Prophylactic and / or therapeutic agent for skin disorder associated with histone deacetylase 4 abnormality

Abstract

To provide a prophylactic and / or therapeutic agent for a skin disorder associated with a histone deacetylase 4 abnormality.SOLUTION: The present invention provides a prophylactic and / or therapeutic agent for a skin disorder associated with a histone deacetylase 4 abnormality, comprising a histone acetyltransferase inhibitor as an active ingredient. This prophylactic and / or therapeutic agent makes it possible to prevent and / or treat a skin disorder associated with a histone deacetylase 4 abnormality.SELECTED DRAWING: Figure 18

Description

[Technical field]

[0001] The present invention relates to an agent for the prevention and / or treatment of skin disorders associated with histone deacetylase 4 abnormalities. [Background technology]

[0002] Pigment cells (melanocytes) are derived from the neural crest during development and are melanin-producing cells. In the skin, melanocytes are mainly present in the basal layer of the epidermis and supply melanin to the surrounding keratinocytes. Although melanocytes play a role in protecting against ultraviolet rays by producing melanin, they also cause DNA damage in response to ultraviolet rays.

[0003] In xeroderma pigmentosum (hereafter simply referred to as XP), the target organ where symptoms are most strongly expressed is the skin, and pigmentation abnormalities are inevitable, so analyzing the UV response of patients' melanocytes is of utmost importance in elucidating the pathology of XP. XP is an autosomal recessive inherited hereditary photosensitivity disease, and is a hereditary DNA repair disorder that results in severe photosensitivity and neurological symptoms. Because the skin of XP patients is congenitally deficient in the ability to repair DNA damage caused by UV rays, severe photosensitivity to sunlight and UV rays (abnormal sunburn, pigmentation abnormalities, skin cancer in exposed areas, etc.) occurs to various degrees depending on the type of disease. For this reason, it is necessary to create a light-blocking environment in all living spaces, which significantly reduces the quality of life of XP patients and their families. Failure to provide proper light blocking will lead to the progression of severe photosensitivity symptoms and freckle-like pigmentation abnormalities, and high frequency of skin cancer despite the young age of the patient. Furthermore, more than half of XP patients suffer from progressive brain and neurodegenerative symptoms of unknown cause, and the severity of these symptoms affects the patient's prognosis. For this reason, there is a strong desire to clarify the pathology of XP and establish a treatment, and the establishment of a treatment is an urgent need for XP patients around the world.

[0004] XP is classified into eight genetically distinct disease types, A to G groups (genetic complementation group) and variant (V) type. In Japan, xeroderma pigmentosum group A (XP-A), which is the most severe type in both skin and neurological symptoms, accounts for approximately 55% of XP cases, while type V, which only has skin symptoms, accounts for approximately 25%. XP-A patients, which are common among Japanese people, have severe photosensitivity symptoms, and show severe sunburn symptoms immediately after birth. Even with low levels of UV rays, they show significantly accelerated photoaging, i.e., severe sunburn reactions, freckle-like pigmented spots of various sizes and tones, and skin cancer in exposed areas at a young age. However, the pathology of XP-A has not yet been elucidated. In XP-A patients, neurological symptoms such as hearing loss, slurred speech, and loss of balance become apparent from around the age of 6, and neurological symptoms such as a tendency to fall gradually begin to appear from around the age of 10, and language function also declines as hearing loss and intellectual disability progresses.

[0005] In XP, the target organ where symptoms are most severe is the skin, and pigmentation abnormalities are inevitable, so research on melanocytes is important in elucidating the pathology. However, since it is difficult to isolate and culture melanocytes from XP patient skin tissue, analysis has been performed using fibroblasts (Non-Patent Documents 1 and 2). On the other hand, melanocytes and fibroblasts have different resistance to ultraviolet light and different responses to DNA damage, so it is important to use melanocytes in XP research (Non-Patent Documents 3 to 5). However, there have been no reports of analysis of the pathology of premature photoaging in XP, in which pigmented and depigmented spots are mixed in exposed areas involving melanocytes, using melanocytes derived from XP patients. Therefore, the present inventors have discovered a method for producing melanocyte stem cells from pluripotent stem cells and establishing melanocytes from the melanocyte stem cells (Patent Document 1, Non-Patent Document 6).

[0006] Histone modification is a representative molecular mechanism involved in epigenetics. Histone modification involves histone acetyltransferase (HAT) and histone deacetylase (HDAC). HAT increases gene expression by acetylating lysine residues on histones. On the other hand, HDAC suppresses gene expression by deacetylating histones. Thus, HAT and HDAC play important roles in gene transcription control, and histone acetylation modification is regulated by the antagonistic action of HAT and HDAC.

[0007] There are 18 types of human HDACs, which are classified into four classes, class I to class IV, and each has its own characteristics. Class I and class II HDACs are hydrolases that have zinc in the enzyme active site, and are characterized by their intracellular localization, with class I localized in the nucleus and class II localized in the nucleus and cytoplasm. HDAC4 belongs to class II. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6624916 [Non-patent literature]

[0009] [Non-Patent Document 1] Bowden et al. Int.J.Mol.Sci.2015,15985-15996 [Non-Patent Document 2] Herman et al. Environ MolMutagen.2014June;55(5): 375-384 [Non-Patent Document 3] Fukumoto et al. GenesCells.2016,Feb;21(2):185-99 [Non-Patent Document 4] Cho-TH et al.PhotodermatolPhotoimmunolPhotomed. 2008 Jun;24(3):110-4. [Non-Patent Document 5] Emri G et al. J Invest Dermatol.2000Sep;115(3):435-40 [Non-Patent Document 6] Hosaka et al. Pigemnt Cell &MelanomaResearch, 2019, 32(5):623-633 Summary of the Invention [Problem to be solved by the invention]

[0010] An objective of the present invention is to provide an agent for preventing and / or treating skin disorders associated with abnormalities in histone deacetylase 4 (HDAC4). [Means for solving the problem]

[0011] As a result of extensive research to solve the above-mentioned problems, the inventors discovered that histone acetyltransferase inhibitors are effective in preventing and / or treating skin disorders associated with histone deacetylase 4 abnormalities, and thus completed the present invention.

