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WO2016063991A1 - Procédé de soin de beauté pour améliorer un état de la peau provoqué par une réduction ou une augmentation de la desquamation des cornéocytes, et procédé d'évaluation - Google Patents

Procédé de soin de beauté pour améliorer un état de la peau provoqué par une réduction ou une augmentation de la desquamation des cornéocytes, et procédé d'évaluation Download PDF

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Publication number
WO2016063991A1
WO2016063991A1 PCT/JP2015/080035 JP2015080035W WO2016063991A1 WO 2016063991 A1 WO2016063991 A1 WO 2016063991A1 JP 2015080035 W JP2015080035 W JP 2015080035W WO 2016063991 A1 WO2016063991 A1 WO 2016063991A1
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Prior art keywords
stratum corneum
serpin
region
promoter
activity
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English (en)
Japanese (ja)
Inventor
雅史 宮井
真実 田中
晃 本山
利彦 日比野
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Shiseido Co Ltd
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Shiseido Co Ltd
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Priority to JP2016555420A priority Critical patent/JP6738280B2/ja
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • 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
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P17/06—Antipsoriatics
    • 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
    • A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
    • 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
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • 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
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/81—Protease inhibitors
    • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
    • G01N2333/8121—Serpins

Definitions

  • the present invention is based on the new knowledge that the expression of serpin B12 is closely involved in exfoliation of the stratum corneum.
  • the present invention relates to a method for evaluating suppression / enhancement and a screening method for a stratum corneum peeling accelerator / a stratum corneum peeling inhibitor.
  • the outermost layer of the epidermis the stratum corneum (SC) consists of keratinized cells (keratinocytes) and a continuous extracellular lipid layer, forming a physiochemical barrier to effectively protect the body from environmental disasters.
  • SCs are created continuously through terminal differentiation of keratinocytes.
  • cell shedding occurs and the SC is maintained at a constant thickness.
  • individual keratinocytes or small aggregates detach from the skin surface. This process is undetectable in healthy people, but in some pathological conditions there is an imbalance between SC creation and ablation.
  • Non-patent Document 1 Lundstrom and Egelrud have reported that chymotrypsin-like serine protease exists in SC and is involved in detachment.
  • This enzyme was purified from human foot SC as a stratum corneum chymotrypsin enzyme, and a cDNA encoding the stratum corneum chymotrypsin enzyme was isolated from a human keratinocyte cDNA library (Non-patent document 2; Non-patent document). 3).
  • the present inventor has shown that not only chymotrypsin enzyme but also trypsin-like serine protease is involved in the SC exfoliation process other than the sole.
  • Non-patent document 4 Non-patent document 5
  • KLK7, hK7 and KLK5, hK5 are involved in stratum corneum detachment
  • KLK8, hK8 is a serine protease active in the human epidermis and has been reported to be involved in the proteolytic cascade in the skin barrier
  • trypsinogens are called cationic trypsinogen, anionic trypsinogen, and mesotrypsinogen based on their equipotential points. According to recent studies, these trypsinogens are encoded by different genes PRSS1, PRSS2, and PRSS3 (Non-patent Document 9). Trypsinogen 1 which is a PRSS1 gene product and trypsinogen 2 which is a PRSS2 gene product are major digestive pancreatic proteases. PRSS3 is a mesotrypsinogen gene and at least two splicing variants are generated (trypsinogen 3 and 4).
  • trypsinogens have the very highly conserved enteropeptidase-activated putative cleavage site sequence DDDDK-I present in pancreatic trypsinogen from various species (Light and Janska, 1989). . Trypsinogen is expressed not only in the pancreas but also in other tissues, including epithelial cells of various tissues and the human brain. Interestingly, mesotrypsin differs from the other two trypsins in substrate specificity and inhibitor sensitivity. Mesotrypsin does not cleave protein substrates very much.
  • the inventor performed cDNA cloning of the trypsinogen gene from a human keratinocyte cDNA library, resulting in the isolation of two splicing isoforms of the mesotrypsinogen gene PRSS3, one of which is identical to trypsinogen 4 (brain trypsinogen), and The other was different only in the exon encoding the N-terminal region and was named trypsinogen 5 (Non-patent Document 10).
  • Both isoforms have the activation sequence DDDDK-I.
  • DDDDK-I is hardly cleaved by trypsin itself because of the presence of a mainly negatively charged residue at the cleavage site.
