JP7264589B2 - Screening method for skin condition-improving agent using thrombin inhibitory action as index, and skin condition-improving agent containing thrombin action inhibitor - Google Patents

Description

TECHNICAL FIELD The present invention relates to the technical field of skin condition improvement, specifically to cosmetic, cosmetic or medical technology.

Since the skin is the outermost layer of the living body, it is constantly exposed to external stimuli. External stimuli include not only physical stimuli such as ultraviolet rays and temperature, but also stimuli by chemical substances and antigenic substances. Japanese cedar pollen that causes hay fever acts via mucous membranes to cause respiratory symptoms and pharyngeal symptoms peculiar to hay fever, and cedar pollen dermatitis is caused by contact with the skin. Cryj1, known as the cedar antigen, is known to activate serine proteases in keratinocytes, and the activated serine proteases mediated the activation of PAR-2 protein, a membrane protein expressed in keratinocytes. and lamellar granules are thought to cause the reduction (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 extracellularly secreted by keratinocytes undergoing apoptosis. Lamellar granules contain intercellular lipids such as ceramide, cholesterol, and fatty acids, and a decrease in lamellar granules leads to a decrease in skin barrier function.

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

JP 2014-152159 A

Arch Dermatol Res 308:49-54, 2016

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

The present inventors conducted intensive research to elucidate the mechanism of the origin of signal transduction that suppresses the secretion of lamellar granules in response to pollen antigens. We found for the first time that the activation of PAR-1 by thrombin or thrombin-like proteins, rather than the activation of PAR-2 by type serine protease, is the origin of signal transduction that inhibits the secretion of lamellar granules. Therefore, the present inventors have invented a new screening method for screening skin condition-improving agents by using the thrombin-suppressing action as an index.

Accordingly, the present invention relates to the following inventions:
[1] A method for screening a skin condition-improving agent, using thrombin inhibitory action as an index.
[2] The screening agent according to item 1, wherein the skin condition improving agent is selected from the group consisting of whitening agents, antipruritic agents, and skin barrier improving agents.
[3] The screening method comprises
contacting prothrombin or thrombin with a candidate agent;
measuring thrombin activity,
selecting a skin condition-improving agent based on the thrombin-inhibiting activity of the candidate agent;
3. The method of item 1 or 2, comprising:
[4] The method according to item 3, wherein the thrombin action is measured by the amount of thrombin substrate degraded or the blood clotting time.
[5] The screening method comprises
culturing PAR-1 expressing cells in medium containing prothrombin or thrombin and a candidate agent;
a step of measuring thrombin activity a step of selecting a skin condition-improving agent based on the thrombin-suppressing action of the candidate drug;
3. The method of item 1 or 2, comprising:
[6] A method according to item 5, wherein the thrombin action is measured by calcium imaging.
[7] A skin condition improving agent containing 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 whitening agents, antipruritic agents, and skin barrier improving agents.
[9] The skin condition-improving agent according to item 7 or 8, wherein the thrombin action inhibitor improves skin condition through suppression of cell action by PAR-1.
[10] A thrombin action inhibitor containing tranexamic acid or a salt thereof.
[11] The thrombin action inhibitor according to item 10, wherein the thrombin action inhibitor suppresses cellular action by PAR-1.
[12] The thrombin action inhibitor according to item 11, wherein the inhibition of thrombin action is achieved by suppressing the activation of prothrombin to thrombin.

By using the inhibitory action of thrombin as an index, it becomes possible to screen skin condition-improving agents.

