JP2012152127A - Skin barrier function recovering agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a skin barrier function recovering agent and a method for screening the same, and a method for recovering the skin barrier function using the same.SOLUTION: There are provided a method for screening the skin barrier function recovering agent with activation of TRPM8, which is one of thermosensitive receptors generally called a transient receptor potential (TRP) family, as a marker; and a skin barrier function recovering agent containing a TRPM8 agonist.

Description

  The present invention relates to a screening method for a skin barrier function recovery agent using TRPM8 receptor activation as an index, a skin barrier function recovery agent containing a TRPM8 agonist, and a skin barrier function recovery method using the same.

  Skin diseases such as atopic dermatitis accompanied by a decrease in skin barrier function are increasing. In recent years, there have been many cases in which the skin is modulated due to mental stress or rough skin in the living environment, and the skin barrier function is lowered. Furthermore, there are cases in which transplanted skin is used during orthopedic surgery and burn surgery, but the skin barrier function is low due to insufficient formation of the skin barrier layer by transplantation, or the skin barrier function decreases by transplantation. It is. As described above, there are many problems such as low skin barrier function through various shapes.

  These reduced skin barrier functions are considered to recover spontaneously. For example, in the case of skin diseases, active clinical treatment is mainly carried out by anti-inflammatory and moisturizing with steroids. However, particularly in the former, side effects are known, and the barrier itself is not improved, and rebound due to discontinuation of external use is also known. From the above, it has been desired to develop a technique for safely promoting the improvement of the skin barrier function.

  During the past decade, several thermosensitive receptors, collectively referred to as the transient receptor potential (TRP) family, have been cloned from the peripheral nervous system. The inventor has previously shown that TRPV1 and TRPV4 are related to the homeostasis of the epidermal permeability barrier (Non-Patent Document 1). TRPV1 is activated by high temperature (43 ° C.) and capsaicin, which activation delays the recovery of damaged skin barrier function. On the other hand, TRPV4 is activated by a certain low temperature (> 33 ° C.) and 4α-phorbol 12,13-didecanoate (4cx-PDD), and the activation promotes the recovery of the barrier function. Other TRP proteins, TRPA1 and TRPM8, are activated at low temperatures. TRPA1 was discovered as a TRP channel that is activated at a temperature below 17 ° C. in nociceptive sensory neurons derived from dorsal root ganglia (Non-patent Document 2). An increase in intracellular calcium and current were observed at 17 ° C. (Non-patent Document 2). TRPA1 is activated by allyl isothiocyanate (Non-patent Document 3) and cinnamaldehyde (Non-patent Document 4).

  Expression of TRPA1 in human epidermal keratinocytes has been reported (Non-patent Document 5). Recently, the present inventor has shown that an increase in intracellular calcium in cultured human keratinocytes is induced at low temperatures (less than 22 ° C.) (Non-patent Document 6). Furthermore, topical application of TRPA1 agonist promoted recovery of epidermal permeability barrier function after destruction (Non-patent Document 7). These results suggest that TRPA1 is present in epidermal keratinocytes and is associated with barrier homeostasis.

