WO2005080977A1 - Method of estimating skin irritation - Google Patents

Abstract

A convenient testing method for estimating a skin irritation, used to predict the degree of skin irritation by a substance to be applied to the skin. There is provided an irritation estimation method for predicting the degree of skin irritation by a test subject, characterized in that the test subject is brought into contact with skin constituting cultured cells, followed by measuring of the amount of substance P in the culture solution.

Description

 Specification

 Skin irritation evaluation method

 Technical field

 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. Background art

 [0002] In recent years, the number of people complaining of sensitive skin has increased, and a large number of hypoallergenic cosmetics, pharmaceuticals, and quasi-drugs for sensitive skin have been put on the market. Possible causes of sensitive skin in modern life include changes in lifestyle (drying of living environment due to cooling and heating, changes in eating habits, etc.), environmental factors such as ultraviolet rays, and stress that is an internal factor. . However, the definition of sensitive skin is not clearly defined in the medical practice or cosmetics industry, and each supplier independently determines and promotes low irritating cosmetics, pharmaceuticals, and quasi-drugs. The fact is that they are sold.

 [0003] Therefore, even if they are marketed as low-irritation products, they cannot be said to be of the same level of irritation, and at present it is not possible to select products with a stimulation level desired by consumers.

 [0004] 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, on the other hand, 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.

[0005] Primary skin irritation can be confirmed by a skin patch test or the like for guinea pigs, porpies, humans and the like. On the other hand, since the stinging stimulus is a sensory stimulus, evaluation by an experimental animal is difficult, and the stimulus sensation evaluation is performed based solely on human sensation. 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. In order to score the sense of sensation, there was a lack of objectivity for various reasons, such as differences in individual sensitivity, effects of seasons, and racial differences, and was not fully satisfactory. Furthermore, since the test is performed by applying the test substance to a specific part of the subject, the number of test subjects is limited by the number of subjects. Therefore, there was a need for a simple stinging test that provided an objective assessment that could be a unified criteria for hypoallergenicity.

 [0006] Substance P (SP) 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. Some of these 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.

[0007] By the way, it has been reported that a physiopathological pattern common to the state of sensitive skin is related to the high ability of skin to release tachykinin, particularly substance P. In addition, the use of substance p antagonists can provide prophylactic and / or Z- or therapeutic effects on sensitive skin, and cabsaicin, which causes the release of tachykinin from epidermal and dermal nerve endings, can be applied to patient skin. A test method for determining whether or not the skin is sensitive by application is known (see Patent Document 1).

[0008] In addition, a method for evaluating a test for irritation of a subject using a three-dimensional tissue culture instead of a patch test, which is a method for evaluating a primary stimulus, is known (see Patent Document 2).

[0009] However, cultured cells that can be used as an alternative to the stinging test (stimulation evaluation test) are not available. The test method used is still known.

 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

 Disclosure of the invention

 Problems to be solved by the invention

[0011] 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.

 Means for solving the problem

[0012] In view of the power and the actual situation, 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.

 [0013] Accordingly, 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 invention's effect

[0014] 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. [0015] In one embodiment of the present invention, first, 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. Next, the analyte is added to the culture solution. After the addition, collect the culture supernatant at a set time, and measure the amount of substance P in the culture supernatant. Using 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. Next, 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. By using this evaluation method, it is possible to accurately and easily screen for subjects related to stinging stimuli, and it can be used as a screening method for stinging stimulus-positive substances and preparations (particularly external preparations). is there.

 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. As described above, 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.

[0017] 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. Here, examples of the surfactant include a cationic surfactant, an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. Examples of 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. These analytes can be used after being dissolved in an appropriate solvent such as purified water or ethanol. The amount of the solvent to be added may vary depending on the type of the solvent, as long as it does not show toxicity to the cells. The added amount of the calorie is preferably 20% or less, more preferably 10% or less of the culture medium. [0018] 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. In particular, 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. One type of skin constituent cells may be used, or a plurality of cells may be used as a mixture. When used for evaluation, 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. Furthermore, it is preferable to use established cells as cultured cells. Such 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. For example, 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 ™ (Toyobo) is an example of a three-dimensional cultured skin (model).

[0019] 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. As a commercially available culture solution of epidermal keratinocytes, KG_2 (manufactured by Kurabo Industries), K-110 (manufactured by Kyokuto Pharmaceutical), EpiLife ™ (manufactured by Cascade) and the like can be used. When skin fibroblasts are used, Fibro cells (Kurabo) can be used as cultured cells, and Medium 106S (Kurabo) can be used as a culture medium. When a commercially available cell or skin model is used, the cell or skin model is used in principle. The instructions in the instruction manual for the model may be followed.

 [0020] 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. As 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. Since 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. However, in some cases, 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

 2,5% CO condition is preferred

 2

 Les ,. Immediately after the treatment, collect the culture supernatant and measure the amount of substance P.

[0021] 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.

 [0022] In the present invention, as for the compounds currently used in cosmetics and various cleaning agents, as a result of examining the amount of substance P in the culture solution at various concentrations, particularly when the concentration of the compound is 0.01-1% by weight. It was found that the substance P content in the range correlated well with the stimulus evaluation in the stinging stimulus test. Therefore, it is particularly preferable to test compounds used as analytes in a concentration range of 0.011% by weight. However, this concentration range varies depending on the compound used as the analyte, the skin constituent cells used in the test, the type of the target product, and the like, and is not limited.

