Prophylactic and / or therapeutic agent for skin disorder

Abstract

Provided is a prophylactic and / or therapeutic agent for photodermatitis caused by light exposure. This prophylactic and / or therapeutic agent for a skin disorder caused by light exposure contains an inhibitor for cellular senescence-associated secretory phenotype factors (SASP factors) as an active ingredient. As a result of exploring responses to light exposure in melanocytes and comprehensively analyzing molecules that can be therapeutic target seeds related to the skin disorder, it was shown that cellular senescence occurs in melanocytes after light exposure. In melanocytes having undergone cellular senescence, enhanced expression of an SASP factor group was observed, and this led to the finding that an inhibitor for SASP factors is effective for preventing and / or treating the skin disorder.

Description

Agents for the prevention and / or treatment of skin disorders

[0001] The present invention relates to a preventive and / or therapeutic agent for skin damage caused by light exposure, comprising a senescence-associated secretory phenotype (SASP) factor inhibitor as an active ingredient.

[0002] This application claims priority to Japanese Patent Application No. 2024-198839, as incorporated herein by reference.

[0003] Melanocytes (pigment cells) are cells that originate from the neural crest during development and produce melanin. In the skin, melanocytes are mainly located in the basal layer of the epidermis and supply melanin to surrounding keratinocytes. In human skin, melanin is thought to have a protective effect against light exposure, especially ultraviolet (UV) rays. On the other hand, exposure to UV rays and other light can also cause DNA damage to melanocytes, and excessive melanin production can lead to various skin disorders such as excessive pigmentation (age spots), melasma, senile lentigines, sun damage, and wrinkles. Even though the occurrence of the aforementioned skin disorders can be prevented by using sunscreen or parasols to block sunlight, people sometimes forget to apply sunscreen, and sunscreen and parasols are often insufficient to prevent sun damage. Such unprevented light exposure can first cause inflammation as skin damage, and in the long term, it can lead to pigment lesions or skin cancer. For example, skin cancer that has already developed requires surgical intervention each time, which places a heavy burden on the patient. In prevention strategies involving shading, the response to emergencies when prevention failed was weak.

[0004] In xeroderma pigmentosum (hereinafter also simply referred to as "XP"), the skin is the target organ where symptoms are most strongly expressed, and pigment abnormalities are particularly invariably present. Therefore, analyzing the UV response of melanocytes in patients is of paramount importance in elucidating the pathogenesis of XP. XP is a hereditary photosensitive disorder inherited in an autosomal recessive manner, and is a hereditary DNA repair disorder that causes severe photosensitivity and neurological symptoms. XP patients' skin congenitally lacks the ability to repair DNA damage caused by exposure to light such as UV, resulting in severe photosensitivity to sunlight and UV (abnormal sunburn, pigment abnormalities, skin cancer in exposed areas, etc.) occurring to varying degrees depending on the disease type. For this reason, it is necessary to create a light-shielding environment throughout the entire living space, and the quality of life (QOL) of XP patients and their families is significantly reduced. If proper light protection is not taken, severe photosensitivity symptoms and freckle-like pigment abnormalities progress, and skin cancer develops at a high frequency even at a young age. Furthermore, more than half of XP patients experience progressive cerebrovascular and neurodegenerative symptoms of unknown cause, and the severity of these symptoms affects the patient's prognosis. Therefore, there is a strong need to elucidate the pathogenesis of XP and establish effective treatments, and the establishment of such treatments is an urgent necessity for XP patients worldwide.

[0005] XP is classified into eight types: genetically distinct groups A through G (genetic complementarity groups) and variant (V). In Japan, approximately 55% of XP cases are classified as group A (xeroderma pigmentosum group A: XP-A), which has the most severe skin and neurological symptoms, while approximately 25% are classified as type V, which only has skin symptoms. XP-A patients, who are more common in Japan, exhibit severe photosensitivity, with intense sunburn symptoms appearing immediately after birth. Even with low levels of UV light, significantly accelerated photoaging is observed, resulting in severe sunburn reactions, freckle-like pigment spots of varying sizes and colors, and skin cancer in exposed areas at a young age. However, the pathogenesis of XP-A remains unclear. In addition, neurological symptoms such as hearing loss, slurred speech, and loss of balance become apparent in XP-A patients around the age of 6, and gradually increase in the likelihood of falls after the age of 10. As hearing loss and intellectual disability progress, language function also declines.

[0006] In XP, the skin is the target organ where symptoms are most strongly expressed, and pigment abnormalities are particularly invariably present, making research on melanocytes crucial for elucidating the pathogenesis. However, because it is difficult to isolate and culture melanocytes from XP patient skin tissue, analyses have been conducted using fibroblasts (Non-Patent Documents 1 and 2). On the other hand, since melanocytes and fibroblasts differ in their resistance to UV and their response to DNA damage, it is important to study XP using melanocytes (Non-Patent Documents 3 to 5). However, there have been no reports to date analyzing the pathogenesis of premature photoaging in XP, in which pigmented and depigmented spots are mixed in the exposed areas where melanocytes are involved, using melanocytes derived from XP patients. Therefore, the present inventors have found a method for producing pigment stem cells from pluripotent stem cells and establishing melanocytes from said pigment stem cells (Patent Document 1, Non-Patent Document 6).

[0007] For skin damage caused by exposure to light such as UV rays, there is a need for more effective preventive and / or therapeutic methods than simply shielding from light.

[0008] Patent No. 6624916

[0009] Bowden et al., Int. J. Mol. Sci. 2015, 15985-15996Herman et al., Environ Mol Mutagen. 2014 June; 55(5): 375-384Fukumoto et al., Genes Cells.2016, Feb; 21(2): 185-99Cho-TH et al., Photodermatol Photoimmunol Photomed. 2008 Jun;24(3): 110-4.Emri G et al. J Invest Dermatol. 2000 Sep; 115(3): 435-40Hosaka et al. Pigment Cell & Melanoma Research, 2019, 32(5): 623-633

[0010] The object of this invention is to provide an agent for preventing and / or treating photodermatological damage caused by light exposure.

[0011] To solve the above problems, the inventors explored the response of melanocytes to light exposure and comprehensively analyzed molecules that could serve as therapeutic targets for skin disorders. As a result, they revealed that cellular senescence occurs in melanocytes after light exposure. Furthermore, after diligent investigation, they confirmed that melanocytes undergoing cellular senescence exhibited increased expression of senescence-associated secretory (SASP) factors. Based on this, they found that SASP factor inhibitors are effective in preventing and / or treating skin disorders, thus completing the present invention.