Denda M., Sokabe T., Tominaga T., et al. Effects of skin surface temperature on epidermal permeability barrier homeostasis. J Invest Dermatol 2007; 127: 654-659 Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures.Cell .; 2003; 112: 819-29. Jordt SE, Bautista DM, Chuang HH, et al. Mustard oils and cannabinoids excite sensory nerve fibers through the TRP channel ANKTM1. Nature. 2004; 427: 260-5. Bandell M, Story GM, Hwang SW, et al. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron. 2004; 41: 849-57. Atoyan R., Shander D., Botchkarvera NV. (2009) Non-neural expression of transient receptor potential type A1 (TRPA1) in human skin.J Invest Dermatol 2009; 129: 2315-5 Tsutsumi M., Denda S., Ikeyama K., et al. Exposure to low temperature induces elevation of intracellular calcium in cultured human keratinocytes.J Invest Dermatol 2010 in press Denda M., Tsutsumi M., Goto M., et al. Topical application of TRPA1 agonists and brief cold exposure accelerate skin permeability barrier recovery.J Invest Dermatol 2010 in press Peier AM, Moqrich A, Hergarden AC, et al. A trp channel that senses cold stimuli and menthol. Cell. 2002; 108: 705-15. Patel T, Ishiuji Y, Yosipovitch G Menthol: a refreshing look at this ancient compound.J Am Acad Dermatol. 2007; 57: 873-8. Sabnis AS, Shadid M, Yost GS, et al. Human lung epithelial cells express a functional cold-sensing TRPM8 variant. Am J Respir Cell Mol Biol. 2008; 39: 466-74. Bodding M., Wissenbach U., Flockerzi V. Characterisation of TRPM8 as a pharmacophore receptor. Cell Calcium 2007; 42: 618-628 Madrid R., Donovan-Rodriguez T., Meseguer V., et al. Contribution of TRPM8 channels to cold transduction in primary sensory neurons and peripheral nerve terminals.J Neurosci 2006; 26: 12512-12525 Denda M., Sato J., Tsuchiya T., et al. Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruption: implication for seasonal exacerbations of inflammatory dermatoses. J Invest Dermatol 1998; 111: 873-878 Denda M, Tsuchiya T. Barrier recovery rate varies time-dependently in human skin.Br J Dermatol 2000; 142: 881-884 Denda M., Fuziwara S., Inoue K. Influx of calcium and chloride ions into epidermal keratinocytes regulates exocytosis of epidermal lamellar bodies and skin permeability barrier homeostasis.J Invest Dermatol 2003; 121: 362-367 Fuziwara S., Inoue K., Denda M. NMDA-type glutamate receptor is associated with cutaneous barrier homeostasis.J Invest Dermatol 2003; 120: 1023-1029 Denda M., Fuziwara S., Hibino T. Expression of voltage-gated calcium channel subunit aC1 in epidermal keratinocytes and effects of agonist and antagonists of the channel on skin barrier homeostasis.Exp Dermatol 2006; 15: 455-460 Denda M., Fuziwara S., Inoue K., et al. Immunoreactivity of VR1 on epidermal keratinocyte of human skin.Biochem Biophys Res Commun 2001; 285: 1250-1252 Inoue K, Koizumi S, Fuziwara S, et al. Functional vanilloid receptors in cultured normal human keratinocytes. Biochem Biophys Res Commun 2002; 291: 124-129 Peier AM, Reeve AJ, Andersson DA, A heat-sensitive TRP channel expressed in keratinocytes. Science. 2002; 296: 2046-9. Chung MK, Lee H, Caterina MJ. Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J Biol Chem. 2003; 278: 32037-46

  This invention is made | formed in view of the said situation, The objective is to provide the method of promoting the improvement of a skin barrier function.

  TRPM8 is activated by low temperature (less than 22 ° C.) and menthol (Non-patent Document 8). Menthol has long been used as an external preparation (Non-patent Document 9). Furthermore, recent studies have shown the expression of cold-sensitive TRPM8 mutants in human lung epithelial cells (Non-patent Document 10). The inventor therefore hypothesized that TRPM8 is present in epidermal keratinocytes and is associated with permeability barrier homeostasis.

  In order to verify this idea, we first performed immunohistochemical studies of mouse skin using TRPM8 antibody and RT-PCR studies using mouse skin and cultured human keratinocytes. Expression of TRPM8 or TRPM8-like protein was confirmed. Next, the effect of topical application of TRPM8 agonist and TRPM8 antagonist on epidermal barrier recovery rate after destruction was evaluated by tape stripping method. Topical application of TRPM8 agonists menthol and WS12 promoted barrier function recovery after tape stripping. The effect of WS12 was inhibited by ruthenium red, a non-selective TRP antagonist, and BCTC, a TRPM8 specific antagonist. Furthermore, the effect of TRPM8 agonists and TRPM8 antagonists on epidermal hyperplasia induced by disruption of barrier function under low humidity was investigated. Topical application of WS12 reduced epidermal proliferation associated with barrier breakdown under low humidity. This effect was inhibited by BCTC. This result indicates that TRPM8 or closely related proteins in epidermal keratinocytes play a role in epidermal permeation barrier homeostasis and epidermal proliferation after barrier injury.

