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WO2005080977A1 - Procédé d'estimation de l'irritation de la peau - Google Patents

Procédé d'estimation de l'irritation de la peau Download PDF

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Publication number
WO2005080977A1
WO2005080977A1 PCT/JP2005/002902 JP2005002902W WO2005080977A1 WO 2005080977 A1 WO2005080977 A1 WO 2005080977A1 JP 2005002902 W JP2005002902 W JP 2005002902W WO 2005080977 A1 WO2005080977 A1 WO 2005080977A1
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WO
WIPO (PCT)
Prior art keywords
substance
skin
amount
irritation
subject
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2005/002902
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English (en)
Japanese (ja)
Inventor
Tsuyoshi Ishii
Yoichi Honma
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rohto Pharmaceutical Co Ltd
Original Assignee
Rohto Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rohto Pharmaceutical Co Ltd filed Critical Rohto Pharmaceutical Co Ltd
Priority to JP2006510292A priority Critical patent/JPWO2005080977A1/ja
Publication of WO2005080977A1 publication Critical patent/WO2005080977A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/5014—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 for testing toxicity
    • 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
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • 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

Definitions

  • the present invention relates to a component used for an external preparation such as a preservative, a surfactant, or an organic acid, and a method for predicting the irritation to the skin of the external preparation using skin constituent cells.
  • Skin irritation includes so-called stinging stimulation and primary skin irritation.
  • Stinging stimulation is a general term for stimuli that cause sensations on the skin, such as “tingling”, “twitching”, “twitching” and “itch”, which are felt when an external preparation such as cosmetics is applied to the skin. Occurs within minutes after application and disappears transiently without inflammatory symptoms.
  • Primary skin irritation is an irritation that causes an inflammatory response to the skin, which occurs within hours after application and is usually accompanied by erythema, edema, and inflammatory symptoms.
  • the stinging test is a test that is used to evaluate the stinging stimulus.This test is useful for confirming the irritation of external preparations such as cosmetics, especially in the development of cosmetics and external preparations. It is widely used. However, the previous stinging test has shown the subject's subjective feeling.
  • Substance P is a neuropeptide consisting of 11 amino acids, is a neurotransmitter of the primary sensory nerve, and is mainly known as a pain information transmitter. Substance P is contained in peripheral nerves and released from nerve endings.Released substance P causes vasodilation and plasma protein leakage, forms erythema and edema, and reduces mast cell decondyles. It promotes the release of histamine and leukotriene, and is a factor that causes primary irritation. Peripheral nerves form a network of sympathetic nerves and unmyelinated sensory nerves (C fibers) mainly in the dermis, and are present particularly around blood vessels, hair follicles and sweat glands.
  • C fibers unmyelinated sensory nerves
  • peripheral nerves extend from the dermis to the epidermis, and nerve endings are also found in the epidermis such as the basal layer and the spinous layer. It is also known that these nerve ending forces adhere to epidermal keratinocytes, melanocytes and Langernon's cells, which are constituent cells of epidermal cells (see Non-Patent Document 1). In addition, it has been reported that epidermal keratinocytes themselves produce substance P and enhance their own substance P production in a photocrine manner (see Non-Patent Document 2), but almost no physiological significance is known. Not.
  • Patent Document 1 JP-A-11-180879
  • Patent Document 2 JP-A-2003-240779
  • Non-Patent Document 1 Nature 1993 (363), 159-163
  • Non-Patent Document 2 Biocnemical and Biophysical Research Communications 1999 (263), 327-333
  • An object of the present invention is to provide a simple skin irritation evaluation test method for estimating the irritability of a substance applied to the skin to the skin.
  • the present inventors have been studying in order to achieve the above object, and in the process, the amount of neuropeptide released from skin constituent cells, particularly the amount of substance P, has been reduced by the stin of the subject.
  • the present inventors have found that the index can be used as an index for predicting the stimulus of the ging, and have completed the present invention.
  • the present invention provides a method for evaluating irritation for predicting a skin irritation sensation of a subject, comprising contacting cultured cells with skin constituents with the subject and measuring the amount of substance P. Is provided.
  • the skin irritation evaluation method of the present invention (hereinafter also referred to as "evaluation method”) is a simple and highly sensitive method for predicting and evaluating skin irritation using the amount of substance P released from skin constituent cells as an index. It is an entirely new evaluation method that provides an in vitro alternative to the stinging test. Since this test method uses skin culture cells and skin tissues that are inferior to human sensation, objective results can be obtained, reproducibility is high, and it can be performed easily. Furthermore, since skin cultured cells and skin tissues are used, it is possible to easily carry out the test at any time without limiting the number of tests in which the number of subjects required to carry out the test is not required.
  • the skin constituent cells are placed in, for example, a culture dish or a culture plate, and an appropriate amount of a nutrient culture solution is added, followed by culturing under commonly used culture conditions.
  • the analyte is added to the culture solution.
  • collect the culture supernatant at a set time and measure the amount of substance P in the culture supernatant.
  • the appropriate stinging positive control treat the skin constituent cells in the same manner as the test subject, and measure substance P in the culture supernatant.
  • the amount of substance P is compared between the case where the stinging positive control is used and the case where the subject is used, and the irritation of the subject is evaluated.
