WO2019131406A1 - Screening method for skin condition improving agents having thrombin-suppressive action as indicator, and skin condition improving agent containing thrombin action inhibitor - Google Patents

Abstract

The purpose of the present invention is to provide a screening method for skin condition improving agents having a novel mechanism of action as an indicator. Screening of skin condition improving agents can be carried out using a thrombin-suppressive action as an indicator.

Description

Screening method for skin condition improving agent using thrombin inhibitory effect as index, and skin condition improving agent containing thrombin action inhibitor

The present invention relates to the technical field of skin condition improvement, and more particularly to cosmetic, cosmetic or medical techniques.

The skin is always exposed to external stimuli because it exists in the outermost layer of the living body. Stimuli from the outside world include physical stimuli such as ultraviolet light and temperature, as well as stimuli by chemical substances and antigenic substances. In addition to causing respiratory symptoms and pharyngeal symptoms specific to hay fever, cedar pollen that causes hay fever acts through mucous membranes, and also causes cedar pollen dermatitis due to contact with the skin. Cryj1, known as a cedar antigen, is known to activate serine proteases in keratinocytes. Activated serine protease is thought to cause the reduction of lamellar granules through activation of PAR-2 protein, a membrane protein expressed in keratinocytes (Non-patent document 1: Arch Dermatol Res 308: 49-54, 2016). Lamellar granules are granules found in the cytoplasm of keratinocytes in the granular layer, and are secreted extracellularly by apoptosis of keratinocytes. Lamellar granules contain intercellular lipids such as ceramide, cholesterol, fatty acids, and the reduction of lamina granules leads to a reduction in skin barrier function.

On the other hand, tranexamic acid developed as a hemostatic agent having an antiplasmin action has been found to bring about a whitening effect on the skin, and a skin external preparation has been developed as a remedy for spots and dry spots (Patent Document 1). Tranexamic acid exerts a coagulant action by inhibiting the action of plasmin, which is a type of trypsin-type serine protease. Plasmin is mainly present in the epidermis in the basal layer and contributes to the activity of melanocytes through prostaglandin production, so suppression of plasmin by tranexamic acid is thought to bring about a whitening effect.

JP, 2014-152159, A

Arch Dermatol Res 308: 49-54, 2016

An object of the present invention is to provide a screening method of a skin condition improving agent using a novel mechanism of action as an index.

The present inventors diligently conducted studies to elucidate the mechanism of the origin of signal transduction that suppresses the secretion of lamellar granules in response to pollen antigens, and trypsin which was considered to be the origin of such signal transduction in the past. For the first time, it was found that activation of PAR-1 by thrombin or a thrombin-like protein, rather than activation of PAR-2 by type-serine protease, was the origin of signal transduction to suppress the secretion of lamellar granules. Therefore, a novel screening method was invented in which a skin condition improving agent can be screened by using the action of suppressing thrombin as an index.

Thus, the present invention relates to the following inventions:
[1] A method of screening a skin condition improving agent, which uses thrombin's inhibitory action as an index.
[2] The screening method according to item 1, wherein the skin condition improving agent is selected from the group consisting of a whitening agent, an itching inhibitor and a skin barrier improving agent.
[3] The screening method is
Contacting prothrombin or thrombin with the candidate agent,
Measuring thrombin activity,
Selecting a skin condition improving agent based on the thrombin inhibitory action of the candidate drug,
The method according to item 1 or 2, comprising
[4] The method according to item 3, wherein the measurement of thrombin activity is measured by the amount of thrombin substrate degradation or blood coagulation time.
[5] The screening method is
Culturing PAR-1 expressing cells in a medium containing prothrombin or thrombin and a candidate agent,
Measuring thrombin activity, selecting a skin condition improving agent based on the thrombin inhibitory action of the candidate drug,
The method according to item 1 or 2, comprising
[6] The method according to item 5, wherein the measurement of thrombin activity is performed by calcium imaging.
[7] A skin condition improving agent comprising a thrombin action inhibitor.
[8] The skin condition improving agent according to item 7, wherein the skin condition improving agent is selected from the group consisting of a whitening agent, an itching inhibitor and a skin barrier improving agent.
[9] The skin condition improving agent according to item 7 or 8, wherein the thrombin action inhibitor improves the skin condition via suppression of cell action by PAR-1.
[10] A thrombin action inhibitor comprising tranexamic acid or a salt thereof.
[11] The thrombin action inhibitor according to Item 10, wherein the thrombin action inhibitor suppresses the cell action by PAR-1.
[12] The thrombin action inhibitor according to Item 11, wherein the inhibition of the thrombin action is exerted by suppressing the degradation of prothrombin to thrombin.
[13] A cosmetic or non-therapeutic method for improving skin condition, which comprises applying a thrombin action inhibitor.
[14] The cosmetic or non-therapeutic method according to item 13, wherein the skin condition improvement is selected from the group consisting of whitening, itching suppression, skin barrier improvement.
[15] The cosmetic or non-therapeutic method according to item 13 or 14 through suppression of cell action by PAR1.
[16] The cosmetic or non-therapeutic method according to any one of items 13 to 15, wherein the thrombin action inhibitor comprises tranexamic acid or a salt thereof.
[17] The cosmetic or non-therapeutic method according to any one of items 13 to 16, wherein the inhibition of the thrombin action is exerted by suppressing the activation of prothrombin to thrombin.
[18] Tranexamic acid or a salt thereof for use in the treatment of dry skin, sensitive skin, scaling, dandruff, atopic dermatitis, psoriasis, eczema via inhibition of the thrombin action.
[19] Tranexamic acid or a salt thereof according to item 18, wherein the inhibition of thrombin action is achieved by suppressing the activation of prothrombin to thrombin.
[20] Tranexamic acid or a salt thereof for use in the treatment of dry skin, sensitive skin, scaling, dandruff, atopic dermatitis, psoriasis, eczema via suppression of cell action by PAR1.
[21] Use of a thrombin action inhibitor in the manufacture of a skin condition improving agent.
[22] The use according to item 21, wherein the skin condition improving agent is selected from the group consisting of a whitening agent, an itching inhibitor and a skin barrier improving agent.
[23] The use according to item 21 or 22, wherein the thrombin action inhibitor improves the skin condition via suppression of cell action by PAR-1.
[24] Use of tranexamic acid or a salt thereof for the production of a thrombin action inhibitor.
[25] The use according to Item 24, wherein the thrombin action inhibitor suppresses the cell action by PAR-1 [26] The inhibition of the thrombin action is exerted by suppressing the activation of prothrombin to thrombin , Use according to item 24 or 25.
[27] A method for improving skin condition, or a method for treating skin disorder or allergic disease, comprising applying a thrombin action inhibitor.
[28] The skin condition improvement is selected from the group consisting of whitening, itching suppression, skin barrier improvement, and the skin disorder is from the group consisting of dry skin, sensitive skin, scale, dandruff, atopic dermatitis, psoriasis, eczema 31. The method according to item 27, wherein the allergic disease selected is hay fever.
[29] The method according to item 27 or 28 through suppression of cell action by PAR1.
[30] The method according to any one of items 27-29, which is performed on a subject having a skin condition caused by PAR1 activation.
[31] The method according to any one of items 27 to 30, wherein the thrombin action inhibitor is tranexamic acid or a salt thereof.