  • enteropeptidase is highly specific for the DDDDK-I sequence (Non-Patent Document 11).
  • the catalytic efficiency of enteropeptidase for bovine trypsinogen is 34,000 times that for bovine trypsin.
  • enteropeptidase is the only enzyme that physiologically converts trypsinogen to trypsin. It has also been found that enteropeptidase is present exclusively in the granular layer of the epidermis. The expression and location of these trypsinogens and their activating enzyme enteropeptidase is consistent with the view that mesotrypsin is involved in terminal differentiation of keratinocytes.
  • LEKTI serine protease inhibitor lympho-epithelial-casal-type 5 inhibitor
  • SPINK5-deficient mice show hyperproliferation of epidermal proteases and symptoms similar to Netherton syndrome through excessive degradation of desmoglen-1.
  • Multiple epidermal kallikreins may be involved in stratum corneum detachment through desmoglen 1 cleavage, and they are regulated by LEKTI.
  • An object of the present invention is to use a novel endogenous inhibitor that inhibits the activity of mesotrypsin, a cosmetic method for improving the skin condition resulting from enhancement / suppression of stratum corneum peeling, and suppression / enhancement of stratum corneum peeling It is to provide an evaluation method and a screening method for a stratum corneum peeling accelerator and a stratum corneum peeling inhibitor.
  • serpin B12 functions as an endogenous trypsin inhibitor that inhibits the activity of mesotrypsin known as a starting protease for stratum corneum detachment.
  • this application encompasses the following inventions: [1] A cosmetic method for improving the skin condition resulting from increased horny layer peeling by enhancing the expression of serpin B12 in the horny layer of a subject. [2] The increased expression of the serpin B12 promotes the core promoter activity in the -1 bp to -150 bp region of the serpin B12 promoter region and / or the silencer activity in the -150 bp to -600 bp region of the serpin B12 promoter region. 2. The method according to 1, which results from inhibition. [3] A cosmetic method for improving the skin condition resulting from the suppression of stratum corneum peeling by suppressing the expression of serpin B12 in the stratum corneum of the subject.
  • the suppression of the expression of the serpin B12 suppresses the core promoter activity in the ⁇ 1 bp to ⁇ 150 bp region of the serpin B12 promoter region and / or the silencer activity in the ⁇ 150 bp to ⁇ 600 bp region of the serpin B12 promoter region. 4. The method according to 3, which is brought about by promotion. [5] The stratum corneum peeling is characterized by measuring the expression level of serpin B12 in a stratum corneum sample collected from the skin of a subject, and using the increase in the expression level of serpin B12 as an index for suppressing stratum corneum peeling. Evaluation method of suppression.
  • the stratum corneum detachment is characterized in that the expression level of serpin B12 in a stratum corneum sample collected from the skin of a subject is measured, and the decrease in the expression amount of serpin B12 is used as an index of enhancement of stratum corneum separation Evaluation method of enhancement.
  • the enhanced exfoliation of the stratum corneum results from a skin condition or skin disease selected from sunburn, dry skin, atopic dermatitis, psoriasis, Netherton syndrome or congenital ichthyosis-like erythroderma 10-12
  • the method in any one of.
  • a method for screening a stratum corneum peeling promoter wherein when a candidate drug is applied to a cultured cell, a drug that inhibits the expression of serpin B12 in the cell is selected as a stratum corneum peeling promoter.
  • Method [15] The method according to 14, wherein an agent that enhances the expression of mesotrypsin in the cell is selected as a stratum corneum peeling promoter.
  • stratum corneum peeling promoter is used for the prevention or treatment of a skin disease selected from ichthyosis vulgaris or warts.
  • a screening method for a stratum corneum peeling inhibitor wherein when a candidate drug is applied to a cultured cell, a drug that enhances the expression of serpin B12 in the cell is selected as a stratum corneum peeling inhibitor.
  • an agent that inhibits the expression of mesotrypsin in the cell is selected as a stratum corneum peeling inhibitor.
  • the stratum corneum peeling inhibitor is used for the prevention, improvement, or treatment of a skin condition or skin disease selected from sunburn, dry skin, atopic dermatitis, psoriasis, Netherton syndrome or congenital ichthyosis-like erythroderma.
