WO2021084760A1 - Procédé d' évaluation d'état de la peau utilisant la protéine par-2 activée dans la peau en tant qu'indicateur, procédé de criblage d'un promoteur ou d'un inhibiteur de l'activation de par-2 et inhibiteur d'activation de par-2 - Google Patents

Procédé d' évaluation d'état de la peau utilisant la protéine par-2 activée dans la peau en tant qu'indicateur, procédé de criblage d'un promoteur ou d'un inhibiteur de l'activation de par-2 et inhibiteur d'activation de par-2 Download PDF

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WO2021084760A1
WO2021084760A1 PCT/JP2019/043162 JP2019043162W WO2021084760A1 WO 2021084760 A1 WO2021084760 A1 WO 2021084760A1 JP 2019043162 W JP2019043162 W JP 2019043162W WO 2021084760 A1 WO2021084760 A1 WO 2021084760A1
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par
active
antibody
activation
skin
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PCT/JP2019/043162
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English (en)
Japanese (ja)
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澄美子 傳田
傳田　光洋
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株式会社 資生堂
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Priority to JP2021554044A priority Critical patent/JP7263538B2/ja
Priority to PCT/JP2019/043162 priority patent/WO2021084760A1/fr
Priority to CN201980101775.9A priority patent/CN114599351A/zh
Publication of WO2021084760A1 publication Critical patent/WO2021084760A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a skin condition evaluation method using active PAR-2 on the skin as an index, and a method for screening a PAR-2 activator or inhibitor using PAR-2 activity as an index. Furthermore, it also relates to a PAR-2 activation inhibitor selected by the screening method according to the present invention.
  • Protease-activated receptor is a type of G protein-conjugated 7-transmembrane receptor that is activated by a specific protease.
  • PAR-1, 3 and 4 are thrombin receptors, while PAR-2 is not activated on thrombin.
  • PAR-2 is known to be activated by trypsin, tryptase, factor VIIa, factor Xa and the like.
  • PAR-2 is activated by cleaving the N-terminal partial peptide of PAR-2 with these proteases to generate a new N-terminal, and the peptide containing this new N-terminal acts as a ligand.
  • PAR-2 is widely distributed in endothelial tissues, and is known to be highly expressed in digestive organs, circulatory organs, respiratory organs, nerves, skin and the like.
  • PAR-2 Protease Activated Receptor-22 of epidermal keratinocytes is involved in the pathophysiology of atopy and psoriasis, and the homeostasis, itchiness, and inflammation of the skin barrier.
  • IP3 inositol trisphosphate
  • NF- ⁇ B Nuclear Factor-kappa B
  • TSLP Thymic Stromal Lymphopoietin
  • Elevated extracellular calcium in the stratum granulosum of the epidermis suppresses the release of lamellar granules and delays barrier recovery.
  • Inflammatory cytokines such as TSLP are thought to stimulate sensory nerves in the skin and cause itching.
  • Patent Document 1 Patent No. 4728248, Patent Document.
  • Patent Document 2 Special Table 2012-591735
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2004-170323.
  • Conventional screening methods for PAR-2 inhibitors use cultured cells expressing PAR-2 to measure intracellular reactions that occur after PAR-2 activation, such as changes in calcium concentration and the amount of cytokines released. However, it was used as an index.
  • PAR-2 activation can be evaluated based on the cleavage state of PAR-2 by using an antibody that detects cleavage of the N-terminal of PAR-2 protein. I found it possible. By using an antibody, it is possible to clarify the site where PAR-2 activation occurs in the epidermis, and by focusing on such a site, a PAR-2 activation inhibitor or a PAR-2 activation promoter is screened. He came to invent the method. Furthermore, by using the screening method, a new PAR-2 activation inhibitor was found. Therefore, the present invention relates to the following:
  • a method for evaluating an epidermal state which comprises detecting active PAR-2 in the epidermis in a skin sample using an anti-active PAR-2 antibody that binds to the N-terminal antigen peptide of active PAR-2.
  • the anti-active PAR-2 antibody binds to active PAR-2 cleaved at a protease cleavage site present in the extracellular N-terminal region, the protease-cleaving site present in the extracellular N-terminal region.