[0012] That is, the present invention comprises the following. 1. A preventive and / or therapeutic agent for skin disorders associated with histone deacetylase 4 abnormality, comprising a histone acetyltransferase inhibitor as an active ingredient. 2. A preventive and / or therapeutic agent for a skin disorder described in the preceding item 1, wherein the skin disorder is a skin disorder associated with histone deacetylase 4 abnormality in melanocytes caused by exposure to light. 3. The preventive and / or therapeutic agent for skin disorders described in the preceding item 1, wherein the skin disorder is a skin disorder caused by photoaging. 4. The preventive and / or therapeutic agent for a skin disorder described in the preceding item 1, wherein the skin disorder is a skin disorder caused by xeroderma pigmentosum. 5. A preventive and / or therapeutic agent for skin disorders associated with histone deacetylase 4 abnormalities, comprising the action of improving abnormalities in histone deacetylase 4 expression. 6. The preventive and / or therapeutic agent for skin disorders described in the preceding item 1, wherein the histone acetyltransferase inhibitor is one or more selected from the group consisting of curcumin, C-646, A-485, anacardic acid, butyrolactone, CTPB and garcinol. 7. A method for screening preventive and / or therapeutic agents for skin disorders, which comprises adding a candidate substance to a melanocyte culture system and selecting a substance that ameliorates cytotoxicity of the melanocytes. 8. The screening method according to the preceding paragraph 7, wherein the substance that ameliorates cytotoxicity in melanocytes is obtained by detecting histone deacetylase 4 and / or a factor associated with cellular senescence-associated secretory phenomenon. 9. The screening method according to the preceding paragraph 7, wherein a substance that ameliorates cytotoxicity in melanocytes is obtained by evaluating the viability of melanocytes. 10. The screening method according to the preceding item 7, wherein a substance that ameliorates cytotoxicity of melanocytes is obtained by evaluating the cell migration ability of melanocytes. 11. The screening method described in the preceding item 7, wherein the melanocytes are melanocytes derived from pluripotent stem cells. 12. The screening method described in the preceding item 11, wherein the pluripotent stem cell-derived melanocytes are pluripotent stem cell-derived melanocytes produced from pluripotent stem cells derived from a patient with xeroderma pigmentosum. 13. The screening method described in the preceding paragraph 7, wherein the cytotoxicity of melanocytes is cytotoxicity caused by exposure to light. 14. The screening method described in the preceding item 13, wherein the cell damage caused by light exposure is a skin disorder associated with histone deacetylase 4 abnormality. 15. A method for testing skin disorders, comprising the step of detecting abnormalities in histone deacetylase 4 and / or cellular senescence-associated secretory factor in skin taken from a subject. 16. The method for testing a skin disorder described in the preceding item 15, wherein the cellular senescence-associated secretory phenomenon factor is one or more selected from MMP-1, MMP-3, TNFα, and IL-6. 17. The method for testing a skin disorder according to the preceding item 15, wherein the detection of abnormalities in histone deacetylase 4 and / or cellular senescence-associated secretory factor is carried out using the following 1) or 2); 1) a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcription product of the histone deacetylase 4 gene and / or the cellular senescence-associated secretory phenomenon factor gene; 2) An antibody that specifically recognizes histone deacetylase 4 and / or senescence-associated secretory factor. 18. A skin disorder testing kit comprising a substance capable of detecting abnormalities in histone deacetylase 4 and / or cellular senescence-associated secretory factor. Effect of the Invention

[0013] Skin disorders associated with histone deacetylase 4 abnormalities can be prevented and / or treated using a preventive and / or therapeutic agent for skin disorders associated with histone deacetylase 4 abnormalities, which contains the histone acetyltransferase inhibitor of the present invention as an active ingredient. [Brief description of the drawings]

[0014] [Figure 1] The figures show melanocytes (HC-iMC) differentiated from iPS cells derived from normal fibroblasts from human skin and melanocytes (XP-A-iMC) differentiated from iPS cells derived from fibroblasts from an XP-A patient (Reference Example 1). [Diagram 2] The expression of MC markers SOX10, MITF, and TYR in each melanocyte is shown. (Reference Example 1) [Diagram 3] Figure 3A shows the mutations in XP-A-iMC and HC-iMC by genomic sequencing. Figure 3B shows that XP-A-iMC does not express XPA protein. (Example 1) [Figure 4]This shows that XP-A-iMC has the properties of XP-A. Figure 4A shows the results of the flow cytometry-based NER method. Figure 4B shows the results of the MTT method. (Example 2) [Diagram 5] Figure 5A shows the protocol for UV irradiation of XP-A-iMC and HC-iMC. Figure 5B shows the difference in genes between XP-A-iMC and HC-iMC affected by UV. Figure 5C shows the probes that increase or decrease after high-dose UV-B irradiation of XP-A-iMC and HC-iMC. (Example 3) [Figure 6] The results of GO analysis to elucidate the molecular mechanisms related to the cellular response of XP-A-iMCs to ultraviolet light are shown in Example 3. [Figure 7] 1 shows the change in HDAC4 expression in XP-A-iMC after ultraviolet irradiation (Example 4). [Figure 8] FIG. 4 shows the results of HDAC4 expression in XP-A-iMC after ultraviolet irradiation (Example 4). [Figure 9] FIG. 1 shows the results of a phase contrast microscope scan of a scratch assay performed on XP-A-iMC after UV irradiation (Example 5). [Figure 10] FIG. 5 shows the results of cell migration assay using XP-A-iMC after UV irradiation (Example 5). [Figure 11] A method for observing cell migration in XP-A-iMC after UV irradiation using digital holographic microscopy is shown (Example 5). [Figure 12] This shows the distance traveled by cells in XP-A-iMCs after UV irradiation, as measured by digital holographic microscopy (Example 5). [Figure 13] This shows the speed of cell migration measured by digital holographic microscopy in XP-A-iMCs after ultraviolet irradiation (Example 5). [Figure 14] 6 shows the results of examining cellular aging of XP-A-iMC after ultraviolet irradiation (Example 6). [Figure 15]1 shows the results of expression of SASP factors by PCR in XP-A-iMCs after ultraviolet irradiation (Example 6). [Figure 16] 1 shows the results of expression of SASP factors in XP-A-iMCs after UV irradiation, as determined by Western blotting (Example 6). [Figure 17] FIG. 7 shows the results of HDAC4 expression in NHEM after ultraviolet irradiation (Example 7). [Figure 18] Example 8 shows the effect of curcumin on XP-A-iMC after UV irradiation. [Figure 19] The effect of C-646 on XP-A-iMC after UV irradiation is shown in Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention relates to an agent for preventing and / or treating skin disorders associated with abnormalities in histone deacetylase 4 (HDAC4), which comprises a histone acetyltransferase (HAT) inhibitor as an active ingredient.