  • a medical method for treating a skin disease caused by suppression of stratum corneum peeling by enhancing the expression of serpin B12 in the stratum corneum of a subject [24] A medical method for treating a skin disease caused by enhancement of stratum corneum detachment by suppressing the expression of serpin B12 in the stratum corneum of a subject. [26] A pharmaceutical composition for treating a skin disease caused by suppression of stratum corneum detachment, comprising a serpin B12 expression enhancer. [27] A pharmaceutical composition for treating a skin disease caused by enhancement of stratum corneum detachment, comprising suppressing the expression of serpin B12.
  • a novel and useful cosmetic method for improving skin condition resulting from enhancement / suppression of stratum corneum peeling, evaluation method for suppression / promotion of stratum corneum peeling, and stratum corneum peeling accelerator / corneal layer peeling suppression An agent screening method can be provided.
  • stratum corneum peeling can be controlled and it can contribute to the prevention, improvement, or treatment of the skin state or skin disease resulting from suppression and enhancement of stratum corneum peeling.
  • Serpin B12 a novel trypsin inhibitor, inhibits the activity of mesotrypsin, the starting protease for stratum corneum peeling, and the activity of KLK5 / 7/8, the stratum corneum peeling enzyme, and is involved in stratum corneum peeling. It is a graph which shows the correlation of serpin B12 (absorbance) of a stratum corneum extract, and mesotrypsin inhibitory activity. It is a graph which shows the inhibitory activity with respect to KLK8 of serpin B12. It is a graph which shows the inhibitory activity with respect to KLK5 / 7 of serpin B12.
  • 3 is a graph showing transcriptional activity due to deletion of a region of ⁇ 1 bp to ⁇ 150 bp in the serpin B12 promoter region. 3 is a graph showing transcriptional activity due to deletion of a region of ⁇ 300 bp to ⁇ 600 bp in the serpin B12 promoter region.
  • Serpin B12 was discovered by High throughput genomic sequence and is known as a trypsin inhibitor belonging to the serpin superfamily. Serpin B12 is reported to be expressed in various organs such as brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary or intestinal tract, and inhibits activities such as trypsin and plasmin (non-patented). Reference 13). However, little is known about the specific action of Serpin B12 in vivo.
  • serpin B12 functions as an endogenous inhibitor of mesotrypsin.
  • Mesotrypsin has an isoelectric point intermediate between cationic trypsinogen and anionic trypsinogen. Unlike these two trypsins, mesotrypsin has low degradability to high molecular weight proteins, so that endogenous mesotrypsin can be controlled so far. Inhibitors were thought to be absent.
  • serpin B12 binds to KLK5 / 7/8, which is a stratum corneum peeling enzyme, and inhibits these activities.
  • the new stratum corneum peeling mechanism discovered this time is shown in FIG.
  • FIG. 1 when the expression of serpin B12 in the stratum corneum is increased, the activities of mesotrypsin and KLK5 / 7/8 are inhibited, so that stratum corneum peeling is suppressed.
  • the expression of serpin B12 in the stratum corneum is suppressed, the activity of mesotrypsin and KLK5 / 7/8 is not inhibited, so that stratum corneum detachment is promoted.
  • a cosmetic method for improving the skin condition resulting from increased horny layer peeling by enhancing the expression of serpin B12 in the horny layer of the subject, and serpin in the horny layer of the subject.
  • a cosmetic method for improving the skin condition resulting from suppression of stratum corneum peeling is provided.
  • Examples of the skin condition resulting from increased exfoliation of the stratum corneum include acutely inflamed skin such as sunburn and rough skin.
  • Examples of skin conditions resulting from suppression of stratum corneum peeling include skin aging caused by chronic inflammation induced by environmental changes such as aging, ultraviolet rays, and drying.
  • a medical method for treating a skin disease caused by increased stratum corneum peeling, and suppressing the expression of serpin B12 in the stratum corneum of the subject Provides a medical method for treating skin diseases resulting from suppression of stratum corneum detachment.
  • Examples of skin diseases caused by increased exfoliation of the stratum corneum include atopic dermatitis, psoriasis, Netherton syndrome and congenital ichthyosis-like erythroderma. For example, ichthyosis vulgaris, warts and the like.
  • the present inventor has recently predicted the upstream region ( ⁇ 1 bp to ⁇ 1500 bp) of the transcription start site in the serpin B12 gene (NM — 080474.2) [see National Center for Biotechnology Information (NCBI)] as the promoter region.