  • the evaluation method according to item 2 wherein the inactive PAR-2 contains an N-terminal amino acid at positions 26 to 36 of SEQ ID NO: 1.
  • the evaluation method according to any one of items 1 to 4 wherein when the detected amount of the active PAR-2 is high, the epidermis is evaluated to be in a rough state.
  • the evaluation of a rough epidermis is to determine at least one epidermis condition selected from the group consisting of an inflammatory condition, rough skin, decreased barrier function, itching, redness, and dryness, according to item 5. Evaluation method.
  • the detection of activated PAR-2 is as follows: The step of contacting the skin sample with the anti-active PAR-2 antibody, The evaluation method according to any one of items 1 to 6, which comprises a step of bringing a labeled secondary antibody into contact with a skin sample and a step of determining the detection intensity of the label in the skin sample. [8] A step of applying a candidate substance in a skin sample, A step of detecting active PAR-2 with an anti-active PAR-2 antibody that binds to the N-terminal antigen peptide of active PAR-2 in a skin sample to which a candidate substance has been applied, and detection of active PAR-2.
  • a method for screening a PAR-2 activation inhibitor which comprises a step of determining a candidate substance for reducing the intensity as a PAR-2 activation inhibitor.
  • the screening method according to item 8 further comprising a step of performing a scratch treatment on the skin sample, and applying the candidate substance to the scratched skin sample.
  • the PAR-2 activation inhibitor is an epidermis condition improving agent.
  • a step of applying a candidate substance in a skin sample A step of detecting active PAR-2 with an anti-active PAR-2 antibody that binds to the N-terminal antigen peptide of active PAR-2 in a skin sample to which a candidate substance has been applied, and detection of active PAR-2.
  • a method for screening a PAR-2 activation promoter which comprises a step of determining a candidate substance for increasing strength as a PAR-2 activation promoter.
  • the screening method according to item 11 wherein the PAR-2 activation promoter is a substance that deteriorates the epidermis condition.
  • the step of detecting the active PAR-2 is A step of contacting a skin sample with the anti-active PAR-2 antibody, and a step of contacting a labeled secondary antibody to detect a label.
  • the screening method according to any one of items 8 to 12, wherein the screening method comprises.
  • the anti-active PAR-2 antibody binds to the active PAR-2 in which the protease cleavage site existing in the extracellular N-terminal region is cleaved, the protease cleavage site existing in the extracellular N-terminal region is bound.
  • the screening method according to item 14, wherein the inactive PAR-2 has an N-terminal amino acid at positions 26 to 36 of SEQ ID NO: 1.
  • the screening method according to any one of items 8 to 15, wherein the activated PAR-2 lacks the N-terminal amino acid up to at least position 36 of SEQ ID NO: 1.
  • a PAR-2 activation inhibitor containing the compound represented by, or a salt thereof.
  • a method for treating or cosmetologically treating inflammatory skin diseases, inflammatory conditions of the skin, rough skin, decreased barrier function, itchiness, redness, and dryness which comprises administering the compound represented by the above, or a salt thereof.
  • PAR-2 activation can be detected in line with PAR-2 activation that occurs in actual skin, and PAR-2 activation can be detected more accurately. It is possible to evaluate the epidermal condition, and in the screening method, it is possible to select a PAR-2 activation inhibitor and / or a PAR-2 activation promoter that acts on the skin.
  • FIG. 1 shows a photograph of a skin sample fluorescently stained with an anti-PAR-2 antibody and an anti-active PAR-2 antibody and taken with a fluorescence microscope.
  • FIG. 2 shows a photograph of a skin sample fluorescently stained with an anti-active PAR-2 antibody and an anti-filaggrin antibody and taken with a fluorescence microscope.
  • FIG. 3 shows a photograph of a skin sample fluorescently stained with an anti-active PAR-2 antibody and an anti-ZO-1 antibody and taken with a fluorescence microscope.