By using the inhibitory effect of thrombin as an index, screening of a skin condition improving agent becomes possible.

FIG. 1A shows the results of calcium imaging in cultured keratinocytes, showing that the addition of Cryj 1 results in fluorescence excitation while the addition of Cryj 1 and tranexamic acid (T-AMCHA) suppresses the excitation. Be The excitation is also suppressed by the addition of Cryj1 and soybean trypsin inhibitor (SBTI), Cryj1 and Bivalirudin. FIG. 1B shows graphically the percentage of activated cells within 2 minutes after reagent introduction in each case. FIG. 2A is a graph showing the activity of the protease in cultured keratinocytes. When adding a fluorescent substrate as a control, including Cryj1, Cryj1 and tranexamic acid (T-AMCHA), Cryj1 and soybean trypsin inhibitor (SBTI), Cryj1 and bivalirudin in the medium, and the reagent-free group (Control) as a control Shows a change in fluorescence of FIG. 2B shows graphically the percentage of activated cells within 2 minutes after reagent introduction in each case. FIG. 3 shows electron micrographs of skin sections after applying a solution containing Cryj1 (FIG. 3A), and Cryj1 and tranexamic acid (T-AMCHA) (FIG. 3B) to the skin. Lamellar granules are indicated by arrows in the photograph. FIG. 4 shows the TEWL values at the application site after application to the skin of a solution containing Cryj1, Cryj1 and tranexamic acid (T-AMCHA), Cryj1 and Bivalirudin, and a reagent-free solution (Control) as a control. FIG. 5A shows the results of gene expression of plasminogen (plasmin) in keratinocytes examined by real-time PCR. And FIG. 5B shows the result of electrophoresis of the amplification product. FIG. 6A shows the results of calcium imaging in cultured keratinocytes. The addition of thrombin shows excitation of fluorescence while the addition of thrombin and tranexamic acid (T-AMCHA) and the addition of thrombin and bivalirudin show that the excitation is suppressed. FIG. 6B graphically shows the proportion of activated cells within 2 minutes after reagent introduction in each case. FIG. 7A shows gene expression of both PAR-1 and PAR-2 when gene expression of PAR family is suppressed (PAR-1 to PAR-4 KD) when thrombin gene expression is suppressed (Thrombin KD). The results of calcium imaging in cultured keratinocytes with and without (PAR-1, 2 DKD) and (ScRNA) are shown. In the case where PAR-1 to PAR-4 gene expression or thrombin gene expression is not suppressed (scRNA), excitation is observed by addition of Cryj1 while in the case where PAR-1 gene expression or thrombin gene expression is suppressed, excitation is caused. I did not see it. FIG. 7B graphically illustrates the percentage of activated cells 2 minutes after Cryj1 addition in the same experimental system. FIG. 8 is a graph showing the enzyme activity as fluorescence using a fluorescent substrate or a chromogenic substrate of each enzyme. FIG. 8A shows that using a substrate for thrombin (F2), thrombin activity was inhibited with bivalirudin while tranexamic acid could not. It is also shown that prothrombin (Pro-F2) has no enzymatic activity. FIG. 8B shows that using a substrate for factor Xa (Fxa), the activity of factor Xa is suppressed by its inhibitory GGACK but not by tranexamic acid. FIG. 8C uses a substrate for thrombin (F2), prothrombin (Pro-F2) and factor Xa. Prothrombin is activated by factor Xa, which results in thrombin activity. Thrombin activity has been shown to be suppressed by Bivalirudin and GGACK, and was shown to be suppressed depending on tranexamic acid in a dose-dependent manner. The above results indicate that tranexamic acid suppresses the activation of prothrombin to thrombin. FIG. 9 shows the results of structural analysis showing the action sites of prothrombin and tranexamic acid in plasminogen.