[0023] 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. Hereinafter, when expressed as the amount of substance P, unless otherwise specified, the amount (protein content) of substance P itself is measured. Yes. When measuring the amount of substance P, it is preferable to immediately collect the culture supernatant immediately after the treatment of the cultured cells and the like and to start the measurement immediately. The measurement is performed within 1 hour, preferably within tens of minutes, more preferably within 30 minutes, more preferably within 10 minutes, more preferably within 5 minutes, particularly preferably within a few minutes to a few seconds from the collection of the supernatant. Start. Considering the time required for the operation, 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. Examples of 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. As 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. As 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.). As an example of 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). Thereafter, a substance P solution labeled with alkaline phosphatase is added, and an anti-substance P polyclonal antibody (Egret) is added, followed by reaction at room temperature. After reacting for 2 hours, wash the plate thoroughly with a detergent solution containing a surfactant, add a substrate (pNPP: p-nitrophenyl phosphate) solution, react for 1 hour at room temperature, and add TSP (trisodium phosphate). After stopping, absorbance Measure (405 nm) and calculate the amount of substance P using the calibration curve prepared from the results obtained from the substance P standard solution.

[0025] As an example of a specific embodiment of the evaluation method of the present invention, 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. In this case, if 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. However, in some cases, 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. Above, 60% or more, or 50% or more, it can be determined that the subject has a stinging stimulus to the skin. This criterion is appropriately selected according to the purpose of the test, the positive control to be used, and the like. In addition, 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.

[0026] In another embodiment of the present invention, a subject can be evaluated without using a positive control as described above. For example, it is possible to evaluate the stinging stimulus of a subject by bringing the subject into contact with cultured skin cells or the like, and using the amount of the substance P derived from the cells detected in the medium as an index. In this method, 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. As a specific example, 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 In this case, 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.

[0027] In this manner, according to the present evaluation method, 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.

 [0028] 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. Examples of 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. Further, as a positive control, a stinging stimulus-positive substance such as phenoxyethanol, methylparaben, or lactic acid, preferably methylparaben may be contained. Specifically, 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. In addition, this kit may contain methyl paraben as a positive control. This kit, for example, 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. Example

 [0029] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

 Example 1 Measurement of Substance P Level and Correlation between the Level and Stinging Stimulation

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.). Thereafter, the number of cells in each well was measured by an ordinary method. The results are shown in Table 1. Phenoxyethanol and methylparaben were used as test subjects. The stimuli of phenoxyethanol and methylparaben are known to be stinging stimuli.

[0031] Stinging Stimulus Word Parity

 For 10 panelists with sensitive skin, apply O.lmr to each subject sample adjusted to a predetermined final concentration by adding water to the area from the outer corner of the eyes to the nostrils, immediately after application, 2.5 minutes, and 5 minutes. Later, a score was obtained according to the following evaluation criteria, and the evaluation was performed using the average of the three times. The evaluation criteria are as follows.

 Rating 0 · · · No stimulation at all

 Rating 1 · · · A little

 Rating 2 · "Feel

 Rating 3 · · · Very Feeling

[Table 1]

[0033] From these results, a correlation was observed between the substance amount and the stinging stimulation, and this evaluation method using epidermal keratinocytes as cultured cells and using the substance amount as an index is useful as an alternative method to the stinging test. I understand.

 Industrial applicability

[0034] 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.

Claims

The scope of the claims  [1] A method for evaluating irritation for predicting skin irritation of a subject, comprising contacting skin constituent cells with the subject and measuring the amount of substance P.  [2] The method of evaluating irritation according to claim 1, wherein the skin irritation is stinging irritation.  [3] (1) a step of adding a subject to a culture solution containing skin constituent cells, (2) a step of culturing skin constituent cells in this state, and (3) measuring the amount of substance P in the culture supernatant 3. The method for evaluating irritation according to claim 1, wherein the method comprises: (4) predicting skin irritation of the subject from measured values of substance P;  [4] The method for evaluating irritancy according to any one of [13] to [13], wherein the skin constituent cells are skin epidermal constituent cells.  [5] The method for evaluating irritation according to any one of [14] to [14], wherein the amount of substance P is measured by an immunological measurement method. 6. The method according to claim 15, wherein the step of predicting the skin irritation of the subject is performed by comparing with a test system using a compound known to have a stinging stimulus as a positive control. Irritation evaluation method described in 1. [7] A measurement kit for predicting skin irritation of a subject, comprising a reagent for measuring skin constituent cells and the amount of substance P. [8] The measurement kit according to claim 7, which comprises, as kit components, an incubator inoculated with epidermal keratinocytes, a culture solution for epidermal keratinocytes, and a reagent for measuring the amount of substance P. [9] The measurement kit according to claim 7 or 8, wherein the reagent for measuring the amount of substance P is an ELISA reagent for measuring the amount of substance P. [10] The kit according to any one of claims 7 to 9, further comprising a compound known to be a stinging stimulator as a positive control as a kit component.