  • FIG. 4 shows the change in fluorescence when the antigen peptide of the anti-active PAR-2 antibody was added when the skin sample was fluorescently stained using the anti-PAR-2 antibody and the anti-active PAR-2 antibody. The photograph taken by the fluorescence microscope is shown. In FIG.
  • FIG. 6B shows a graph in which the fluorescence intensity is quantified in the photograph of FIG. 6A.
  • FIG. 7A shows the full-length amino acid sequence of human PAR-2
  • FIG. 7B shows the full-length amino acid sequence of mouse PAR-2.
  • One aspect of the present invention relates to a method for evaluating an epidermal state using an anti-active PAR-2 antibody that binds to an N-terminal antigen peptide of active PAR-2.
  • the state of rough epidermis which is the epidermis state caused by the activation of PAR-2, can be evaluated. More specifically, the rough epidermis includes an inflammatory condition, rough skin, decreased barrier function, itching, redness, and dryness.
  • the evaluation method according to the present invention includes detecting the active PAR-2 of the epidermis in a skin sample using an anti-active PAR-2 antibody that binds to the N-terminal antigen peptide of the active PAR-2.
  • PAR-2 refers to protease receptor 2 (Protease activated receptor-2).
  • the amino acid sequence of PAR-2 is represented by, for example, SEQ ID NO: 1 in humans (FIG. 7A) and by SEQ ID NO: 2 in mice (FIG. 7B).
  • peptides human: SLIGKV, mouse: SLIGRL
  • the organism from which PAR-2 is derived may be any organism, but mammals such as humans are preferred.
  • PAR-2 is not limited to those having the above amino acid sequences, but also homologues (homologs and splicing variants), variants, derivatives, matures and amino acids thereof, as long as their biological functions are equivalent to these. Modified products and the like can be included.
  • Active PAR-2 refers to PAR-2 cleaved at a protease cleavage site present in the extracellular N-terminal region of PAR-2.
  • PAR-2 in which the protease cleavage site existing in the extracellular N-terminal region of PAR-2 is not cleaved is called inactive PAR-2.
  • the protease cleavage site existing in the extracellular N-terminal region of PAR-2 is between the 36th and 37th positions of SEQ ID NO: 1, and the N-terminal amino acid up to at least the 36th position is cleaved.
  • S (serine) at position 37 of SEQ ID NO: 1 is exposed as a new N-terminal.
  • Intracellular signals are induced by binding of a peptide containing a new N-terminal (FIGS. 7A and B, underlined) to the extracellular second loop.
  • protease cleavage site existing in the N-terminal region of PAR-2 is cleaved by an enzyme such as trypsin or tryptase, but it is not intended to be limited thereto.
  • active PAR-2 and inactive PAR-2 are the same except that they are cleaved at a protease cleavage site existing in the extracellular N-terminal region, active PAR-2 can be detected with an antibody. When doing so, it is necessary to use an antibody that can identify the N-terminus of active PAR-2.
  • an antibody capable of identifying the N-terminal of active PAR-2 can bind to the N-terminal antigenic peptide of active PAR-2.
  • an antigenic peptide means a peptide containing SLIGKV (SEQ ID NO: 3) in the case of humans, and is preferably a peptide having an N-terminal portion of SLIGKV.
  • SLIGKV SEQ ID NO: 3
  • SLIGRL SEQ ID NO: 4
  • the anti-active PAR-2 antibody used in the present invention binds to active PAR-2 cleaved at a protease cleavage site present in the extracellular N-terminal region, while cleaving protease present in the extracellular N-terminal region.
  • a protease cleavage site present in the extracellular N-terminal region
  • cleaving protease present in the extracellular N-terminal region Refers to an antibody that does not bind to inactive PAR-2 whose site has not been cleaved.
  • Such an antibody can be produced according to a conventional method using an antigen peptide containing SLIGKV.
  • an antigen peptide containing SLIGKV as an example, a peptide having 10 to 20 residues starting with SLIGKV from the sequence of SEQ ID NO: 1 can be used.
  • an anti-active PAR-2 antibody there is a goat anti-PAR-2N19 antibody (sc-8206) sold by Santa Cruz.
  • an anti-inactive PAR-2 antibody can be used for the purpose of detecting the inactive PAR-2.