  • the method of the present invention can also be used as a primary screening method before performing a stinging test by a stinging stimulus evaluation method using humans.
  • the number of tests for the stinging stimulus evaluation method using humans as subjects is limited due to problems such as the number of subjects. Therefore, the method of the present invention can be used as a primary screening method for excluding a clearly stimulus-positive subject from an experimental subject in vivo in advance. This will enable efficient development of hypoallergenic products with more secure safety.
  • the subject that can be evaluated by the present invention is not particularly limited as long as it is a substance or composition widely applied to the skin, but various preservatives, fragrances, surfactants, dyes, solubilizers, gels Agents, thickeners, emulsifiers, fats and oils, amino acids, saccharides, water-soluble polymers, organic acids, base components and compositions containing these.
  • the surfactant include a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant.
  • the composition include various external preparations, for example, cosmetics, medicinal external preparations, various cleaning agents (detergent, stone, hair cleaning agent, liquid cleaning agent) and the like.
  • the skin constituent cells used in the present invention include epidermal keratinocytes (epidermal keratinocytes), skin fibroblasts, Langernon's cells, melanocytes, hair matrix cells, and hair, which are skin cells. Papillary cells and the like.
  • skin keratinocytes which are easily susceptible to external stimuli and are preferred by skin epidermal constituent cells (eg, epidermal keratinocytes, Langerhans cells, melanocytes, etc.) which are cells constituting the epidermal part of the skin are particularly preferable.
  • skin epidermal constituent cells eg, epidermal keratinocytes, Langerhans cells, melanocytes, etc.
  • One type of skin constituent cells may be used, or a plurality of cells may be used as a mixture.
  • the skin constituent cells may be monolayer cultured cells or multilayer cultured cells, or may be three-dimensional cultured skin composed of a plurality of skin constituent cells. Human skin-derived cells are preferred since they can be used for the purpose of the power stinging test.
  • Cultured cells can be used as skin constituent cells, or they may be prepared from skin tissue by a conventional method.
  • cultured cells can be established by a method known to those skilled in the art, or can be obtained from commercial products.
  • Some epidermal keratinocytes are commercially available as cultured cells, and they can be easily obtained.
  • primary and secondary culture cells of human epidermal keratinocytes, HaCaT keratinocyte strain, and the like can be used.
  • As commercially available cultured cells of epidermal keratinocytes for example, Epidercell NHEK (F), Epidercell NHEK (B) (both manufactured by Kurabo Industries) can be used.
  • TESTSKIN TM Toyobo
  • TESTSKIN TM is an example of a three-dimensional cultured skin (model).
  • the skin constituent cells used in the test are cultured under a normal culture condition using an appropriate medium, and those that have survived before contact with the subject can be used, and those that have become subconfluent It is preferable to use As the medium, supplementary reagents (insulin, epidermal growth factor, hydration, etc.) suitable for commonly used basic media (iso-osmotic pH equilibration solution mixed with salts, amino acids, sugars, vitamins and other trace essential nutrients) are used. Mouth cortisone, pituitary pituitary extract, etc.) or serum.
  • KG_2 manufactured by Kurabo Industries
  • K-110 manufactured by Kyokuto Pharmaceutical
  • EpiLife TM manufactured by Cascade
  • KG_2 manufactured by Kurabo Industries
  • K-110 manufactured by Kyokuto Pharmaceutical
  • EpiLife TM manufactured by Cascade
  • Skin fibroblasts are used, Fibro cells (Kurabo) can be used as cultured cells, and Medium 106S (Kurabo) can be used as a culture medium.
  • Medium 106S (Kurabo) can be used as a culture medium.
  • the cell or skin model is used in principle. The instructions in the instruction manual for the model may be followed.
  • the treatment of the skin constituent cells by the subject is preferably carried out, for example, by culturing the skin constituent cells and adding the subject to a culture solution.
  • a culture solution a medium, an isotonic buffer, a physiological saline, or the like can be used as long as the cells can survive the cells.
  • the stinging stimulus is a stimulus that occurs within a few minutes after application
  • the treatment time of the subject and skin constituent cells is 0.5 to 60 minutes, preferably 1 to 30 minutes, more preferably 1 to 10 minutes, and most preferably. One five minutes is fine.
  • the present invention can be implemented by performing processing longer or shorter than the above.
  • the treatment conditions are not particularly limited as long as the cells do not die and the substance P is released into the culture solution, but the culture during the treatment is usually performed at a temperature in the range of room temperature to about 40 ° C, preferably About 35 40 ° C, 3-7% CO condition, especially 37 ° C
  • the concentration of the analyte to be added varies depending on the purpose of use and the type of the analyte. Usually, the final concentration is preferably 0.000001 to 10% by weight, particularly preferably 0.001 to 1% by weight. . In general, if the concentration of such a compound is also high, it exhibits some effect including toxicity on cells, so that a concentration of 10% by weight or more is not preferable. A low concentration of less than 0.000001% by weight is not preferable because it is unlikely to be used and is difficult to formulate.
  • the amount of substance P can be measured more easily with better reproducibility by measuring a force protein that can be expressed by the amount of substance P mRNA or the amount of protein.
  • a force protein that can be expressed by the amount of substance P mRNA or the amount of protein.
  • the amount (protein content) of substance P itself is measured. Yes.
  • the culture is usually performed within 0.5 minutes to 1 hour, preferably within 110 minutes, even more preferably within 110 minutes, and most preferably within 15 minutes. What is necessary is just to start measuring the amount of substance P during the cleaning.
  • the measurement method is not particularly limited, and a commonly used general measurement method is used.
  • the amount of substance P may be measured directly by measuring the amount in the supernatant of the culture solution, but can also be measured indirectly by using an antibody such as ELISA or immunochromatography.
