Pharmaceutical composition for treating hereditary symmetrical pigmentation disorder, model mouse for hereditary symmetrical pigmentation disorder, and method for screening compounds for treating hereditary symmetrical pigmentation disorder

Abstract

Provided are a pharmaceutical composition for treating dyschromatosis symmetrica hereditaria, a dyschromatosis symmetrica hereditaria model mouse, and a screening method for a compound for treating dyschromatosis symmetrica hereditaria. This pharmaceutical composition for treating dyschromatosis symmetrica hereditaria contains, as an active ingredient, at least one compound selected from the group consisting of JAK inhibitors and STAT inhibitors.

Description

[Technical Field]

[0001] The disclosure of the present application relates to a pharmaceutical composition for treating hereditary symmetrical pigmentation disorders, a mouse model for hereditary symmetrical pigmentation disorders, and a method for screening a compound for treating hereditary symmetrical pigmentation disorders. [Background technology]

[0002] Dyschromatosis symmetrica hereditaria (DSH) is a pigmentary disorder characterized by the appearance of pigmented and white patches on the dorsum of both hands and feet. In humans, the causative gene is the gene encoding double-stranded RNA-specific adenosine deaminase 1 (ADAR1 or ADAR). Mouse double-stranded RNA-specific adenosine deaminase 1 is referred to as "Adar1." Model cells, regardless of their origin, may also be referred to as "Adar1." It is known that converting adenosine in RNA to inosine results in the production of proteins that are not consistent with the DNA information. It has been shown that heterozygous mutations in the ADAR1 gene cause DSH (see Non-Patent Document 1).

[0003] DSH rarely causes neurological complications, but does not cause serious, life-threatening symptoms. However, because it develops in childhood and symptoms appear on exposed areas, it can cause significant psychological stress for patients due to cosmetic issues. Therefore, there is a need for the establishment of therapeutic drugs and treatment methods, but there are currently no effective therapeutic drugs or treatment methods.

[0004] In research into disease treatment, it is common to prepare model animals such as mice that have the disease, use the model animals to examine the pathology of the disease, and administer test compounds to the model animals to screen for therapeutic compounds. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Yoshinori Miyamura et al., “Mutations of the RNA-Specific Adenosine Deaminase Gene (DSRAD) Are Involved in Dyschromatosis Symmetrica Hereditaria”, Am.J.Hum.Genet.73:693-699,2003 Summary of the Invention [Problem to be solved by the invention]

[0006] However, simply knocking out Adar1 does not result in mice displaying the phenotype seen in DSH patients (hereinafter sometimes referred to as the "DSH phenotype"), making it unusable for DSH research.

[0007] The disclosure of the present application has been made to solve the above-mentioned problems, and as a result of intensive research, the following new findings have been found: (1) DSH can be treated by using a JAK inhibitor or a STAT inhibitor as an active ingredient; (2) when a heterozygous Adar1 knockout mouse, in which Adar1 function has been lost throughout the body, is administered a specific substance or a gene that produces the substance is expressed, the mouse exhibits a DSH phenotype; (3) a homozygous Adar1 knockout mouse, in which Adar1 function has been lost specifically in melanocytes, exhibits a DSH phenotype; and (4) by using the knockout mouse (hereditary symmetrical pigmentary disorder model mouse) or cells (model cells) in which Adar1 function has been reduced or lost, screening for compounds for treating DSH can be performed.

[0008] That is, an object of the disclosure of the present application is to provide a pharmaceutical composition for treating hereditary symmetrical pigmentation disorders, a model mouse for hereditary symmetrical pigmentation disorders, and a method for screening a compound for treating hereditary symmetrical pigmentation disorders. [Means for solving the problem]

[0009] The disclosure of the present application relates to a pharmaceutical composition for treating hereditary symmetrical pigmentation disorders, a model mouse for hereditary symmetrical pigmentation disorders, and a method for screening a compound for treating hereditary symmetrical pigmentation disorders, which are shown below.