FIG. 1A shows the results of calcium imaging in cultured keratinocytes, showing that the addition of Cryj1 causes fluorescence excitation, while the addition of Cryj1 and tranexamic acid (T-AMCHA) suppresses the excitation. be Excitation is also suppressed by the addition of Cryj1 and soybean trypsin inhibitor (SBTI) or Cryj1 and Bivalirudin. FIG. 1B graphically shows the percentage of cells activated within 2 minutes after reagent introduction in each case. FIG. 2A is a graph showing protease activity in cultured keratinocytes. Cryj1, Cryj1 and tranexamic acid (T-AMCHA), Cryj1 and soybean trypsin inhibitor (SBTI), Cryj1 and bivalirudin were included in the medium, respectively, and a reagent-free group (Control) was used as a control, and a fluorescent substrate was added. shows the fluorescence change of FIG. 2B graphically shows the percentage of cells activated within 2 minutes after reagent introduction in each case. FIG. 3 shows electron micrographs of skin sections after Cryj1 (FIG. 3A) and a solution containing Cryj1 and tranexamic acid (T-AMCHA) (FIG. 3B) were applied to the skin. Lamellar granules are indicated by arrows in the photograph. FIG. 4 shows the TEWL values at the application sites after applying solutions containing Cryj1, Cryj1 and tranexamic acid (T-AMCHA), Cryj1 and bivalirudin, and a reagent-free solution (Control) as a control to the skin. FIG. 5A shows the results of examining gene expression of plasminogen (plasmin) in keratinocytes by real-time PCR. And FIG. 5B shows the result of electrophoresis of the amplified product. FIG. 6A shows the results of calcium imaging in cultured keratinocytes, with the addition of thrombin showing excitation of fluorescence, whereas the addition of thrombin and tranexamic acid (T-AMCHA) and the addition of thrombin and bivalirudin resulted in fluorescence excitation. is suppressed. FIG. 6B graphically shows the percentage of cells activated within 2 minutes after reagent introduction in each case. FIG. 7A shows that when thrombin gene expression is suppressed (Thrombin KD), when PAR family gene expression is suppressed (PAR-1 to PAR-4 KD), both PAR-1 and PAR-2 gene expression is suppressed. Calcium imaging results in suppressed (PAR-1,2 DKD) and unsuppressed (scRNA) cultured keratinocytes are shown. When PAR-1 to PAR-4 gene expression or thrombin gene expression was not suppressed (scRNA), addition of Cryj1 caused excitation, whereas when PAR-1 gene expression or thrombin gene expression was suppressed, excitation was observed. was not seen. FIG. 7B graphically shows the percentage of activated cells 2 minutes after addition of Cryj1 in the same experimental system. FIG. 8 is a graph showing enzyme activity by fluorescence using a fluorogenic substrate or a chromogenic substrate for each enzyme. FIG. 8A shows that using substrates for thrombin (F2), thrombin activity was inhibited by bivalirudin, but not by tranexamic acid. Also, prothrombin (Pro-F2) is shown to have no enzymatic activity. FIG. 8B uses substrates for Factor Xa (Fxa) and shows that the activity of Factor Xa is inhibited by GGACK, which has its inhibitory effect, but not by tranexamic acid. FIG. 8C uses substrates for thrombin (F2), prothrombin (Pro-F2) and factor Xa are used. Prothrombin is activated by Factor Xa, resulting in thrombin activity. It has been shown that thrombin activity is suppressed by bivalirudin and GGACK, and that it is dose-dependently suppressed by tranexamic acid. These results indicate that tranexamic acid inhibits the activation of prothrombin to thrombin. FIG. 9 shows the results of structural analysis showing the sites of action of prothrombin and tranexamic acid on plasminogen.

In one aspect of the present invention, the present invention relates to a method of screening for a skin condition improving agent, using thrombin inhibitory action as an index.

A skin condition refers to the condition of the skin. Exacerbated skin conditions include itch induction, pigmentation, rough skin, skin barrier function deterioration, blemishes, 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. Accordingly, skin condition improving agents include whitening agents, anti-itch agents, and skin barrier improving agents.