  Therefore, in a first aspect, the present invention provides a screening method for a skin barrier function restoring agent that indicates activation of TRPM8 receptor. Preferably, the TRPM8 receptor is a keratinocyte TRPM8 receptor.

  In a second aspect, the present invention provides a skin barrier function restoring agent containing a TRPM8 agonist. Such a skin barrier function restoring agent may be a pharmaceutical composition or a cosmetic composition. The TRPM8 agonist is selected from the group consisting of menthol, icilin, eucalyol, WS-12 (((1R, 2S, 5R) -N- (4-methoxyphenyl) -5-methyl-2- (1-methylethyl ) Cyclohexanecarboxamide), WS-23 (2-isopropyl-N, 2,3-trimethylbutyramide), WS-3 (N-methyl-P-methane-3-carboxamide), WS-148 (phosphine oxide) CPS-113 (N-Substituted Allyl Alkyl Cyclo® Alkyl-Carboxamide), CPS-369, Geraniol, Linarool, Fresco Rat, MGA, PMD-38, Contact P and Hydroxyl-citronellal The skin barrier function restoration | recovery agent of Claim 2 which is one or more compounds chosen from these.

  In a third aspect, the present invention provides a method for restoring skin barrier function, which comprises applying a TRPM8 agonist to the skin. Such skin barrier function recovery may be therapeutic, prophylactic, or cosmetic.

  According to the present invention, it is possible to provide a more effective skin barrier function restoring agent without any side effects, for example, by completely new means in the prior art.

The result of the immunohistological study of the epidermis of a hairless mouse is shown. FIG. 1A: immunostaining with TRPM8 antibody; FIG. 1B: superimposed image with nuclear staining: FIG. 1C: negative control in the absence of primary antibody; bars are 20 μm). The result of the RT-PCR test of a hairless mouse (FIG. 2A) and a cultured human keratinocyte (FIG. 2B) is shown. Evaluation of the effect of TRPM8 agonist on intact skin of hairless mice. Effect of topical application of WS12 on epidermal proliferation induced by barrier breakdown under low humidity. FIG. 4A: Untreated control; FIG. 4B: Barrier function under low humidity (ethanol applied); FIG. 4C: After breaking the barrier function under low humidity, WS12 solution was applied; FIG. 4D: Low humidity After breaking the barrier function below, WS12 and BTCT were applied. The graph which quantified the result shown in FIG.

As described above, the present invention provides a screening method for a skin barrier function restoring agent, wherein a compound that activates cellular TRPM8 receptor is selected as a useful drug. Such cells are preferably keratinocytes.
TRPM8 can form excitatory ion channels or TRPM8 channels that can be activated by low temperature or cold-inducing compounds. The activated TRPM8 channel gates the influx of Ca ²⁺ ions through the channel, resulting in membrane depolarization. The amino acid sequence and base sequence of TRPM8 are known, and the A amino acid sequence and base sequence of human TRPM8 are listed in GenBank Y090109.
The activation of the TRPM8 receptor is not particularly limited. For example, calcium imaging (in this case, intracellular calcium concentration increases when the receptor is activated), membrane potential measurement by batch clamp (In this case, if the receptor is activated, depolarization of the cell membrane is observed).

  The TRPM8 receptor referred to in the present invention may be a receptor present in mammalian cells, preferably skin cells constituting the stratum corneum, epidermis, basement membrane, dermis, etc., most preferably in the surface layer of keratinocytes. The cell stratum corneum cells are preferably derived from humans, but may be derived from any mammals such as mice, rats, guinea pigs, rabbits and pigs.