In one aspect of the present invention, the present invention relates to a method of screening for a skin condition improving agent, which is based on the inhibitory effect of thrombin.

Skin condition refers to the condition of the skin. Deteriorated skin conditions include itching, pigmentation, rough skin, reduced skin barrier function, spots, wrinkles, sagging and the like. In the present invention, improvement of skin condition mainly relates to whitening, suppression of itching, and / or improvement of skin barrier function. Therefore, skin condition improving agents include skin lightening agents, itching inhibitors, and skin barrier improving agents.

Thrombin is a type of serine protease involved in blood coagulation and is also called factor IIa. In addition to catalyzing the reaction of fibrinogen to fibrin, the coagulation system also activates factor V, factor VIII and factor IX. It is present in blood as prothrombin (factor II) and is activated by the action of factors Xa and Va to form thrombin.

The inhibitory action of thrombin may be such that the action of thrombin or a thrombin-like protein may be inhibited as a result, and may include, for example, inhibition of activation of prothrombin to thrombin, in addition to inhibition of the active center of thrombin. Furthermore, in the present invention, it is particularly preferable that the action of thrombin on keratinocytes be suppressed, and the action on keratinocytes is exerted mainly through the activation of PAR-1 which is a thrombin receptor. Thus, the inhibitory action of thrombin may be PAR-1 inhibitory action. Thrombin-like protein refers to a protein that activates PAR-1, like thrombin. As such a thrombin-like protein, a homologue or ortholog of thrombin may be used, or a non-human-derived protein may be used. The thrombin-like protein may also be a partial peptide having the thrombin activity of human or non-human prothrombin. In the screening method of the present invention, by using a thrombin-like protein having PAR-1 activation activity as thrombin does, thrombin inhibition activity can also be used as an index. The activity of thrombin may be directly measured or may be measured indirectly by observing the downstream phenomenon controlled by thrombin, using the inhibitory effect of thrombin as an index. As a direct measure of thrombin action, a substance serving as a thrombin substrate can be introduced, and the action of thrombin can be determined based on the amount of degradation of the substrate. As an example of the indirect measurement of the suppressive action of thrombin, a test using blood and a test using cultured cells can be mentioned.

Substrates of thrombin used to measure the effects of thrombin include peptides or proteins that can be degraded by thrombin. When such a peptide or protein is labeled with a fluorescent substance and a quencher and the fluorescent substrate can be detected only when it is cleaved by thrombin, or when it is labeled with a chromogenic substance and cleaved by thrombin Chromogenic substrates that allow only detection of absorbance are commercially available, and it is preferable to use such fluorescent substrates and chromogenic substrates.

As an example of the screening method of the skin condition improving agent of the present invention, the following:
Contacting thrombin with a candidate agent,
Measuring thrombin activity,
The method includes a step of selecting a skin condition improving agent based on the thrombin inhibitory action of the candidate agent.

The step of contacting thrombin with the candidate agent is performed by adding the candidate agent to a solution containing thrombin or preparing a solution containing thrombin and the candidate agent. In the step of measuring the thrombin activity, for example, a thrombin substrate for measuring the thrombin action is added, and the thrombin activity is measured based on the amount of degradation of the substrate. When the above-mentioned fluorescent substrate or chromogenic substrate is used, the amount of decomposition of the substrate can be determined by measuring fluorescence or absorbance. If the thrombin activity is reduced when the candidate agent is added as compared to the thrombin activity in the control, it can be determined that the candidate agent has a thrombin inhibitory effect. Candidate agents having thrombin inhibitory activity can be selected as skin condition improving agents. The thrombin activity of the control may be previously determined in an experimental system not containing only the candidate drug, or may be compared at the same time by experiment.

The screening method of the present invention can use prothrombin, which is a precursor of thrombin, instead of thrombin. In that case, substances for converting prothrombin to thrombin, such as factor IXa and complexes thereof, are also included.

Thrombin and prothrombin used in the present invention may be those purified from blood, or may be those from which recombinantly expressed proteins are produced in cultured cells. In yet another embodiment, blood may be used as it is or as a solution containing thrombin and / or prothrombin. When using blood, thrombin activity can be determined based on blood clotting time. In that case, the candidate drug can be selected as a substance having a thrombin inhibitory action when the blood coagulation time is extended. A specific measurement technique of thrombin activity in blood can be performed based on prothrombin time measurement method or thrombotest. In the blood, blood coagulation is achieved by the cooperation of several factors. Thus, a candidate drug selected only by prolonging blood clotting time may not have a thrombin inhibitory effect. In that case, a test using a substrate of thrombin can also be performed to further confirm whether the thrombin inhibitory effect is actually achieved.