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

The inhibitory action of thrombin may result in inhibition of the action of thrombin or a thrombin-like protein, and may include, for example, inhibition of the active center of thrombin as well as inhibition of activation of prothrombin to thrombin. Furthermore, in the present invention, it is particularly preferred that the action of thrombin on keratinocytes is suppressed, and the action on keratinocytes is exhibited mainly through the activation of the thrombin receptor PAR-1. Therefore, the inhibitory effect of thrombin may be a PAR-1 inhibitory effect. A thrombin-like protein is a protein that activates PAR-1 like thrombin. Such thrombin-like proteins may be homologues or orthologues of thrombin, or non-human-derived proteins. The thrombin-like protein may also be a partial peptide having thrombin activity of human or non-human prothrombin. In the screening method of the present invention, by using a thrombin-like protein having a PAR-1 activating action like thrombin, the thrombin inhibitory action can be used as an index. Using the inhibitory action of thrombin as an indicator may be performed by directly measuring the activity of thrombin, or indirectly by observing downstream phenomena controlled by thrombin. As a direct measurement of the action of thrombin, a substance that serves as a substrate for thrombin is introduced, and the action of thrombin can be determined based on the amount of degradation of the substrate. Examples of indirect measurement of the inhibitory action of thrombin include tests using blood and tests using cultured cells.

Thrombin substrates used to measure the action of thrombin include peptides or proteins that can be cleaved by thrombin. Such peptides or proteins may be labeled with a fluorophore and a quencher to allow detection of fluorescence only when cleaved by thrombin, or labeled with a chromogenic substrate and quenched by thrombin. Chromogenic substrates are commercially available that enable detection of only absorbance, and it is preferable to use such fluorogenic or chromogenic substrates.

As an example of the screening method for the skin condition improving agent of the present invention, the following:
contacting thrombin with a candidate agent;
measuring thrombin activity,
selecting a skin condition-improving agent based on the thrombin-inhibiting activity of the candidate agent.

The step of contacting thrombin with the drug candidate is performed by adding the drug candidate to a solution containing thrombin or by preparing a solution containing thrombin and the drug candidate. In the step of measuring thrombin activity, for example, a thrombin substrate for measuring thrombin action is added, and thrombin activity is measured based on the amount of degradation of the substrate. When a fluorogenic or chromogenic substrate as described above is used, the amount of degradation of the substrate can be determined by measuring fluorescence or absorbance. A candidate agent can be determined to have thrombin-inhibiting activity if thrombin activity is reduced upon addition of the candidate agent relative to thrombin activity in controls. Candidate agents with thrombin-suppressing activity can be selected as skin condition-improving agents. Control thrombin activity may be predetermined in an experimental system that does not contain the candidate agent alone, or may be compared in parallel experiments.

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

Thrombin and prothrombin used in the present invention may be purified from blood or may be recombinantly expressed proteins produced in cultured cells. In yet another embodiment, blood may be used as is or processed 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 effect when the blood clotting time is prolonged. A specific method for measuring thrombin activity in blood can be based on the prothrombin time measurement method or the thrombotest. In blood, blood coagulation is achieved through the cooperation of multiple factors. Therefore, it is possible that candidate agents selected solely for prolongation of blood clotting time do not have thrombin inhibitory activity. In that case, a test with a substrate for thrombin may be carried out to further confirm that a thrombin-suppressing effect is indeed achieved.

PAR-1 (protease-activated receptor-1) is a receptor that mediates cellular actions by thrombin. PAR-1 is reported to be expressed on the cell membrane of platelets, vascular endothelial cells, and vascular smooth muscle cells. It is activated by cleavage of the N-terminal peptide of the extracellular region by thrombin, and transmits a signal for thrombin aggregation in platelets. As an example of the screening method of the present invention, cells expressing PAR-1 can be used to determine the inhibitory effect of thrombin.

Specifically, the method for screening skin condition-improving agents using thrombin inhibitory action as an indicator according to the present invention is as follows:
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-inhibiting activity of the candidate agent.

Here, thrombin activity can be measured by measuring the activity of PAR-1-expressing cells. For example, when PAR-1 is activated, the intracellular concentration of Ca ²⁺ increases, so Ca ²⁺ imaging enables measurement of thrombin activity. More specifically, when PAR-1-expressing cells are cultured in a medium containing thrombin and a candidate drug and Ca ²⁺ imaging is performed, if thrombin-induced increase in intracellular Ca ²⁺ concentration is suppressed, thrombin Candidate agents can be selected as substances that inhibit the action. Here, thrombin may be added to the medium, or may be generated 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 addition of any antigen, eg Cryj1. The substance inhibiting thrombin action thus selected may be an inhibitor of PAR-1, ie a PAR-1 antagonist. Materials thus selected may act as skin condition improvers, ie, lightening agents, antipruritic agents, and barrier function improvers.