[0012] In other words, the present invention comprises the following: 1. A preventive and / or therapeutic agent for skin damage caused by light exposure, comprising a cellular senescence-associated secretory factor (SASP factor) inhibitor as an active ingredient. 2. The preventive and / or therapeutic agent according to item 1, wherein the SASP factor is one or more selected from IL1B (Interleukin 1B), IL6 (Interleukin 6), CXCL8 (chemokine (CXC motif) ligand 8), MMP1 (matrixmetalloproteinase-1), MMP3 (matrixmetalloproteinase-3), and CCL20 (CC motif chemokine ligand 20). 3. The preventive and / or therapeutic agent according to item 1, wherein the SASP factor inhibitor as an active ingredient is an agent having an inhibitory effect on one or more SASP factors selected from IL1B, IL6, CXCL8, MMP1, MMP3, and CCL20. 4. 1. The preventive and / or therapeutic agent according to paragraph 1, wherein the SASP factor inhibitor is a JAK (Janus kinase) inhibitor. 5. The preventive and / or therapeutic agent according to paragraph 4, wherein the JAK inhibitor is one or more selected from the group consisting of baricitinib, upadacitinib, tofacitinib, peficitinib, filgotinib, abrocitinib, ritrecitinib, and delgocitinib. 6. The preventive and / or therapeutic agent according to paragraph 1, wherein the SASP factor inhibitor is one or more selected from the group consisting of curcumin and curcumin analogs. 7. The preventive and / or therapeutic agent according to paragraph 6, wherein the curcumin analog is EF24. 8. The preventive and / or therapeutic agent for skin disorders according to paragraph 1, wherein the skin disorder caused by light exposure is a skin disorder due to photoaging. 9. A preventive and / or therapeutic agent for the skin disorder described in paragraph 8 above, wherein the skin disorder caused by photoaging is skin cancer due to photoaging. 10. A preventive and / or therapeutic agent for the skin disorder described in paragraph 1 above, wherein the skin disorder caused by light exposure is skin disorder due to xeroderma pigmentosum.11. A screening method for preventive and / or therapeutic agents for skin disorders, characterized by adding a candidate substance to a culture system of light-exposed melanocytes and / or a culture system of cisplatin-stimulated melanocytes, and selecting a substance that can suppress the expression of SASP factors secreted from the melanocytes or suppress the activity of SASP factors. 12. The screening method according to item 11, wherein the SASP factors are one or more selected from IL1B, IL6, CXCL8, MMP1, MMP3, and CCL20. 13. The screening method according to item 11, wherein the melanocytes are melanocytes derived from pluripotent stem cells. 14. The screening method according to item 13, wherein the melanocytes derived from pluripotent stem cells are melanocytes derived from pluripotent stem cells produced from pluripotent stem cells derived from a patient with xeroderma pigmentosum.

[0013] 15. A method for preventing and / or treating skin damage caused by light exposure, comprising administering an inhibitor of cellular senescence-associated secretory factors (SASP factors). 16. The method for prevention and / or treatment according to item 15, wherein the SASP factors are one or more selected from cytokines, chemokines, etc., including IL1B, IL6, CXCL8, MMP1, MMP3, and CCL20. 17. The method for prevention and / or treatment according to item 15, wherein the SASP factor inhibitor is an agent having an inhibitory effect on one or more SASP factors selected from cytokines, chemokines, etc., including IL1B, IL6, CXCL8, MMP1, MMP3, and CCL20. 18. The method for prevention and / or treatment according to item 15, wherein the SASP factor inhibitor is a JAK inhibitor. 19. 20. The method of prevention and / or treatment according to paragraph 18, wherein the JAK inhibitor is one or more selected from the group consisting of baricitinib, upadacitinib, tofacitinib, peficitinib, filgotinib, abrocitinib, ritrecitinib, and delgocitinib. 21. The method of prevention and / or treatment according to paragraph 20, wherein the SASP factor inhibitor is one or more selected from the group consisting of curcumin and curcumin analogs. 22. The method of prevention and / or treatment according to paragraph 15, wherein the curcumin analog is EF24. 23. The method of prevention and / or treatment according to paragraph 15, wherein the skin disorder caused by light exposure is a skin disorder due to photoaging. 24. The preventive and / or therapeutic method described in paragraph 15, wherein the skin disorder caused by the aforementioned light exposure is a skin disorder due to xeroderma pigmentosum.

[0014] The present invention provides a preventive and / or therapeutic agent for skin disorders containing the SASP factor inhibitor as an active ingredient, which can prevent and / or treat skin disorders caused by light exposure. Furthermore, the present invention is superior in that it is a senomorphic agent that acts with a mild effect, rather than a senolytic agent that has a strong effect of removing all senescent cells. Since the senescent cells that exhibit cellular senescence in the skin are melanocytes, the side effects of removing all senescent melanocytes would be serious.

[0015] Figure 1A is a conceptual diagram showing that both DNA damage caused by light exposure and DNA damage caused by cisplatin treatment are types of DNA damage that form crosslinks within the DNA strand, and furthermore, that the repair mechanism is the same nucleotide excision repair (NER) pathway. Therefore, Figure 1B shows that the cisplatin-based screening system of the present invention can be used as a substitute for a screening system for searching for candidate molecules that show therapeutic effects against DNA damage caused by light exposure. Figure 2A shows the experimental protocol for confirming the effects of UV-B irradiation on HC-iMC and XP-iMC. Figure 2B is a photograph showing the results of visualizing γH2AX, a DNA damage marker, using an immunofluorescence microscope. Figure 2C is a photograph showing the results of staining UV-B irradiated XP-iMC with SA-β-gal (senescence-related acid β-galactosidase). Figure 2D shows the results of visually measuring the proportion of two populations: SA-β-gal positive senescent cells and SA-β-gal negative non-senescent cells. Figure 2E shows the results of RT-qPCR to confirm the expression levels of the SASP factors IL1B, IL6, CXCL8, MMP1, MMP3, and TNF-α in UV-B irradiated XP-iMCs. Figure 2F shows the results of Western blotting to confirm the protein expression levels of the SASP factors IL1B, IL6, MMP1, and MMP3 in UV-B irradiated XP-iMCs. (Example 1) Figure 3A shows the experimental protocol to confirm the effects of cisplatin on HC-iMCs and XP-iMCs. Figure 3B is a photograph showing the results of immunofluorescence microscopy to visualize γH2AX, a DNA damage marker. Figure 3C is a photograph showing the results of SA-β-gal staining of cisplatin-treated XP-iMCs. Figure 3D shows the results of flow cytometry to estimate the proportion of two populations: β-gal-positive senescent cells and β-gal-negative non-senescent cells stained with the cell senescence detection kit SPiDER-βGal (Dongrentang, SG03). Figure 3E shows the results of RT-qPCR analysis of the expression levels of the SASP factors IL1B, IL6, CXCL8, MMP1, and CCL20 in cisplatin-treated XP-iMCs.(Example 2) Figure 4A shows the screening protocol using cisplatin of the present invention. Figure 4B shows the results of confirming the induction of cellular senescence when XP-iMCs were cultured with each candidate substance in the presence of cisplatin according to the protocol in Figure 4A. (Example 3) The results of confirming non-senescent cells and senescent cells when XP-iMCs were cultured with each candidate substance in the presence of cisplatin according to the protocol in Example 3 are shown. Figure 5A shows XP-iMCs that were not treated with cisplatin or any of the candidate substances, Figure 5B shows XP-iMCs treated with cisplatin alone, and Figure 5C shows the results for XP-iMCs treated with cisplatin in addition to EF24, curcumin, baricitinib, or upadacitinib. (Example 4) Figure 6A is a photograph showing the results of SA-β-gal staining when XP-iMCs were cultured in the presence of each candidate substance in the presence of cisplatin according to the protocol of Example 3. Figure 6B shows the results of confirming the expression levels of the SASP factors IL1B, CXCL8, MMP1 and CCL20 genes by RT-qPCR for XP-iMCs treated with EF24, curcumin, baricitinib or upadacitinib in addition to cisplatin. (Example 5) Figure 7A is a photograph showing the results of SA-β-gal staining for XP-iMCs that were treated with UV-B irradiation of various intensities and cultured for 3 or 6 days. Figure 7B shows the results of visually measuring the proportion of two populations: SA-β-gal positive senescent cells and SA-β-gal negative non-senescent cells. Figure 7C is a figure showing the results of confirming the expression levels of the SASP factors IL1B, IL6, CXCL8, MMP1, MMP3 and CCL20 genes by RT-qPCR under different UV-B irradiation conditions. (Example 6) Figure 8A shows the experimental protocol for confirming the effects of UV-B irradiation on XP-iMCs. Figure 8B is a photograph showing the results of staining each XP-iMC treated with EF24, curcumin, baricitinib, or upadacitinib with SA-β-gal. Figure 8C shows the results of visually measuring the proportion of two populations: SA-β-gal positive senescent cells and SA-β-gal negative non-senescent cells.(Example 7) This figure shows the experimental protocol for confirming the effects of a JAK inhibitor and a curcumin analog (EF24) on light-induced skin damage in vivo. (Example 8) Figure 10A is 6 kJ / m. 2 This is a photograph showing erythema in mouse skin treated with delgocitinib under UV-B irradiation for various time intervals. Figure 10B is a graph showing the degree of skin erythema quantified using a pigment meter. (Example 8, Figure 11A is 6 kJ / m 2 This is a photograph showing erythema in mouse skin treated with EF24 under UV-B irradiation for various durations. Figure 11B is a graph showing the degree of skin erythema quantified using a pigment meter. (Example 8) Figure 12A shows the collection of mouse skin sections. Figure 12B shows the results of confirming the degree of expression of various SASP-related genes in skin sections collected from mice treated with delgocitinib under UV-B irradiation. (Example 9)