[0016] The present inventors have explored the response of melanocytes to light exposure, comprehensively analyzed molecules that could be therapeutic target seeds for skin disorders, and identified HDAC4 as a gene that epigenetically controls melanocytes. Furthermore, they have clarified that cellular senescence occurs in melanocytes after light exposure using immunofluorescence antibody technique. Furthermore, it is believed that cellular senescence affects the senescence-associated secretory phenotype (SASP) factor group, and in fact, they have confirmed the increase in expression of many SASP factors. That is, light exposure reduces the expression of HDAC4 in the skin, which relatively causes acetylation of histones, and it is believed that the accompanying increase in expression of SASP-related genes leads to various skin disorders.

[0017] Therefore, the present inventors have found for the first time that ameliorating abnormalities in HDAC4 expression may be applicable to the prevention and / or treatment of skin disorders. As used herein, senescence-associated secretory phenomenon (SASP) factors include, for example, MMP-1, MMP-3, TNFα, IL-6, and the like.

[0018] As used herein, "HDAC4 abnormality" refers to a quantitative abnormality in histone deacetylase 4 (HDAC4), and in particular refers to a decrease in HDAC4 expression after light exposure compared to before light exposure. "Expression" may be at the gene level or protein level. The preventive and / or therapeutic agent of the present invention is characterized by having an effect of improving abnormality in HDAC4 expression.

[0019] In the present specification, the term "skin disorder" particularly refers to skin disorders associated with HDAC4 abnormality, and examples of such disorders include skin disorders associated with HDAC4 abnormality in melanocytes caused by light exposure, skin disorders caused by photoaging, and skin disorders caused by xeroderma pigmentosum. Here, "photoaging" refers to changes in the appearance and function of the skin observed as a result of repeated light exposure, and broadly includes skin disorders that may occur due to light exposure. Specific skin disorders are not limited to skin disorders that may occur in XP patients, but also include skin disorders that may occur in non-XP patients due to light exposure. For example, skin disorders include pigmented lesions with a mixture of pigmented and depigmented spots, dryness and atrophy of the skin, reduced elasticity of the skin, formation of wrinkles and sagging, skin cancer occurring at a young age, formation of freckles, pigmentation, darkening of the skin, increased yellowing, reduced skin barrier function, reduced stratum corneum function, and the like, but are not limited thereto.

[0020] As explained in the Background Art section, "xeroderma pigmentosum (XP)" in this specification is a hereditary photosensitive disease inherited in an autosomal recessive manner. In this specification, XP is used as a concept including all eight disease types, which are genetically distinct groups A to G (genetic complementation group) and variant (V) type. It is particularly suitable for group A (XP-A), which is the most severe type in terms of both cutaneous and neurological symptoms. Since XP inevitably causes pigmentation abnormalities, research on melanocytes is important in advancing the elucidation of the pathology.

[0021] In this specification, "light exposure" is not limited to normal light exposure in daily life or light exposure exceeding this (e.g., sunburn), but also includes slight light exposure that may cause skin damage in XP patients. In this specification, "light" is not particularly limited as long as it is "light" that contains ultraviolet rays, and examples of "light" include sunlight, ultraviolet rays (e.g., UV-B, UV-C, UV-A), and illumination light, and particularly refers to sunlight and ultraviolet rays. The wavelength of light is not particularly limited, but is, for example, 0 to 3000 nm, preferably 10 to 400 nm, more preferably 200 to 380 nm, and particularly preferably 280 to 315 nm. The dose is not particularly limited, but is, for example, 10 J / m 2 ~500 J / m 2 , preferably 30 to 200 J / m 2 , more preferably 150 J / m 2 It is.

[0022] In the present specification, the term "HAT inhibitor" literally refers to a substance that inhibits or suppresses the enzymatic activity of histone acetyltransferase (HAT). By inhibiting or suppressing the enzymatic activity of HAT, a relative balance between HDAC4 and HAT is achieved, and abnormalities in HDAC4 are improved. Examples of HAT inhibitors include one or more selected from the group consisting of curcumin, C-646, A-485, anacardic acid, butyrolactone, CTPB, and garcinol. The preventive and / or therapeutic agent of the present invention contains a HAT inhibitor as an active ingredient. In the present specification, the HAT inhibitor contained as an active ingredient may be a compound such as curcumin, C-646, A-485, anacardic acid, butyrolactone, CTPB, and garcinol, as well as a derivative of the compound, a pharma- ceutically acceptable salt of the compound or a derivative of the compound, or a hydrate. In particular, curcumin, C-646, and A-485 are preferable. Curcumin (UPAC name: (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is a fat-soluble polyphenol that is found in high concentrations mainly in turmeric, but is also found in cardamom, cloves, cumin, pepper, coriander, paprika, mace, etc. Potential medical uses of curcumin include antitumor, antioxidant, antiamyloid, and anti-inflammatory effects, and it has been actively researched in recent years.

[0023] The preventive and / or therapeutic agent of the present invention is used as a pharmaceutical composition. The pharmaceutical composition means a composition suitable for administration in medical applications, and may contain one or more pharma- ceutical acceptable carriers and / or pharma-ceutical acceptable excipients in addition to the HAT inhibitor as an active ingredient. The pharmaceutical composition as the preventive and / or therapeutic agent of the present invention can be prepared by a commonly used method using pharmaceutical carriers, excipients, etc. that are commonly used in the art. The administration may be in any form of oral administration using tablets, pills, capsules, granules, powders, liquids, etc., or parenteral administration using intra-articular, intravenous, intramuscular injections, suppositories, eye drops, eye ointments, transdermal liquids, ointments, transdermal patches, transmucosal liquids, transmucosal patches, inhalants, etc. As the solid composition for oral administration according to the present invention, tablets, powders, granules, sustained release agents, etc. are used. In such solid compositions, one or more active ingredients are mixed with at least one pharma- ceutically acceptable carrier and / or pharma- ceutically acceptable excipient.