Tranexamic acid is known to exhibit blood coagulation and antiallergic effects based on its antiplasmin action, and has been developed as a hemostatic agent and anti-inflammatory agent. On the other hand, it has been found to have a whitening effect and is also being developed as a whitening agent. The whitening action and antiallergic action are also considered to be based on the antiplasmin action of tranexamic acid, but the action mechanism has not yet been elucidated.

Cryj1, a cedar pollen antigen, has been reported to cause a decrease in lamellar granules through the action of a serine protease. The serine protease was then thought to exert cellular actions via PAR-2.

Our studies showed that the cellular action of Cryj1 on keratinocytes was suppressed by administration of tranexamic acid (FIGS. 1A-B). This action of tranexamic acid was found to be due to the inhibition of extracellular serine protease activity (FIGS. 2A and 2B). In fact, transdermal administration tests showed that tranexamic acid prevented Cryj1-induced reduction of lamellar granules (Fig. 3A-B), and also inhibited Cryj1-induced increase in TEWL (decreased skin barrier function). (Fig. 4).

Since the cellular actions of Cryj1 are based on the activation of serine proteases, the suppression of cellular actions by tranexamic acid is also mediated by PAR-2, a type of serine protease substrate known to date. It was thought that there was. However, it was shown that plasminogen, the precursor of plasmin that contributes to PAR-2 activation, is absent in keratinocytes (Fig. 5). In keratinocytes, PAR-2 knockdown did not alter keratinocyte responsiveness to Cryj1, whereas PAR-1 knockdown reduced keratinocyte responsiveness to Cryj1. The results indicated that PAR-1 is important for cellular actions by Cryj1 (Fig. 6). Since PAR-1 is known as a thrombin receptor, it was predicted that the inhibitory effect of tranexamic acid on Cryj1-mediated cell action was based on antithrombin action, not antiplasmin action.

Indeed, administration of thrombin to cultured keratinocytes exerted cellular effects (Fig. 7A). Tranexamic acid also inhibited its cellular actions, as did the thrombin inhibitor bivalirudin (FIGS. 7A-B). When thrombin was knocked down in keratinocytes, the cellular action of Cryj1 was lost (FIGS. 8A-B), revealing that the cellular action of Cryj1 is caused by the interaction between thrombin and PAR-1. . It was also found that tranexamic acid suppresses cell action by suppressing such thrombin action. As a result of more detailed analysis of the thrombin-suppressing action of tranexamic acid, it was found that tranexamic acid does not act on thrombin or factor Xa, which is a thrombin activator (Figs. 9A and 9B), but acts on prothrombin. (Fig. 9C). Therefore, it was shown that the cellular action in keratinocytes by tranexamic acid is achieved by suppressing the activation of prothrombin.

As newly discovered by the present inventors, tranexamic acid has an anti-plasmin effect as well as an anti-thrombin effect. However, no expression of plasminogen is seen in keratinocytes. In addition, PAR-1 is expressed in keratinocytes, and it has been shown that cell activation by Cryj1, an antigenic substance, is mediated by the PAR-1 pathway. Since the target of cell action by plasmin is PAR-2, the action of tranexamic acid on the skin is not due to the conventionally known antiplasmin action, but is likely based on the antithrombin action found in the present invention. is high.

In another aspect of the invention, the invention relates to a skin condition improving agent comprising a thrombin action inhibitor. Thrombin inhibitors have been well studied in the field of hematology, and a number of compounds have been developed as thrombin inhibitors. For example, dabigatran and argatroban have been developed as drugs having a competitive inhibitory effect on thrombin, and compounds such as SCH503458 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 preferred from the viewpoint of direct action on the skin. By transdermal administration, even a drug that causes side effects in systemic administration can be tolerated from the viewpoint of improving skin conditions. The skin-improving agent of the present invention can be used as a whitening agent, an anti-itch agent, and a skin-barrier improving agent. The skin-improving agent of the present invention can improve skin conditions through suppression of PAR-1.