[0016] The present invention relates to a preventive and / or therapeutic agent for skin damage caused by light exposure, comprising an inhibitor of cellular senescence-associated secretory phenomena (SASP) factors as an active ingredient.

[0017] The inventors investigated the response of melanocytes to light exposure and comprehensively analyzed molecules related to skin damage. They concluded that melanocytes exposed to light are affected by the SASP factor group, and confirmed an increase in the expression of numerous SASP factors. In other words, it is thought that various skin disorders occur due to increased expression of SASP-related genes caused by light exposure. Melanocytes (also called pigment cells or melanin cells) are cells that have tyrosinase activity and produce melanin. They possess intracellular organelles (melanosomes) for melanin synthesis, and are dendritic cells whose dendrite shape changes depending on the environment.

[0018] In this specification, "skin disorders" include skin disorders caused by light exposure, skin disorders caused by photoaging, etc., and also include skin disorders experienced by XP patients. Here, "photoaging" refers to changes in the appearance and function of the skin that are observed as a result of repeated light exposure, and broadly includes skin disorders that can occur due to light exposure. Specific skin disorders include, but are not limited to, pigmented lesions in which pigmented and depigmented spots are present, dry or atrophied skin, decreased skin elasticity, formation of wrinkles and sagging, formation of freckles, hyperpigmentation, darkening of the skin, increased yellowing, decreased skin barrier function, decreased stratum corneum function, and skin cancer that develops at a young age.

[0019] In this specification, “light exposure” is not limited to normal or excessive light exposure in daily life (e.g., sunburn), but also includes slight light exposure that may cause skin damage to, for example, XP patients. In this specification, “light” is not particularly limited as long as it includes ultraviolet (UV) light, but examples include sunlight, UV (e.g., UV-B, UV-C, UV-A), and illumination light, and particularly refers to sunlight and UV. 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 That is the case.

[0020] Cellular senescence-associated secretion (SASP) factors are proteins produced and secreted in conjunction with cellular senescence, and include inflammatory cytokines, chemokines, extracellular matrix-degrading enzymes (Matrix metalloproteinases: MMPs), and growth factors. Examples of inflammatory cytokines include IL1B (Interleukin 1B), IL6 (Interleukin 6), and TNF-α (Tumor Necrosis Factor α); examples of chemokines include CCL20 (CC motif chemokine ligand 20) and CXCL8 (chemokine (CXC motif) ligand 8); examples of extracellular matrix-degrading enzymes include MMP1 (matrix metalloproteinase-1) and MMP3 (matrix metalloproteinase-3); and examples of growth factors include VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor), and HGF (hepatocyte growth factor). In this specification, the term SASP factors is not limited to the factors specifically listed above, but may refer to any SASP factors that can be secreted from cells due to skin damage or photoaging caused by light exposure.

[0021] The SASP factor inhibitor included as an active ingredient in the light exposure-induced skin damage prevention and / or treatment agent of the present invention may have the effect of suppressing at least one or more SASP factors selected from, for example, IL1B, CXCL8, MMP1, and CCL20. Furthermore, in addition to the SASP factors IL1B, CXCL8, MMP1, and CCL20 listed above, it may also have the effect of suppressing any of the SASP factors selected from IL6, TNF-α, CXCL8, MMP3, VEGF, PDG, and HGF. Examples of SASP factor inhibitors having such an effect include JAK (Janus kinase) inhibitors. JAK is a type of kinase, and there are types such as JAK1, JAK2, JAK3, and TYK2. JAK plays an important role in intracellular signal transduction. In particular, it is activated by cytokine binding. ATP (adenosine triphosphate) is used for JAK activity. JAK inhibitors inhibit JAK-mediated signal transduction by inhibiting the ATP binding site. Examples of JAK inhibitors used herein include baricitinib, upadacitinib, tofacitinib, peficitinib, filgotinib, abrocitinib, ritrecitinib, and delgocitinib. Curcumin (UPAC name: (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is an example of a drug that can suppress the SASP factor through a mechanism different from that of JAK inhibitors. Curcumin is a lipid-soluble polyphenol that is mainly found in high concentrations in turmeric and is also contained in cardamom, cloves, cumin, pepper, coriander, paprika, mace, etc. Many analogues of curcumin have been developed, and EF24 (3,5-bis(benzylidene)-4-piperidone, Cas No. 342808-40-6) is particularly preferred as a SASP factor inhibitor according to this specification.The above-mentioned JAK inhibitors, curcumin, and their analogues may be derivatives of the compound, pharmaceutically acceptable salts of the compound or its derivatives, or hydrates.