[0010] (1) A pharmaceutical composition for treating hereditary symmetric pigmentation disorder, comprising as an active ingredient at least one compound selected from the group consisting of a JAK inhibitor and a STAT inhibitor. (2) The JAK inhibitor is selected from Tofacitinib, Itacinitib, Solcitinib, AZD1480, Ruxolitinib, JAK3-IN-6, Curcumol, Peficitinib, Cerdulatinib, FM-381, Filgotinib, and Delgocitinib; The STAT inhibitor is selected from napabucasin, ochromycinone, fludarabine, nifuroxazide, C188-9, and AS1517499; A pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to (1) above. (3) The JAK inhibitor is Delgocitinib. A pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to (2) above. (4) The JAK inhibitor is Ruxolitinib. A pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to (2) above. (5) The JAK inhibitor is tofacitinib. A pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to (2) above. (6) The JAK inhibitor is peficitinib. A pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to (2) above. (7) The JAK inhibitor is cerdulatinib. A pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to (2) above. (8) A heterozygous Adar1 knockout mouse in which the function of Adar1 (Adenosine Deaminase Acting on RNA1) is lost throughout the body, exhibiting a phenotype of variegated skin and / or hair color; Adar1 heterozygous knockout mice. (9) The Adar1 p150 is knocked out. The Adar1 heterozygous knockout mouse described in (8) above. (10) Adar1 KO:Dct-LacZ Tg mice were obtained by crossing Adar1 heterozygous knockout mice, which lack the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout the body, with Dct-LacZ transgenic mice. (11) The Adar1 p150 is knocked out. Adar1 KO:Dct-LacZ Tg mice described above (10). (12) Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mice obtained by crossing the Adar1 KO:Dct-LacZ Tg mice described in (10) or (11) above with K14-Scf Tg mice. (13) Adar1 homozygous knockout mice, in which the function of Adar1 (Adenosine Deaminase Acting on RNA1) is lost specifically in melanocytes. (14) exhibiting a variegated hair color phenotype; The Adar1 homozygous knockout mouse described in (13) above. (15) A step of administering a test compound to the knockout mouse according to any one of (8) to (14) above; a test compound selection step of selecting a test compound that suppresses depigmentation; A method for screening a compound for treating hereditary symmetrical pigmentation disorder, comprising: (16) A DSH model cell preparation step of preparing cells in which Adar1 is inactivated; a control cell preparation step of preparing control cells in which Adar1 is not inactivated; administering a test compound and interferon and / or a Toll-like receptor ligand to the DSH model cells; administering a test compound and interferon and / or a Toll-like receptor ligand to the control cells; a step of comparing the number of viable cells after administering a test compound and interferon and / or a Toll-like receptor ligand to the DSH model cells with the number of viable cells after administering a test compound and interferon and / or a Toll-like receptor ligand to the control cells; a test compound selection step of selecting a test compound based on the comparison result of the viable cell number comparison step; A method for screening a compound for treating hereditary symmetrical pigmentation disorder, comprising: [Effects of the Invention]

[0011] The pharmaceutical composition for treating hereditary symmetrical pigmentation disorders disclosed in the present application can be used to treat hereditary symmetrical pigmentation disorders. Furthermore, the model mice and model cells for hereditary symmetrical pigmentation disorders disclosed in the present application can be used to screen for compounds for treating hereditary symmetrical pigmentation disorders. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a photograph, substituted for a drawing, showing the Adar1 heterozygous knockout mouse produced in Example 1. [Figure 2] Figure 2 is a photograph in place of a drawing. Figure 2A is a photograph of the Adar1 heterozygous knockout mouse generated in Example 2, and Figure 2B is a photograph of a wild-type mouse on a C57BL / 6 background for comparison. [Figure 3] FIG. 3 is a photograph, substituted for a drawing, showing the Adar1 heterozygous knockout mouse produced in Example 3. [Figure 4] FIG. 4 is a photograph, substituted for a drawing, showing the Adar1 homozygous knockout mouse produced in Example 4. [Figure 5] Figure 5 is a photograph in place of a drawing. Column A of Figure 5 is a photograph of the control cells and DSH model cells prepared in Example 5. Columns B and C of Figure 5 are photographs of the control cells and DSH model cells administered with Ruxolitinib and Tofacitinib in Example 6. [Figure 6] Figure 6 is a photograph in place of a drawing. Figure 6A is a photograph of DSH model mice receiving transdermal administration of tofacitinib in Example 7, and Figure 6B is a photograph of DSH model mice receiving transdermal administration of DMSO in Comparative Example 1. [Figure 7] Figure 7 is a photograph in place of a drawing. Figure 7A is a photograph showing oral administration of tofacitinib to DSH model mice in Example 8, and Figure 7B is a photograph showing oral administration of DMSO to DSH model mice in Comparative Example 2. [Figure 8] Figure 8 is a photograph in place of a drawing. Column A of Figure 8 is a photograph of the control cells and DSH model cells prepared in Example 5. Column B of Figure 8 is a photograph of the control cells and DSH model cells when delgocitinib was administered in Example 10. [Figure 9] Figure 9 is a photograph in place of a drawing. Figure 9B is a photograph showing transdermal administration of delgocitinib to DSH model mice in Example 11, and Figure 9A is a photograph showing transdermal administration of DMSO to DSH model mice in Comparative Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] The pharmaceutical composition for treating DSH, the hereditary symmetrical pigmentation disorder mouse model (Adar1 knockout mouse), and the screening method for a compound for treating DSH disclosed in the present application are described in detail below.

