WO2003053466A1

WO2003053466A1 - Skin barrier function repair accelerators

Info

Publication number: WO2003053466A1
Application number: PCT/JP2002/012945
Authority: WO
Inventors: Shigeyoshi Fujiwara; Mitsuhiro Denda; Kaori Inoue
Original assignee: Shiseido Company, Ltd.
Priority date: 2001-12-13
Filing date: 2002-12-11
Publication date: 2003-07-03
Also published as: JPWO2003053466A1

Abstract

It is intended to provide skin barrier function repair accelerators containing an excitation cell receptor antagonist or a regulatory cell receptor agonist and a method of screening a substance having an activity of accelerating the repair of skin barrier function.

Description

Description Skin PARIA function recovery promoter Technical field

TECHNICAL FIELD The present invention relates to a skin parer function recovery promoter containing an excitatory cell receptor agonist or an inhibitory cell receptor agonist, and a method for screening a substance having a skin parer function recovery promoting activity. Background art

It is known that the loss of moisture from the skin is more active in healthy skin symptoms in various skin diseases such as atopic dermatitis, psoriasis, and contact dermatitis. ing. This so-called increase in transepidermal water loss (TEWL) is attributed to a decrease in components that are thought to play a role in retaining moisture in the skin and as one of the main functions. Has been considered.

Conventionally, NMF (Natural Moisturizing Fac) has been used as an active ingredient that has the effect of improving and preventing skin diseases and rough skin from the viewpoint of replenishing the skin with components that are responsible for water retention and skin parietal function. amino acids as tor), lipids as keratinocyte intercellular lipids, and other mucopolysaccharides such as hyaluronic acid, or similar substances, are also highly safe. It is blended in.

Recently, it has been reported that a specific substance that activates the biosynthesis of a component in the skin responsible for one function of the skin has an effect of improving skin roughness (Japanese Patent Application Laid-Open No. 912/1990). No. 952).

Improvement of rough skin · There is not much research on substances with preventive effects. However, studies on substances that have an effect of improving or recovering skin barrier function are not sufficient, and the relationship between the effect of improving skin barrier function and the effect of improving and preventing skin roughness is not clear. Therefore, a substance having the effect of improving and preventing rough skin does not always have the effect of improving the skin parer function.

On the other hand, it has been reported that when the function of the skin barrier is reduced, the skin function of the skin is reduced, and abnormal skin reproductivity occurs. In particular, in the case of the elderly, it takes a long time to recover the decreased skin barrier function, and new skin is effective to prevent abnormal skin proliferation due to a decrease in skin function with aging. Development of a barrier function recovery accelerator was requested.

By the way, it has recently been found that mammalian skin cell surfaces also have an equivalent of various neuronal receptors found on the nerve cell surface (enever, et.al. The Journal of Investigative Dermatology ( 1999), Vol.112, pp, 337-341). The mechanism of action of these excitatory and inhibitory receptors in neurons responsible for signaling at the chemical synapse of neurons has been largely elucidated, but their function in skin cells is largely unknown. Not studied.

Skin cells, like nerve cells, consume energy and create potential differences inside and outside the cell membrane. This potential difference is mainly due to the opening and closing of Ca ²⁺ , Na ⁺ , and CI ion channels by various receptors on the cell surface. Japanese Patent Application Laid-Open No. 2000-290135 ^discloses a Ca ²⁺ channel inhibitor or a metal salt having a Ca ²⁺ channel inhibitory action, for example, manganese salt, strontium Salt, lanthanum salt, cobalt salt, zinc salt, magnesium salt, iron salt, norium salt, jill thiazem and its salt, verapamil and its salt, difedipin and its It is disclosed that salt and the like can restore damaged skin barrier function in a very short time. The mechanism and mechanism of action by which such metal salts promote the recovery of skin barrier function is not known, but between the loading of external potential by metal salts and the promotion of skin barrier recovery function. It can be assumed that there is some relationship between The inventor of the present invention has found that skin cells have receptors present on the surface of nerve cells, and that skin barrier cells can be recovered by changes in intracellular and extracellular potentials. Investigate the involvement of excitatory and inhibitory receptors in the recovery of skin paria function, and from the viewpoint of the mechanism of action of such cell receptors, can a new, novel skin paria function recovery promoter be discovered? We examined whether. Disclosure of the invention

As a result of intensive studies, the present inventors have found that antagonists of excitatory cell receptors or agonists of inhibitory cell receptors are effective in promoting the recovery of skin parier function.

Accordingly, the present invention provides, in the first aspect, an agent for promoting the recovery of skin pariar function, which comprises an agonist of an excitatory cell receptor or an agonist of an inhibitory cell receptor.

In one aspect, the excitatory cell receptor is a glutamate receptor

, ATP receptor, heat-stimulated receptor, and adrenaline β2 receptor.

In a further aspect, the glutamate receptor is an NMD A (N-methyl-D-aspartate) receptor.

In a further aspect, the NMDA receptor antagonist is MK-801 (dizocilpine) or D-AP5 (D- (1-)-2-amimino 5-phosphonopentanoic acid). In a further aspect, the ATP receptor is P2X (capriogenic purino receptor), preferably a P2X3 receptor (modotropic purino receptor subtype 3).

In a further aspect, the antagonist of the ATP receptor is suramin, PPADS (pyridoxal phosphate-16-azophenyl).

2, 2, 4, or 1-disulfonic acid) or TNP-ATP (trinitrophone ATP).

In a further aspect, the heat-stimulated receptor is VR-1 (vanilloid receptor subtype 1).

In a further aspect, the VR-1 antagonist is capsazepine.

In a further aspect, the antagonist of the adrenerin 32 receptor is ICI-118, 551 ((chi) -1-1_ [2,3-— (dihid mouth_7-methyl-1H— Indene 4- (yl) oxy] — 3 — [(1-methylethyl) amino] -12-butanol).

In another aspect, the inhibitory cell receptor is a GABA (γ-aminobutyric acid) receptor or a glycine receptor.

In a further aspect, the GA receptor is a type I GAB receptor (a C1-channel-incorporated bicycline-sensitive receptor).

In another aspect, the agonist of the type A-GABA receptor is GABA, muscimol or isogpasin.

Preferably, the skin barrier function recovery promoter is an external preparation for skin.

In a second aspect, the present invention relates to a method for screening a skin-parallel function-recovery activity promoting substance, which comprises suppressing or suppressing the excitatory activity of an excitatory cell receptor. Select a candidate substance that promotes inhibitory activity, apply the substance to mammalian skin, and A substance that reduces the transepidermal water loss or a substance that reduces or suppresses the increase in the thickness of the skin subjected to dry stimulation.