In another aspect of the invention, the invention relates to a thrombin action inhibitor comprising tranexamic acid or a salt thereof. Tranexamic acid has been developed as a blood coagulant, anti-inflammatory agent, and whitening agent through its antiplasmin action. On the other hand, its effect on thrombin action is completely unknown. Such thrombin action inhibitors may be used as skin barrier function improvers. In addition, since it suppresses reactions caused by allergens, it can be used as an anti-allergic agent, an anti-itch agent, and an anti-inflammatory agent. Skin conditions that may be improved or treated with thrombin action inhibitors include cosmetic problems such as dry skin, sensitive skin, scaling and dandruff. Furthermore, thrombin action inhibitors can be used to treat skin diseases such as atopic dermatitis, psoriasis and eczema, as well as allergic diseases such as hay fever.

In the field of hematology, inhibition of the action of thrombin results in an anticoagulant action of the blood, which is the opposite action to the antiplasmin action that has hitherto been known as the action of plasmin. The action of thrombin is roughly divided into a cell action mediated by PAR, which is a thrombin receptor, and a fibrin clot formation action caused by acting on fibrinogen. Thrombin action in the present invention mainly refers to action mediated by PAR-1. Therefore, thrombin action inhibitors can be referred to as inhibitors of thrombin action via PAR, preferably PAR-1. The thrombin inhibitory action may be an antagonistic action on the PAR-1 receptor, or may be exhibited by inhibiting the decomposition of prothrombin into thrombin.

The skin condition-improving agents screened by the present invention can be incorporated into functional foods, cosmetics, quasi-drugs, and pharmaceuticals for the purpose of whitening, suppressing itching, and improving the skin barrier. Cosmetics to be blended include sunscreens, lotions, serums, beauty creams, aftercare lotions, sun oils, etc., but can be blended into any cosmetics as long as they are applied to the skin. . Pharmaceuticals include anti-inflammatory agents for external use on the skin, oral agents for anti-inflammatory agents, and the like. In addition, it is preferably formulated as an external preparation for skin that can be directly applied to the skin. The skin condition-improving agent of the present invention can optionally contain optional compounding ingredients used in cosmetics, pharmaceuticals, etc., as long as the effects are not impaired. Examples of the optional ingredients include oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, UV absorbers, moisturizers, fragrances, and various medicinal effects. Ingredients, preservatives, pH adjusters, neutralizers, and the like. Other medicinal ingredients, such as anti-inflammatory ingredients and whitening ingredients, may also be included.

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

The embodiments of the invention described below are for illustrative purposes only and are not intended to limit the scope of the invention. The technical scope of the present invention is limited only by the description of the claims. Modifications of the present invention, such as additions, deletions and replacements of constituent elements of the present invention, can be made without departing from the gist of the present invention.

Example 1: Suppression of Cell Activation by Cryj1 and Activation by Addition of Tranexamic Acid or Bivalirudin cultured for days. For the confluent culture, the medium was replaced with EPILIFE-KG2+Ca1.8 mM (sold by Kurabo Industries), cultured for 28-48 hours, and then EPILIFE-KG2+Ca1 containing 5 μM Fura2 AM (sold by life technology). 0.8 mM (sold by Kurabo) and cultured for 1 hour. Then, the buffer was replaced with a buffer (pH 7.4) containing 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl ₂ , 1.2 mM MgCl ₂ , 25 mM HEPES, and 10 mM D-glucose. or tranexamic acid (T-AMCHA) (marketed by LKT Laboratories) (10 mM), bivalirudin (marketed by PROSPEC) (100 ng/ml), or soybean trypsin inhibitor (SBTI) (1 μM) at a concentration of was added to the buffer. Fluorescence was measured with a fluorescence microscope to determine changes in intracellular calcium concentration. The results are shown in FIG. 1(A). Intracellular calcium concentration values approximately 2 minutes after addition of Cryj1 were calculated using Aqua Cosmos software and shown in FIG. 1(B) (p<0.0001). It was shown that cells activated by the addition of Cryj1 alone were suppressed by the addition of tranexamic acid, bivalirudin or soybean trypsin inhibitor.