[0022] In aged skin, melanocytes are often detected as senescent cells, but keratinocytes are reported not to be detected (Victorelli et al., EMBO J 38, e101982.10.15252 / embj.2019101982.(2019), Waaijer et al., J Gerontol A Biol Sci Med Sci71, 1022-1028. 10.1093 / gerona / glv114. (2016)). Furthermore, previous studies have shown that melanocytes are resistant to UV-induced apoptosis, suggesting that melanocytes are a major component of UV-resistant senescent cells in aged skin (Fukumoto et al., Genes Cells 21, 185-199. 10.1111 / gtc.12330.(2016)). Subsequently, senescent melanocytes produce SASP factors such as IL6 (Interleukin 6) and IL8 (Interleukin 8) / CXCL8 (CXC motif ligand 8), which have been reported to induce paracrine signaling senescence in neighboring cells consisting of keratinocytes and fibroblasts (Victorelli et al., 2019). Chemicals that target senescent cells or the senescence process are called anti-aging agents, and there have been reports of senolytic agents that selectively remove senescent cells via apoptosis (Di Micco et al., Nat Rev Mol Cell Biol 22, 75-95. 10.1038 / s41580-020-00314-w (2021), Wang et al., J Biochemistry. 10.1093 / jb / mvae015. (2024)). The aforementioned chemical substances that target senescent cells or the aging process are senescent cell scavengers and have a potent effect of completely removing or killing senescent cells.

[0023] However, since the senescent cells exhibiting cellular senescence in the skin are melanocytes, the side effects of completely removing senescent melanocytes would be severe. There have been no reports on the prevention and / or treatment of skin damage caused by light exposure that focus on melanocyte senescence. Furthermore, there is a need for drugs that act with a mild effect rather than killing all damaged cells (senomorphic agents).

[0024] The preventive and / or therapeutic agent for light-induced skin disorders of the present invention is used as a pharmaceutical composition. A pharmaceutical composition means a composition suitable for administration in medical use and may contain one or more pharmaceutically acceptable carriers and / or pharmaceutically acceptable excipients in addition to a SASP factor inhibitor as an active ingredient. The pharmaceutical composition as a preventive and / or therapeutic agent of the present invention can be prepared by commonly used methods using pharmaceutical carriers, excipients, etc., commonly used in the art. Administration may be by oral administration in the form of tablets, pills, capsules, granules, powders, liquids, etc., or by parenteral administration in the form of injections such as intra-articular, intravenous, intramuscular, etc., suppositories, eye drops, eye ointments, transdermal solutions, ointments, transdermal patches, transmucosal solutions, transmucosal patches, inhalants, etc. As a solid composition for oral administration according to the present invention, tablets, powders, granules, sustained-release agents, etc., can be used. In such a solid composition, one or more active ingredients are mixed with at least one pharmaceutically acceptable carrier and / or pharmaceutically acceptable excipient.

[0025] Examples of pharmaceutically acceptable carriers as used herein include various organic or inorganic carrier substances commonly used as pharmaceutical materials, such as excipients, lubricants, binders, and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, and buffering agents in liquid formulations. Furthermore, as needed, appropriate amounts of additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, and wetting agents may also be used.

[0026] Examples of pharmaceutically acceptable excipients as used herein include isotonic agents, bulking agents, preservatives and bactericides, binders, antioxidants, solubilizers, solubilizers, suspending agents, fillers, pH adjusters, stabilizers, absorption enhancers, release rate regulators, colorants, plasticizers, and adhesives. Specifically, examples include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, and light anhydrous silicic acid. Examples of lubricants include magnesium stearate, calcium stearate, talc, and colloidal silica. Examples of binders include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, and sodium carboxymethylcellulose.

[0027] Examples of disintegrants include starch, carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, sodium carboxymethyl starch, and L-hydroxypropylcellulose. Examples of solvents include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and olive oil. Examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate. Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzethonium chloride, and glyceryl monostearate; and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. Examples of isotonic agents include glucose, D-sorbitol, sodium chloride, glycerin, and D-mannitol. Examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates. Examples of preservatives include para-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Examples of antioxidants include sulfites, ascorbic acid, and α-tocopherol.

[0028] The dosage of the SASP factor inhibitor as the active ingredient varies depending on the patient, route of administration, symptoms, weight, age, etc., and can be selected as appropriate. The SASP factor inhibitor as the active ingredient may also be administered in combination with other drugs as appropriate.

[0029] The present invention also extends to a screening method for agents that prevent and / or treat skin damage caused by light exposure. The screening method involves adding candidate substances to a melanocyte culture system and selecting substances that improve the cellular damage of the melanocytes. Melanocytes are responsible for protecting themselves from light through melanin production, but the expression of SASP factors increases in melanocytes themselves when exposed to light such as UV. Therefore, for example, substances that can suppress the expression of SASP factors and / or the activity of SASP factors can be selected. In the screening, substances that improve cellular damage of melanocytes can also be selected by comparing the suppression of SASP factor expression or the suppression of SASP factor activity in melanocytes exposed to light and / or stimulation equivalent to light exposure with the case where there is no light exposure and / or stimulation equivalent to light exposure.

[0030] Considering that DNA repair for DNA damage caused by light exposure and DNA damage caused by cisplatin treatment are both types of DNA damage that form crosslinks within the DNA strand, and furthermore, that the repair mechanism is the same nucleotide excision repair (NER) pathway, and that many XP patients are caused by mutations in NER-related genes (Lehmann, et al., Orphanet J Rare Dis 6, 70. 10.1186 / 1750-1172-6-70 (2011)), the inventors developed a screening method using cisplatin as a stimulus equivalent to the aforementioned light exposure (see Figures 1A and 1B). This screening method offers high reproducibility and allows for adjustment of the cisplatin dosage, so cisplatin can be used as an aging inducer in place of light exposure such as ultraviolet (UV) treatment. By adding candidate substances to a culture system of melanocytes treated with cisplatin and culturing them, candidate substances that can suppress the expression of SASP factors and / or the activity of SASP factors can be selected, thereby screening for SASP factor inhibitors that can be used as preventive and / or therapeutic agents for skin damage caused by light exposure according to the present invention. The amount of cisplatin that can be used in the screening method of the present invention is not particularly limited, as long as it is at a concentration that can reflect the light exposure to the cells, but can be appropriately set in the range of 0.001 to 10 μM, preferably in the range of 0.01 to 5 μM, more preferably in the range of 0.1 to 3 μM, and even more preferably in the range of 0.5 to 1 μM.

[0031] The selection of candidate substances capable of suppressing the expression of SASP factors and / or the activity of SASP factors can be carried out by methods such as preparing RNA (e.g., total RNA, mRNA) fractions from cells used in screening or their culture supernatants, and detecting the transcripts of genes of factors related to SASP factors contained in the fractions; detecting them by immunological methods using antibodies that specifically recognize SASP factors; or detecting them by mass spectrometry. The preparation of RNA fractions can be done using methods that are already known or any methods that may be developed in the future. For example, it can be done using known methods such as guanidine-CsCl ultracentrifugation or AGPC, and high-purity total RNA can be prepared quickly and easily from minute samples using commercially available RNA extraction kits (e.g., RNeasy Mini Kit; QIAGEN, etc.). Methods for detecting the transcripts of SASP factor genes in RNA fractions include, for example, PCR (RT-PCR, competitive PCR, real-time PCR, etc.) and hybridization (Northern blotting, dot blotting, DNA chip analysis, etc.). Immunological methods include, for example, Western blotting, ELISA, FIA, and RIA. In this specification, antibodies include, but are not limited to, native antibodies such as polyclonal antibodies and monoclonal antibodies (mAbs), chimeric antibodies that can be produced using genetic engineering techniques, humanized antibodies and single-chain antibodies, and their binding fragments. Preferably, the antibody is a polyclonal antibody, a monoclonal antibody, or a binding fragment thereof. A binding fragment means a region of the aforementioned antibody that has 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. Preferably, it is IgG or IgM, and more preferably IgG considering the ease of purification, etc. Furthermore, in the present invention, a commercially available anti-SASP factor antibody or a kit containing an anti-SASP factor antibody may be used as the antibody that specifically recognizes the SASP factor.