PAR-1 (protease-activated receptor-1) is mentioned as a receptor which transmits the cellular action by thrombin. PAR-1 has been reported to be expressed on cell membranes by platelets, vascular endothelial cells, and vascular smooth muscle cells. Thrombin activates the cleavage of the extracellular domain N-terminal peptide, and in platelets, transmits a signal of thrombin aggregation. As an example of the screening method of the present invention, PAR-1 expressing cells can be used to determine the inhibitory effect of thrombin.

Concretely, the screening method of the skin condition improving agent which made the thrombin suppression effect which concerns on this invention a parameter | index is the following as an example:
Culturing PAR-1 expressing cells in a medium containing thrombin and a candidate agent,
Measuring thrombin activity, and selecting a skin condition improving agent based on the thrombin inhibitory action of the candidate drug.

Here, thrombin activity can be measured by measuring the activity of PAR-1 expressing cells. As one example, when PAR-1 is activated, the cell concentration of Ca ²⁺ in cells increases, and therefore, it is possible to measure thrombin activity by performing Ca ²⁺ imaging. More specifically, when PAR-1 expressing cells are cultured in a medium containing thrombin and a candidate agent and Ca ²⁺ imaging is performed, thrombin is inhibited when the increase in intracellular Ca ²⁺ concentration by thrombin is suppressed. Candidate agents can be selected as agents that inhibit action. Here, thrombin may be added to the medium, or it may be produced by activating its precursor prothrombin. Prothrombin may be added or may be expressed and secreted by the cells themselves. Activation of prothrombin may be performed by the addition of any antigen, such as Cryj1. The substance that suppresses the thrombin action thus selected may be an inhibitor of PAR-1, ie, a PAR-1 antagonist. The substance thus selected can act as a skin condition improving agent, that is, a whitening agent, an itching inhibitor and a barrier function improving agent.

Tranexamic acid is known to exert a coagulant action and an antiallergic action based on an antiplasmin action, and is developed as a hemostat and an anti-inflammatory agent. On the other hand, an effect on whitening has been found, and is also developed as a whitening agent. It is believed that the skin lightening action and the antiallergic action are also based on the antiplasmin action that tranexamic acid has, but its mechanism of action has not been elucidated yet.

It has been reported that Cryj1, an antigen of cedar pollen, causes the reduction of lamellar granules through the action of serine protease. And the serine protease was thought to exert a cell action via PAR-2.

Our studies showed that the cellular action of Cryj1 on keratinocytes was suppressed by the administration of tranexamic acid (FIG. 1A-B). The action of tranexamic acid was found to be due to the suppression of extracellular serine protease activity (Fig. 2A-B). And indeed, in transdermal administration studies, tranexamic acid has been shown to prevent the reduction of lamellar granules by Cryj1 (Fig. 3A-B) and also to suppress the increase of TEWL (reduction of skin barrier function) by Cryj1. Was done (Figure 4).

Since the cell action by Cryj1 is based on the activation of serine protease, the suppression of the cell action by tranexamic acid is also a cell action through PAR-2 which is a kind of a substrate of serine protease conventionally known. It was considered to be. However, it was shown that plasminogen, a precursor of plasmin that contributes to PAR-2 activation, was not present in keratinocytes (FIG. 5). In addition, in keratinocytes, even when PAR-2 was knocked down, there was no change in the responsiveness of keratinocytes to Cryj1, while PAR-1 knockdown reduced the responsiveness of keratinocytes to Cryj1. From the results, it was shown that PAR-1 is important for the cell action by Cryj1 (FIG. 6). Since PAR-1 is known as a thrombin receptor, it was predicted that the suppressive action of the cell action by Trajexamic acid by Cryj 1 is based not on the antiplasmin action but on the antithrombin action.

In fact, when thrombin was administered to cultured keratinocytes, the cellular action was exerted (FIG. 7A). In addition, tranexamic acid inhibited its cellular action in the same manner as the thrombin inhibitor Bivalirudin (FIGS. 7A-B). In keratinocytes, the cell action by Cryj1 was lost when knockdown of thrombin was performed (Fig. 8A-B), and it became clear that the cell action by Cryj1 is caused by the interaction between thrombin and PAR-1 . And, tranexamic acid was found to suppress the cell action by suppressing such a thrombin action. As a result of more detailed analysis of the thrombin inhibitory action of tranexamic acid, it is found that tranexamic acid acts on prothrombin while it does not act on thrombin or factor Xa which is a thrombin activating factor (Fig. 9A and B). Shown (FIG. 9C). Therefore, it was shown that the cell action in keratinocytes by tranexamic acid is achieved by suppressing the activation of prothrombin.

As the present inventors have newly found, tranexamic acid has antithrombotic activity as well as antiplasmin activity. However, expression of plasminogen is not seen in keratinocytes. In addition, in keratinocytes, PAR-1 is expressed, and it has been shown that activation of cells by the antigenic substance Cryj1 is mediated through the PAR-1 pathway. Since the target of the cell action by plasmin is PAR-2, the action of tranexamic acid on the skin is not due to the antiplasmin action conventionally known, but the probability based on the antithrombin action found in the present invention Is high.