  The TRPM8 agonist refers to a chemical substance that activates TRPM8, and various natural and synthetic compounds are known as TRPM8 agonists, and without particular limitation, the TRPM8 agonist is menthol, icilin, eucalyol, WS -12 (((1R, 2S, 5R) -N- (4-methoxyphenyl) -5-methyl-2- (1-methylethyl) cyclohexanecarboxamide), WS-23, WS-30, WS-148, CPS -113, CPS-369, geraniol, linalool, frescolat, MGA, PMD-38, contact P and hydroxyl-citronellal.

  The state desired to restore the skin barrier function in the present invention refers to, for example, inflammation, preferably external stimulation such as ultraviolet irradiation stimulation, thermal stimulation (heating and cooling), chemical drug stimulation, osmotic pressure stimulation, oxidative stimulation, etc. It means a state in which the skin barrier function is reduced due to stress.

  The skin barrier function recovery method according to the present invention can be carried out by applying a composition containing a TRPM8 agonist to a skin site requiring recovery of the barrier function, for example, applying it locally. Such a composition may be applied to the site as a pharmaceutical composition or as a cosmetic, for example, as a skin external preparation.

  The pharmaceutical or cosmetic composition according to the present invention is usually used by containing the above TRPM8 agonist in an aqueous solvent such as water or ethanol. The blending amount of the TRPM8 agonist according to the present invention is not particularly limited, and for example, it is applied as a solution having a concentration in the range of 1 μM to 10 mM, preferably 10 μM to 1 mM, more preferably about 100 μM. In addition, when preparing the chemical | medical agent which concerns on this invention as a bath agent, since it is normally diluted about 100 to 1000 times at the time of use, it is preferable that a mixing | blending is prescribed by the high density | concentration which considered it. As said aqueous solvent, a lower alcohol is preferable and content of a lower alcohol is 20-80 mass% in the composition of this invention, More preferably, it is 40-60 mass%.

  Further, the pharmaceutical or cosmetic composition according to the present invention, in addition to the TRPM8 agonist, which is the essential component, is a component that is usually used in an external preparation for skin such as cosmetics and pharmaceuticals, such as other whitening agents, moisturizers, antioxidants, Oily components, ultraviolet absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, and the like can be appropriately blended as necessary.

  Other metal sequestering agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, caffeine, tannin, verapamil, tranexamic acid and the like according to the use of the composition Derivatives, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, Other whitening agents such as kojic acid, sugars such as glucose, fructose, mannose, sucrose, and trehalose, vitamins A such as retinoic acid, retinol, retinol acetate, and retinol palmitate can be appropriately blended.

  The pharmaceutical or cosmetic composition according to the present invention is applied to an external preparation such as cosmetics, pharmaceuticals, quasi drugs, etc., for example, lotion, cream, milky lotion, lotion, pack, bath preparation, ointment, hair lotion. As long as it is conventionally used for external preparations for skin, such as hair nick, hair liquid, shampoo, rinse, hair nourishing and hair restorer, any form may be used.

  Next, the present invention will be described in more detail with reference to examples.

Materials and Methods Methods All hairless mice (HR-1, Hoshino Test Animal Breeding), 7-10 weeks old, were used for all experiments. Measurement of skin barrier function, barrier disruption, and application of test samples were performed under anesthesia using Nembutal. This research is approved by the Ethics Committee of Shiseido Research Center in accordance with the guidelines of the National Institutes of Health.

reagent
L-Menthol and Ruthenium Red were purchased from Sigma (St Louis, MI, USA). (1R, 2S, 5R) -N- (4-Methoxyphenyl) -5-methyl-2- (1-methylethyl) cyclohexanecarboxamide (WS12) was purchased from Tocris (Ellisville, MI, USA). N- (4-t-butylphenyl) -4- (3-chloropyridin-2-yl) tetrahydropyrazine-1 (2H) -carboxamide (BCTC) was purchased from Enzo Life Science (Plymouth Meeting, PA, USA) did. WS12 has been reported as a TRPM8 agonist (11) and BCTC has been reported as a TRPM8 antagonist (12).