[0014] First, Adar1 knockout mice will be described. The Adar1 knockout mice disclosed in the present application include Adar1 heterozygous knockout mice and Adar1 homozygous knockout mice. Below, Adar1 heterozygous knockout mice and Adar1 homozygous knockout mice will be described in more detail.

[0015] (First embodiment of Adar1 heterozygous knockout mouse) In the first embodiment, heterozygous knockout mice of Adar1 are first produced by completely eliminating Adar1 function. There are no particular limitations on the method for producing heterozygous knockout mice of Adar1 function, as long as the Adar1 function is eliminated. For example, the mice may be produced by the following procedure.

[0016] (a) The gene to be knocked out (Adar1) in the mouse genome is cloned in whole or in part, or cloned genomic DNA is purchased. A base sequence is then created that includes the gene (or part of the gene) and surrounding regions. Mutations are introduced or parts are deleted to inactivate the gene. In addition to the deletions and other inactivation modifications, a marker gene that provides an observable difference (such as color, fluorescence, or antibiotic resistance genes) is also incorporated. Genetic information for mouse Adar1 is publicly known and can be obtained, for example, from GenBank Gene ID: 56417. There are no particular limitations on the region of Adar1 to be knocked out. For example, since Adar1 has isoforms such as p110, which is localized in the nucleus, and p150, which is localized in the cytoplasm, the p150 isoform may be specifically deleted. (b) Embryonic stem cells are isolated from mouse blastocysts (early mouse embryos, consisting of a spherical, undifferentiated cell mass surrounded by extraembryonic cells). Embryonic stem cells, such as those from brown mice, can be cultured in vitro. (c) The base sequence prepared in (a) above is introduced into the embryonic stem cells obtained in (b) above using a method such as electroporation. Next, using the marker gene introduced in (a) above, embryonic stem cells that have actually undergone recombination with the new base sequence (heterozygous type) are isolated. (d) The homologously recombined embryonic stem cells isolated in (c) above are injected into a blastocyst, for example, of a black mouse, and the blastocyst is then injected into the uterus of a female mouse, giving birth to a baby mouse. This baby mouse will be a chimera, with part of its body derived from the original blastocyst and the other part containing two cells derived from the genetically modified embryonic stem cells. As a result, its coat will be brown and black. (e) Among the chimeric mice, only those whose germ cells (eggs or sperm) are derived from genetically engineered cells are used. Specifically, by mating a chimeric mouse with a black mouse, the newly born mice with completely brown bodies are derived from embryonic stem cells. The heterozygosity of the altered gene among these mice is confirmed using PCR or other methods, and heterozygous Adar1 knockout mice are obtained. These Adar1 heterozygous knockout mice are then backcrossed to produce black mice.

[0017] Adar1 heterozygous knockout mice may be used according to the above procedure or obtained from institutions such as Mutant Mouse Regional Resource Centers (MMRRC) (Stock No. 034620-JAX).