[0032] Further evaluation methods include assessing the survival rate of melanocytes used in screening, or evaluating the cell migration ability of melanocytes, to select substances that improve melanocyte cell damage.

[0033] Candidate substances used in the screening method of the present invention may be high-molecular-weight compounds or low-molecular-weight compounds. Examples of high-molecular-weight compounds are not particularly limited, but include 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, and may also be substances containing both low-molecular-weight and high-molecular-weight compounds.

[0034] Cultureable melanocytes used for screening can be produced, 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 cells (EG cells), but iPS cells are preferred. Methods for inducing differentiation from pluripotent stem cells into melanocytes can be any known methods or methods to be developed in the future. Specifically, the methods described in the examples or the method described in Japanese Patent No. 6624916 can be applied. Pluripotent stem cells may be iPS cells that have already been established, or they can be produced by any known methods or methods to be developed in the future. Specifically, examples include the method described in the examples, Cell, 131(5): 861-872 (2007), Cell, 126(4): 663-676 (2006), and the method described in Non-Patent Literature 6. Pluripotent stem cells can be produced, for example, from cells derived from XP patients. Methods for producing pluripotent stem cells derived from XP patients can be any known methods or any methods developed in the future. Specifically, the methods described in the examples or the method described in Japanese Patent Application Publication No. 2021-17405 can be applied. The culture environment can be any known environment and is not particularly limited, but generally cells can be cultured under conditions of 37±1℃ and 5±1% CO2.

[0035] Regarding the melanocytes used for screening and the melanocytes prepared from pluripotent stem cells derived from XP patients, the mechanism of action can be analyzed by analyzing the expression of SASP factors when the selected drug is treated with cells, by light exposure or cisplatin induction. For example, DNA repair for senescent cells by light exposure or cisplatin induction, such as DNA damage caused by light exposure and DNA damage caused by cisplatin treatment, is all carried out through the nucleotide excision repair (NER) pathway. Therefore, it is considered that the cytotoxicity of cisplatin-treated cells reflects the cytotoxicity by light exposure (see Fig. 1A). And it is considered that the NER pathway is suppressed after administration of the SASP factor inhibitor.

[0036] The present invention also extends to a method for examining skin disorders, including a step of detecting an abnormality of SASP factors in a skin specimen collected from a subject. The step of detecting an abnormality of SASP factors is not particularly limited as long as it is a step of detecting an abnormality of SASP factors by a method capable of detecting the abnormality. For example, a step of preparing an RNA (e.g., total RNA, mRNA) fraction from a skin specimen of a subject and detecting a transcript of a gene of a factor related to the SASP factor contained in the fraction, a step of detecting by an immunological technique using an antibody that specifically recognizes the SASP factor, a step of detecting by a mass spectrometry method, etc. can be mentioned. Specifically, in the inspection method of the present invention, the abnormality of the SASP factor can be detected by using the following 1) or 2). 1) A nucleic acid probe or nucleic acid primer capable of specifically detecting a transcript of the SASP factor gene, 2) An antibody capable of specifically recognizing the SASP factor.

[0037] In the inspection method of the present invention, when the SASP factor detected from the skin specimen of the subject is higher than that of the control, it can be determined that the possibility of the above-mentioned skin disorder is high. Therefore, in the inspection method of the present invention, after the above 1) and 2), when the SASP factor is detected at a higher value than the control in the subject, it can be determined that the subject has a skin disorder or is likely to have a skin disorder.

[0038] In the inspection method of the present invention, the "subject" includes not only those with unknown skin disorders or suspected skin disorders, but also those with unknown or suspected XP, as well as those with skin disorders.

[0039] The present invention further extends to a kit for skin disorder inspection, which contains substances capable of detecting abnormalities of SASP factors. Examples of substances capable of detecting abnormalities of SASP factors include nucleic acid probes or nucleic acid primers capable of specifically detecting transcription factors of SASP factor genes, antibodies capable of specifically recognizing SASP factors, and the like. In this specification, "primers, probes, etc." can also be modified as long as their functions are not significantly impaired. Examples of modifications include labeling substances, fluorescent dyes, enzymes, proteins, radioisotopes, chemiluminescent substances, biotin, and the like. Primers, probes, etc. can also be used after being immobilized on an arbitrary solid phase. In this specification, the "solid phase" is not particularly limited as long as it can immobilize polynucleotides, and examples include glass plates, nylon membranes, microbeads, silicon chips, capillaries, or other substrates. The kit of the present invention may further contain reaction buffers, dNTPs, heat-resistant DNA polymerases, competitor nucleic acids, fluorescent reagents, competitor antibodies, labeled secondary antibodies, blocking solutions, and the like.

[0040] The present invention also extends to a method for inspecting skin disorders caused by light exposure. By detecting abnormalities of SASP factors in skin specimens collected from a subject, skin disorders can be predicted in advance. Abnormalities of SASP factors include enhanced gene expression of one or more SASP factors selected from IL1B, IL6, CXCL8, MMP1, and CCL20.

[0041] To facilitate the understanding of the present invention, reference examples and examples are shown below to specifically explain the present invention. Needless to say, the present invention is not limited thereto. Note that the "reference examples" show the experimental content leading to the completion of the present invention, and the method for preparing iPS cell-derived melanocytes, which is important in the screening and effect confirmation experiments of preventive and / or therapeutic agents for skin disorders of the present invention.

[0042] (Reference Example 1) Production of iPS cell-derived melanocytes In this reference example, after producing XP disease-specific pigment stem cells, they were differentiated into melanocytes (MC) to produce iPS cell-derived melanocytes.

[0043] (Creation of XP disease-specific pigment stem cells) For the establishment of human iPS cell lines, normal fibroblasts derived from human skin (TIG-120 cells; JCRB0542) and fibroblasts derived from XP-A patients (XP3OS) were used. Human iPS cell lines were established from these two fibroblast cell lines using the CytoTune-iPS2.0SendaiReprogramming Kit (ID Pharma Co., Ltd.) (Non-patent Literature 6). Subsequently, human iPS cells were seeded on non-adherent culture dishes (50-200 cells / ml) and cultured for two weeks in primate ES / iPS cell medium to form embryoid bodies. Subsequently, embryoid bodies were seeded on adherent culture dishes coated with fibronectin, and melanocytes were cultured in 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 nMTPA (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). Cells were cultured in a medium supplemented with Supplement (HMGS) (Invitrogen) to induce differentiation into melanocytes. During this time, the medium was changed every 2-3 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-inducing factors. Seven days after differentiation induction was stopped, pigment stem cells were observed in the medium.