In one aspect, the selection of a substance that suppresses the excitatory activity of the excitatory cell receptor or promotes the inhibitory activity of the inhibitory cell receptor is performed by selecting a substance that is a calcium ion or sodium ion in the cell. Or, evaluate the effect on chloride ion concentration.

In another aspect, the excitatory cell receptor is a glutamate receptor

An ATP receptor, a heat-stimulated receptor or an adrenaline j32 receptor, and the inhibitory cell receptor is a γ-aminobutyric acid receptor or a glycine receptor.

Furthermore, the present invention relates to a method for screening a substance for promoting skin pariole function recovery activity, which comprises causing a substance which is a candidate for a skin parier function recovery activity promoter to act on a mammalian cell, and thereafter, It is intended to provide a method comprising measuring a chlorine ion concentration and selecting a substance which increases the intracellular chloride ion concentration as a substance promoting the recovery of the skin parer function.

Preferably, human epidermal keratinocytes are used as the cells.

Furthermore, the present invention relates to a method of screening a substance for promoting skin pariole function recovery activity, which comprises causing a substance that is a candidate for a skin parier function recovery activity promoter to act on mammalian cells, and then in the cell. Providing a method comprising measuring calcium or sodium ion concentration and selecting a substance which reduces the intracellular calcium or sodium ion concentration as a substance which promotes the recovery of skin barrier function. .

Preferably, human epidermal keratinocytes are used as the cells.

In a third aspect, the present invention relates to a method for improving skin, comprising: It is intended to provide a method characterized by applying a skin external preparation containing an agonist of an inciting cell receptor or an agonist of an inhibitory cell receptor to promote the recovery of the skin parier function. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the relationship between D-glutamic acid, L-glutamic acid and L-aspartic acid and the recovery of skin parier function.

Fig. 2 is a graph showing the relationship between the excitatory receptors antagonist MK-801 and agonist NMDA and the recovery of skin paria function. Fig. 3 shows that MK-801 and NMDA are simultaneously applied to the skin. It is a rough showing skin PARIA function recovery when applied.

FIG. 4 is a Draf showing the relationship between the excitatory receptor antagonist D—AP5 and the recovery of skin parier function.

FIG. 5 is a graph showing the relationship between the excitatory receptors antagonist MK-801 and agonist NMDA and the thickness of the skin subjected to the dry stimulus.

FIG. 6 is a graph showing the relationship between the excitatory receptors antagonist MK-801 and agonist NMDA and intracellular Ca ²⁺ concentration. FIG. 7 is a graph showing the relationship between the excitatory receptor, an ionotropic ATP receptor P2X3, and skin barrier recovery.

Figure 8 shows the function of the skin barrier when suramin, PPADS, and TNP—ATP, the antagonists of the ionotropic ATP receptor excitatory receptor, are applied to the skin. It is a graph which shows recovery.

FIG. 9 is a graph showing the relationship between ptsazepine, which is an antagonist of the excitatory receptor VR-1, and cabsaicin, which is an agonist, and the recovery of skin paria function. FIG. 10 is a graph showing the relationship between the antagonist and agonist of each of adrenalin j31 and β2 receptors, which are excitatory receptors, and the recovery of the skin barrier function.

FIG. 11 is a graph showing the relationship between the type I GAB II receptor, which is an inhibitory receptor, and the recovery of skin parer function.

FIG. 12 is a graph showing the recovery of skin parier function when GABA, an inhibitory receptor agonist, and bicuclimetmet bromide, an agonist, are simultaneously applied to the skin.

FIG. 13 is a graph showing the relationship between the inhibitory receptor agonist GABA and antagonist BM and the thickness of the skin subjected to the dry stimulus.FIG. 14 shows the inhibitory receptor agonist GABA and 4 is a graph showing the relationship between antagonist BM and intracellular C 1— concentration.

FIG. 15 is a graph showing the relationship between glycine receptors, which are excitatory receptors, which are inhibitory receptors, and skin barrier recovery.

FIG. 16 is a graph showing the relationship between the influx of chloride ions or calcium ions into cells and the recovery of skin barrier function.

FIG. 17 is a histological observation photograph showing the relationship between the influx of chloride ions or calcium ions into cells and the recovery of skin parer function. (A) is the result of the control with only the acetone treatment (magnification X7,500), (B) is the result of the C 1 -ionophore treatment (magnification X7,500), and (D) is the enlarged view of (B). (Magnification X75,000), (C) is the result of Ca ²⁺ ionophore treatment (magnification X7,500), (E) is an enlarged view of (C) (magnification X75,000). Quantification of the area of the lipid part observed in the histological observation photographs of (A) the control only treated with acetone, (B) the C1-ionophore treatment, and (C) the Ca2 ⁺ ionophore treatment in 7 It is a graph which shows a comparison. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the configuration of the present invention will be described in detail.

Xingyuan cell receptor

The term "excitatory cell receptor" as used herein refers to a skin cell, i.e., a cell constituting the stratum corneum, epidermis, basement membrane, dermis, etc. Excitatory receptor Such excitation is caused by the Ca ²⁺ and Na + ions flowing into the cells by binding of the agonist to the receptor. Such excitable cell receptors that are currently found in skin cells include, for example, glutamate (NMDA) receptor, ATP receptor, heat-stimulated receptor, and adrenaline] 8 Receptor, acetylcholine • Nicotinic acid-like receptor, serotonin receptor and the like. In the present invention, glutamate (NMDA type) receptor, ATP receptor, heat-stimulated receptor, for example, VR-1, or adrenaline] 32 receptor is preferred. It is to be understood that the present invention is not limited to the above-mentioned excitatory receptors and the excitatory receptors presently present in skin cells, but extends to other receptors and receptors whose presence is to be found in the future. It is.

The activity of each of these excitatory receptors is suppressed by the presence of a specific antagonist around cells. Antagonists of glutamate (NMDA type) receptor include MK_801, D-AP5, D-AP7, Conantokin T, (R) -CPP and the like. In the present invention, MK-801 or 0-5 is preferred. Antagonists of the ATP receptor include suramin, PPADS, TNP-STP and the like. Antagonists of VR-1 which is a thermal stimulus receptor include cabsazepine and the like. ICI — 118, 551, alprenolol, (sat) monoatenolol, S (one) athenolol, bromo Acetylalprenolol methane, (Sat)-CGP-12 17 7 A, ICI 18 946, practrol, pronetanol, DL-propranolol hydrochloride, propranolol, R-(+ ) Probranolol, S — (1) Propranolol, sotalol, S — (1) Lithride, etc. Acetylcholine. Antagonists of nicotinic acid-like receptors include benzoquinodinum, conde 1 phine, ο; Antagonists of serotonin receptors include MDL-7222, Y-251300, metoclopramide and the like. It is to be understood that the present invention is not limited to the above-mentioned antagonists or antagonists of excitatory receptors presently present in skin cells, but also to other and future antagonists whose presence is to be found. .