[0024] The pharma- ceutically acceptable carriers in the present specification include various organic or inorganic carrier substances commonly used as pharmaceutical ingredients, such as excipients, lubricants, binders and disintegrants in solid preparations, or solvents, solubilizers, suspending agents, isotonicity agents and buffers in liquid preparations. Furthermore, if necessary, conventional additives such as preservatives, antioxidants, colorants, sweeteners, adsorbents and wetting agents can also be used appropriately and in appropriate amounts.

[0025] The pharma- ceutically acceptable excipients in the present specification include, for example, isotonicity agents, bulking agents, preservatives / bactericides, binders, antioxidants, solubilizers, solubilizing agents, suspending agents, fillers, pH regulators, stabilizers, absorption enhancers, release rate control agents, colorants, plasticizers, adhesives, etc. Specific examples include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, etc.

[0026] Examples of lubricants include magnesium stearate, calcium stearate, talc, colloidal silica, etc. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methyl cellulose, sodium carboxymethyl cellulose, etc.

[0027] Examples of disintegrants include starch, carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, sodium carboxymethylstarch, and L-hydroxypropylcellulose.

[0028] Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil, and the like.

[0029] Examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate.

[0030] Examples of suspending agents include surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzethonium chloride, and glycerin monostearate; and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose.

[0031] Examples of isotonicity agents include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, and the like.

[0032] Examples of the buffer include buffer solutions of phosphates, acetates, carbonates, citrates, and the like.

[0033] Examples of preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0034] Antioxidants include, for example, sulfites, ascorbic acid, α-tocopherol, and the like.

[0035] The dosage of the HAT inhibitor as an active ingredient varies depending on the subject, administration route, symptoms, body weight, age, etc., and can be appropriately selected. The HAT inhibitor as an active ingredient may be administered in combination with other drugs as appropriate.

[0036] The present invention also encompasses a method for screening for a preventive and / or therapeutic agent for skin disorders. The screening method can be characterized by adding a candidate substance to a melanocyte culture system and selecting a substance that ameliorates cytotoxicity of the melanocytes.

[0037] "Melanocytes" (also called pigment cells or melanocytes) are cells that have tyrosinase activity and produce melanin. They have intracellular organelles (melanosomes) for melanin synthesis and are morphologically dendritic cells whose dendrite shape changes depending on the environment.

[0038] Melanocytes can be prepared, for example, from pluripotent stem cells. Examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic tumor cells (EC cells), and embryonic germ stem cells (EG cells), with iPS cells being preferred. The method of inducing differentiation from pluripotent stem cells to melanocytes can be any method known per se or any method developed in the future. Specifically, the method described in the Examples or the method described in Japanese Patent No. 6624916 can be applied. The pluripotent stem cells may be prepared from iPS cells that have already been established, or by any method known per se or any method developed in the future, specifically, for example, the method described in the Examples, Cell, 131(5): 861-872 (2007), Cell,126(4):663-676(2006), the method described in Non-Patent Document 6, and the like. The pluripotent stem cells can be prepared, for example, from cells derived from XP patients. The method for producing pluripotent stem cells derived from XP patients can be any method known per se or any method to be developed in the future. Specifically, the method described in the Examples or the method described in JP 2021-17405 A can be applied. The culture environment may be any known environment per se and is not particularly limited, but generally, cells can be cultured under conditions of 37±1°C and 5±1% CO2.

[0039] In the present specification, the "cytotoxicity of melanocytes" is preferably cytotoxicity caused by, for example, light exposure. Melanocytes are responsible for protection from light by melanin production, but melanocytes themselves also suffer DNA damage due to ultraviolet rays, and abnormal expression of the gene HDAC4, which controls DNA acetylation, occurs. The "cytotoxicity caused by light exposure" is preferably skin damage associated with HDAC4 abnormality. In the screening method of the present invention, a substance that improves cytotoxicity of melanocytes can be selected by a method known per se or any method to be developed in the future. For example, a substance that improves cytotoxicity of melanocytes can be selected by a method such as detection of HDAC4 and / or SASP factors, evaluation of the viability of melanocytes, or evaluation of the cell migration ability of melanocytes. These selection methods can select a substance that improves cytotoxicity of melanocytes by comparing with and without light exposure. The candidate substance to be subjected to the screening method of the present invention may be a polymeric compound or a low molecular weight compound. Examples of polymeric compounds are not particularly limited, but include, for example, proteins and nucleic acid substances, specifically, antibodies, antibody fragments, peptides, such as siRNA or shRNA. Examples of low molecular weight compounds are not particularly limited. Furthermore, the substance may contain a low molecular weight compound and a high molecular weight compound.

[0040] The present invention also encompasses a method for testing a skin disorder, comprising the step of detecting abnormalities in HDAC4 and / or SASP factors in skin sampled from a subject.

[0041] The step of detecting an abnormality in HDAC4 and / or SASP factors is not particularly limited as long as it is a step of detecting an abnormality in HDAC4 and / or SASP factors by a method capable of detecting the abnormality in HDAC4 and / or SASP factors. For example, the step of preparing an RNA (e.g., total RNA, mRNA) fraction from the skin of a subject and detecting a transcription product of the HDAC4 gene and / or SASP factor gene contained in the fraction, the step of detecting by an immunological method using an antibody that specifically recognizes HDAC4 and / or SASP factors, the step of detecting by mass spectrometry, etc. Specifically, in the test method of the present invention, the detection of an abnormality in HDAC4 and / or SASP factors can be performed using the following 1) or 2). 1) A nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcription product of the HDAC4 gene and / or the SASP factor gene; 2) An antibody capable of specifically recognizing HDAC4 and / or SASP factors.