In another aspect of the present invention, the present invention relates to a skin condition improving agent comprising a thrombin action inhibitor. Thrombin action inhibitors have been well studied in the field of hematology, and several compounds are being developed as thrombin action inhibitors. For example, dabigatran and argatroban are being developed as agents having a competitive inhibitory effect on thrombin, and compounds such as SCH 503458 and E5555 have also been developed as PAR-1 antagonists. Thrombin action inhibitors known in the art of hematology are known, and these thrombin action inhibitors can be developed as skin condition improving agents. The thrombin action inhibitor can be used by any administration route, but transdermal administration is preferable from the viewpoint of acting directly on the skin. By using transdermal administration, even drugs that cause side effects in systemic administration can be tolerated from the viewpoint of improving the skin condition. The skin improving agent of the present invention can be used as a whitening agent, an itching inhibitor and a skin barrier improving agent. The skin improving agent of the present invention can improve skin condition via suppression of PAR-1.

In another aspect of the present invention, the present invention relates to a thrombin action inhibitor comprising tranexamic acid or a salt thereof. Tranexamic acid has been developed as a blood clotting agent, anti-inflammatory agent and whitening agent through antiplasmin action. On the other hand, the effect on thrombin action is not known at all. Such a thrombin action inhibitor can be used as a skin barrier function enhancer. Moreover, since the reaction by an allergen is suppressed, it can be used as an antiallergic agent, an itching inhibitor, and an antiinflammatory agent. Skin conditions that may be ameliorated or treated by thrombin action inhibitors include cosmetic problems such as dry skin, sensitive skin, scaling, dandruff and the like. In addition, thrombin action inhibitors can be used to treat skin diseases such as atopic dermatitis, psoriasis, eczema and allergic diseases such as hay fever.

In the field of hematology, the inhibition of the thrombin action is an anticoagulant action of the blood, which is the opposite of the antiplasmin action which has hitherto been known as the action of plasmin. The thrombin action is roughly divided into a cell action mediated by PAR, which is a thrombin receptor, and a fibrin thrombus action caused by acting on fibrinogen. The thrombin action in the present invention mainly refers to an action via PAR-1. Thus, the thrombin action inhibitor can be said to be an inhibitor of thrombin action via PAR, preferably PAR-1. The thrombin inhibitory action may be an antagonistic action on the receptor of PAR-1, or may be exerted by suppressing the degradation of prothrombin to thrombin.

According to the present invention, the skin condition improving agent screened may be added to functional foods, cosmetics, quasi-drugs and medicines for the purpose of whitening, itching suppression and skin barrier improvement. Examples of cosmetics to be blended include sunscreens, lotions, essences, beauty creams, aftercare lotions, sun oils, etc. However, they can be formulated into any cosmetic as long as they are applied to the skin. . The medicines include anti-inflammatory external skin preparations and anti-inflammatory oral drugs. Moreover, it is preferable to mix | blend as a skin external preparation which can be directly applied to skin. The skin condition improving agent of the present invention can be appropriately blended, as necessary, with any compounding component used for cosmetics, medicines, etc., as long as the effect is not impaired. Examples of the optional components include oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, UV absorbers, moisturizers, perfumes, various medicinal effects. Ingredients, preservatives, pH adjusters, neutralizing agents, etc. may be mentioned. As other medicinal ingredients, for example, an anti-inflammatory ingredient, a whitening ingredient and the like may be contained.

All documents mentioned herein are incorporated herein by reference in their entirety.

The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, for example, additions, deletions and replacements of the constituent features of the present invention can be made without departing from the spirit of the present invention.

Example 1: Suppression of cell activation by Cryj1 and activation by addition of tranexamic acid or bivalirudin Cultured keratinocytes (commercially available: Kurabo) were cultured in EPI LIFE-KG2 medium (commercially available: Kurabo) in a humidified atmosphere at 37 ° C. It was cultured for a day. The culture medium was replaced with EPI LIFE-KG 2 + Ca 1.8 mM (commercially available: Kurabo), and cultured for 28 to 48 hours, followed by EPI LIFE-KG 2 + Ca1 containing 5 μM Fura2 AM (commercially available: life technology). The culture was replaced with .8 mM (commercially available: Kurabo) and cultured for 1 hour. Then, it is replaced with a buffer (pH 7.4) containing NaCl 150 mM, KCl 5 mM, CaCl ₂ 1.8 mM, MgCl ₂ 1.2 mM, HEPES 25 mM, D-glucose 10 mM, and 100 ng / ml of Cryj1 (commercial source: HAYASHIBARA) Or tranexamic acid (T-AMCHA) (commercially available: LKT Laboratories) (10 mM), bivalirudin (commercially available: PROSPEC) (100 ng / ml), or soybean trypsin inhibitor (SBTI) (1 μM) Was added to the buffer. The fluorescence was measured by a fluorescence microscope to measure changes in intracellular calcium concentration. The results are shown in FIG. 1 (A). The intracellular calcium concentration value about 2 minutes after addition of Cryj1 was calculated using Aqua Cosmos software, and is shown in FIG. 1 (B) (p <0.0001). It was shown that cells activated by Cryj1 alone addition were suppressed by the addition of tranexamic acid, bivalirudin or soybean trypsin inhibitor.