Inhibitory neuronal receptor

As used herein, the term "inhibitory cell receptor" refers to an inhibitory receptor present on the surface of cells constituting skin cells, for example, keratinocytes, that induces cells from an excited state to a suppressed state. Such suppression is caused by the entry of C1- into cells by binding of the agonist to the receptor. Such suppressor cell receptors that are currently found in skin cells include, for example, GABA receptors, glycine receptors, and the like. It is to be understood that the present invention is not limited to inhibitory receptors present in skin cells at present, but extends to receptors found in the future.

The activity of such inhibitory receptors is suppressed by the binding of their respective agonists. Examples of GABA receptor agonists include GABA, muscimol, isogpasin, TACA or THIP, and the like. In the present invention, GABA, muscimol or isogpasin Is preferred. Glycine receptor agonists include glycine, alanine, hypotaurine, serine, taurine and the like. In the present invention, glycine is preferred. It is to be understood that the present invention is not limited to the agonists described above and those of the inhibitory receptors presently present in skin cells, but also extends to other and future agonist receptors.

Skin parer function recovery promotion effect test

In the present invention, the effect of promoting the recovery of skin parer function can be evaluated by various methods. For example, the effect of tape stripping on the skin of mammals (eg, humans, mice, rats, rabbits, etc.) can be evaluated. The process of restoring the function of the damaged skin barrier to its original state is evaluated quantitatively or qualitatively by evaluating the amount of transepidermal water loss (TEWL) as an index. Can be measured. Such a measurement can be performed, for example, as described below.

1. Measure the transdermal water loss (TEWL) near the back of the hairless mouse using a water loss measurement device. The value at this time is assumed to be a TEWL recovery rate of 100%.

2. Destroy the skin barrier by peeling off the stratum corneum of the hairless mouse using cellophane tape or the like. At this time, it is preferable to repeat this operation until the value of TEWL becomes about 800 to 900. The value obtained by subtracting the measured value before peeling the horny layer from the measured value after peeling off the horny layer is defined as the deepest damage state, that is, the recovery rate of 0%.

3. Place the test sample at an appropriate concentration (eg, 1 mM) in an appropriate amount (eg, 100 μl) on an appropriate substrate, eg, plastic wrap, and affix it to the back of the mammal for an appropriate amount of time. After 5 minutes, remove it.

4. After a suitable time (eg, 0, 2, 4, 6 hours), the amount of water loss Measure TEWL using a measuring device. As in the case of removing the stratum corneum, the recovery rate is calculated by subtracting the TEWL value before the removal of the stratum corneum from the measured value at each time.

That is, the recovery rate can be determined according to the following equation:

Recovery rate (%)

(TEWL at each time after removing stratum corneum)-(TEWL before removing stratum corneum)

== X100

(TEWL immediately after removing the stratum corneum) — (TEWL before removing the stratum corneum)

In the present invention, “promoting the recovery of the skin barrier function” means that the transepidermal water loss (TEWL) immediately after the tape stripping of the skin is 0% and the value before the tape stripping is 10%. A value of 0% means that the value of TEWL at each measurement time clearly has a significant difference when compared with the control, and has an effect of promoting the τ EWL recovery rate. According to J Invest Dermatol, 86; 598, 1986), the skin is treated with a Cot ton ball impregnated with a 4% aqueous solution of sodium dodecyl sulfate (SDS) to make a judgment. different.

Skin thickness measurement test

The effect of promoting the recovery of skin parrier function can also be determined based on the measurement of skin thickness. When a dry stimulus is applied to the skin, the skin tissue is damaged and the skin parrier function is reduced, and as a result, the thickness of the skin increases due to abnormal proliferation of epidermal cells. Therefore, a decrease in the function of skin Paria can be measured by histological observation of skin thickness. Such a histological observation may be performed, for example, as follows. Mammals, preferably hairless mice, are bred in advance in dry conditions, for example in an environment at room temperature and a humidity of 10% or less, until the TEWL reaches a desired level, for example, about 2.5 to 3.5 mg / cni ² / h. Use a ton to break down the skin pariers. Next Then, the test substance is dropped on the treated skin, and after that, the skin is cultivated in the above-mentioned environment for an appropriate time to give a dry stimulus, and then the skin of the treated portion is collected, and the skin sample is stained or the like. The thickness of the epidermis is measured by microscopic observation.

The agonist of the excitatory cell receptor or the agonist of the suppressive cell receptor according to the present invention significantly promotes the recovery of the skin pariar function, thereby significantly suppressing abnormal epidermal proliferation caused by dry stimulation. However, it can even reduce the thickness of the skin.

Screening method

A method for screening the substance for promoting skin PAR recovery is to select a substance that is a candidate for an agonist of an exciting cell receptor or an agonist of an inhibitory cell receptor, and apply the substance to mammalian skin. This can be achieved by selecting a substance that reduces the transepidermal water loss of the skin. As mentioned at the beginning, the excitatory receptor binds to the agonist and causes the influx of Ca ²⁺ and Na + ions into the cell, thereby inducing the cell to an excitable state, The receptor binds the agonist to the cell, causing the influx of C 1 -ions into the cell, and inducing the cell from an excited state to a suppressed state. Antagonist, an excitatory receptor, has the opposite effect to agonist, i.e., it causes a decrease in intracellular Ca ²⁺ and Na + ions, so the candidate substance is intracellular Ca2 ⁺ or Na +. It can be selected by evaluating the decrease of the ion and the increase of the C1-ion. In addition, since the inhibitory receptor agonist causes an increase in intracellular C 1 -ion, candidate substances include an increase in intracellular C 1 -ion, Ca ²⁺ and It can be selected by evaluating the decrease in Na ⁺ ion.

The concentration of intracellular Ca ²⁺ , Na + ions, and CI ions can be measured by conventional methods. For example, in Japanese Patent Application No. 2001-180366, A method for screening receptor agonists by assessing calcium ion concentration on the cell surface is described. Specifically, in this application, it is a stimulus receptor on the surface of epithelial cells.

VR1 receptor (vanilloid receptor subtype 1) and P2X receptor agonists increase intracellular calcium ion concentration.

A method for screening the agonist of the receptor by evaluating the increase in the calcium ion concentration in epithelial cells due to the effect of a test substance is disclosed. As an indicator of intracellular cr ion, for example, a predetermined quinoline derivative is often used. Such a quinoline derivative is used as an indicator of cr ion based on its decay-causing function of fluorescence due to the presence of a haptogenide ion. Such quinoline derivatives include SPQ (6-Methoxy-N- (3-sulfopropyl) quinolinium 'monohydrate) and MQA E (N-Ethoxycarbonylmethyl-6-methoxyquinodium bromide). , DiH—MEQ (6-Methoxy N-ethylquinoline iodide) and the like. DiH-MEQ is available from Molecular Probes as its oxidized form, MEQ (6-Methoxy-1-N-ethylquinoline). Therefore, for example, selection of a test substance that is a candidate for an antagonist of the excitatory cell receptor or an agonist of the inhibitory cell receptor is performed by evaluating the intracellular calcium ion concentration as described below. be able to.