  • the core promoter was present in the -1 bp to -150 bp region
  • the silencer was present in the region from -150 bp to -600 bp, and in particular, from -150 bp to -300 bp.
  • the region was found to have a strong silencer function.
  • the core promoter activity is promoted in the region of ⁇ 1 bp to ⁇ 150 bp of the serpin B12 promoter region and / or the silencer activity is inhibited in the region of ⁇ 150 bp to ⁇ 600 bp of the serpin B12 promoter region, particularly in the region of ⁇ 150 bp to ⁇ 300 bp.
  • the silencer activity is inhibited in the region of ⁇ 150 bp to ⁇ 600 bp of the serpin B12 promoter region, particularly in the region of ⁇ 150 bp to ⁇ 300 bp.
  • core promoter activity means an activity for controlling initiation of gene transcription
  • siencer activity means an activity for suppressing gene transcription
  • the expression level of serpin B12 in a stratum corneum sample collected from the skin of a subject is measured, and the increase in the expression level of serpin B12 is used as an index for suppressing stratum corneum peeling.
  • the method for evaluating suppression of stratum corneum peeling, and the expression level of serpin B12 in a stratum corneum sample collected from the skin of the subject are measured, and the decrease in the expression level of serpin B12 is used as an index of enhancement of stratum corneum peeling.
  • the method for evaluating the enhancement of stratum corneum peeling is provided.
  • the stratum corneum sample can be collected by an arbitrary method.
  • the stratum corneum such as cocoon may be shaved with a file to collect the stratum corneum powder.
  • it can also extract
  • Tape stripping is a method of collecting a stratum corneum sample by applying a piece of adhesive tape to the skin surface layer, peeling it off, and attaching the skin stratum corneum to the peeled adhesive tape. If the tape stripping method is used, it is possible to measure the expression of the serpin B12 only by taking one tape of the stratum corneum, and it is possible to evaluate the stratum corneum thickening using the serpin B12 as an index.
  • the preferred method of tape stripping is to first clean the surface of the skin with, for example, ethanol to remove sebum, dirt, etc., and lightly place a piece of adhesive tape cut to an appropriate size (eg 2 ⁇ 5 cm) on the skin surface, This is done by applying a uniform force to the entire tape and pressing it flat, and then peeling off the adhesive tape with a uniform force.
  • the adhesive tape may be a commercially available cellophane tape or the like.
  • Scotch Superstrength Mailing Tape manufactured by 3M
  • cellophane tape (Cellotape (registered trademark); Nichiban Co., Ltd.) or the like can be used.
  • Serpin B12 in the skin stratum corneum sample adhering to the adhesive tape can be obtained by removing the tape piece from a suitable extract such as Tris-buffer (pH 8.0) (0.1 M Tris-HCl, 0.14 M NaCl, 0.1% Tween). It can be isolated and extracted from the tape by dipping in -20) and extracting the stratum corneum.
  • Tris-buffer pH 8.0
  • the expression of serpin B12 preferably uses an antibody specific for serpin B12, and is a well-known method in the art, for example, an immunostaining method using a fluorescent substance, a dye, an enzyme, etc., Western blotting It can be carried out by various methods such as ELISA, immunoassay, for example, ELISA and RIA.
  • the antibody specific for serpin B12 used in the ELISA may be a monoclonal antibody or a polyclonal antibody. Methods for producing monoclonal and polyclonal antibodies are well known to those skilled in the art. For example, Lunstrum et al., J Biol. Chem. 1986, 261: 9042-9048; Hurle et al. J J Cell Science 1994, 107: 2623-2634.
  • the sandwich immunoassay method is particularly preferable.
  • RNA is quantified by quantitative polymerase chain reaction (PCR), for example, real-time polymerase chain reaction (RT-PCR). Selection of suitable primers for RT-PCR can be performed by methods well known to those skilled in the art.
  • PCR quantitative polymerase chain reaction
  • RT-PCR real-time polymerase chain reaction
  • the expression level of serpin B12 in a stratum corneum sample is 30% or more, preferably 50% or more, more preferably 70% or more, and most preferably 90% or more compared to the control value, “the stratum corneum peeling is suppressed. It is good to judge that.
  • the expression level of serpin B12 in the stratum corneum sample is 30% or more, preferably 50% or more, more preferably 70% or more, and most preferably 90% or more compared to the control value, It may be determined that it has been enhanced.