Recovery of barrier function The epidermal barrier function was evaluated by measuring the transdermal moisture transpiration (TEWL) using an electric moisture meter (Meeco, Warrington, PA, USA) as reported previously (Non-patent Document 13). For the barrier function recovery experiment, as previously reported (Non-patent Document 13), the skin on both sides was repeatedly tape peeled until TEWL was 7-10 mg / cm ² / h. One point on each flank was measured and the effect was evaluated using 8 mice for each treatment. Next, TEWL was measured at the same location after 1, 3, 6, and 24 hours after barrier failure. Barrier destruction was performed from 7:00 am to 8:00 am, and the barrier recovery rate was immediately evaluated to avoid the influence of circadian rhythm (Non-Patent Document 14). The results of barrier function recovery are expressed in percentage of recovery because the degree of barrier destruction varies from day to day. The percentage recovery of each mouse was calculated according to the following formula:

Formula: (TEWL Immediately after Destruction-TEWL at Predetermined Time) / (TEWL Immediately After Destruction-Baseline TEWL) x 100%

Cell culture Normal human epithelial keratinocytes derived from foreskin were purchased from Kurashiki Spinning Co., Ltd. (Osaka) and cultured in EPILIFE-KG2 (Kurashiki Spinning Co., Ltd.). Keratinocytes were seeded on collagen-coated coverslips (Nalge Nunc, Naperville, Illinois, USA) and used within 5 days.

Immunohistochemistry Skin samples were fixed for 10 minutes by adding 4% paraformaldehyde in PBS, washed with PBS solution, subjected to blocking treatment using blocking solution for 30 minutes at room temperature, and rabbit anti-TRPM8 antibody (100: 1, Abeam, Cambridge, MA, USA) was added and incubated at room temperature for 1 hour. The skin sample was then washed 3 times for 5 minutes with a PBS solution containing 0.05% Tween 20, diluted to 500: 1 with Alexa Fluor 594 containing Hoechst 33528 (1: 1000, Dojindo, Kumamoto). Binding with labeled anti-rabbit IgG goat antibody (Molecular Probes, OH, USA) for 30 minutes. Thereafter, the plate was washed 3 times for 5 minutes with a PBS solution containing 0.05% Tween20. Finally, skin samples were treated with Fluoromount Plus (Diagnostic Biosystems, Pleasanton, CA, USA).

Reverse Transcript Polymerase Chain Reaction Assay Total RNA, mouse skin sample and cultured human keratinization using ISOGEN (Wako Pure Chemical Industries, Osaka) containing phenol and guanidine thiocyanate according to manufacturer's instructions Isolated from cells. The total RNA preparation was reverse transcribed using Superscript II and analyzed by PCR. The specific primers used are as follows.

Mouse version 1:
5'-cttggcagaacagctactgg-3 '(forward 2092-2111) (SEQ ID NO: 1)
5'-atgtggatgagccttagcgt-3 '(reverse 2715-2696) (SEQ ID NO: 2)
Version 2:
5'-aggttcccagtgacgtggatag-3 '(forward 2919-2940) (SEQ ID NO: 3)
5'-aggttcccagtgacgtggatag-3 '(reverse 3509-3490) (SEQ ID NO: 4)
Version 3:
5'-aggcagtggtacatgaacgga-3 '(forward 2528-2548) (SEQ ID NO: 5)
5'-ggtggagagcatgtagatgcac-3 '(reverse 3079-3058) (SEQ ID NO: 6)

Human version 1:
5'-ctggagaatggcttgaacctac-3 '(forward 1472-1493) (SEQ ID NO: 7)
5'-tctcgggaaatctctccatacc-3 '(reverse 2106-2085) (SEQ ID NO: 8)
Version 2:
5'-atggacatcccactgtcgaag-3 '(forward 843-863) (SEQ ID NO: 9)
5'-ggaagttcgcaaccagtttcc-3 '(reverse 1634-1614) (SEQ ID NO: 10)

  The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a reference gene.