[0042] The RNA fraction can be prepared using known techniques such as guanidine-CsCl ultracentrifugation and AGPC, but high-purity total RNA can be prepared quickly and easily from a small amount of sample using a commercially available RNA extraction kit (e.g., RNeasy Mini Kit; manufactured by QIAGEN, etc.). Methods for detecting the transcription products of the HDAC4 gene and / or SASP factor gene in the RNA fraction include, for example, PCR (RT-PCR, competitive PCR, real-time PCR, etc.) and hybridization (Northern blot, dot blot, DNA chip analysis, etc.). Immunological techniques include, for example, Western blotting, ELISA, FIA, RIA, etc. In the present specification, the antibody includes, but is not limited to, natural antibodies such as polyclonal antibodies and monoclonal antibodies (mAbs), chimeric antibodies that can be produced using gene recombination techniques, humanized antibodies, single-chain antibodies, and binding fragments thereof. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody, or a binding fragment thereof. The binding fragment means a partial region of the above-mentioned antibody having specific binding activity, and specifically includes, for example, F(ab')2, Fab', Fab, Fv, sFv, dsFv, sdAb, etc. (Exp.Opin.Ther. Patents, Vol.6, No.5, p.441-456, 1996). The class of the antibody is not particularly limited, and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. IgG or IgM is preferable, and IgG is more preferable in consideration of ease of purification, etc. In the present invention, it is also preferable to use a commercially available anti-HDAC4 antibody, anti-SASP factor antibody, or a kit containing anti-HDAC4 antibody and anti-SASP factor antibody as an antibody that specifically recognizes HDAC4 and / or SASP factor.

[0043] In the testing method of the present invention, when HDAC4 is detected at a lower level in a subject compared to a control, or when SASP factors are detected at a higher level in a subject compared to a control, it can be determined that the subject has a high possibility of having a skin disorder as described above. Thus, in addition to the above steps 1) and 2), the testing method of the present invention may also include a step of 3) determining that the subject has a skin disorder or is highly likely to have a skin disorder when HDAC4 is detected at a lower level in a subject compared to a control, or when SASP factors are detected at a higher level in a subject compared to a control.

[0044] In the testing method of the present invention, the "subject" includes those who are unknown to have a skin disorder, those suspected of having a skin disorder, etc., as well as those who are unknown to have XP, those suspected of having XP, etc.

[0045] The present invention also relates to a skin disorder test kit containing a substance capable of detecting abnormalities in HDAC4 and / or SASP factors. Examples of substances capable of detecting abnormalities in HDAC4 and / or SASP factors include nucleic acid probes or nucleic acid primers capable of specifically detecting transcription factors of HDAC4 genes and / or SASP factor genes, and antibodies capable of specifically recognizing HDAC4 and / or SASP factors. In the present specification, the "primers or probes, etc." may be modified as long as their functions are not significantly impaired. Examples of modifications include labels, fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, etc. The primers or probes, etc. may be immobilized on any solid phase for use. In the present specification, the "solid phase" is not particularly limited as long as it is capable of immobilizing polynucleotides, and examples of such include glass plates, nylon membranes, microbeads, silicon chips, capillaries, and other substrates. The kit of the present invention may further comprise a reaction buffer solution, dNTPs, a heat-stable DNA polymerase, a competitor nucleic acid, a fluorescent reagent, a competitor antibody, a labeled secondary antibody, a blocking solution, and the like. EXAMPLES

[0046] In order to aid in the understanding of the present invention, the present invention will be specifically described below with reference to the following Reference Examples and Examples, but it goes without saying that the present invention is not limited to these. Note that the "Reference Examples" show the experimental details leading to the completion of the present invention.

[0047] (Reference Example 1) Preparation of iPS-derived melanocytes In this reference example, XP disease-specific melanocyte stem cells were produced, and then differentiated into melanocytes (MCs), producing iPS-derived melanocytes.

[0048] (Creation of XP disease-specific pigment stem cells) Human iPS cell lines were established using normal human skin-derived fibroblasts (TIG-120 cells; JCRB0542) and fibroblasts derived from an XP-A patient (XP3OS). XP3OS cells (JCRB0303) are known to have the Japanese founder mutation of XPA (c.390-1G>C:g.15148G>C). These cell lines were obtained from the JCRB Bioresource Bank (Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM) (Nacalai) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (LifeTechnologies). Human iPS cell lines were established from these two fibroblast cell lines using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (ID Pharma Co., Ltd.) (Non-Patent Document 6). Thereafter, human iPS cells were seeded onto a non-adherent culture dish (50 to 200 cells / ml) and cultured in a medium for primate ES / iPS cells for two weeks to form embryoid bodies. The embryoid bodies were then seeded on fibronectin-coated adhesion culture dishes and cultured in Human Melanocyte Maintenance Medium (Medium 254 medium (Invitrogen)) supplemented with 3 μM CHIR99021 (Stemgent), 50 ng / ml SCF (R&D), 100 nM ET-3 (American Peptide Company), 500 μM dbcAMP (Sigma), 50 nM TPA (12-tetradecanoylphorbol 13-acetate (Sigma), 4 ng / ml bFGF (Wako), 100 μM L-ascorbic acid (Sigma), 0.05 μM dexamethasone (Sigma), 1 mg / ml linoleic acid-bovine serum (Sigma), and 1X insulin-transferrin-sodium selenite (Sigma). The cells were cultured in a medium supplemented with Human Melanocyte Growth Supplement (HMGS) (Invitrogen) to induce differentiation into melanocytes. During this period, the medium was changed every 2 to 3 days.Fourteen days after differentiation induction, spindle-shaped cells containing dark brown granules were observed under a microscope, so differentiation induction was stopped and the cells were cultured in melanocyte maintenance medium that did not contain differentiation inducers. Seven days after differentiation induction was stopped, melanocyte stem cells were observed in the medium.

[0049] (Induction of differentiation into XP disease-specific melanocytes) The pigment stem cells described above were differentiated into MCs about one week after adding 3 μM CHIR99021 every two days. Hereinafter, cells differentiated into melanocytes from fibroblast-derived iPS cells derived from XP-A patients are referred to as XP-A-iMCs, and cells differentiated into melanocytes from normal fibroblast-derived iPS cells derived from human skin are referred to as HC-iMCs.