Example 2 Measurement of Serine Protease Activity Cultured keratinocytes (sold by Kurabo Industries) were cultured in EPILIFE-KG2 medium (sold by Kurabo Industries) at 37° C. in a humidified atmosphere for 2 days. After the culture became confluent, the medium was replaced with EPILIFE-KG2+Ca1.8mM (sold by Kurabo Industries), and after culturing for 28 to 48 hours, a serine protease activity measurement reagent (sold by AAT Bioquest) was added to the medium. bottom. Cryj1 (100 ng/ml), Cryj1 (100 ng/ml) + tranexamic acid (10 mM), Cryj1 (100 ng/ml) + soybean trypsin inhibitor (SBTI) (1 μM), and Cryj1 (100 ng/ml) to cultures +bivalirudin (100 ng/ml) was added to each, 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 reagent addition was calculated using Aqua Cosmos software and shown in FIG. 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) were applied to the abdomen of a human subject (female, 34 years old) for 20 minutes. , post-application epidermal sections were obtained. The cross section of the obtained skin sample was photographed with an electron microscope (sold by Hitachi High-Technologies) (Fig. 3). In electron micrographs taken, lamellar granules (LG) in the granular layer of the epidermis are indicated by arrows. Skin to which Cryj1 was applied alone had fewer lamellar granules than skin to which Cryj1 and tranexamic acid were applied.

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 on the abdomen of a human subject (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 sites was measured using an electronic balance after each application. The results are shown in FIG.

Example 5 Expression and Culture of Plasmin and Plasminogen in Keratinocytes Keratinocytes (marketed by Kurabo Industries) were cultured in EPILIFE-KG2 medium at 37° C. in a humidified atmosphere for 2 days. Confluent cultures were harvested, and cDNA was prepared using Trizol (marketed by NIPPON GENE) according to the product instructions for VILO Master Mix (marketed by Invitrogen). Real-time PCR was performed using 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. However, no plastonogen expression was observed (Fig. 5).

Example 6: Cryj1-reactive in PAR family knockdown keratinocytes PAR family (PAR-1, PAR-2, PAR-3, PAR-4) siRNA (sold by GE Dharmacon) in cultured keratinocytes (sold by Kurabo) Alternatively, gene expression was knocked down by introducing thrombin siRNA (marketed by GE Dharmacon). scRNA (marketed by GE Dharmacon) was introduced as a control. The introduced cultured keratinocytes were cultured in EPILIFE-KG2 (marketed by Kurabo Industries) in a humidified atmosphere at 37° C. for 1 day. For the confluent culture, the medium was replaced with EPILIFE-KG2+Ca 1.8 mM (sold by Kurabo Industries), cultured for 1 to 2 days, and then EPILIFE-KG2+Ca1 containing 5 μM Fura2 AM (sold by life technology). 0.8 mM (sold by Kurabo) and cultured for 1 hour. Then, the buffer was replaced with a buffer (pH 7.4) containing 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl ₂ , 1.2 mM MgCl ₂ , 25 mM HEPES, and 10 mM D-glucose. was added alone to the buffer at a concentration of Fluorescence was measured with a fluorescence microscope to determine 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 shown in FIG. 7(B). Knockdown of PAR-1 abolished keratinocyte responsiveness to Cryj1. It was also shown that cell activation by the addition of Cryj1 did not occur in thrombin-knockdown keratinocytes.