[0018] As shown in the Examples below, simply losing the function of Adar1 results in the Adar1 heterozygous knockout mouse's body hair remaining black and not exhibiting the DSH phenotype. On the other hand, as a result of extensive research, we have newly discovered that administering mouse interferon (m-Ifn) or a Toll-like receptor ligand to the Adar1 heterozygous knockout mouse according to the first embodiment, generated by the above procedure, results in mottled body hair color and the DSH phenotype. Note that the "mottled" body hair color of the Adar1 heterozygous knockout mouse according to the first embodiment means that the hair in the lesioned area is white throughout the entire lesion, or that white and black hair are mixed together, compared to the black hair in the surrounding non-lesional area.

[0019] Interferons are proteins secreted by animal cells in response to the invasion of foreign substances, such as pathogens (especially viruses) and tumor cells, and are substances that suppress viral and cellular proliferation. Toll-like receptors (Toll-like receptors) are receptor proteins present on the cell surface of animals that detect various pathogens and activate innate immunity. The interaction between loss of Adar1 function and exogenously administered immune or inflammatory substances, or the interaction between reduced or lost Adar1 function and the endogenous expression of immune or inflammatory substances induced by ligand administration, or in other words, the interaction between reduced or lost ADAR1 function and hyperimmune or hyperinflammatory conditions, is thought to cause Adar1 heterozygous mice to exhibit a DSH phenotype.

[0020] The interferon to be used is not particularly limited as long as Adar1 heterozygous mice exhibit the DSH phenotype. Examples include m-Ifnα, m-Ifnβ, m-Ifnω, m-Ifnε, m-Ifnκ, m-Ifnζ, m-Ifnγ, and m-Ifnλ. Other examples include homologs of human type 1 interferons (IFNα, IFNβ, IFNω, IFNε, and IFNκ), type 2 interferon (IFNγ), and type 3 interferon (IFNλ).

[0021] Furthermore, there are no particular limitations on the Toll-like receptor ligand, as long as Adar1 heterozygous mice exhibit a DSH phenotype. For example, commercially available Toll-like receptor ligands such as Pam3CSK4 (synthetic tripalmitoylated lipopeptide), histone, zymosan (a cell wall component derived from yeast), MALP-2, Poly(I:C) (Polyinosinic-polycytidylic acid sodium salt), lipopolysaccharide (LPS) (a membrane component of Escherichia coli), paclitaxel, Lipid A, Flagellin (a protein derived from Salmonella typhimurium), Gardiquimod, Imiquimod / R-837 (an imidazoquinoline amine analogue of guanosine), Imidazoquinoline Resiquimod / R-848 (a low-molecular-weight imidazoquinoline compound), Loxoribine, CpG ODN 2006, CpG ODN 1668, and Profilin may be used.

[0022] The method of administration of these substances may be either local administration to the skin or systemic administration by oral administration or injection.

[0023] The skin of wild-type adult mice does not contain pigment cells (melanocytes) that produce brown or black pigment (melanin), but pigment cells are present in the hair follicles. Therefore, when screening for a compound for treating DSH using the Adar1 heterozygous mouse of the first embodiment, changes in body hair can be observed after administering a test compound. Furthermore, DSH is inherited in an autosomal dominant manner, and only one allele of Adar1 is mutated (heterozygous). The knockout mouse of the first embodiment also has the effect of exhibiting a phenotype under the same genetic conditions as human DSH.

[0024] (Second embodiment of Adar1 heterozygous knockout mouse) Next, a second embodiment of an Adar1 heterozygous knockout mouse will be described. The Adar1 heterozygous knockout mouse according to the second embodiment differs from the Adar1 heterozygous knockout mouse according to the first embodiment in that, instead of administering interferon or a Toll-like receptor ligand, the Adar1 heterozygous knockout mouse is crossed with a transgenic mouse expressing LacZ; otherwise, the Adar1 heterozygous knockout mouse is the same as the Adar1 heterozygous knockout mouse according to the first embodiment. Therefore, the second embodiment will mainly describe the differences from the first embodiment, and redundant explanations of matters already explained in the first embodiment will be omitted. Therefore, it goes without saying that matters already explained in the first embodiment can be employed in the second embodiment, even if they are not explicitly explained in the second embodiment.