Epidermal hyperplasia induced by barrier disruption at low humidity All animals were individually housed in 7.2 liter cages. The relative humidity in the cage was kept below 10% using dry air, as previously reported (13). All temperatures were the same (22-25 ° C.), and the outside air was circulated 100 times per hour. Air was not directly applied to the animals. During the experiment, animal behavior was not restricted. The NH ₃ level was always less than 1 ppm. As previously reported (Non-Patent Document 13), the animals were initially raised for 48 hours under dry air conditions, and then the skin on both sides was treated with a cotton ball soaked with acetone. This procedure was terminated when TEWL reached 2.5-3.5 mg / cm ² / h. Immediately after breaking the barrier, 100 μL of WS12 ethanol solution (2 μM) or a mixture of BCTC (10 μM) and WS12 (2 μM) was applied to one side of the treated area. Ethanol was applied to the other side. The animals were again raised for 48 hours under dry conditions. After the experiment, animals were euthanized with diethyl ether inhalation and skin samples were removed from the treated areas. One hour before euthanasia, 20 μL bromodeoxyuridine (BrdU) 10 mM solution per gram body weight was injected intraperitoneally. Untreated control mice were also treated with BrdU at the same time. After fixation with 4% paraformaldehyde, the full thickness skin sample was embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin. On each section, 5 spots were selected at random, the thickness of the epidermis was measured with an optical micrometer, and the average value was obtained by calculation. Sections were immunostained with anti-BrdU antibody for evaluation of DNA synthesis. Five points were randomly selected from each section. The number of immunostained cells per 1 mm of epidermis was counted and the average value was calculated. Measurement was performed in a blinded manner.

Statistics The results were expressed as mean ± standard deviation. Statistical significance of differences was determined by analysis of variance using the least significant difference method.

Results FIG. 1 shows the results of an immunohistological study of the epidermis of hairless mice. Immunoreactivity to TRPM8 antibody was observed in keratinocytes (FIG. 1A: immunostaining with TRPM8 antibody; FIG. 1B: superimposed image with nuclear staining: FIG. 1C: negative control in the absence of primary antibody; bar Is 20 μm). An immunohistochemical study of the skin of hairless mice revealed immunoreactivity to TRPM8 antibody through the living layer of the epidermis. FIG. 2 shows the results of RT-PCR tests on the epidermis of hairless mice (FIG. 2A) and cultured human keratinocytes (FIG. 2B). From the results of RT-PCR of mouse epidermis (FIG. 2A) and human keratinocytes (FIG. 2B), it was confirmed that mRNA encoding a TRPM8-like protein is expressed in epidermal keratinocytes.

  Next, the effect of TRPM8 agonist on intact skin of hairless mice was evaluated. The permeation barrier was broken by tape peeling and immediately 100 μL of the test solution was applied. Topical application of menthol (1 mM) and WS12 (2 μM) promoted barrier function recovery. Acceleration by WS12 was inhibited by pretreatment with ruthenium red (100 μM) or BCTC (10 μM) (FIG. 3).

  Topical application of WS12 (2 μM) prevented epidermal hyperplasia induced by barrier breakdown under low environmental humidity. This effect was inhibited by BCTC (10 μM) (FIG. 4). Representative sections are shown in FIGS. 4A-D. FIG. 4A shows a hyperproliferative epidermis that has been water treated after acetone treatment at low humidity. Gray dots represent BrdU positive cells. An increase was seen in the epidermal basal layer (FIG. 4A). Topical application of WS12 prevented epidermal hyperplasia (FIG. 4B). The effect of WS12 was inhibited by BCTC (FIG. 4C). FIG. 4D shows an untreated control. FIG. 5 shows the number of BrdU positive cells in the epidermis of animals. These results suggest that activation of TRPM8 or TRPM8-like protein is effective in preventing epidermal hyperproliferative responses induced by barrier damage.