Example 7: Activation of cultured keratinocytes by addition of thrombin Cultured keratinocytes (sold by Kurabo) were cultured in EPILIFE-KG2 medium (sold by Kurabo) in a humidified atmosphere at 37°C for 2 days. For the confluent culture, the medium was replaced with EPILIFE-KG2+Ca1.8mM (sold by Kurabo Industries), cultured for 28-48 hours, and then EPILIFE-KG2+Ca1 containing Fura2 AM (sold by life teqnology) at 5 μM. 0.8 mM (sold by Kurabo) and cultured for 1 hour. Then, the solution was replaced with a buffer (pH 7.4) containing 150 mM NaCl, 5 mM KCl, 1.8 mM CaCl ₂ , 1.2 mM MgCl ₂ , 25 mM HEPES and 10 mM D-glucose, and 100 pg/ml of thrombin (distributor: Nacalai Tesque) was added. ml concentrations were added to the buffer either alone or in combination with tranexamic acid (T-AMCHA) (10 mM) or bivalirudin (Prospec) (100 ng/ml). Fluorescence was measured with a fluorescence microscope to determine changes in intracellular calcium concentration. The results are shown in FIG. 6(A). Intracellular calcium concentration values about 2 minutes after the addition of thrombin were calculated using Aqua Cosmos software and shown in FIG. 6(B) (tranexamic acid: p<0.0001, bivalirudin: p<0.0001). It was shown that addition of thrombin activated the cells and that addition of thrombin was prevented by simultaneous addition of tranexamic acid or bivalirudin.

Example 8: Identification of mechanism of action of tranexamic acid to suppress thrombin action Thrombin (F2) (40 ng) (sold by Nacalai Tesque), prothrombin (pro-F2) (40 ng) (sold by Invitrogen), thrombin + tranexam Solutions containing acid (100 µM) (F2 + TAMCHA) and thrombin + bivalirudin (1 µg) (F2 + Biv) were prepared respectively, thrombin fluorescent substrate (distributor: Cosmo Bio) (20 µM) was added, and fluorescence was measured with a fluorometer. bottom. The results are shown in FIG. 8(A). Bivalirudin is a drug that exerts an antithrombin effect by acting on the active center of thrombin. While thrombin activity was suppressed by bivalirudin, tranexamic acid did not suppress thrombin activity.

Factor Xa (FXa) (9.5 ng) (sold by abcam), Factor Xa (9.5 ng) + GGACK (sold by abcam) (100 μM) (FXa + GGACK), Factor Xa (9.5 ng) + GG tranexam A solution containing acid (10 mM) (FXa+T-AMCHA) was prepared respectively, a fluorescent substrate for Factor Xa (marketed by abcam) was added, and fluorescence was measured with a fluorometer. The results are shown in FIG. 8(B). GGACK is a drug that acts on the active center of factor Xa and exerts a factor Xa inhibitory effect. While factor Xa activity was suppressed by GGACK, tranexamic acid did not suppress factor Xa activity.

To the prothrombin (Pro-F2) (40 ng)+Factor Xa (FXa) solution was further added tranexamic acid (T-AMCHA) at 10 mM, 1 mM and 100 μM, respectively. To yet another prothrombin (Pro-F2) (40 ng) + factor Xa (FXa) solution, bivalirudin (1 μg) and GGACK (10 μM) were added, respectively. A chromogenic substrate for thrombin (manufactured by Rossix) was added to these test solutions, and absorbance was measured with an absorptiometry 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 of FIGS. 8(A) to (C), tranexamic acid was shown to suppress the activation of prothrombin to thrombin without suppressing the activities of thrombin and factor Xa.

Example 9: Structural Analysis Three- dimensional structural data (PDB ID: 1CEB) of plasminogen (KR1 domain) to which tranexamic acid is bound and three-dimensional structural data of prothrombin (PDB ID: 5EDM) were obtained from the protein data bank (PDB). The three-dimensional structures were compared using the analysis software Chimera (Fig. 9). The results showed that the conformation of plasminogen (KR1 domain) to which tranexamic acid is bound overlaps very well with the two KR domains in prothrombin. This suggested that tranexamic acid binds to the KR domain of prothrombin.

Example 10: Screening Method (1) A solution containing thrombin (F2) and a drug candidate is injected into each well of a 96-well plate. Next, a thrombin fluorescent substrate (distributor: Cosmo Bio) is added. Fluorescence after addition is measured with a plate reader and compared with a control to determine the thrombin activity inhibitory effect of the candidate agent. Candidate agents with thrombin activity inhibitory action can be screened as skin condition improving agents, preferably whitening agents, anti-itch agents, and skin barrier improving agents.