  • the anti-inactive PAR-2 antibody can generate an N-terminal region of inactive PAR-2, for example, a peptide at positions 26 to 36 of SEQ ID NO: 1 as an antigen.
  • an anti-PAR-2 antibody can be used for the purpose of detecting active PAR-2 and inactive PAR-2 without distinguishing them.
  • the anti-PAR-2 antibody can be produced by using a region other than the extracellular N-terminal region as an antigen peptide.
  • Antibodies are used in the broadest sense in the present invention, and monoclonal antibodies, polyclonal antibodies, and modified antibodies thereof are also included as long as the desired specific binding property is shown.
  • the antibody in the present invention may be any animal-derived antibody such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, and a camel antibody.
  • the modified antibody include a protein containing a part of the antibody and an antibody fragment capable of binding to an antigen.
  • antibody fragments include Fab fragment, Fv fragment, F (ab') 2 fragment, Fab'fragment, or scFv.
  • a direct method for directly labeling an antibody for detection for example, a direct method for directly labeling an antibody for detection, an indirect method for detecting a secondary antibody that recognizes an antibody with a label, and a complex-forming substance such as biotin-avidin are used.
  • the amplified amplification method can be used.
  • an antibody is biotinylated and detection is possible by using labeled avidin or streptavidin.
  • the detection of active PAR-2 can be performed on a skin sample.
  • the skin sample may be a skin sample collected from the skin or a skin sample obtained by a three-dimensional skin model.
  • Epidermal cells proliferate in the basal layer, differentiate into the stratum spinosum, the stratum granulosum, and finally shed from the skin surface. Enucleated in the process of differentiation into the stratum corneum, resulting in dead cells.
  • Active PAR-2 is expressed in the outermost layer of the epidermis where living epidermal cells are present, that is, from the stratum granulosum to the stratum granulosum, more preferably in the outer layer of the stratum granulosum.
  • the granular layer can be classified into three layers, SG1 to SG3, from the outside to the inside. It is said that tight junctions are formed in the SG2 layer of the stratum granulosum.
  • the expression pattern of active PAR-2 is also consistent with the expression pattern of ZO-1, which constitutes a tight junction. Therefore, in immunohistochemical staining, when detecting active PAR-2 in the skin, from the viewpoint of specifying the location, co-staining with a protein such as ZO-1 that can determine the location, or nuclear staining for discriminating enucleation. Co-staining is preferred.
  • the method for evaluating the epidermis state of the present invention can evaluate the epidermis state according to the abundance of active PAR-2. More specifically, it can be evaluated that the higher the abundance of active PAR-2, the worse the epidermal condition. More preferably, when the abundance of active PAR-2 is large in the region from the granular layer to the stratum granulosum of the epidermis, it can be evaluated that the epidermis state is poor.
  • the amount of active PAR-2 stained in the stratum granulosum to stratum granulosum region is measured by image processing software (for example, manufactured by Nikon Corporation).
  • the epidermis state can be determined by comparing with an appropriately set threshold value.
  • the epidermal state may be evaluated by the abundance ratio of active PAR-2 and inactive PAR-2 or the abundance ratio of active PAR-2.
  • the abundance ratio of active PAR-2 to inactive PAR-2 is, for example, to an antibody that binds to active PAR-2 but not to inactive PAR-2, and to active PAR-2.
  • the abundance ratio may be determined by using an antibody that does not bind but binds to the inactive PAR-2.
  • the abundance ratio of active PAR-2 is, for example, for antibodies that bind to active PAR-2 but not to inactive PAR-2, and to both active PAR-2 and inactive PAR-2. It can be determined using an antibody that can bind.
  • a further aspect of the present invention relates to a method for screening a PAR-2 activation inhibitor or a PAR-2 activation promoter.
  • This screening method is specifically described in the following steps: The process of applying a candidate substance in a skin sample, A step of detecting active PAR-2 with an anti-active PAR-2 antibody that binds to the N-terminal antigen peptide of active PAR-2 in a skin sample to which a candidate substance has been applied, and detection of active PAR-2.