  • the control value is not particularly limited as long as it indicates the expression level of serpin B12 in a healthy stratum corneum. For example, a corner at a corresponding site collected from a healthy person having a good statistically significant number of skin conditions. It may be an average value of the expression level of serpin B12 in the layer sample.
  • the expression level of mesotrypsin in the stratum corneum sample can be further measured, and the decrease in the expression level can be used as an index for suppressing stratum corneum peeling.
  • the expression level of mesotrypsin in the stratum corneum sample can be measured, and the increase in the expression level can be used as an index of the enhancement of stratum corneum peeling.
  • the silencer activity in the region of 300 bp can be measured, and the increase in the core promoter activity and / or the decrease in the silencer activity can be used as an index for suppressing stratum corneum detachment.
  • the silencer activity in the region of ⁇ 300 bp can be measured, and the decrease in the core promoter activity and / or the increase in the silencer activity can be used as an index for the enhancement of stratum corneum detachment.
  • a method for cloning a transcription regulatory region, a method for preparing a plasmid, and a method for measuring transcription control activity are well known in the art.
  • the regulatory control activity can be measured by, for example, a luciferase assay, and the same technique as in Example 11 below can be used.
  • the present invention provides a screening method for a stratum corneum peeling promoter, wherein when a candidate drug is applied to a cultured cell, a drug that inhibits the expression of serpin B12 in the cell is used as the stratum corneum peeling promoter.
  • a method characterized by selection, and a screening method for a stratum corneum peeling inhibitor, wherein when a candidate drug is applied to a cultured cell, a drug that enhances the expression of serpin B12 in the cell is used as a stratum corneum peeling inhibitor A method characterized by selecting is provided.
  • Serpin B12 is expressed by dissolving a candidate drug in water or a medium (for example, EpiLife (registered trademark) medium), adding it to a screening system containing cultured cells, and using a method well known in the art as described above. It can be determined by measuring the expression level of serpin B12 in the cultured cells.
  • the cultured cells are skin cells, typically epidermal cells, and particularly preferably keratinocytes.
  • the cultured cells may be derived from humans or other animals such as rats, mice, rabbits and the like.
  • the addition amount of the candidate drug cannot be generally specified, but the concentration is about 1 ng / ml to about 1 mg / ml, preferably about 10 ng / ml to 100 ⁇ g / ml, more preferably about 100 ng / ml to 10 ⁇ g / ml. .
  • the addition of the candidate drug is preferably performed in the presence of calcium chloride and / or S100A8 or S100A8 / A9.
  • the culture conditions are not particularly limited, but the incubation is preferably performed under 5% CO 2 , typically at 30 to 37 ° C. for 1 to 14 hours, preferably at 34 to 37 ° C. for 2 to 7 hours.
  • the expression of serpin B12 in cultured cells is 30% or more, preferably 50% or more, more preferably 70% or more, and most preferably 90% or more compared to the control value, It may be determined as “agent”.
  • “corneal layer peeling inhibitor” Judgment may be made.
  • the control value is not particularly limited as long as it indicates the expression level of serpin B12 in a healthy stratum corneum. For example, a culture at a corresponding site collected from a healthy person having a good statistically significant number of skin conditions. It may be the average value of the expression level of serpin B12 in the cell.
  • an agent that enhances the expression of mesotrypsin in the cells may be selected as a stratum corneum peeling promoter, and in the screening method for the stratum corneum peeling inhibitor, An agent that inhibits the expression of mesotrypsin in the cells may be selected as a stratum corneum peeling inhibitor.
  • the agent that inhibits the core promoter activity in the -1 bp to -150 bp region of the serpin B12 promoter region and / or the -150 bp to -600 bp region of the serpin B12 promoter region may be selected as a stratum corneum peeling promoter, and in the screening method for a stratum corneum peeling inhibitor, a serpin B12 promoter region of ⁇ 1 bp to The agent that promotes the core promoter activity in the region of ⁇ 150 bp and / or the agent that inhibits the silencer activity in the region of ⁇ 150 bp to ⁇ 600 bp of the Serpin B12 promoter region, particularly the region of ⁇ 150 bp to ⁇ 300 bp, is exfoliated. It may be selected as an inhibitor.
  • the stratum corneum peeling accelerator and the stratum corneum peeling inhibitor obtained by the screening method of the present invention are caused by the improvement of the skin condition resulting from the suppression and enhancement of the above-mentioned stratum corneum peeling and the suppression and enhancement of the stratum corneum peeling. It can be used for the treatment of skin diseases.