Example 2 Measurement of Serine Protease Activity Cultured keratinocytes (commercially available: Kurabo) were cultured in EPI LIFE-KG2 medium (commercially available: Kurabo) under a humidified atmosphere at 37 ° C. for 2 days. For the culture that became confluent, replace the medium with EPI LIFE-KG2 + Ca 1.8 mM (commercially available: Kurabo), culture for 28 to 48 hours, and then add a serine protease activity measuring reagent (commercially available: AAT Bioquest) to the medium did. For cultures, Cryj1 (100 ng / ml), Cryj1 (100 ng / ml) + tranexamic acid (10 mM), Cryj1 (100 ng / ml) + soybean trypsin inhibitor (SBTI) (1 μM), and Cryj 1 (100 ng / ml) + Bivalirudin (100 ng / ml) was added respectively, and a reagent-free group was used as a control. After addition of the reagent, fluorescence (wavelength: 545 nm) was measured with a fluorescence microscope to measure serine protease activity. The results are shown in FIG. 2 (A). The percentage of serine protease substrate degradation at about 1 minute after addition of the reagent was calculated using Aqua Cosmos software and is shown in FIG. 2 (B) (tranexamic acid: p <0.0001, SBTI: p <0.0001, Bivalirudin: p <0.0001).

Example 3: Effect of Cryj1 and tranexamic acid on human skin Cryj1 (1 μg / ml), Cryj1 (1 μg / ml) + tranexamic acid (100 mM) was applied for 20 minutes to the abdominal position of a human subject (female, 34 years old) , Epidermal section after application was obtained. The cross section of the skin sample was photographed with an electron microscope (commercially available: Hitachi High-Technologies) for the skin sample obtained (FIG. 3). In the electron micrographs taken, lamellar granules (LG) in the granular layer of the epidermis are indicated by arrows. The skin applied with Cryj1 alone had reduced lamellar granules as compared to the skin applied with Cryj1 and tranexamic acid.

Example 4: Effects of Cryj1 and tranexamic acid and bivalirudin on human skin Tape stripping with cellophane tape (TS) + Cryj1 (1 μg / ml), TS + Cryj1 (1 μg / ml) + tranexam at the abdominal position of human subjects (female 34 years old) Acid (100 mM), TS + Cryj1 (1 μg / ml) + bivalirudin (1 μg / ml) were applied for 2 hours, 4 hours and 6 hours, and TEWL at the application site was measured using an electronic balance after each application. The results are shown in FIG.

Example 5: Expression of plasmin and plasminogen in keratinocytes Culture Keratinocytes (commercially available: Kurabo) were cultured in EPI LIFE-KG2 medium at 37 ° C. in a humidified atmosphere for 2 days. The confluent culture was recovered, and cDNA was prepared using Trizol (commercially available as NIPPON GENE) according to the product description of VILO Master Mix (commercially available as Invitrogen). The following primer set for amplification of plasminogen, PLG-Fw: ggatgtgcctcggtagctc (SEQ ID NO: 1), PLG-Rv: gaacaattggctcccacag (SEQ ID NO: 2) and a primer set for internal standard Gapdh amplification, are used for real-time PCR Although it did, expression of plasinogen was not seen (FIG. 5).

Example 6: The PAR1 (PAR-1, PAR-2, PAR-3, PAR-4) siRNA (commercially available: GE Dharmacon) in Cryj1-reactive cultured keratinocytes (commercially available: Kurabo) in PAR family knockdown keratinocytes Alternatively, gene expression was knocked down by introducing thrombin siRNA (commercially available: GE Dharmacon). As a control, scRNA (commercially available: GE Dharmacon) was introduced. The introduced cultured keratinocytes were cultured in EPI LIFE-KG2 (commercially available: Kurabo) under a 37 ° C. humidified atmosphere for 1 day. EPI LIFE-KG2 + Ca1 was used to replace the culture medium with EPI LIFE-KG2 + Ca 1.8 mM (commercially available: Kurabo), and cultured for 1 to 2 days, and then containing Fura2 AM (commercially available: life technology) at 5 μM. The culture was replaced with .8 mM (commercially available: Kurabo) and cultured for 1 hour. Then, it is replaced with a buffer (pH 7.4) containing NaCl 150 mM, KCl 5 mM, CaCl ₂ 1.8 mM, MgCl ₂ 1.2 mM, HEPES 25 mM, D-glucose 10 mM, and 100 ng / ml of Cryj1 (commercial source: HAYASHIBARA) Added alone to the buffer at a concentration of The fluorescence was measured by a fluorescence microscope to measure changes in intracellular calcium concentration. The results are shown in FIG. 7 (A). The intracellular calcium concentration value about 2 minutes after addition of Cryj1 was calculated using Aqua Cosmos software, and is shown in FIG. 7 (B). Knockdown of PAR-1 abolished the reactivity of keratinocytes to Cryj1. It was also shown that activation of cells by the addition of Cryj1 did not occur in thrombin knockdown keratinocytes.