  • Candidate substances include arbitrary substances, and for example, a compound library for searching for pharmaceuticals or cosmetics, or a library of animal and plant extracts can be used. Furthermore, a cosmetic material library and the like can be selected. Active PAR-2 in skin samples that differ only in that no candidate substance has been applied can be detected and used as a control. The controls may be tested in parallel or in advance.
  • the screening method of the present invention may include a step of scratching a skin sample.
  • the scratch treatment is a treatment that imitates scratching the skin with a fingernail, and can be performed by bringing a spatula or the like into contact with a skin sample and reciprocating it one to a plurality of times, for example, 50 times.
  • the scratch treatment can cause invisible damage to the epidermis of the skin sample. It has been found that PAR-2 activation occurs when scratch treatment is performed. Therefore, in the method for screening a PAR-2 activation inhibitor, scratch treatment can activate PAR-2 in a skin sample, which is useful for screening a PAR-2 activation inhibitor.
  • the candidate substance screened as a PAR-2 activation inhibitor after the scratch treatment is a substance capable of suppressing the PAR-2 activation immediately after the scratch treatment, and has an immediate effect at the time of scratching. It can be used as a PAR-2 activation inhibitor.
  • a PAR-2 activation inhibitor can be used as a barrier recovery function promoter, an inflammation inhibitor, and an itch inhibitor applied at the time of scratching.
  • the detection of active PAR-2 is more specifically described in the following step: A step of contacting a skin sample with the anti-active PAR-2 antibody, This can be done by contacting the labeled secondary antibody with the skin sample and by performing a step of determining the detection intensity of the label on the skin sample.
  • the skin sample can be contacted with the anti-active PAR-2 antibody, for example, for 1 to 20 hours at room temperature or under cooling.
  • a step of fixing the skin sample in advance may be performed prior to this contact step.
  • Immobilization of the skin sample may be performed by any method, but can be performed, for example, by incubating the skin sample in an ethanol or paraformaldehyde-containing solution for 16 to 24 hours at room temperature or under cooling.
  • the secondary antibody is an antibody that binds to the anti-active PAR-2 antibody, and can be selected depending on the organism from which the anti-active PAR-2 antibody, which is the primary antibody, is derived.
  • the label may be any label, but a fluorescent label may be used as an example. When a fluorescent label is used, the contact step with the secondary antibody is performed in the dark and can be contacted at room temperature for 1 hour, for example. Contact is carried out, for example, by incubating in a solution containing each antibody.
  • the detection intensity can be determined by appropriately selecting an apparatus according to the label.
  • a fluorescence microscope can be used, and fluorescence can be detected by using an excitation light and a filter that match the fluorescent label.
  • a washing step may be further included before and after the contact step with the primary antibody or the secondary antibody.
  • the present invention relates to a PAR-2 activation inhibitor, which comprises a compound represented by, or a salt thereof.
  • the PAR-2 activation inhibitor comprises N-methyl-trans-4- (aminomethyl) cyclohexanecarboxamide or a salt thereof.
  • Patent Document 4 Japanese Patent Laid-Open No. 9-227367.
  • the PAR-2 activation inhibitor screened in the screening method of the present invention can be used as a rough skin inhibitor, for example, an inflammation inhibitor, a rough skin inhibitor, a barrier function enhancer, and an itch inhibitor. Since the PAR-2 activation inhibitor of the present invention has an effect of suppressing inflammation against scratches, it is a drug for treating or preventing inflammatory skin diseases such as atopic dermatitis, psoriasis, and contact dermatitis. Alternatively, it can be used as a cosmetic composition and can be administered to a subject in need of suppression of PAR-2 activation. A subject requiring suppression of PAR-2 activation is, for example, a subject in which itching is induced by inflammation or scratching.
  • the PAR-2 activation inhibitor can be incorporated into cosmetics, quasi-drugs, and pharmaceuticals.
  • cosmetics containing the PAR-2 activation inhibitor of the present invention include, but are not limited to, lotions, milky lotions, beauty essences, creams, foundations, etc., but the direct purpose is to improve the skin. It is intended to include everything that is not applied to the skin, but includes, for example, sunscreens, insect repellents, hair removers, hair restorers, shaving lotions, after-shaving lotions and the like.