  • a method for directly measuring substance P include, but are not limited to, high performance liquid chromatography.
  • the method of indirect measurement is not limited to the ability to use a commonly used competitive method or sandwich attestation method, which is preferably an immunological measurement method using an anti-substance P antibody.
  • a labeled antibody a direct label (a label such as colloidal gold or a fluorescent substance) or an enzyme-labeled antibody can be used.
  • Antibodies used for the measurement can be prepared by using commercially available antibodies or by methods known to those skilled in the art.
  • the anti-substance P antibody a monoclonal antibody or a polyclonal antibody can be used, and a commercially available product can be obtained from Santa Cruz or Zymmet.
  • a commercially available substance P amount measurement kit can be used.Substance P ELISA (R & D Systems Inc.), Substance P, EIA Kit (Cayman Chemical Company), Substance P, EIA High Sensitivity Kit ( Peninsula Laboratories, Inc.).
  • the method of measuring the amount of substance P by ELISA using the competitive method add the culture supernatant and the substance P standard solution to a 96-well plate coated with an appropriate amount of anti-magpie polyclonal antibody (goat).
  • a test system using a compound known to cause stinging as a positive control and a test system using a test subject are used in a medium.
  • a method of comparing the amount of substance P may be mentioned.
  • the amount of substance P in the medium of the test system using the subject is equal to or greater than the amount of substance P in the medium of the test system using the positive control, the subject is stinging to the skin. Is determined.
  • the amount of substance P in the culture medium of the test system using the subject may be 90% or more, 80% or more, and 70% or more of the amount of substance P in the culture medium of the test system using the positive control.
  • a positive control can be appropriately selected depending on the purpose of a force test in which phenoxyethanol, methylparaben, lactic acid, and the like can be used, the cultured cells to be used, and the like.
  • a subject in another embodiment, can be evaluated without using a positive control as described above.
  • cultured skin constituent cells and the like are treated in the same manner as described above, the culture supernatant is collected, and the amount of substance P in the supernatant is measured.
  • the cells and skin model, culture conditions, and the method for measuring and evaluating substance P that can be used in this embodiment are substantially the same as described above.
  • the case of the evaluation methods using normal human epidermal keratinocytes are preferably, substance P amount detected on the upper brass five minutes after the culture supernatant harvested force of about 2.5 pg / l X 10 5 cells or more
  • the stinging stimulus is false positive, and when it is about lOpg / 1 ⁇ 10 5 cells or more, it can be determined that the stinging stimulus is positive.
  • the evaluation method of this embodiment is particularly useful as a primary screening before a stinging test in humans.
  • the power-off value can be set appropriately as desired.
  • the screen of the positive substance or the positive preparation of the stinging stimulus using the release amount of substance P in the skin constituent cells by the subject as an index is obtained.
  • a method is provided.
  • the evaluation method of the present invention does not rely on a conventional sensory judgment method, can provide an objective and quantitative evaluation for each subject, and is useful as a simple screening method.
  • the present invention also provides a measurement kit for predicting skin irritation of a subject.
  • This kit contains, for example, cultured skin constituent cells that release substance P upon contact with a stinging stimulus-positive substance, and reagents for measuring the amount of substance P.
  • reagents for measuring the amount of substance P include anti-substance P antibody, labeled anti-substance P antibody, labeled substance P (solution), and a known amount of substance P to be used as a control standard. Power that is not limited to these.
  • a stinging stimulus-positive substance such as phenoxyethanol, methylparaben, or lactic acid, preferably methylparaben may be contained.
  • the kit composition includes an incubator inoculated with epidermal keratinocytes, a culture solution for epidermal keratinocytes, and a reagent for measuring the amount of substance P.
  • the substance P amount measurement reagent is, for example, a substance P amount measurement ELISA reagent.
  • this kit may contain methyl paraben as a positive control.
  • This kit stimulates cells by adding a culture solution in which a test sample is dispersed or dissolved to a plate prepared by seeding an appropriate amount of epidermal keratinocytes on a 96-well plate, and stimulating cells after a certain period of time. It can be used by measuring the amount of P using an ELISA reagent for measuring the amount of substance P.
  • a measurement kit for another embodiment of the present invention (evaluation method without using a comparison control) can also be constituted according to the above, and a method for constructing such a kit is known to those skilled in the art.
  • the substance P amount was measured by the ELISA method.
  • Normal human epidermal keratinocytes (Epidercell NHEK (B)) were seeded on a 24-well plate at 2 x 10 5 cells / well, and 37 ° C using 1 ml of epidermal keratinocyte culture medium KG-2 (Kurabo). And cultured under 5% CO. When the medium becomes subconfluent, replace the medium and adjust to the specified final concentration in the well. Each of the test samples was added in an amount of 10/1. The culture supernatant was collected 5 minutes and 10 minutes thereafter. Substance P in the collected culture supernatant was quantified by substance P ELISA (manufactured by R & D Systems Inc.).
  • This evaluation method uses the amount of substance released from the skin constituent cells in the culture solution as an index. It is a method for predicting the skin irritation of a subject, and it is particularly useful for the development of external preparations because components and compositions that may cause skin troubles can be easily selected in vitro. Furthermore, according to the present invention, skin irritation of an external preparation such as a preservative, a surfactant or an organic acid and a cosmetic containing them can be evaluated in vitro with high sensitivity.