1) Stimulation of cells by test substance

First, epidermal cells, for example, epidermal keratinocytes, are cultured in a suitable cell culture medium, for example, KGM medium according to a conventional method. The day before the measurement of calcium ion, inoculate the cultured cells into a 96-well plate at an appropriate cell concentration (for example, about 2 × 10 ⁵ cells / well). On the next day, gently pipet the above culture solution so that the cells do not detach. Absorb. Next, an appropriate buffer, for example, BBS (Balanced salt solution) and a calcium-sensitive fluorescent dye, for example, Fluo 3-AM (Dainippon Pharmaceutical) are added to the cultured epidermal keratinocytes (for example, Incubate (for example, at 37 ° C for 60 minutes) under appropriate conditions, and transfer this fluorescent dye into the above cultured keratinocytes. Is taken. Other pigments include calcium-sensitive fluorescent dyes such as Quin 2, Quin 2—AM, Fura_2, Fura-2_AM, Indo—l, Fluo-3, Rhod-2. For example, a calcium-sensitive photoprotein such as E. coli or a reagent used for nuclear magnetic resonance with ¹⁹ F such as 5-FBAPTA may be used. After the uptake, the cells are washed, freshly buffered with the same buffer (BBS) is added, and the mixture is allowed to stand (for example, 15 minutes). Thereafter, the buffer is removed from the cultured epidermal keratinocytes, and a test substance dissolved in the same buffer is added to the cells to stimulate the cells. As a control, add the same buffer in which the test substance is not dissolved to the cells.

2) Measurement of intracellular calcium ion concentration

Fluorescence is measured in a conventional manner using a fluorescence microplate reader at excitation and emission wavelengths corresponding to the colorants, for example, over time.

The intracellular calcium ion concentration can be calculated according to the following equation.

C a ²⁺ M) = (AF-F _fflin / F _nax -AF) K d

F _nin : Fluorescence value of dye when _EDTA is added and _Ca ²⁺ is blocked (minimum fluorescence intensity)

F _max: fluorescence values of the dye in the excess presence of C a ²⁺ (Yabu壌cell membranes Ri by the surfactant) (the maximum fluorescence intensity)

K d: dissociation constant between the dye and C a ²⁺ (for example, 390 nM when the dye is F 1 uo 3-AM) Thus, if the intracellular calcium ion concentration is significantly reduced, the test substance is expected to be an agonist of an excitatory cell receptor or an agonist of an inhibitory cell receptor.

In addition, the selection of a test substance that is a candidate for an agonist of an excitatory cell receptor or an agonist of an inhibitory cell receptor is performed by using, for example, DiH-MEQ as described below. It can also be done by evaluating the concentration.

1) Stimulation of cells by test substance

Stimulation of cells with a test substance can be performed in the same manner as described for the evaluation of intracellular calcium concentration.

2) Measurement of intracellular chloride ion concentration

Dilute yellow oil, DiH-MEQ (6-Methoxy-N-ethylquinoline iodide), in a suitable physiological buffer (eg PBS) and fill it with 25-50 μ 5 DiH-MEQ. Prepare solution. Note that DiH—MEQU can be prepared as described in Molecular Probes, Inc., Molecular Probes Product Information (Revised January 31, 2001) MP06886. If necessary, DiH-MEQ may be prepared in DMSO (dimethylsulfoxide) into a concentrated stock solution (25-50 mM), and the stock solution may be diluted in physiological buffer.

Incubate the stimulated cells with DiH_MEQU filling solution for 5-10 minutes.

The cells are washed with physiological buffer (about twice) and incubated at 37 ° C for 15 minutes to disperse the dye uniformly, then the maximum excitation and emission wavelength of the MEQ (eg, about 34 Observe with a fluorescence microscope using filters for 4 nm and about 450 nm. Significant loss of fluorescence causes an increase in intracellular C1-ion This means that the test substance is expected to be a candidate for the agonist of the excitatory cell receptor or the agonist of the inhibitory cell receptor. The substance selected as the agonist of the excitatory cell receptor or the agonist of the inhibitory cell receptor selected in this manner was subjected to the above-mentioned effect of promoting the recovery of skin parier function, thereby reducing the amount of transdermal water loss. Antagonists of excitatory cell receptors and agonists of inhibitory cell receptors can be screened by selecting substances that reduce or suppress the increase in the thickness of the skin subject to the dry stimulus. You can lean.

The substance for promoting recovery of the function of the skin paria of the present invention, that is, the agonist of the excitatory cell receptor or the agonist of the inhibitory cell receptor is, for example, an ointment, a cream, an emulsion, a lotion, a pack, a bath agent, etc. It can be incorporated into cosmetics, pharmaceuticals, and quasi-drugs, and can be applied to the skin, preferably as an external preparation for skin. The blending amount is not particularly limited, but based on the total amount of these bases, 0.001 mM to: LM, preferably 0.01 to: L 0 0 mM, more preferably 0.1 to: L will be around 0 mM. Example

Test method for promoting recovery of skin parrier function

In each of the subsequent experiments, the effect of promoting the recovery of skin pari function was based on the skin pari function destroyed by applying tape stripping to the skin of hairless mice (Type HR-1, HOSHINO, Japan). The process of recovery to the state was evaluated using the transepidermal water loss (TEWL) as an index, as follows.

1. Measure transepidermal water loss (T EWL) near the back of the hairless mouse using a water loss measuring device ME ECO (Meeco, Warrington, PA, USA). The value at this time is assumed to be a TE WL recovery rate of 100%. 2. The skin is destroyed by peeling off the stratum corneum of the hairless mouse using cellophane tape. At this time, this operation is repeated until the value of TEWL becomes approximately 800 to 900. The value obtained by subtracting the measurement value before peeling off the stratum corneum from the measurement value after peeling off the stratum corneum is defined as the deepest damage state, that is, the recovery rate of 0%.

3. Place the test sample on a plastic wrap at 100 μm with ImM, affix it to the back, and peel it off after 5 minutes.

4. After 0, 2, 4, 6 or 0, 1, 3, 6 hours, measure TEWL by MEECO. As in the case of removing the stratum corneum, the TEWL value before the removal of the stratum corneum is subtracted from the measured value at each time to determine the recovery rate.