  • Example 1 Immunohistochemical staining of PAR-2 and active PAR-2 on skin Human abdominal skin obtained from KAC was incubated in PBS containing 4% paraformaldehyde for 24 hours at 4 ° C. to fix samples. After washing with PBS, it was replaced with PBS containing 30% sucrose, and a frozen block was prepared together with OCT compound. This was sliced with a microtome to prepare a skin section having a thickness of 6 ⁇ m, which was used for staining.
  • the primary antibody was diluted 300-fold goat anti-PAR-2N19 antibody (Santa Cruz, sc-8206) and Incubated for 16 hours in blocking solution containing 300-fold diluted rabbit anti-PAR-2H99 antibody (Santa Cruz, sc-5597).
  • Active PAR-2 (N19) was localized in the outermost layer of the epidermis, whereas PAR-2 was widely distributed in the stratum spinosum to the stratum granulosum of the epidermis, and was particularly abundant in the stratum granulosum.
  • Example 2 Identification of the localized region of active PAR-2 As in Example 1, active PAR-2 was detected using a goat anti-PAR-2N19 antibody (Santa Cruz, sc-8206) as the primary antibody. At the same time, a rabbit anti-filaggrin antibody (Santa Cruz, sc-30229) was used instead of the rabbit anti-PAR-2H99 antibody, and the same secondary antibody was used to examine the localization with filaggrin (Fig. 2). .. Filaggrin is present in the outermost layer to the stratum corneum of unenucleated cells, while active PAR-2 is localized only in the outermost layer of unenucleated cells, completely with the localization of filaggrin. Did not match.
  • Example 1 As in Example 1, a goat anti-PAR-2N19 antibody (Santa Cruz, sc-8206) was used as the primary antibody to detect active PAR-2, and a 500-fold diluted mouse was used instead of the rabbit anti-PAR-2H99 antibody.
  • An anti-ZO1-1 antibody (Thermo Fisher Scientific, 339100) was used, and an Alexa488-labeled anti-mouse IgG antibody (Thermo Fisher Scientific) was used as the secondary antibody to examine localization with tight junctions. Activated PAR-2 was shown to be localized outside the tight junction layer.
  • Example 3 Examination of specificity of anti-active PAR-2 antibody Similar to Example 1, goat anti-PAR-2N19 antibody (Santa Cruz, sc-8206) and rabbit anti-PAR-2H99 antibody (Santa) were used as primary antibodies. Using Cruz, sc-5597) and the same secondary antibody, skin sections were stained. The goat anti-PAR-2N19 antibody was mixed with 5 times the weight of the antigen peptide (sequence: N-terminal portion containing SLIGKV (SEQ ID NO: 3)) (Santa Cruz, sc-8206P), treated at room temperature for 30 minutes, and then centrifuged. The supernatant was added to the skin section together with the rabbit anti-PAR-2H99 antibody and incubated. The shooting results are shown in FIG. Incubation with the antigenic peptide eliminated the fluorescence from the goat anti-PAR-2N19 antibody, but did not change the fluorescence from the rabbit anti-PAR-2H99 antibody (FIG. 4).
  • Example 4 Activation of PAR-2 antibody in epidermis by trypsin treatment
  • the abdominal skin of a 51-year-old woman obtained from KAC (7th day after storage at 4 ° C after surgery) was used as a medium for maintaining human skin tissue (Biopredic, Inc., The dermis was immersed in MIL218) and organ culture was performed at 37 ° C. for 1 day.
  • a ring of silicone adhesive was formed on the skin and the following solution was applied into the ring: Water, 0.05% soy trypsin inhibitor (SBTI), 50 ⁇ M trypsin + 0.05% soy trypsin inhibitor Agent (Try 50 ⁇ M + SBTI), 10 ⁇ M trypsin (Try 10 ⁇ M), 100 ⁇ M trypsin (Try 100 ⁇ M). Sigma (T-0303) was used as the trypsin, and Sigma (T6522) was used as the soybean trypsin inhibitor. Incubation was carried out for 30 minutes in a 37 ° C. 5% CO 2 atmosphere so that the upper surface of the skin was not covered with the medium.