[0050] XP-A-iMCs and HC-iMCs were observed under a phase-contrast microscope. As a result, XP-A-iMCs and HC-iMCs had a long, spindle-shaped morphology with dendrites, similar to normal human epidermal melanocytes (NHEM) (Figure 1). In addition to visual observation of cell morphology, to ensure greater accuracy, observation of pellets obtained by centrifugation and immunohistochemical staining were performed. The pellets obtained by centrifugation were visually black, indicating that they had differentiated into MCs. Immunohistochemical staining confirmed that the cells had the properties of MCs, based on the expression of MC markers SOX10, MITF, and TYR (Figure 2).

[0051] (Example 1) Confirmation of mutations by genomic sequencing In this example, it was confirmed whether XP-A-iMC correctly contained the Japanese founder mutation (c.390-1G>C:g.15148G>C).

[0052] Genomic DNA was isolated from XP-A-iMCs using the Gentra Puregene Cell Kit (Qiagen, Hilden). To confirm the presence or absence of the Japanese XP-A founder mutation g.15148G>C in the XPA gene (c.390-1 G>C), the region containing this mutation was amplified with the following PCR primers: 5'-GCTGTGTGCCTAAGTT-3' (forward) (SEQ ID NO: 1) 5'-TGCAAAACTAGGAAAAGTTT-3' (reverse) (SEQ ID NO: 2) Ex-Taq (Takara Bio)

[0053] The PCR products were resolved on an agarose gel, purified using a QIAquick Gel Extraction Kit (Qiagen), and sequenced with the same forward primer using an Applied Biosystems 3500 Genetic Analyzer (Applied Biosystems) (Figure 3A). Furthermore, Western blotting confirmed that XP-A-iMCs did not express the XPA protein (Figure 3B).

[0054] Example 2: Confirmation that XP-A-iMC has the properties of XPA In this example, the nature of XP, which has impaired DNA repair and is therefore vulnerable to ultraviolet light, was examined using the flow cytometry-based NER method (Nakano et al., 2018, J Invest Dermatol, 138:467-47) and the MTT (proliferation / survival) method. The flow cytometry-based NER method is a DNA repair test that is also used in clinical diagnosis, and is a method that involves analysis by flow cytometry using a monoclonal antibody against (6-4)PP after UV-C irradiation.

[0055] (Flow cytometry-based NER method) We evaluated DNA lesion repair in XP-A-iMCs and HC-iMCs by flow cytometry-based NER. Specifically, XP-A-iMCs and HC-iMCs were seeded on a 10 cm dish and irradiated with 30 J / m on the following day. 2The cells were irradiated with UV-C and XP-A-iMCs and HC-iMCs were harvested immediately or 6 hours after irradiation. They were then stained with mouse primary monoclonal antibody against (6-4)PP (1:1,000, Cosmo BioCo.,Ltd.) for 1.5 hours at 22°C, resuspended in PBS-TB containing AlexaFluor 488 antibody (1:200, Thermo Fisher Scientific) for 1 hour at 22°C, and further resuspended in PBS containing 5 μg / ml propidium iodide (Thermo Fisher) to stain the XP-A-iMCs and HC-iMCs. After staining, the cells were analyzed using a FACSVerse flow cytometer (BD Biosciences). All data were analyzed using FlowJo software (BD Life Sciences). The removal rate of DNA lesions was calculated based on the mean fluorescence intensity. The fluorescence intensity of XP-A-iMCs and HC-iMCs not irradiated with UV was used as the background, and the fluorescence intensity of the cells immediately after UV irradiation was set to 100%. The decrease in fluorescence intensity was measured after 6 hours.

[0056] (MTT method) Cell viability was confirmed by the MTT assay. Specifically, XP-A-iMC, HC-iMC, and NHEM were evaluated 24 hours after UV-B irradiation using CellProliferationKit I (MTT assay) (Roche) according to the manufacturer's protocol. The medium was replaced with phenol red-free medium (Lifeline CellTechnology) immediately before reaction with the MTT reagent. The final product was then transferred to a 96-well plate and measured at absorbance of 595 nm and 650 nm using an Emax precision microplate reader (MolecularDevices). Cell viability was expressed relative to that of non-UV-irradiated cells.

[0057] The results of the flow cytometry-based NER method are shown in Figure 4A. Figure 4A shows representative histograms showing the relative signal intensity of (6-4)PP in unirradiated, immediately after UV-C irradiation, and 6 hours after UV-C irradiation. In HC-iMC and XP-A-iMC, 96.5% and 6.1% of (6-4)PP were restored within 6 hours after UV-C irradiation, respectively. Similar results were obtained from three independent experiments.

[0058] The results of the MTT assay are shown in Figure 4B. The cell viability 24 hours after irradiation was compared for low doses (10 J / m 2 , 20 J / m 2 ) or high dose (100 J / m 2 , 200 J / m 2 ) (Figure 4B). For reference, the viability of commercially available NHEM was also evaluated (Gaddameedhi S et al. 2010). The viability of HC-iMC was almost the same as that of NHEM, and was 200 J / m 2 was significantly lower than baseline at 100 J / m 2 On the other hand, the decrease was ≥ 20 J / m 2 It was significantly decreased by UV-B irradiation.

[0059] (Example 3) Research into the pathology of XP using XP-A-iMC In this example, we analyzed the UV response of XP-A-iMCs, comprehensively explored the response of melanocytes to UV stimulation, and sought to identify molecules (drug discovery target molecules) that are directly involved in the pathogenesis of XP and could serve as therapeutic target seeds.

[0060] XP-A-iMCs and HC-iMCs were seeded in 10 cm dishes and incubated with low-dose (30 J / m 2 ) or high dose (150 J / m 2 ) UV-B irradiation, then incubated at 37°C for 4 or 12 hours (Figure 5A), and then comprehensive transcriptional profile analysis was performed. Gene transcription profiling was performed using the GeneChip TMThe Human Genome U133 Plus 2.0 Array (Affymetrix) was used according to the manufacturer's protocol. Data were analyzed using the Robust Multichip Average (RMA) method with GeneSpring 13.1.1 software (Agilent Technologies, Inc.). Differences in genes affected by UV-B between XP-A-iMCs and HC-iMCs were revealed.