  • Step 1 Desalting Treatment Purification was performed under low temperature conditions (4 ° C.) using an AKTAprime (GE Healthcare) system. The desalting column used for the desalting treatment was Hipprep 26/10 Desalting (GE Healthcare) and was performed under the conditions of the product manual. Tris-HCl (pH 8.0) was used as an elution buffer.
  • Step 2 Anion exchange For purification, the system of AKTAprime (GE Healthcare) was used under low temperature conditions (4 ° C). For anion exchange, purification using an anion exchange column Hiprep 16/10 Q XL (GE Healthcare) was performed. The work was performed under the conditions of the product manual.
  • Tris-HCl (pH 8.0) + NaCl was used as an elution buffer, and elution was performed with a NaCl concentration varied from 0 mM to 500 mM.
  • Step 3 Hydroxyapatite Purification was performed under low temperature conditions (4 ° C.) using a Pharmacia LKB controller LCC-501 Plus (Pharmacia) system. Using a hydroxyapatite column CHT2-I (Bio-Rad), it was performed under the conditions of the product manual. A phosphate buffer (pH 6.8) was used as an elution buffer, and elution was performed with a concentration change of 0 mM to 500 mM.
  • Step 4 Strong Cation Exchange Purification was performed at low temperature conditions (4 ° C.) using a Pharmacia LKB controller LCC-501 Plus (Pharmacia) system. Using Mono S 5/50 GL (GE Healthcare), it was performed under the conditions of the product manual. As the elution buffer, phosphate buffer (pH 6.0) + NaCl was used, and elution was carried out while changing the concentration from 0 mM to 1000 mM. Step 5: Difference in isoelectric point For purification, a system of Pharmacia LKB controller LCC-501 Plus (Pharmacia) was used under low temperature conditions (4 ° C.). Using Mono P 5/50 GL (GE Healthcare), it was performed under the conditions of the product manual.
  • Step 6 Gel Filtration Purification was performed at low temperature conditions (4 ° C.) using a Pharmacia LKB controller LCC-501 Plus (Pharmacia) system. Using Mono P 5/50 GL (GE Healthcare), it was performed under the conditions of the product manual. PBS (DULBECCO'S phosphate buffer ED SALINE (Sigma)) was used as an elution buffer.
  • the mesotrypsin inhibitory fraction derived from the stratum corneum extract was collected and subjected to the following tests.
  • (1) CBB staining The mesotrypsin inhibition fraction derived from the stratum corneum extract was subjected to SDS-PAGE using 5-20% e-PAGEEL (Cat. No. E-T520L, ATTO), and then only the gel Was placed in a container, washed with MilliQ water for 5 minutes ⁇ 3 times, and stained with CBB Stain One Super (Cat. No. 11642-31, nacalai quest).
  • (2) Western blot analysis Samples obtained by purification were subjected to SDS-PAGE using 5-20% e-PAGEEL (Cat.
  • the mixture was subjected to a fused silica trap column (100 ⁇ m inner diameter ⁇ 1 cm length, JupiterProteo C14, 10 ⁇ m, Pumen, 10 ⁇ m, 10 ⁇ m, Pm, 10 ⁇ m, Pm, 10 ⁇ m, Pm, 10 ⁇ m, Pm, 10 ⁇ m, Pm, 10 ⁇ m, Pm). Torrance, CA).
  • the trap column was desalted by flowing a gradient initial buffer (0.1% formic acid, 5% acetonitrile / distilled water) for 30 minutes, and then a fused silica analytical column (100 ⁇ m inner diameter ⁇ 12 cm length) by switching the two-way valve.
  • Serpin B12 KLK (KLK5 / 7/8) Inhibitory Activity
  • Serine protease buffer 0.1 M Tris-HCl (pH 8.0), 0.1% Tween 20
  • Serpin B12 inhibitory activity was measured.
  • the activity of KLK8 was measured using mesotrypsin (PRSS3) and purified recombinant-KLK8.
  • PRSS3 mesotrypsin
  • the activity of KLK5 was measured using recombinant-KLK5 (Cat. No. 1108-SE, R & D systems)
  • the activity of KLK7 was measured using purified recombinant-KLK7.
  • Boc-Gln-Ala-Arg-MCA (Cat. No.