  • the surface was washed with water, fixed with 4% paraformaldehyde (PFA) in the same manner as in Example 1, replaced with 30% sucrose, an OCT block was prepared, and a section having a thickness of 6 ⁇ m was prepared with a microtom. Sections were used as primary antibodies in blocking solutions of 300-fold diluted goat anti-PAR-2N19 antibody (Santa Cruz, sc-8206) and 300-fold diluted rabbit anti-PAR-2H99 antibody (Santa Cruz, sc-5597) for 16 hours. Incubated.
  • PFA paraformaldehyde
  • Example 5 Suppression of active PAR-2 in skin sample after scratch treatment
  • the abdominal skin (7 days after surgery) of a 40-year-old woman obtained from KAC was put into a medium for maintaining human skin tissue (Biopredic).
  • the cells were cultured at 37 ° C. for 1 day.
  • the skin was scratched 50 times by applying a metal spatula to the skin.
  • Silicone adhesive was formed into a ring on the scratched area and the following solution was applied into the ring: Water, 1% Tranexamic acid (LKT Laboratories), 0.7. % N-methyl-trans-4-aminomethylcyclohexanecarboxamide hydrochloride (CAS No. 38697-94-8).
  • a sample was obtained by incubating for 1 hour in a 37 ° C.
  • the fluorescence intensity of the active PAR-2 was measured using NIS-elements software (manufactured by Nikon Corporation) and shown as a graph (FIG. 6B). No change was observed in the fluorescence intensity of the inactive PAR-2 (H99). Immediately after the scratch treatment, the fluorescence intensity of the active PAR-2 was low, but the fluorescence intensity increased significantly 1 hour after the scratch treatment.

Abstract

Dans des procédés d'évaluation ou des procédés de criblage classiques des effets inhibiteurs de PAR-2, l'activation de PAR-2 est évaluée indirectement en utilisant, en tant qu'indicateur, des réactions intracellulaires se produisant dans des cellules cultivées exprimant PAR-2 après l'activation de PAR-2. Ainsi, avec ces procédés classiques, on se trouve confronté à un problème, à savoir que l'activation de PAR-2 dans des tissus réels tels que la peau ne peut pas être évaluée avec précision. Les présents inventeurs ont découvert que, en utilisant un anticorps capable de détecter le clivage de l'extrémité N-terminale de la protéine PAR-2, l'activation de PAR-2 peut être évaluée sur la base de l'état clivé de PAR-2. L'évaluation de l'activation de PAR-2 dans la peau à l'aide de cet anticorps fait la clarté sur le site où l'activation de PAR-2 se produit en raison d'une trypsinisation ou d'un stimulus externe. Ainsi, la présente invention concerne un nouveau procédé de criblage d'un inhibiteur de l'activation de PAR-2 ou d'un promoteur de l'activation de PAR-2, qui attire l'attention sur le site précité dans la peau à l'aide de l'anticorps qui permet de détecter le clivage de l'extrémité N-terminale de la protéine PAR-2.
PCT/JP2019/043162 2019-11-01 2019-11-01 Procédé d' évaluation d'état de la peau utilisant la protéine par-2 activée dans la peau en tant qu'indicateur, procédé de criblage d'un promoteur ou d'un inhibiteur de l'activation de par-2 et inhibiteur d'activation de par-2 WO2021084760A1 (fr)

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PCT/JP2019/043162 WO2021084760A1 (fr) 2019-11-01 2019-11-01 Procédé d' évaluation d'état de la peau utilisant la protéine par-2 activée dans la peau en tant qu'indicateur, procédé de criblage d'un promoteur ou d'un inhibiteur de l'activation de par-2 et inhibiteur d'activation de par-2
CN201980101775.9A CN114599351A (zh) 2019-11-01 2019-11-01 以皮肤中的活性型par-2为指标的皮肤状态评价方法、par-2激活促进剂或抑制剂的筛选方法和par-2激活抑制剂

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