[0044] (Induction of differentiation into XP disease-specific melanocytes) The pigment stem cells described above differentiated into melanocytes (MCs) approximately one week after the addition of 3 μM CHIR99021 every two days. Hereafter, cells differentiated into melanocytes from iPS cells derived from fibroblasts of XP patients with a single nucleotide mutation in the XPA gene will be referred to as XP-iMC, and cells differentiated into melanocytes from iPS cells derived from normal human skin fibroblasts will be referred to as the healthy control line HC-iMC.

[0045] (Example 1) Effects of UV-B irradiation on HC-iMC and XP-iMC In this example, HC-iMC and XP-iMC derived from XP patients with single nucleotide mutations in the XPA gene were cultured according to the protocol shown in Figure 2A, with a UV-B irradiation of 15 mJ / cm³ on day 1 of culture. 2 We performed UV-B irradiation and confirmed the induction of cellular senescence using UV-B irradiation-induced DNA damage markers, SA-β-gal (senescence-related acid β-galactosidase) as an aging marker, and the gene expression levels of SASP factors.

[0046] For both HC-iMC and XP-iMC, the DNA damage marker γH2AX was visualized using immunofluorescence microscopy, and the effects of UV-B irradiation were examined. As a result, γH2AX staining was observed in XP-iMC when irradiated with UV-B, confirming that DNA damage had occurred (Figure 2B).

[0047] XP-iMCs were stained with SA-β-gal, and SA-β-gal-positive cells were visually detected to examine the effect of UV-B irradiation on the induction of cellular senescence. The results showed that SA-β-gal staining was observed when UV-B irradiation was performed (Figure 2C). Based on these results, the proportions of two populations—SA-β-gal-positive senescent cells and SA-β-gal-negative non-senescent cells—were estimated. The results showed that the SA-β-gal positivity rate in UV-B irradiated XP-iMCs was over 50% (Figure 2D).

[0048] Furthermore, the expression levels of IL1B, IL6, CXCL8, MMP1, MMP3, and TNF-α, among the SASP factors, were measured by RT-qPCR using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, KK4600) and the 7300 Real-Time PCR System (Applied Biosystems) in XP-iMCs irradiated with UV-B. The expression levels of the target genes were normalized using the mRNA levels of β2-microglobulin (B2M) or the GAPDH gene as an internal control. When the expression levels of each gene in XP-iMCs not irradiated with UV-B were set to 1 and observed relatively, the gene expression levels of IL1B, IL6, CXCL8, MMP1, MMP3, and TNF-α were all high, confirming upregulation of SASP factors (Figure 2E).

[0049] The sequences of the RT-qPCR primers for each gene are as follows: • B2M forward: 5'-GGCATTCCTGAAGCTGACA-3' (SEQ ID NO: 1) • B2M reverse: 5'-CTTCAATGTCGGATGGATGAAAC-3 (SEQ ID NO: 2) • GAPDH forward: 5'-TGTTGCCATCAATGACCCCTT-3' (SEQ ID NO: 3) • GAPDH reverse: 5'-CTCCACGACGTACTCAGCG-3' (SEQ ID NO: 4) • IL1B forward: 5'-AGCTCGCCAGTGAAATGATGG-3' (SEQ ID NO: 5) • IL1B reverse: 5'-GTCCTGGAAGGAGCACTTCAT-3' (SEQ ID NO: 6) • IL6 forward: 5'-ACATCCTCGACGGCATCTCA-3' (SEQ ID NO: 7) • IL6 reverse: 5'-TCACCAGGCAAGTCTCCTCA-3' (SEQ ID NO: 8) • CXCL8 forward: 5'-GTTTTTGAAGGGCTGAGAATTC-3' (SEQ ID NO: 9) ・CXCL8 reverse: 5'-CCCTACAACAGACCCACAATAC-3' (SEQ ID NO: 10) ・MMP1 forward: 5'-AATAGTGGCCCAGTGGTTGA-3' (SEQ ID NO: 11) ・MMP1 reverse: 5'-GGCTGCTTCATCACCTTCAG-3' (SEQ ID NO: 12) ・MMP3 forward: 5'-CACTCACAGACCTGACTCGGTT-3' (SEQ ID NO: 13) ・MMP3 reverse: 5'-AAGCAGGATCACAGTTGGCTGGG-3' (SEQ ID NO: 14) ・TNF-α forward: 5'-ATCTTCTCGAACCCCGAGTGA-3' (SEQ ID NO: 15) ・TNF-α reverse: 5'-CGGTTCAGCCACTGGAGCT-3' (SEQ ID NO: 16)

[0050] The expression of IL6, MMP1, and MMP3 in UV-B irradiated and unirradiated XP-iMCs was examined by Western blot analysis. The results showed increased expression of SASP factors (Figure 2F).

[0051] (Example 2) Effects of cisplatin on HC-iMC and XP-iMC In this example, HC-iMC and XP-iMC were cultured in a culture system containing 1 μM cisplatin on day 1 of culture according to the protocol shown in Figure 3A. The induction of cellular senescence using DNA damage markers, SA-β-gal and SPiDER-βGal as senescence markers, and the gene expression levels of SASP factors were confirmed. Each measurement method followed the method of Example 1.

[0052] For both HC-iMC and XP-iMC, the DNA damage marker γH2AX was visualized using immunofluorescence microscopy to examine the effects of cisplatin presence or absence. As a result, in the XP-iMC system treated with cisplatin, γH2AX staining was observed on day 5 of culture, confirming that DNA damage had occurred (Figure 3B).

[0053] SA-β-gal positive cells were stained from XP-iMCs, and the effect of cisplatin addition was examined. As a result, SA-β-gal staining was observed when cisplatin was added (Figure 3C). Quantification from the staining image of SPiDER-βGal positive cells showed that the SA-β-gal positivity rate of XP-iMCs was over 80% after cisplatin addition (Figure 3D).

[0054] Furthermore, the expression levels of the SASP factors IL1B, CXCL8, MMP1, and CCL20 were measured in XP-iMCs in a culture system supplemented with cisplatin. The sequences of the RT-qPCR primers for the IL1B, CXCL8, and MMP1 genes are as shown in Example 1, and the sequence of the RT-qPCR primer for the CCL20 gene is as follows: • CCL20 forward: 5'-CTCCTGGCTGCTTTGATGTC-3' (SEQ ID NO: 17) • CCL20 reverse: 5'-TGCTTGCTGCTTCTGATTCG-3' (SEQ ID NO: 18)

[0055] When the expression levels of each gene in XP-iMC in a system without cisplatin were set to 1 and observed relatively, the gene expression levels of IL1B, CXCL8, MMP1, and CCL20 were all elevated, confirming increased expression of SASP factors (Figure 3E).

[0056] From the results of Examples 1 and 2, for HC-iMC and XP-iMC, regarding the DNA damage marker, the positive rate of SA-β-gal, and the gene expression level of SASP factors when cultured in a culture system containing 15 mJ / cm 2 of UV-B irradiation and 1 μM cisplatin, it was confirmed that similar results were shown.