[0061] The number of probes whose gene expression levels changed significantly (10-fold or more) in XP-A-iMCs and HC-iMCs is shown in Figure 5B. When examining the number of probes whose expression levels changed significantly (10-fold or more), it was found that the expression levels of 27 probes (15 upregulated and 12 downregulated) changed when HC-iMCs were irradiated with low-dose UV-B. However, although the expression levels changed by more than 10-fold 4 hours after irradiation compared to before irradiation, it was found that almost all genes returned to the pre-irradiation level 12 hours after irradiation. On the other hand, when XP-A-iMCs were irradiated with low-dose UV-B, the expression levels of 72 probes (34 upregulated and 38 downregulated) changed significantly 4 hours after irradiation, which was a significantly higher number than that observed in HC-iMCs.

[0062] Next, HC-iMC was exposed to high-dose UV-B (150 J / m 2 ) and picked up 26 or 55 probes whose expression increased or decreased by 10-fold or more 4 hours after irradiation. The gene expression levels of these probes in HC-iMC and XP-A-iMC 4 or 12 hours after irradiation are shown in Figure 5C. In HC-iMC, all expression levels returned to the original level after 12 hours. On the other hand, in XP-A-iMC, it was found that the expression levels of almost all probes did not return to the basal level even after 12 hours, but remained up- or down-regulated (Figure 5C). It was found that a characteristic of XP-A-iMC is that the gene expression levels do not return to the pre-irradiation levels even after 12 hours after irradiation. (Figure 5C)

[0063] To clarify the molecular mechanisms related to the cellular response to UV-B in XP-A-iMCs in more detail, GO analysis (p<0.05) was performed. From the results of genes (n=49) whose expression levels were specifically increased in XP-A-iMCs 12 hours after high-dose UV-B irradiation, the main GO term categories in XP-A-iMCs were cell proliferation and cell death control, followed by programmed cell death control (p=0.004), apoptotic process control (p=0.004), cell proliferation control (p=0.004), response to stimuli (p=0.004), and cell death control (p=0.007). On the other hand, the main GO term categories in XP-A-iMCs suggested by the 132 specifically decreased expression genes were cell division (p=3.15 x 10 -10 ), mitotic cell cycle (p=3.88x10 -9 ), chromosome segregation (p=8.64 x 10 -8 ), mitotic cell cycle process (p=3.12 x 10 -7 ) and cell cycle process (p=2.04x10 -6 ) were included (Figure 6).

[0064] (Example 4) Identification of Histone Deacetylase 4 (HDAC4) In this example, among the 132 gene probes whose expression was specifically decreased in XP-A-iMC, histone deacetylase 4 (HDAC4) was identified as a gene that strongly exhibited characteristic behavior (Figure 7). HDAC4 plays an important role in histone modification, and is known from previous reports to be important in cell proliferation, cell cycle progression, differentiation, and development, and is also involved in autophagy and mitophagy. This certainly explains the pathology observed in XP, and it is possible that this gene epigenetically controls melanocytes. HDAC4 behavior was decreased in both MCs 4 hours after UV-B irradiation and recovered in NHEM and HC-iMCs 12 hours after, but in XP-A-iMC, HDAC4 expression tended not to recover and continued to decrease 12 hours after UV irradiation (Figure 7). HDAC4 expression in XP-A-iMC was confirmed from multiple angles using RT-PCR and Western blotting (Figures 8A, B).

[0065] (Example 5) Examination of cell migration ability of XP-A-iMC In this example, the cell migration ability of XP-A-iMCs after ultraviolet irradiation was examined using a scratch assay and digital holographic microscopy.

[0066] The scratch assay and digital holographic microscopy analysis revealed that XP-A-iMCs and HC-iMCs were seeded in a 60 mm dish and, when they reached confluence, were exposed to high doses of radiation (150 J / m 2 ) UV-B irradiation, followed by scratching with a small scraper to create a gap. The gap was then filled by cell migration and observed daily under a phase-contrast microscope (Figure 9). The results of cell migration assay using the scratch assay are shown in Figure 10. It was found that cell migration was significantly impaired in XP-A-iMCs with reduced HDAC4 compared to HC-iMCs.

[0067] Cell migration was also verified using a digital holographic microscope (Figure 11). 2 The HoloMonitor was used to image and track the movements of XP-A-iMC and HC-iMC after UV-B irradiation. (R) M4 microscope (Phase Holographic ImagingAB) was used. XP-A-iMC and HC-iMC (1×10 4 ) were seeded in a 6-well plate and exposed to a high dose (150 J / m 2) were irradiated with UV-B and the migration was tracked for 72 hours. Figure 12 shows the distance migrated by the cells, and Figure 13 shows the speed of cell migration. It was confirmed that the migration ability was reduced in XP-A-iMCs with reduced HDAC4 after UV-B irradiation. This suggests that the phenotype of XP-A-iMCs may cause pigmentation and depigmentation, which are skin symptoms characteristic of XP-A patients. In addition, since it was shown in Example 2 that the survival rate of XP-A-iMCs is reduced by light irradiation, it may be possible to obtain a substance that improves cytotoxicity of melanocytes by adding a candidate substance to a melanocyte culture system and evaluating the survival rate and cell migration ability of melanocytes.

[0068] (Example 6) Study of XP-A-iMC in cellular senescence In this example, whether cellular senescence occurs in XP-A-iMCs after ultraviolet irradiation was examined using an immunofluorescence antibody technique.

[0069] XP-A-iMCs and HC-iMCs were seeded in a 60 mm dish and, when they reached approximately 70% confluence, were incubated with high-dose (150 J / m 2 ) UV-B irradiation, 24 hours later, we examined the expression of γH2AX, which is an indicator of DNA damage and an indicator of cellular senescence. A significant increase in γH2AX expression was confirmed in XP-A-iMC compared to HC-iMC by immunofluorescence, indicating that cellular senescence was promoted (Figure 14).

[0070] Furthermore, we hypothesized that melanocytes undergoing cellular senescence have changes in the senescence-associated secretory phenotype (SASP) factors that induce inflammation, and we used RT-PCR to verify the increased expression of a number of SASP factors. 2), increased expression of many SASP factors was confirmed in XP-A-iMCs in which HDAC4 expression was reduced (Fig. 15). Furthermore, expression of SASP factors was confirmed by Western blotting (Fig. 16). It has been reported in other types of cancer that increased expression of SASP factors causes chronic inflammation, but in XP-A patients, increased expression of SASP factors causes chronic inflammation, which is thought to be the cause of the skin symptoms, which are a marked acceleration of photoaging, including an increase in the incidence of characteristic photocarcinogenesis. Therefore, it was suggested that the skin symptoms of XP could be comprehensively suppressed by inhibiting the decrease in HDAC4. On the other hand, even in NHEM, the skin symptoms were not suppressed after irradiation with a significantly high dose (500 J / m 2 When UV-B of 150 J / m was irradiated to XP-A-iMC, 2 ), an increase in the expression of SASP factors was observed (Figure 15).