  • 3D Keratinocyte Starter Kit Cat. No. PR3D-K-50, CELLnTEC
  • the attached cell insert was placed in a cell culture dish (60 mm), The cells were infiltrated with Progenitor Cell Targeting (PCT) medium CnT-Prime medium (Cat. No. CnT-PR) for various epithelial cells accurately reproducing the niche environment. Thereafter, a suspension of cultured cells obtained by transfecting NHEK with pCMV-HA / Serpin B12 was added to each insert, and CnT-Prime medium was added to the cell culture dish (60 mm). The dishes were placed in a CO 2 incubator and cultured for 2 days until the cells were confluent.
  • PCT Progenitor Cell Targeting
  • CnT-Prime medium Cat. No. CnT-PR
  • CnT-Prime 3D Barrier medium (Cat. No. CnT-PR-3D, CELLnTEC) for keratinocyte three-dimensional culture that enables rapid cell differentiation and multilayering. 2 Placed in incubator and cultured overnight. As an air exposure operation for multilayering the epidermis layer, all media inside and outside the insert were removed with an aspirator, and a differentiation medium CnT-Prime 3D Barrier medium was added outside the insert. Thereafter, the differentiation medium CnT-Prime 3D Barrier medium was changed every 3 days only outside the insert.
  • serpin B12 is enhanced, residual nuclear components are observed in the stratum corneum cells, and the stratum corneum becomes thick. On the other hand, when serpin B12 is suppressed, it is confirmed that the stratum corneum becomes very thin. (FIG. 6).
  • the keratinocyte transfection was carried out in the same manner as above except for the addition amount of transfection of serpin B12 (0.5 ⁇ g / ml, 1.0 ⁇ g / ml or 2.0 ⁇ g / ml) and the culture period after transfection (3 days).
  • the concentration dependency of serpin B12 in differentiation (keratinization) was examined. As a result, it was confirmed that there was a significant correlation between the expression level of serpin B12 and keratinocyte keratinization (FIG. 7).
  • the stratum corneum becomes very thick and the residual core component is observed in the stratum corneum, while the granular layer is not flattened. Recognize.
  • keratinocytes were cultured in a serum-free medium, and after confluence, 1.5 mM calcium was added, and further cultured for 2 days. Then, the following drugs were added and cultured for 24 hours, followed by treatment with Isogen (Nippon Gene).
  • Isogen Natural Gene
  • the mRNA was prepared by the usual method. Specifically, 100 ⁇ l of chloroform was added and shaken up and down until chloroform and Isogen were mixed. It was allowed to stand at room temperature for 2 to 3 minutes, and then centrifuged at 4 ° C. and 12000 g for 15 minutes. The supernatant was transferred to a new tube and 250 ⁇ l of isopropanol was added. Shake up and down and mix for 10 minutes at room temperature.
  • Regions are -1500 bp (-1 bp to -1500 bp), -1200 bp (-1 bp to -1200 bp), -900 bp (-1 bp to -900 bp), -600 bp (-1 bp to -600 bp), -300 bp (-1 bp to- 300 bp) and ⁇ 150 bp ( ⁇ 1 bp to ⁇ 150 bp) were amplified by polymerase chain reaction (PCR) using the primers of SEQ ID NOs: 5 to 11 below.
  • PCR polymerase chain reaction
  • the target plasmid was purified from the transformant.
  • QIAprep Spin Miniprep Kit (Cat. No. 27106, QIAGEN) and EndoFree Plasmid Maxi Kit (Cat. No. 12362, QIAGEN) were used.
  • human keratinocyte primary culture cells Normal Human Epidermal Keratinocytes (NHEK)
  • FuGENE R
  • HD Transfection Reagent Cat. No. E2311, Promega
  • -1500 bp (-1 bp to -1500 bp), -1200 bp (-1 bp to -1200 bp), -900 bp (-1 bp to -900 bp), -600 bp (-1 bp to -600 bp), created for identification of the transcriptional regulatory region, Using the pGL4.12 plasmid containing the region of -300 bp (-1 bp to -300 bp) as a template, site-directed mutagenesis was performed to remove the -1 bp to -150 bp region ( ⁇ -1 bp to -150 bp).
  • Amplification was carried out by the reaction method (PCR) using the primers of SEQ ID NOS: 12 to 13 below.
