imaging was performed and the brightness values ​​of the FFN206 signal were observed. As a result, it was found that Merkel cells responded to Sandalore stimulation (Figures 8 and 9). Figures 8 and 9 show that after the influx of Sandalore stimulation at 300 s, the brightness value continued to decrease beyond the response line calculated from baseline. Figure 8(A) shows the change in brightness value in the mouse epidermis, and it was observed that Sandalore activates Merkel cells via OR2AT4 expressed in Merkel cells, promoting the release of neurotransmitters.

Claims

1. The following formula: 【Chemistry 1】 [In the formula, R １ and R ２ They either form a double bond together, or R １ and R ２ They form a cyclopropyl group together; R ３ and R ４ They are either identical or different, independently selected from hydrogen, methyl, or R ３ and R ４ They form a double bond together; R ５ and R ６ are the same or different and are independently selected from hydrogen, methyl, or R ５ and R ６ form a double bond together; or R ５ and R ６ form a cyclopropyl group together; R ７ is methyl or ethyl; R ８ [is hydrogen or methyl] A Merkel cell activator containing the compound represented by [formula].

2. The activator according to claim 1, wherein the compound is sandalol or bramanol.

3. An activator according to claim 1 or 2, which enhances the sense of touch.

4. The following formula: 【Chemistry 2】 [In the formula, R １ and R ２ They either form a double bond together, or R １ and R ２ They form a cyclopropyl group together; R ３ and R ４ They are either identical or different, independently selected from hydrogen, methyl, or R ３ and R ４ They form a double bond together; R ５ and R ６ They are either identical or different, independently selected from hydrogen, methyl, or R ５ and R ６ They form a double bond together; or R ５ and R ６ They form a cyclopropyl group together; R ７ is methyl or ethyl; R ８ [is hydrogen or methyl] A synaptic vesicle enhancer in Merkel cells, containing the compound represented by [formula].

5. The enhancer according to claim 4, wherein the compound is sandalol or bramanol.

6. The enhancer according to claim 4 or 5, wherein the enhancer improves the efficiency of neurotransmission between Merkel cells and nerve cells.

7. An enhancer according to any one of claims 4 to 6, which enhances the sense of touch.

8. An enhancer according to any one of claims 4 to 7, which enhances collagen production.

9. The following formula: 【Transformation 3】 [In the formula, R １ and R ２ They either form a double bond together, or R １ and R ２ They form a cyclopropyl group together; R ３ and R ４ They are either identical or different, independently selected from hydrogen, methyl, or R ３ and R ４ They form a double bond together; R ５ and R ６ They are either identical or different, independently selected from hydrogen, methyl, or R ５ and R ６ They form a double bond together; or R ５ and R ６ They form a cyclopropyl group together; R ７ is methyl or ethyl; R ８ [is hydrogen or methyl] A neurotransmitter release enhancer in Merkel cells, comprising the compound represented by [the formula shown].

10. The neurotransmitter release enhancer according to claim 9, wherein the compound is sandalol or bramanol.

11. A neurotransmitter release enhancer according to claim 9 or 10, which enhances the sense of touch.

12. A neurotransmitter release enhancer according to any one of claims 9 to 11, which enhances collagen production.

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