[0071] Example 7: Decreased expression of HDAC4 in NHEM In this example, HDAC4 expression in NHEM was confirmed. NHEM were seeded in a 60 mm dish and irradiated with a significantly high dose (500 J / cm 2 ) UV-B irradiation, and 24 hours later, HDAC4 expression was confirmed using RT-PCR (Figure 17). It was also confirmed that the pathological condition in which HDAC4 expression is significantly reduced by UV exposure was also observed in melanocytes from healthy subjects when the amount of UV light was significantly increased, suggesting that the reduction in HDAC4 may be generally important in photoaging.

[0072] (Example 8) Improvement of HDAC4 expression by curcumin XP-A-iMCs were seeded in a 60 mm dish and, when they reached approximately 70% confluence, were incubated with a high dose (150 J / m 2 After irradiating the cells with UV-B at 150 J / m, curcumin was added to the cell culture medium and cultured for 24 hours, after which the expression of HDAC4 was confirmed using RT-PCR. Dimethyl sulfoxide (DMSO), the solvent for curcumin, was used as a control. 2We found that the expression of HDAC4, which had been decreased by UV-B irradiation, was significantly restored (Figure 18). We also confirmed that the effect was dependent on the curcumin concentration.

[0073] (Example 9) Improvement of HDAC4 expression by C-646 XP-iMCs were seeded in a 60 mm dish and, when they reached approximately 70% confluence, were incubated with a high dose (150 J / m 2 After irradiating the cells with 150 J / m UV-B, C-646 was added to the cell culture medium and cultured for 24 hours, after which the expression of HDAC4 was confirmed by RT-PCR (Figure 19). Dimethyl sulfoxide (DMSO), the solvent for C-646, was used as a control. 2 ) and found that the expression of HDAC4, which had been decreased by UV-B irradiation, was significantly restored. [Industrial Applicability]

[0074] According to the preventive and / or therapeutic agent for skin disorders associated with histone deacetylase 4 abnormality, which contains the histone acetyltransferase inhibitor of the present invention as an active ingredient, it is possible to prevent and / or treat skin disorders associated with histone deacetylase 4 abnormality, and it is very useful not only for XP patients but also for many skin diseases caused by photoaging, for example. It is particularly useful for XP patients, since an effective treatment method has not yet been established. In addition, it is possible to screen for a new preventive and / or therapeutic agent for skin disorders focusing on HDAC4 and melanocytes.

Claims

1. A preventive and / or therapeutic agent for skin disorders associated with histone deacetylase 4 abnormality, comprising a histone acetyltransferase inhibitor as an active ingredient.

2. The preventive and / or therapeutic agent for skin disorders according to claim 1, wherein the skin disorder is a skin disorder associated with histone deacetylase 4 abnormality in melanocytes caused by exposure to light.

3. The preventive and / or therapeutic agent for skin disorders according to claim 1 , wherein the skin disorder is a skin disorder caused by photoaging.

4. The preventive and / or therapeutic agent for a skin disorder according to claim 1 , wherein the skin disorder is a skin disorder caused by xeroderma pigmentosum.

5. The preventive and / or therapeutic agent for skin disorders associated with histone deacetylase 4 abnormalities according to claim 1, which has the effect of improving abnormalities in histone deacetylase 4 expression.

6. The method for preventing and / or treating skin disorders according to claim 1, wherein the histone acetyltransferase inhibitor is one or more selected from the group consisting of curcumin, C-646, A-485, anacardic acid, butyrolactone, CTPB and garcinol.

7. A method for screening for a preventive and / or therapeutic agent for skin disorders, which comprises adding a candidate substance to a culture system of melanocytes and selecting a substance that ameliorates cytotoxicity of the melanocytes.

8. The screening method according to claim 7, wherein the substance that ameliorates cytotoxicity of melanocytes is obtained by detecting histone deacetylase 4 and / or senescence-associated secretory phenomenon factor.

9. The screening method according to claim 7, wherein the substance that ameliorates cytotoxicity of melanocytes is obtained by evaluating the viability of melanocytes.

10. The screening method according to claim 7, wherein the substance that ameliorates cytotoxicity of melanocytes is obtained by evaluating the cell migration ability of melanocytes.

11. The method of claim 7, wherein the melanocytes are melanocytes derived from pluripotent stem cells.

12. The method of screening according to claim 11, wherein the pluripotent stem cell-derived melanocytes are pluripotent stem cell-derived melanocytes produced from pluripotent stem cells derived from a patient with xeroderma pigmentosum.

13. The screening method according to claim 7, wherein the cytotoxicity of melanocytes is cytotoxicity caused by exposure to light.

14. The screening method according to claim 13, wherein the cell damage caused by light exposure is a skin disorder associated with histone deacetylase 4 abnormality.

15. A method for testing a skin disorder, comprising the step of detecting abnormalities in histone deacetylase 4 and / or cellular senescence-associated secretory factor in skin sampled from a subject.

16. The method for testing a skin disorder according to claim 15, wherein the cellular senescence-associated secretory phenomenon factor is one or more selected from the group consisting of MMP-1, MMP-3, TNFα, and IL-6.

17. The method for testing a skin disorder according to claim 15, wherein the detection of abnormalities in histone deacetylase 4 and / or cellular senescence-associated secretory factor is carried out using the following 1) or 2): 1) a nucleic acid probe or a nucleic acid primer capable of specifically detecting a transcription product of the histone deacetylase 4 gene and / or the cellular senescence-associated secretory phenomenon factor gene; 2) An antibody that specifically recognizes histone deacetylase 4 and / or senescence-associated secretory factor.

18. A skin disorder testing kit comprising a substance capable of detecting abnormalities in histone deacetylase 4 and / or cellular senescence-associated secretory factor.

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