Discussion The present inventor recently showed that exposure to low temperatures (<22 ° C.) induces an increase in intracellular calcium in cultured human keratinocytes (Non-patent Document 6). Furthermore, topical application of TRPA1 agonist or short-term cold exposure promoted recovery of barrier function (Non-patent Document 7). These results suggest that TRPA1 in epidermal keratinocytes is associated with epidermal permeability barrier homeostasis. Previous reports have shown that TRPA1 was activated at temperatures below 17 ° C (Non-patent Document 2). Recent studies by the inventor have shown that the increase in intracellular calcium in cultured human keratinocytes begins at about 22 ° C. (Non-patent Document 6). Therefore, it was assumed that TRPM8 is also present in epidermal keratinocytes. Results of immunostaining and reverse transcription polymerase chain reaction confirmed the expression of TRPM8 or closely related similar proteins in keratinocytes. Furthermore, similar to the inventors' previous discovery of TRPA1 (Non-Patent Document 7), topical application of TRPM8 agonists promoted barrier function recovery, which was inhibited by TRPM8 antagonists. These results suggest that both TRPA1 and TRPM8 are present in epidermal keratinocytes and are involved in barrier homeostasis. However, the mechanism is still unknown.

  The inventor has previously shown that calcium influx blocks lamellar secretions between the granule layer and the stratum corneum, resulting in delayed recovery of the barrier function (Non-Patent Document 15). Furthermore, calcium permeation channels such as NMDA receptor channels and voltage-dependent positive calcium channels delayed recovery of the barrier function (Non-patent Documents 16 and 17). These results suggest that an increase in intracellular calcium delayed barrier function recovery. However, although TRP family members are calcium permeation channels, their activity does not necessarily delay the recovery of barrier function. For example, activation of TRPV4 promotes the activity of barrier function, but activation of TRPV1 delays it (Non-patent Document 1). As described above, activation of TRPA1 and TRPM8 promoted restoration of barrier function. Thus, activation of some TRPs may induce electrophysiological phenomena in keratinocytes that are different from other calcium channels.

  For a long time, peripheral nerve fibers have been recognized to be involved in the skin warming system. However, since the inventor first reported expression of TRPV1 in epidermal keratinocytes, expression of other temperature sensitive TRP, TRPV3, TRPV4, and TRPA1 in epidermal keratinocytes has been demonstrated ( Non-patent documents 20, 21, 5, 6). The results of this study indicate that TRPM8 or closely related proteins are present in epidermal keratinocytes. Thus, epidermal keratinocytes appear to have a wide range of temperature sensing systems.

  Menthol, a TRPM8 agonist, is used locally as an itching, disinfectant, analgesic and cooling agent (Non-patent Document 9). Our results show that TRPM8 agonists promote recovery of barrier function and reduce epidermal proliferation. Thus, activation of TRPM8 may also reduce the inflammatory response. Thermosensitive receptors may become new targets for itching and disinfectants.

Claims (3)

  A screening method for a skin barrier function restoring agent that indicates activation of TRPM8 receptor.   A skin barrier function-restoring agent containing a TRPM8 agonist.   The TRPM8 agonist is menthol, icilin, eucalyol, WS-12 (((1R, 2S, 5R) -N- (4-methoxyphenyl) -5-methyl-2- (1-methylethyl) cyclohexanecarboxamide), WS-23 (2-isopropyl-N, 2,3-trimethylbutyramide), WS-3 (N-ethyl-P-methane-3-carboxamide), WS-148 (phosphine oxide) CPS-113 (N -Substituted allylalkylcyclo (R) alkyl-carboxamide), CPS-369, geraniol, linalool, frescolat, MGA, PMD-38, contact P and hydroxyl-citronellal 1 or The skin barrier function restoration | recovery agent of Claim 2 which is a some compound.