That is, the recovery rate is calculated according to the following formula:

Recovery rate (%)

(TEWL at each time after removing stratum corneum) — (TEWL before removing stratum corneum)

= = X 100

(TEWL immediately after removal of stratum corneum)-(TEWL before removal of stratum corneum)

1) Examination of excitatory receptors

Example 1

Examination of the relationship between glutamate receptor and recovery of skin parrier function

Experiment a) D-, L-glutamic acid and L-aspartic acid are known to be neurotransmitters that bind to glutamate receptors in vivo and exert a neuroexcitatory action.

The aqueous solutions of D-glutamic acid (Wako Pure Chemical Co., Ltd.), L-glutamic acid (Wako Pure Chemical Co., Ltd.) and L-aspartic acid (Wako Pure Chemical Co., Ltd.) were prepared as described in the above test method. Each hairless mouse was applied to the epidermis, and a TEWL test was performed. Figure 1 shows the results.

As is evident from the results in Fig. 1, D-glutamic acid promotes the recovery of skin pallia, while L-glutamic acid and L-aspara Formic acid delayed skin recovery. Therefore, it was suggested that glutamate receptor, which is an excitatory receptor, is involved in the recovery of paria from skin.

Experiment b) The aqueous solutions of MK-801 (Tocris), an antagonist of NMD type A glutamate receptor, and NMDA (Tocris), an agonist, were prepared as described in the above test method. Each was applied to the epidermis of a hairless mouse, and a TEWL test was performed. Figure 2 shows the results.

From the results in Figure 2, it was revealed that Antagonist MK-801 promotes skin barrier recovery, while agonist NMDA slows the recovery of the barrier.

Experiment c) Next, MK-801 and NMDA were mixed, applied to the epidermis of a hairless mouse as described in the above test method, and a TEWL test was performed. Figure 3 shows the results.

From the results shown in FIG. 3, it was also confirmed that when MK-801 and NMDA were simultaneously applied to the epidermis of mice, the delay effect of the barrier effect of NMDA was eliminated by MK_801.

Experiment d) For the NMDA-type glutamate receptor antagonist D-AP5 (Tocris), the aqueous solution was applied to the epidermis of hairless mice as described in the above test method, and the TEWL test was performed. went. Fig. 4 shows the results.

From the results in FIG. 4, it was clarified that Antagonist D—AP5 promoted skin reparation.

Summarizing the above results, inhibition of the excitatory cell receptor, NMD type A glutamate receptor, can promote the restoration of skin pariole function, and NMDA type glutamate Receptor antagonists are found to be effective in restoring skin Paria function It became.

Example 2

Skin thickness measurement test

As mentioned above, when a dry stimulus is applied to the skin, the skin tissue is damaged and the skin parer function is reduced. As a result, the thickness of the skin increases due to abnormal epidermal proliferation. Therefore, the effect of Antagonist MK-801, an excitatory acid receptor having the effect of promoting the recovery of skin function on skin, on the skin thickness was observed histologically.

Hairless mice were bred in advance in an environment at a temperature of 22 to 25 ° C and a humidity of 10% or less. The barrier on the back skin of the hairless mouse was destroyed using acetone until the TEWL became 2.5 to 3.5 mg / cm ² / h. Then, an aqueous solution of MK_801, NMDA or AMPA (α-amino-3-hydroxy-15-methinole-4-isoxazolepropionic acid) in 200 mM was dropped on the skin 200 μl each, Thereafter, the skin was bred in the above environment for 48 hours to give a dry stimulus, and the skin of the treated portion was collected. After fixing with 4% paraformaldehyde, The cells were embedded in raffin, cut into 4 μπ μ sections, stained with hematoxylin and eosin, and the epidermal thickness was measured with a microscope. ΑΜ Ρ is a ligand specific to the ionotropin glutamate receptor, similar to NMDA. The control is uncoated with any solution. The results are shown in Fig. 5. The results in Fig. 5 show that the application of 8-801 significantly reduces the thickness of the skin compared to the control, and thus the antagonist of the excitatory acid receptor It was demonstrated that 有意 -801 significantly suppressed the abnormal epidermal proliferation caused by the dry stimulus and improved the skin parrier effect. Surprisingly, the application of the excitatory acid receptor agonist NMD II significantly increased the skin thickness compared to the control. Therefore, it was revealed that the abnormal epidermal proliferation caused by the dry stimulus was significantly promoted and the skin condition was deteriorated. Even more surprising, AMP A, like NMDA, is a ligand specific for the ionotropic glutamate receptor, but does not significantly increase skin thickness compared to control. It was shown to behave differently from NMDA.

Example 3

Effect of NMDA glutamate receptor agonist and antagonist on intracellular calcium ion concentration

Next, the effects of agonist NMDA and antagonist MK-801 of the NMD type A glutamate receptor, which are excitatory cell receptors, on the intracellular calcium concentration were examined.

Cells and their culture

Normal human epidermal keratinocytes (NHEK) were obtained from cryopreserved primary passage cells (Kurabo) from neonatal foreskin. The cells were placed on a collagen-coated cover glass, pituitary gland extract (0.4% by volume), human recombinant epidermal growth factor (0.1 _ng / ml), insulin (10 / xg / ml), human Serum-free epidermal keratinocytes consisting of Humedia — KB 2 (Kurabo Co., Ltd.) containing drocortisone (0.5 μg / ml), gentamicin (50 μg / ml) and amphotericin-I B (50 ng / ml) A Cultured in culture medium. Ca2 ⁺ imaging of single epidermal keratinocytes

Changes in [C a ^{2 +} ] in a single cell were determined by the fura _2 method (Grynkicwicz, G. et.al. (Ι98ΰ A new generation of Ca indicators with g reat ly improved fluid escene properties, J. Biol. 260, 3440-3450) (Koizumi, S. et. Al. (1997) Inhi bit ion by ATP of calcium oscillation in rat cultured hippoca mpal neurons. Br. J. Pharmacol. 122, 5: L-58). Briefly, the culture medium of cells grown on force glass was first replaced with a BSS buffer of the following composition: (all in mM) NaCl 150; KC 15; CaCl ₂ 1.8; MgCl ₂ 1.2; N_2- Hydroxicetyl piperazine-N 2-ethanesulfonic acid (HEPES) 25; D-glucose 10 (pH 7.4) ₀ The cells were converted to 5 μM dye fura-2 acetomethyl methyl ester (fur a-2AM) (molecular The cells were loaded with fura-2 by incubating in BSS for 45 minutes at room temperature (20-22 ° C) at room temperature (Probes). The cells were then washed with BSS buffer and incubated for a further 15 minutes to deesterify the loaded dye.

Next, the BSS buffer was removed from the cells, and a test substance (NMDA or MK-801 + NMDA: lmM, respectively) dissolved in the buffer was added to the cells, and the cells were tested for about 30 minutes. The substance was stimulated.