[0057] (Example 3) Screening of candidate substances by melanocytes treated with cisplatin In this example, for XP-iMC, screening was performed on a total of 15 substances including each substance known as a senolytic agent as a candidate substance for the preventive and / or therapeutic agent for skin disorders of the present invention according to the protocol shown in FIG. 4A. Induction of cellular senescence with SPiDER-βGal (or βGal) as a senescence marker when cultured for 6 days in a culture system containing 1 μM cisplatin and each candidate substance on the first day of culture was confirmed. Induction of cellular senescence was stained with the cellular senescence detection kit SPiDER-βGal (Tongrentang, SG03) by the same method as in Example 1, and BD FACSCanto TM II flow cytometry system (BD Biosciences) was used to detect and confirm positive cells of SA-β-gal, which is a senescence-related marker.

[0058] As a result, it was confirmed that the induction of cellular senescence by cisplatin was suppressed by baricitinib, EF24 (3,5-bis(benzylidene)-4-piperidone), curcumin, and upadacitinib. On the other hand, it was also confirmed that there are substances that cannot suppress the induction of cellular senescence by cisplatin even though they are known substances as senolytic agents.

[0059] (Example 4) Effects of JAK inhibitors, curcumin, and curcumin analogs on cellular senescence In Example 3, each candidate substance that suppressed the induction of cellular senescence by cisplatin was used to confirm its inhibitory effect on the induction of senescent cells having SPiDER-βGal (or βGal) activity. First, it was confirmed that XP-iMCs that were not treated with either cisplatin or any of the candidate substances had a high proportion of non-senescent cells (Figure 5A), while XP-iMCs treated with cisplatin alone had a high proportion of senescent cells (Figure 5B). On the other hand, in XP-iMCs treated with EF24, a curcumin analog that suppresses the induction of cellular senescence by cisplatin, or with baricitinib or upadacitinib, both JAK inhibitors, in addition to cisplatin, it was confirmed that the number of senescent cells possessing SPiDER-βGal (or βGal) activity was significantly reduced compared to when treated with cisplatin alone (Figure 5C).

[0060] (Example 5) Effects of JAK inhibitors, curcumin, and curcumin analogs on skin disorders XP-iMCs treated with cisplatin and the curcumin analog EF24, curcumin, or the JAK inhibitor baricitinib or upadacitinib were stained with SA-β-gal and the cell morphology was observed. As a result, when treated with cisplatin alone, a large number of SA-β-gal positive cells were observed. On the other hand, when treated with the above candidate substances, it was confirmed that the staining intensity of SA-β-gal was low. However, the cell morphology could be confirmed, suggesting that the above substances do not completely remove senescent cells (senolytic) but rather act senomorphically (inhibit senescent cells) (Figure 6A). Upregulation of the genes for the SASP factors IL1B, CXCL8, MMP1, and CCL20 was observed in XP-iMCs in a culture system supplemented with cisplatin. However, suppression of the upregulation of the genes for each SASP factor was observed by applying one of the above-mentioned JAK inhibitors, curcumin, or curcumin analogs (Figure 6B).

[0061] Based on these results, JAK inhibitors, curcumin, and curcumin analogs can prevent and / or treat skin damage caused by light exposure.

[0062] (Example 6) Expression of each SASP factor when light irradiation conditions are changed In this example, XP-iMCs derived from XP patients with a single nucleotide mutation in the XPA gene were cultured at 30 J / m² on day 1 of culture. 2 or 40 J / m 2 We confirmed the induction of cellular senescence, using SA-β-gal as a senescence marker, after UV-B irradiation and culture for 3 or 6 days. Cells that were not irradiated with UV-B were used as a control. As a result, it was observed that the number of SA-β-gal-positive cells increased in a dose-dependent manner, and also increased in a culture duration-dependent manner (Figure 7A, B).

[0063] Furthermore, the expression levels of the SASP factors IL1B, IL6, CXCL8, MMP1, MMP3, and CCL20 were confirmed by RT-qPCR under varying UV-B irradiation conditions. As a result, compared to the control group without UV-B irradiation, the expression level was 30 J / m² on day 1 of culture in the affected group. 2 or 40 J / m 2 When cells were irradiated with UV-B and cultured for 3 or 6 days, high levels of expression were observed for all SASP factors (Figure 7C). In particular, very high levels of SASP factor expression were observed on day 3 of culture, regardless of the irradiation dose.

[0064] (Example 7) Effects of JAK inhibitors, curcumin, and curcumin analogs on skin damage caused by light irradiation. In this example, XP-iMCs derived from XP patients with a single nucleotide mutation in the XPA gene were cultured according to the protocol shown in Figure 8A, with a dose of 40 J / m² on day 1 of culture. 2 We performed UV-B irradiation and confirmed the induction of cellular senescence using the DNA damage marker and SA-β-gal as the aging marker three days after UV-B irradiation.

[0065] XP-iMCs treated with UV-B irradiation only and no other drug treatments were used as a control. Each XP-iMC treated with EF24, curcumin, baricitinib, or upadacitinib was then stained with SA-β-gal, and SA-β-gal-positive cells were visually detected (Figure 8B). The results showed that the number of SA-β-gal-positive cells was significantly lower in the cells treated with each drug compared to the control XP-iMCs. Specifically, while the SA-β-gal positivity rate in the control XP-iMCs was over 50%, it was significantly suppressed to 40% or less in the XP-iMCs treated with each drug (Figure 8C).

[0066] As a result, JAK inhibitors, curcumin, and curcumin analogs, which are SASP factor inhibitors, were observed to have a clear improvement effect on skin damage caused by light irradiation.

[0067] (Example 8) Effects of JAK inhibitors and curcumin analogs (EF24) on in vivo skin damage caused by light irradiation In this example, the effects of JAK inhibitors and curcumin analogs (EF24) on in vivo skin damage caused by light irradiation were confirmed. Delgocitinib ointment 0.5% was used, and approximately 0.1 g was applied. EF24 was used as a curcumin analog, dissolved in DMSO at a concentration of 100 μM, and 50 μL was applied.

[0068] 6 kJ / m³ for mice (Albino hairless mice (Hos:HR-1), 10-13 weeks old, etc.) 2 The mice were irradiated once with UV-B on day 0. Simultaneously with irradiation, approximately 0.1 g of delgocitinib ointment 0.5% or 50 μL of EF24 100 μM was applied to the skin of the mice. Application was performed twice daily from day 0 to day 3. Experiments were conducted in two control groups: one without UV-B irradiation and another in the vehicle group that received UV-B irradiation and was treated only with petrolatum or DMSO (Figure 9).

[0069] 6 kJ / m³ for mice 2The mice were irradiated once with UV-B light on day 0. Simultaneously with irradiation, approximately 0.1 g of 0.5% delgocitinib ointment was applied to the skin of the mice. Application was performed twice daily from day 0 to day 3. A control group was set up without UV-B irradiation, and a vehicle group was set up with UV-B irradiation and only petrolatum applied. Representative images of the back skin (2 × 2 cm area) of the experimental mice are shown (Figure 10A). In the control group, there were no changes in the skin at 0, 24, 48, and 72 hours. On the other hand, in the vehicle group, erythema was observed from 24 hours after irradiation and showed a tendency to worsen towards 72 hours. After 72 hours, the erythema had spread to the entire irradiation field, and scaling was also observed. Topical application of delgocitinib significantly suppressed erythema formation. Figure 10B shows the changes in erythema levels measured using a portable skin colorimeter (DCC1001 (Delfin Technologies)). The erythema levels also quantified the visually observed trend, and the erythema levels in the delgocitinib topical group after 72 hours were significantly lower than those in the vehicle group, confirming the suppression of erythema.