Example 7 Activation of Cultured Keratinocytes by Addition of Thrombin Cultured keratinocytes (commercially available: Kurabo) were cultured in EPI LIFE-KG2 medium (commercially available: Kurabo) under a humidified atmosphere at 37 ° C. for 2 days. For the culture that became confluent, replace the medium with EPI LIFE-KG2 + Ca 1.8 mM (commercially available: Kurabo), culture for 28 to 48 hours, and then EPI LIFE-KG2 + Ca1 containing 5 μM Fura2 AM (commercially available: life teqnology) The culture was replaced with .8 mM (commercially available: Kurabo) and cultured for 1 hour. Then, it is replaced with a buffer (pH 7.4) containing NaCl 150 mM, KCl 5 mM, CaCl ₂ 1.8 mM, MgCl ₂ 1.2 mM, HEPES 25 mM, D-glucose 10 mM, and thrombin (commercially available: Nacalai Tesque) at 100 pg / hour. Buffer was added at a concentration of ml alone, or in combination with tranexamic acid (T-AMCHA) (10 mM) or bivalirudin (commercially available: PROSPEC) (100 ng / ml). The fluorescence was measured by a fluorescence microscope to measure changes in intracellular calcium concentration. The results are shown in FIG. 6 (A). The intracellular calcium concentration value about 2 minutes after the addition of thrombin was calculated using Aqua Cosmos software, and is shown in FIG. 6 (B) (tranexamic acid: p <0.0001, bivalirudin: p <0.0001). The addition of thrombin indicated that the cells were activated and that the addition of thrombin was blocked by the simultaneous addition of tranexamic acid or bivalirudin.

Example 8 Identification of Mechanism of Thrombinamic Acid Thrombin Action Inhibition Thrombin (F2) (40 ng) (commercially available: Nacalai Tesque), prothrombin (pro-F2) (40 ng) (commercially available: Invitrogen), thrombin + tranexam Prepare solutions containing acid (100 μM) (F2 + TAMCHA) and thrombin + bivalirudin (1 μg) (F2 + Biv) respectively, add thrombin fluorescent substrate (commercially available: Cosmo Bio) (20 μM), and measure fluorescence with a fluorometer did. The results are shown in FIG. 8 (A). Bivalirudin is an agent that acts on the active center of thrombin to exert an antithrombin action. While thrombin activity was suppressed by bivalirudin, tranexamic acid did not suppress thrombin activity.

Factor Xa (FXa) (9.5 ng) (marketing source: abcam), Factor Xa (9.5 ng) + GGACK (marketing source: abcam) (100 μM) (FXa + GGACK), factor Xa (9.5 ng) + GG Tranexam Solutions containing acid (10 mM) (FXa + T-AMCHA) were prepared, respectively, a fluorescent substrate for factor Xa (commercially available: abcam) was added, and the fluorescence was measured with a fluorometer. The results are shown in FIG. 8 (B). GGACK is an agent that exerts a factor Xa inhibitory action on the active center of factor Xa. While the activity of factor Xa was suppressed by GGACK, tranexamic acid did not suppress factor Xa activity.

To the prothrombin (Pro-F2) (40 ng) + factor Xa (FXa) solution, tranexamic acid (T-AMCHA) was further added at 10 mM, 1 mM and 100 μM, respectively. Bivalirudin (1 μg) and GGACK (10 μM) were added to another prothrombin (Pro-F2) (40 ng) + factor Xa (FXa) solution, respectively. To these test solutions, a chromogenic substrate for thrombin (commercially available: Rossix) was added, and the absorbance was measured with an absorptiometric system. The results are shown in FIG. 8 (C). Tranexamic acid was shown to suppress thrombin activity in a concentration dependent manner.

Referring to the results in FIGS. 8 (A) to (C), it was shown that tranexamic acid did not inhibit the activity of thrombin or factor Xa, but inhibited the activation of prothrombin to thrombin.

Example 9: Structural analysis From conformational data bank (PDB), conformational data (PDB ID: 1CEB) of plasminogen (KR1 domain) bound to tranexamic acid and conformational data (PDB ID: 5EDM) of prothrombin It acquired and compared three-dimensional structure using analysis software Chimera (FIG. 9). As a result, it was shown that the conformation of plasminogen (KR1 domain) to which tranexamic acid is bound very well overlaps with two KR domains in prothrombin. This suggests that tranexamic acid binds to the KR domain of prothrombin.

Example 10 Screening Method (1) A solution containing thrombin (F2) and a candidate drug is injected into each well of a 96-well plate. Next, a thrombin fluorescent substrate (commercially available: Cosmo Bio) is added. The fluorescence after addition is measured with a plate reader and compared with a control to determine the thrombin activity suppressing effect of the candidate drug. Candidate agents having a thrombin activity suppressing action can be screened as a skin condition improving agent, preferably a skin lightening agent, an itching inhibitor, and a skin barrier improving agent.

(2) Inject each well of the 96-well plate a solution containing prothrombin (pro-F2), factor IXa, and a candidate drug. Next, a thrombin chromogenic substrate (commercially available: Rossix) is added. The absorbance after addition is measured with a plate reader and compared with a control to determine the thrombin activity suppressing effect of the candidate drug. Candidate agents having a thrombin activity suppressing action can be screened as a skin condition improving agent, preferably a skin lightening agent, an itching inhibitor, and a skin barrier improving agent.

(3) Culture Keratinocytes (commercially available: Kurabo) are cultured for 2 days in an EPI LIFE-KG2 medium at 37 ° C. in a humidified atmosphere. For the culture that became confluent, replace the medium with EPI LIFE-KG2 + Ca 1.8 mM (commercially available: Kurabo), culture for 28 to 48 hours, and then EPI LIFE-KG2 + Ca1 containing 5 μM Fura2 AM (commercially available: life teqnology) Replace with 8 mM (marketing source: Kurabo). Add Cryj1 (commercially available: HAYASHIBARA) and a candidate drug to the culture medium. The fluorescence is measured with a fluorescence microscope to measure changes in intracellular calcium concentration. A candidate agent can be determined to have a thrombin activity suppressive effect when calcium concentration is reduced as compared to a control. Candidate agents having a thrombin activity suppressing action can be screened as a skin condition improving agent, preferably a skin lightening agent, an itching inhibitor, and a skin barrier improving agent.