The cover glass was mounted on an inverted fluorescence microscope (TMD-130; Nikon) equipped with a 75 W xenon lamp and a band pass filter of 34O and 36OnM, and the measurement was performed at room temperature. The image data was recorded using a high-sensitivity silicon intensifier and target camera (C-2741-08; Hamamatsu Photonitas Co., Ltd.), and a Ca ²⁺ analysis system (Full Sawa Laboratories Appliance) And prayed for each other. Figure 6 shows the results.

The results in FIG. 6 indicate that NMDA-type glutamate receptor agonist NMDA exerts an effect of significantly increasing the intracellular Ca ²⁺ concentration. It was also suggested that antagonist MK-801 offset such effects of NMDA, and that MK-801 significantly reduced intracellular Ca ²⁺ concentration. Therefore, it was demonstrated that the test substance can be identified in vitro as an excitatory receptor agonist or antagonist through measurement of increase and decrease of intracellular Ca ²⁺ concentration. Was done.

Example 4

Examination of the relationship between ion channel type ATP receptor and restoration of skin barrier function

Experiment a) The presence of the ion channel type ATP receptor P2X3 in the skin has been demonstrated. Therefore, it is considered that these receptors may also induce excitement in the epidermis.

An aqueous solution of ATP (Wako Pure Chemical Industries, Ltd.), an agonist of P2X3, was applied to the epidermis of hairless mice as described in the above test method, and a TEWL test was performed. Figure 7 shows the results.

The results in FIG. 7 revealed that ATP delayed skin barrier recovery. Therefore, by suppressing the activity of the excitatory cell receptor, the ion channel-type ATP receptor P2X3, it is possible to promote the restoration of skin parietal function, and consequently the antagonism of the ATP receptor Is thought to be effective in restoring the function of Paria.

Experiment b) Next, each of the aqueous solutions of the ATP receptor antagonists PP ADS (Sigma), TNP-ATP (Molecular Probes) and suramin (Sigma) was subjected to the above tests. Hairless mice were applied to the epidermis as described in the method and subjected to a TEWL test. Figure 8 shows the results. In addition, PPADS and TNP-ATP show specificity for P2X3-type receptor, but suramin is an ATP receptor antagonist which does not show specificity for P2X3-type receptor.

From the results shown in Fig. 8, it can be seen that PP AD S, TNP-ATP and suramin all restored the barrier function of the skin, and that the ATP receptor antagonist was effective in restoring the function of the skin paria. It has been proven. In addition, PPADS and TNP specific for P2X3 type receptor are significantly different in recovery rate between ATP and non-specific suramin. The absence of a difference indicated that the ATP receptor antagonist was effective in restoring skin barrier function irrespective of specificity for the P2X3-type receptor.

Example 5

Examination of the relationship between thermoreceptor VR-1 and recovery of skin paria function

As described above, it is known that the heat receptor VR-1 is expressed on the epithelial cell surface. Therefore, it was examined whether these receptors also induce excitation in the epidermis.

An aqueous solution (1 mM) of capsaicin (Tocris), which is an agonist of VR-1, an aqueous solution (ImM) of antagonist, and a mixed aqueous solution (1 mM each) of cabsaicin and forcepsazepine Hairless mice were applied to the epidermis as described in the test method, and a TEWL test was performed. Figure 9 shows the results.

From the results in Fig. 9, it was found that cabsaicin (CPC) delayed the recovery of the skin barrier, but that the antagonist, ptsazepine (CPZ), promoted the recovery of the skin function. In addition, when cubsaicin was combined with forcepsazepine (CPC + CPZ), the effect of skin force-based recovery on forcepsazepine was reduced. Therefore, it was shown that antagonist of VR-1 which is a heat receptor is effective for restoring skin parrier function.

Example 6

Examination of the relationship between adrenaline ι3 receptor and recovery of skin parier function

Next, the relationship between the [adrenerin] 3 receptor and the recovery of the skin pariar function was also examined.

Axenaterol hemi-fumarate (Tocris) aqueous solution (ImM), an agonist of adrenaline i31 receptor, Betataxol hydrochloride hemihydrate (Tocris) aqueous solution (ImM), an antagonist And key A mixed aqueous solution of Samoterol hemi-fumarate and betataxol hydrochloride hemihydrate (ImM each) was applied to the skin of hairless mice as described in the above test method, and a TEWL test was performed.

Also, procaterol hydrochloride (Tocris) aqueous solution (ImM), which is an agonist of the drenerin β2 receptor, and IC 1-118,551 (Tocris) aqueous solution (1 mM), which is an agonist. A mixed aqueous solution (1 mM each) of ICI-118 and 551 with hydrochloric acid and potassium hydroxide was applied to the epidermis of hairless mice as described in the above test method, and a TEWL test was performed. Was.

The results are shown in FIG.

From the results in Figure 10, the agonist propoterol hydrochloride (] 32G) delays the recovery of the skin barrier, but the antagonist ICI-118, 55 1 (β 2 N) promoted the recovery of skin paria function. Also, when ICI-118, 551 is combined with pro-titanium hydrochloride (] 32 G + Ν), skin parer function recovery based on IC 1-118, 551 is achieved. The effect has decreased. Therefore, it was shown that antagonist of the adrenaline J32 receptor is effective in restoring the skin pariar function. On the other hand, with respect to the adrenaline 1 receptor, neither the agonist (JSIG) nor the antagonist (1N) showed a significant effect on the recovery of skin paria monofunction.

Examination of inhibitory receptors

Example 7

Examination of the relationship between GABA receptor and recovery of skin parrier function

GABA receptor is an inhibitory neuronal receptor and has a C1-channel. Bicucliline-sensitive (A-type GABA), G-protein coupled (B-type GABA) and C1-channel Insensitive There is a gender type (C-type GAB A).

Experiment a) GAB A (Wako Pure Chemical Industries, Ltd.), Muscimol (MUS) (Tocris), Isogpasin (Tocris) ), And its antagonist, biculine metbromide (BM) (ICN BioMedical), were applied to the epidermis of hairless mice as described in the test method above, and a TEWL test was performed. Was. Further, GAB A and biculine met bromide were mixed, applied to the epidermis of hairless mice as described in the above test method, and subjected to a TEWL test. The results are shown in Figures 11 and 12.

From the results of Figs. 11 and 12, the avatars GABA, musmol (MUS) and isogpasin (IG) promote barrier recovery of the skin, while the antagonist vikcuri It was found that methmetromide (BM) did not promote the restoration of parier function.

Experiment b) In addition, GABA and biculine metromide were simultaneously applied to the mouse epidermis, and a TEWL test was performed. Figure 12 shows the results.

From the results in FIG. 12, it was also confirmed that GABA A promoted the recovery of the parer function, and that it was inhibited by viklimmetromide.