(2) Inject a solution containing prothrombin (pro-F2), factor IXa, and drug candidates into each well of a 96-well plate. A chromogenic substrate for thrombin (supplied by Rossixs) is then added. Absorbance after addition can be measured with a plate reader and compared to a control to determine the thrombin activity inhibitory effect of the candidate drug. Candidate agents with thrombin activity inhibitory action can be screened as skin condition improving agents, preferably whitening agents, anti-itch agents, and skin barrier improving agents.

(3) Cultured keratinocytes (sold by Kurabo Industries) are cultured in EPILIFE-KG2 medium at 37° C. in a humidified atmosphere for 2 days. For the confluent culture, the medium was replaced with EPILIFE-KG2+Ca1.8mM (sold by Kurabo Industries), cultured for 28-48 hours, and then EPILIFE-KG2+Ca1 containing Fura2 AM (sold by life teqnology) at 5 μM. .8 mM (sold by Kurabo). Cryj1 (marketed by HAYASHIBARA) and candidate agents are added to the medium. Fluorescence is measured with a fluorescence microscope to measure changes in intracellular calcium concentration. A candidate agent can be determined to have an inhibitory effect on thrombin activity if calcium concentration is decreased compared to controls. Candidate agents with thrombin activity inhibitory action can be screened as skin condition improving agents, preferably whitening agents, anti-itch agents, and skin barrier improving agents.

(4) Cultured keratinocytes (sold by Kurabo Industries) are cultured in EPILIFE-KG2 medium at 37° C. in a humidified atmosphere for 2 days. For confluent cultures, medium is replaced with EPILIFE-KG2+Ca 1.8 mM, cultured for 24-48 hours, and then replaced with EPILIFE-KG2+Ca 1.8 mM containing 5 μM Fura2 AM (marketed by life teqnology). Thrombin or prothrombin and the candidate drug are added to the medium. Fluorescence is measured with a fluorescence microscope to measure changes in intracellular calcium concentration. A candidate agent can be determined to have an inhibitory effect on thrombin activity if the calcium concentration is reduced compared to controls. Candidate agents with thrombin activity inhibitory action can be screened as skin condition improving agents, preferably whitening agents, anti-itch agents, and skin barrier improving agents.

Claims (11)

A screening method for an agent for improving cosmetic skin conditions worsened by allergens, using thrombin inhibitory action as an index. The screening method according to claim 1, wherein the skin condition exacerbated by the allergen is a skin condition caused by a decrease in lamellar granules. 3. The screening method according to claim 1 or 2, wherein the improving agent is selected from the group consisting of antipruritic agents and skin barrier improving agents. The screening method is
contacting prothrombin or thrombin with a candidate agent;
measuring thrombin activity,
a step of selecting a cosmetic skin condition-improving agent that has been aggravated by the allergen, based on the thrombin-inhibiting action of the candidate agent;
The method according to any one of claims 1 to 3, comprising 5. The method of claim 4, wherein the measurement of thrombin activity is determined by the amount of thrombin substrate degraded or blood clotting time. The screening method is
culturing PAR-1 expressing cells in medium containing prothrombin or thrombin and a candidate agent;
a step of measuring the inhibitory effect on PAR-1 receptor activation a step of selecting a skin condition improving agent based on the PAR-1 inhibitory effect of the candidate drug;
The method according to any one of claims 1 to 3, comprising 7. The method according to claim 6, wherein the inhibitory effect on PAR-1 receptor activation is measured by calcium imaging. An agent for improving cosmetic skin conditions worsened by allergens, comprising an inhibitor of prothrombin-to-thrombin activation or PAR-1 receptor activation ( excluding tranexamic acid), wherein The improving agent is selected from the group consisting of antipruritic agents and skin barrier improving agents. 9. The skin condition-improving agent according to claim 8, wherein the inhibitor of PAR-1 receptor activation improves skin conditions aggravated by allergens through suppression of cell action by PAR-1. An inhibitor of PAR-1 receptor activation , comprising tranexamic acid or a salt thereof. An inhibitor of the decomposition of prothrombin into thrombin, comprising tranexamic acid or a salt thereof.

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