(4) Culture Keratinocytes (commercially available: Kurabo) are cultured in EPI LIFE-KG2 medium for 2 days under a humidified atmosphere at 37 ° C. For confluent cultures, medium is replaced with EPI LIFE-KG2 + Ca 1.8 mM and cultured for 24-48 hours, then replaced with EPI LIFE-KG2 + Ca 1.8 mM containing 5 μM Fura2 AM (marketed by life sciences). Thrombin or prothrombin and the candidate agent are added to the culture medium. The fluorescence is measured with a fluorescence microscope to measure changes in intracellular calcium concentration. The candidate agent can be determined to have a thrombin activity suppressive effect when the calcium concentration is decreased as compared to the control. Candidate agents having a thrombin activity suppressing action can be screened as a skin condition improving agent, preferably a skin lightening agent, an itching inhibitor, and a skin barrier improving agent.

Claims (31)

A screening method of a skin condition improving agent using the inhibitory effect of thrombin as an index. The screening method according to claim 1, wherein the skin condition improving agent is selected from the group consisting of a whitening agent, an itching inhibitor and a skin barrier improving agent. The screening method is
Contacting prothrombin or thrombin with the candidate agent,
Measuring thrombin activity,
Selecting a skin condition improving agent based on the thrombin inhibitory action of the candidate drug,
The method according to claim 1 or 2, comprising The method according to claim 3, wherein the measurement of thrombin activity is measured by the amount of thrombin substrate degradation or blood coagulation time. The screening method is
Culturing PAR-1 expressing cells in a medium containing prothrombin or thrombin and a candidate agent,
Measuring thrombin activity, selecting a skin condition improving agent based on the thrombin inhibitory action of the candidate drug,
The method according to claim 1 or 2, comprising The method according to claim 5, wherein the measurement of thrombin activity is performed by calcium imaging. A skin condition improving agent comprising a thrombin action inhibitor. The skin condition improving agent according to claim 7, wherein the skin condition improving agent is selected from the group consisting of a whitening agent, an itching inhibitor and a skin barrier improving agent. The skin condition improving agent according to claim 7 or 8, wherein the thrombin action inhibitor ameliorates the skin condition via suppression of cell action by PAR-1. A thrombin action inhibitor comprising tranexamic acid or a salt thereof. The thrombin action inhibitor according to claim 10, wherein the thrombin action inhibitor suppresses the cell action by PAR-1. The thrombin action inhibitor according to claim 11, wherein the inhibition of the thrombin action is achieved by suppressing the degradation of prothrombin to thrombin. A cosmetic or non-therapeutic method for improving skin condition comprising applying a thrombin action inhibitor. 14. A cosmetic or non-therapeutic method according to claim 13, wherein the skin condition improvement is selected from the group consisting of whitening, itching control, skin barrier improvement. The cosmetic or non-therapeutic method according to claim 13 or 14 through suppression of cell action by PAR1. The cosmetic or non-therapeutic method according to any one of claims 13 to 15, wherein the thrombin action inhibitor comprises tranexamic acid or a salt thereof. The cosmetic or non-therapeutic method according to any one of claims 13 to 16, wherein the inhibition of the thrombin action is effected by suppressing the activation of prothrombin to thrombin. Tranexamic acid or a salt thereof for use in the treatment of dry skin, sensitive skin, scaling, dandruff, atopic dermatitis, psoriasis, eczema via inhibition of the thrombin action. The tranexamic acid or a salt thereof according to claim 18, wherein the inhibition of thrombin action is achieved by suppressing the activation of prothrombin to thrombin. Tranexamic acid or a salt thereof for use in the treatment of dry skin, sensitive skin, scaling, dandruff, atopic dermatitis, psoriasis, eczema via suppression of cell action by PAR1. Use of a thrombin action inhibitor in the manufacture of a skin condition improving agent. 22. The use according to claim 21, wherein the skin condition improving agent is selected from the group consisting of a whitening agent, an itching inhibitor and a skin barrier improving agent. The use according to claim 21 or 22, wherein the thrombin action inhibitor improves the skin condition through suppression of cell action by PAR-1. Use of tranexamic acid or a salt thereof for the production of a thrombin action inhibitor. The use according to claim 24, wherein the thrombin action inhibitor suppresses the cell action by PAR-1. 26. The use according to claim 24 or 25, wherein the inhibition of thrombin action is effected by suppressing the activation of prothrombin to thrombin. A method for improving skin condition, or a method for treating skin disorder or allergic disease, comprising applying a thrombin action inhibitor. Said skin condition improvement is selected from the group consisting of whitening, itching suppression, skin barrier improvement, and said skin disorder is selected from the group consisting of dry skin, sensitive skin, scale, dandruff, atopic dermatitis, psoriasis, eczema 28. The method of claim 27, wherein the allergic disease is hay fever. The method according to claim 27 or 28 through suppression of cell action by PAR1. The method according to any one of claims 27-29, which is performed on a subject having a skin condition caused by PAR1 activation. The method according to any one of claims 27 to 30, wherein the thrombin action inhibitor is tranexamic acid or a salt thereof.

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