Summarizing the above, activation of the inhibitory cell receptor, C1-channel-incorporated victorin-sensitive receptor, can promote the restoration of skin parier function. It has been clarified that agonist, a type of viklin-sensitive receptor, is effective in restoring the paria function of the skin.

Example 8

Leather Jt thickness measurement test A skin thickness measurement test was performed on agonist GABA as an inhibitory receptor in the same manner as in Example 2. That is, histological observation was made of the effect of GABA, which has an effect of promoting the recovery of skin function on skin, on skin thickness.

Hairless mice were bred in advance in an environment at a temperature of 22 to 25 ° C and a humidity of 10% or less. The barrier on the back skin of the hairless mouse was ruptured using acetone until the TEWL became 2.5 to 3.5 mg / cm ² / h. Next, a 1 mM aqueous solution of GABA, a mixed aqueous solution of GABA and BM (ImM each), or water was added dropwise to the skin at 200 μΐ each, and then raised for 48 hours in the above environment. After applying the dry stimulus, the skin of the treated area was collected. After fixation with 4% paraformaldehyde, it was embedded in paraffin, cut into 4-im sections, stained with hematoxylin-eosin, and the thickness of the epidermis was measured with a microscope. The controls are uncoated with any solution. Figure 14 shows the results.

From the results shown in Fig. 14, it can be seen that when water was applied, the skin thickness was significantly increased, and the epidermal proliferation abnormality caused by the dry stimulus was significantly promoted. 'It was shown that the growth did not occur and that the abnormal epidermal proliferation caused by the dry stimulation could be significantly suppressed. It was also clarified that when BM was mixed with GAB A, the skin thickness increased to the same level as when water was applied, and that the effect of GAB A on suppressing abnormal epidermal proliferation was eliminated by BM.

Example 9

Effect of agonist and antagonist of GABAA receptor on intracellular chloride concentration

Next, we investigated how the inhibitory cell receptors GABA receptor agonist (GABA) and antagonist (BM) affect the intracellular chloride concentration. In the same manner as in Example 3, normal human epidermal keratinocytes (NHEK) were cultured in a serum-free epidermal keratinocyte culture medium, and the C1-imaging was performed.

Briefly, C 1 -imaging was performed by dissolving DiH—MEQ (Molekiura Iprobe) in PBS to prepare a 25-50 solution of 0111—1 ^ £ Q. In addition, DiH-MEQ was prepared as described in Molecular Probes Product Information (Revised January 31, 2001) MP06886 from Molecular Probes' MEQ force. If necessary, prepare DiH-MEQ into a concentrated stock solution (25-50 mM) using DMSO (dimethylsulfoxide), and transfer the stock solution to physiological buffer. May be diluted.

To stimulate the cells with the test substance, the cells are incubated with the DiH-MEQ-filled solution for 5 minutes, then GABA or GABA + BM (1 mM aqueous solution each) is added to the cells, and the cells are incubated for about 5 minutes. Incubation.

The cells were washed with physiological buffer (about twice) and incubated at 37 ° C for 15 minutes to uniformly disperse the dye. Then, fluorescence microscopy was performed using filters for the maximum excitation and emission wavelengths of MEQ (344 nm and 44 nm, respectively). Figure 14 shows the results.

From the results in Figure 14, the GABA receptor agonist GAB A exerts a significant increase in intracellular C1-concentration, and the antagonist BM counteracts such an effect of GAB A It was shown that. Therefore, it was demonstrated that whether the test substance was an inhibitory receptor agonist or antagonist could be identified in vitro by measuring the increase or decrease of the intracellular C1-concentration.

Example 10 Investigation of the relationship between daricin receptor and recovery of skin parier function Glycine (Wako Pure Chemical Industries, Ltd.), an agonist of daricin receptor that is recognized to be present in epidermal cells, was Hairless mice were applied to the epidermis as described in the test method, and a TEWL test was performed. Figure 15 shows the results.

From the results shown in Fig. 15, it was confirmed that arginist, daricin, promotes skin barrier recovery. Therefore, activation of the inhibitory cell receptor daricin receptor can promote the recovery of skin barrier function, and as a result, the glycine receptor agonist also promotes skin barrier function. It proved to be effective for recovery. Example 1 1

This example demonstrates the relationship between the influx of various ions into cells and the recovery of skin paria function.

C 1 -ionophore (chloride ionophore 1) (Fluka, Switzerland) or Ca ^{2 +} ionophore (ionomycin) (Wako Pure Chemical Industries, Ltd.) was used as described in the above test method and the skin of hairless mice And subjected to a TEWL test. Figure 16 shows the results.

C 1 -ionophore is a compound that promotes the influx of chloride ions into cells. As is evident from the results in FIG. 16, the skin parrier function was significantly restored in the skin of mice treated with C 1 -ionophore as compared to the case where only control (water) was applied. Therefore, it was clarified that the influx of chloride ions into cells restored the function of the skin barrier, and by measuring the increase or decrease of intracellular C1-concentration, the substance that promotes the recovery of skin function was in vitro. It has been confirmed that ro can be screened.

In contrast, in the skin of mice treated with Ca ²⁺ ionophore, which promotes the influx of calcium ions into cells, control (water) The skin parrier function was significantly reduced as compared with the case where only the skin was applied. Therefore, it was clarified that the influx of calcium ions into the cells reduced the function of the skin barrier.

Furthermore, the relationship between the influx of ions into cells and the recovery of skin parrier function was confirmed by histological observation.

Specifically, hairless mice were raised in advance in an environment at a temperature of 22 to 25 ° C and a humidity of 10% or less, as in Example 8, until the TEWL reached 2.5 to 3.5 mg / cm ² / h. and Yabu壌the path Ria one hairless mouse dorsal skin with seton, then CI - droplet 1 mu Micromax aqueous or C a ² + Ionofoa of 1 mu Micromax aqueous solution to 2 0 0 μ 1 skin respective Ionofoa After that, the animals were bred for 48 hours in the above environment to give a dry stimulus, and then the skin of the treated portion was collected. After fixing with 4% paraformaldehyde, the cells were embedded in paraffin, cut into 4 μιη sections, stained with hematoxylin-eosin, and the epidermal thickness was measured with a microscope. Controls were not coated with any solution. Figure 17 shows the results.

Fig. 17 (A) shows the result of the control with only the acetate treatment (magnification X7,500), (B) shows the result of the C 1 -ionophore treatment (magnification X7, 500), and (D) shows the result. (B) Magnified view (magnification X75,000), (C) Ca2 ⁺ ionophore treatment result (magnification X7,500), (E) Magnified view of (C) (magnification Χ75,000) Is shown.