[0070] 6 kJ / m³ for mice 2The mice were irradiated once with UV-B on day 0. Simultaneously with irradiation, 50 μL of EF24 100 μM was applied to the skin of the mice. Application was performed twice a day from day 0 to day 3. A control group was set up without UV-B irradiation, and a vehicle group was set up with UV-B irradiation and only DMSO applied. Representative images of the back skin (2 × 2 cm area) of the mice after the experiment are shown (Figure 11A). In the control group, there were no changes in the skin at 0, 24, 48, and 72 hours. On the other hand, in the vehicle group, erythema was observed from 24 hours after irradiation and showed a tendency to worsen towards 72 hours. After 72 hours, the erythema had spread to the entire irradiation field, and scaling was also observed. Topical application of EF24 significantly suppressed erythema formation. Figure 11B shows the changes in erythema levels measured using a portable skin colorimeter (DCC1001 (Delfin Technologies)). The erythema levels also quantified the visually observed trend, and the erythema levels in the EF24 topical application group after 72 hours were significantly lower than those in the vehicle group, confirming the suppression of erythema.

[0071] (Example 9) Effects of JAK inhibitors and curcumin analogs (EF24) on the expression of each SASP factor in vivo by light irradiation In this example, the effects of JAK inhibitors and curcumin analogs (EF24) on the expression of SASP factors in vivo by light irradiation were confirmed.

[0072] In this example, mice were injected with 6 kJ / m³ using the experimental method of Example 8. 2The mice were irradiated once with UV-B on day 0. Simultaneously with irradiation, approximately 0.1 g of delgocitinib ointment 0.5% or 50 μL of EF24 100 μM was applied to the skin of the mice. Application was performed twice daily from day 0 to day 3. Experiments were conducted in a control group (no UV-B irradiation) and a vehicle group (UV-B irradiation with only petrolatum or DMSO application). Subsequently, 2 cm × 2 cm skin samples were collected from the mice (Figure 12A). The expression levels of the p21, Il1b, Mmp1a, Cxcl1, and Tnf genes were confirmed in the collected skin samples by RT-qPCR using the same method as in Example 1. UV-B irradiation was observed to increase the expression of the aging marker p21 and the SASP factors Il1b, Mmp1a, Cxcl1, and Tnf in the mouse skin. These increased expression levels were suppressed by topical application of delgocitinib.

[0073] The sequences of the RT-qPCR primers for each gene are as follows: • p21 Forward: 5'-TCGCTGTCTTGCACTCTGGTGT-3' (SEQ ID NO: 19) • p21 Reverse: 5'-CCAATCTGCGCTTGGAGTGATAG-3' (SEQ ID NO: 20) • Cxcl1 Forward: 5'-TCCAGAGCTTGAAGGTGTTGCC-3' (SEQ ID NO: 21) • Cxcl1 Reverse: 5'-AACCAAGGGAGCTTCAGGGTCA-3' (SEQ ID NO: 22) • Il1b Forward: 5'-TGGACCTTCCAGGATGAGGACA-3' (SEQ ID NO: 23) • Il1b Reverse: 5'-GTTCATCTCGGAGCCTGTAGTG-3' (SEQ ID NO: 24) • Mmp1a Forward: 5'-AGGAAGGCGATATTGTGCTCTCC-3' (SEQ ID NO: 25) • Mmp1a Reverse: 5'-TGGCTGGAAAGTGTGAGCAAGC-3' (SEQ ID NO: 26) ・Tnfα Forward: 5'-GGTGCCTATGTCTCAGCCTCTT-3' (SEQ ID NO: 27) ・Tnfα Reverse: 5'-GCCATAGAACTGATGAGAGGGAG-3' (SEQ ID NO: 28)

[0074] The present invention's SASP factor inhibitor, as an active ingredient, is a preventive and / or therapeutic agent for skin damage caused by photoexposure, and is highly useful as it can be applied not only to XP patients but also to many skin diseases caused by photoaging, for example. It is particularly useful for XP patients, as effective treatment methods have not yet been established. Furthermore, by treating melanocytes with cisplatin, it is possible to screen for preventive and / or therapeutic agents for skin damage that focus on SASP factors and melanocytes. The present invention's SASP factor inhibitor, as an active ingredient, is a preventive and / or therapeutic agent for skin damage caused by photoexposure, and because it acts senomorphically (inhibits senescent cells) rather than senolytically (completely removes senescent cells), it acts gently and can be used preventively, making it very useful.

Claims

1. A preventive and / or therapeutic agent for skin damage caused by light exposure, comprising an inhibitor of cellular senescence-associated secretory factors (SASP factors) as an active ingredient.

2. The prophylactic and / or therapeutic agent according to claim 1, wherein the SASP factor is one or more selected from IL1B (Interleukin 1B), IL6 (Interleukin 6), CXCL8 (chemokine (CXC motif) ligand 8), MMP1 (matrixmetalloproteinase-1), MMP3 (matrixmetalloproteinase-3), and CCL20 (CC motif chemokine ligand 20).

3. The preventive and / or therapeutic agent according to claim 1, wherein the SASP factor inhibitor as the active ingredient has an inhibitory effect on one or more SASP factors selected from IL1B, IL6, CXCL8, MMP1, MMP3, and CCL20.

4. The preventive and / or therapeutic agent according to claim 1, wherein the SASP factor inhibitor is a JAK (Janus kinase) inhibitor.

5. The prophylactic and / or therapeutic agent according to claim 4, wherein the JAK inhibitor is one or more selected from the group consisting of baricitinib, upadacitinib, tofacitinib, peficitinib, filgotinib, abrocitinib, ritrecitinib, and delgocitinib.

6. The preventive and / or therapeutic agent according to claim 1, wherein the SASP factor inhibitor is one or more selected from the group consisting of curcumin and curcumin analogs.

7. The preventive and / or therapeutic agent according to claim 6, wherein the curcumin analog is EF24.

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

9. The preventive and / or therapeutic agent for a skin disorder according to claim 8, wherein the skin disorder caused by the light exposure is skin cancer due to photoaging.

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

11. A screening method for preventive and / or therapeutic agents for skin disorders, characterized by adding candidate substances to a culture system of melanocytes exposed to light and / or a culture system of melanocytes stimulated with cisplatin, and selecting substances that can suppress the expression of SASP factor secreted from the melanocytes or suppress the activity of SASP factor.

12. The screening method according to claim 11, wherein the SASP factor is one or more selected from IL1B, IL6, CXCL8, MMP1, MMP3, and CCL20.

13. The screening method according to claim 11, wherein the melanocyte is a melanocyte derived from pluripotent stem cells.

14. The screening method according to claim 13, 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.

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