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Abstract

Procédé de test commode servant à estimer une irritation de la peau, utilisé pour prédire le degré d'irritation de la peau par une substance devant être appliquée sur la peau. Il est fourni un procédé d'estimation de l'irritation servant à prédire le degré d'irritation de la peau par un sujet testé, caractérisé en ce qu'on met en contact le sujet testé avec des cellules constituant la peau mises en culture, ce après quoi on mesure la quantité de substance P dans la solution de culture.
PCT/JP2005/002902 2004-02-24 2005-02-23 Procédé d'estimation de l'irritation de la peau Ceased WO2005080977A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006510292A JPWO2005080977A1 (ja) 2004-02-24 2005-02-23 皮膚刺激性評価方法

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JP2004048501 2004-02-24
JP2004-048501 2004-02-24

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WO2005080977A1 true WO2005080977A1 (fr) 2005-09-01

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05336996A (ja) * 1992-06-12 1993-12-21 Koken Co Ltd 培養細胞を用いた細胞毒性試験法
JPH06180311A (ja) * 1991-01-28 1994-06-28 Procter & Gamble Co:The 皮膚刺激能の評価方法
JP2002062294A (ja) * 2000-08-23 2002-02-28 Shiseido Co Ltd 皮膚表面状態の測定方法及び装置
JP2003240779A (ja) * 2002-02-15 2003-08-27 Toyobo Co Ltd 刺激性試験方法
JP2003238986A (ja) * 2002-02-22 2003-08-27 Shiseido Co Ltd サブスタンスｐ増加抑制剤

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06180311A (ja) * 1991-01-28 1994-06-28 Procter & Gamble Co:The 皮膚刺激能の評価方法
JPH05336996A (ja) * 1992-06-12 1993-12-21 Koken Co Ltd 培養細胞を用いた細胞毒性試験法
JP2002062294A (ja) * 2000-08-23 2002-02-28 Shiseido Co Ltd 皮膚表面状態の測定方法及び装置
JP2003240779A (ja) * 2002-02-15 2003-08-27 Toyobo Co Ltd 刺激性試験方法
JP2003238986A (ja) * 2002-02-22 2003-08-27 Shiseido Co Ltd サブスタンスｐ増加抑制剤

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAE S.J. ET AL: "Autocrine induction of substanc P mRNA and peptide in cultured normal human keratinocytes.", BBRC., vol. 263, 1999, pages 327 - 333, XP002992158 *
KATAYAMA I. ET AL: "Stress response, tachykinin, and cutaneous inflammation.", JID SYMPOSIUM PROCEEDINGS., vol. 6, no. 1, November 2001 (2001-11-01), pages 81 - 86, XP002992157 *
TOYODA M. ET AL: "Skin and the Nervous System", JPN. J. DERMATOL, vol. 109, no. 5, 1999, pages 725 - 737, XP002992156 *

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