It is the stratum corneum that prevents the loss of water from living organisms in mammals. The stratum corneum is constantly renewed, and in healthy skin these structures continue to form without interruption. The lamellar granules containing lipids are formed in the granular layer, and lipids inside the lamellar granules are released extracellularly by exocytosis as the cells become keratinized, forming a layered structure of lipids in the intercellular space (Mitsuhiro Denda) , "Skin" Vol. 41, No. 5, pp. 518-523, October 1999). The lipid layer is a major component of the stratum corneum, which is responsible for the function of the skin paria. The thicker the lipid layer, the higher the parier function.

When C 1 -ionophore treatment was performed (B), the lipid layer (indicated by △ in (B)) was significantly thickened compared to the control (A). Therefore, histological observations revealed that the influx of chloride ions into the cells restored the function of the skin panel. In control, a large number of exocytosis (shown by arrows in (A)) of lipid-containing lamellar granules was observed. Referring to (C), which shows the results of Ca ²⁺ ionophore treatment, it was observed that the lipid layer (indicated by the arrow in (C)) became thinner, and the influx of calcium ions into the cells caused the skin to lose its thickness. It became clear that the function of Paria was reduced. In particular, when comparing (D) showing an enlarged view of the result of the C 1 -ionophore treatment with (E) showing an enlarged view of the result of the Ca ^{2 +} ionophore treatment, the difference in the thickness of the lipid layer can be clearly seen ((D )), The lipid layer is indicated by *, and in (E), an arrow is indicated). In addition, in (C), a large number of lipid-containing lamella granules were observed (indicated by (△) in (C)). FIG. 18 shows (A) the control in which only acetone was treated in FIG. (B) The results of quantitative comparison of the area of the lipid portion observed in the histological observation photographs of the treatment with C 1 -ionophore and the treatment with (C) Ca ^{2 +} ionophore. When treated with C 1 -ionophore, the lipid area increases significantly compared to the control, and when treated with Ca ^{2 +} ionophore, the lipid area decreases significantly compared to the control. It was clarified quantitatively.

Therefore, according to histological observation, an increase in intracellular chloride ion concentration leads to an increase in lipid layer and, consequently, recovery of skin pariole function, and conversely, an increase in intracellular calcium ion concentration decreases skin pariole function. It was clearly shown that Thus, histological observations support that it is possible to screen effective substances with one function of skin barrier through measurement of various ion concentrations in cells. Industrial applicability

From the above results, it has been clarified that the agonist of the excitatory cell receptor and the agonist of the inhibitory cell receptor are effective in promoting the recovery of the skin parrier function. According to the present invention, there is provided a novel and effective skin barrier function recovery promoter.

Claims

The scope of the claims

1. An agent for promoting the recovery of skin pariar function, which comprises an agonist of an excitatory cell receptor or an agonist of an inhibitory cell receptor.

2. The skin pall function according to claim 1, wherein the excitatory cell receptor is selected from the group consisting of a glutamate receptor, an ATP receptor, a heat-stimulated receptor and an adrenaline) 32 receptor. Recovery promoter.

3. The agent of claim 2, wherein the glutamate receptor is an NMD A (N_methyl_D-aspartate) receptor.

4. The NMDA receptor antagonist is MK-801 (dizosylvin) or D-AP5 (D- (1-)-2-amino_5-phosphonopentanoic acid). The skin para-functional recovery promoter according to the above.

5. The skin barrier function recovery promoter according to claim 2, wherein the ATP receptor is a P 2 X receptor (inotropic purino receptor).

6. The antagonist of the ATP receptor is suramin, PPADS (pyridoxal phosphate-16-azophenyl 2,4,4, disulfonic acid) or TNP-ATP (trinitrophene). 3. The agent for promoting the recovery of skin paria monofunction according to claim 2, which is Lou ATP).

7. The skin barrier monofunctional recovery promoter according to claim 2, wherein the thermal stimulus receptor is VR-1 (vanilloid receptor subtype 1).

8. The skin barrier function recovery promoter according to claim 7, wherein the antagonist of VR_1 is ptsazepine.

9. Antagonist of the adrenerin] 32 receptor is ICI — 118, 55 1 (Sat) 1 1 — [2,3- 3. The skin paria monofunctional recovery promoter according to claim 2, which is 1 H-indene-4-yl) -3-[(1-methylethyl) amino] -12-butanol. 4.

10. The agent for promoting the recovery of skin parer function according to claim 1, wherein the inhibitory cell receptor is a GAB A (τ; -aminobutyric acid) receptor or a daricin receptor.

11. The skin barrier function recovery promoter according to claim 10, wherein the GABΑ receptor is a Α type_GABΑ receptor (a bicycline-sensitive receptor having a built-in Cr channel).

12. The skin PARIA function recovery promoter according to claim 11, wherein the agonist of the type A-GAB A receptor is GAB A, muscimol or isogpasin.

1 3. The skin barrier function recovery promoter according to any one of claims 1 to 12, which is a skin external preparation.

1 4. A method for screening a substance that promotes the recovery of cutaneous parier function, which selects a substance that suppresses the excitatory activity of an excitatory cell receptor or that promotes the inhibitory activity of an inhibitory cell receptor. It is necessary to select a substance that reduces the transdermal water loss of the skin by applying the substance to the skin of a mammal or that reduces or suppresses the increase in the thickness of the skin exposed to dryness. Comprising a method.

1 5. The selection of a candidate substance that suppresses the excitatory activity of the excitatory cell receptor or promotes the inhibitory activity of the inhibitory cell receptor is performed by selecting a calcium ion or a sodium ion in the cell. 15. The screening method according to claim 14, wherein the method is performed by evaluating an influence on a chlorine ion concentration.

1 6. The excitatory cell receptor is a glutamate receptor, an ATP receptor, a heat-stimulated receptor or an adrenaline 32 receptor; 15. The screening method according to claim 14, wherein the cell receptor is a γ-aminobutyric acid receptor or a daricin receptor.

17. A method for screening a skin-parer function recovery activity promoting substance, in which a candidate substance for a skin-parer function recovery activity promoting substance is allowed to act on mammalian cells, and then the intracellular chlorine ion concentration is measured. And selecting the substance that increases the intracellular chloride ion concentration as a substance for promoting the function of restoring the function of the skin pariers.

18. A method for screening a skin barrier function recovery activity promoting substance, in which a substance that is a candidate for a skin barrier function recovery activity promoting substance is allowed to act on a mammalian cell, and thereafter, the calcium or sodium in the cell is used. A method for measuring the concentration of calcium ions and selecting a substance that reduces the concentration of intracellular calcium or sodium ions as a substance for promoting the activity of restoring the skin parier function.

19. A method for improving the skin, characterized in that a skin external preparation containing an agonist of an excitatory cell receptor or an agonist of an inhibitory cell receptor is applied to the skin to promote the recovery of the skin barrier function. The method.