  • Forward primer for -1 bp to -150 bp deletion AATGACCTGTCTAGCCTCGAGGATATC (SEQ ID NO: 12)
  • Reverse primer for -1 bp to -150 bp deletion AGGCTAGCAGGGTCATTATCATCCCTG (SEQ ID NO: 13)
  • PrimeSTAR® Mutagenesis Basal Kit (Cat. No. R046A, TAKARA) was used for the site-directed mutagenesis polymerase chain reaction. Thereafter, competent cells were transformed with the plasmid.
  • C8540-03, Invitrogen was used as the competent cell. Thereafter, the target plasmid was purified from the transformant. For purification of the plasmid, QIAprep Spin Miniprep Kit (Cat. No. 27106, QIAGEN) and EndoFree Plasmid Maxi Kit (Cat. No. 12362, QIAGEN) were used. Using the purified plasmid, human keratinocyte primary culture cells (Normal Human Epidermal Keratinocytes (NHEK)) were transfected with FuGENE (R) HD Transfection Reagent (Cat. No. E2311, Promega). After transfection, the cells were cultured for 48 hours in an incubator.
  • NHEK Normal Human Epidermal Keratinocytes
  • R HD Transfection Reagent
  • the transcriptional regulatory region of serpin B12 was determined by conducting a luciferase assay using Dual-Luciferase® Reporter Assay System (Cat. No. E1980, Promega). PGL4.74 [hRluc / TK] (Cat. No. E6921, Promega) was used as a standard for the luciferase assay. As shown in FIG. 12, transcriptional activity was hardly observed due to deletion of the -1 bp to -150 bp region, indicating that the -1 bp to -150 bp region functions as a core promoter.
  • site-directed mutagenesis was amplified by polymerase chain reaction (PCR) using primers of SEQ ID NOs: 14 to 15 below. .
  • LGK-101, TOYOBO was used for the ligation reaction, and One Shot (R) OmniMAX TM 2 T1R Chemically Competent E. coli (Cat. No. C8540-03, Invitrog) was used as the competent cell. ) was used. Thereafter, the target plasmid was purified from the transformant. For purification of the plasmid, QIAprep Spin Miniprep Kit (Cat. No. 27106, QIAGEN) and EndoFree Plasmid Maxi Kit (Cat. No. 12362, QIAGEN) were used.
  • human keratinocyte primary culture cells Normal Human Epidermal Keratinocytes (NHEK)
  • FuGENE (R) HD Transfection Reagent Cat. No. E2311, Promega
  • the cells were cultured for 48 hours in an incubator. Thereafter, a transcriptional regulatory region of serpin B12 was determined by performing a luciferase assay using Dual-Luciferase® Reporter Assay System (Cat. No. E1980, PROMEGA).
  • PGL4.74 [hRluc / TK] (Cat. No. E6921, PROMEGA) was used as a standard for the luciferase assay.
  • the region of ⁇ 150 bp to ⁇ 600 bp functions as a silencer.
  • the -300 bp to -600 bp region affects the promoter region as a silencer region, but alone has no function as a silencer.
  • the region of ⁇ 150 bp to ⁇ 300 bp is considered to function as a silencer alone.

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Abstract

L'invention concerne un procédé de soin de beauté pour améliorer un état de la peau provoqué par l'augmentation ou la réduction de la desquamation des cornéocytes, un procédé pour évaluer la réduction ou l'augmentation de la desquamation des cornéocytes, et un procédé de criblage d'un agent d'activation de la desquamation des cornéocytes ou d'un agent de réduction de la desquamation des cornéocytes, dans tous lesquels un nouvel inhibiteur endogène capable d'inhiber l'activité de la mésotrypsine est utilisé. La présente invention repose sur la découverte selon laquelle la serpine B12 peut agir comme un inhibiteur de trypsine endogène qui inhibe l'activité de la mésotrypsine qui est connue comme une protéase impliquée dans l'initiation de la desquamation des cornéocytes.
PCT/JP2015/080035 2014-10-24 2015-10-23 Procédé de soin de beauté pour améliorer un état de la peau provoqué par une réduction ou une augmentation de la desquamation des cornéocytes, et procédé d'évaluation Ceased WO2016063991A1 (fr)

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WO2022025109A1 (fr) * 2020-07-29 2022-02-03 味の素株式会社 Composition d'atténuation des rides

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JPWO2022025109A1 (fr) * 2020-07-29 2022-02-03

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