[0025] Transgenic mice expressing LacZ may express LacZ throughout the body or may be limited to the skin or a portion of the skin cells. The Dct-LacZ transgenic mice shown in the following examples express LacZ under the control of the Dct promoter, i.e., mice that express LacZ specifically in pigment cells. Examples of transgenic mice expressing LacZ throughout the body include ROSA26-lacZ (Soriano P. Nat Genet. 1999 Jan;21(1):70-1) and CAG-lacZ (C57BL / 6-Tg(Cag-LacZ)11Miya, Animal Resources and Development Research Division, Life Resources Research and Support Center, Kumamoto University, CARD ID 1455).

[0026] The Adar1 heterozygous knockout mouse according to the second embodiment is obtained by crossing an Adar1 heterozygous knockout mouse that does not exhibit the DSH phenotype, which has been produced by the same procedure as in the first embodiment, with a Dct-LacZ transgenic mouse. The obtained knockout mouse according to the second embodiment (hereinafter sometimes referred to as an "Adar1 KO:Dct-LacZ Tg mouse"), unlike the first embodiment, exhibits the DSH phenotype without administration of interferon or a Toll-like receptor ligand.

[0027] LacZ is known as a reporter gene and produces β-galactosidase, which breaks down lactose into glucose and galactose. Although the mechanism of action in Adar1 KO:Dct-LacZ Tg mice is unclear, the DSH phenotype is also exhibited through a mechanism different from that of the hyperimmune or hyperinflammatory state of the first embodiment. The "patchy" DSH phenotype observed in the Adar1 knockout mice of the second embodiment refers to a mixture of white and black body hair, similar to the Adar1 knockout mice of the first embodiment. However, while the Adar1 heterozygous knockout mice of the first embodiment exhibit a DSH phenotype only in areas administered with interferon or Toll-like receptor ligands, the Adar1 heterozygous knockout mice of the second embodiment exhibit a different effect in that the DSH phenotype is exhibited over a wider area.

[0028] The Adar1 KO:Dct-LacZ Tg mice according to the second embodiment exhibit a DSH phenotype without the administration of interferon or Toll-like receptor ligands. Therefore, the Adar1 knockout mice do not require the external administration of substances to induce a hyperimmune state, which results in easy handling and enables the implementation of the method for screening compounds for treating DSH, which will be described later, at low cost.

[0029] (Third embodiment of Adar1 heterozygous knockout mouse) Next, a third embodiment of an Adar1 heterozygous knockout mouse will be described. The Adar1 heterozygous knockout mouse according to the third embodiment is obtained by crossing the Adar1 heterozygous knockout mouse according to the second embodiment (Adar1 KO:Dct-LacZ Tg mouse) with a K14-Scf Tg mouse (the obtained mouse may be referred to as an "Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse"). The Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse according to the third embodiment is obtained by crossing the Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse with a K14-Scf Tg mouse, thereby introducing pigment cells into the skin of the Adar1 KO:Dct-LacZ Tg mouse according to the second embodiment. Therefore, the Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse according to the third embodiment exhibits a phenotype in which the skin also develops white and black (brown) "mottled" patches in addition to the phenotype according to the second embodiment.

[0030] When the screening method is performed using the Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse according to the third embodiment, in addition to the effects described in the first and second embodiments, it is possible to screen for DSH therapeutic compounds by observing changes in skin color. Furthermore, the DSH phenotype of the body hair of the mouse according to the third embodiment is a patchy DSH phenotype in which the white and black hair areas are more clearly separated (polka dot pattern), compared to the phenotype of the mouse according to the second embodiment in which white and black hair are finely mixed together (salt and pepper hair-like pattern). Therefore, when evaluating the hair color, large white spots are formed, making it easier to assess the efficacy of DSH therapeutic compounds.

[0031] (Embodiment of Adar1 homozygous knockout mouse) Adar1 homozygous knockout mice can be generated by using the Cre-lox system to conditionally knockout Adar1, if the knockout site is limited, such as in pigment cells. If the genetic background of the mice is black, it will be possible to determine whether the DSH phenotype appears.

[0032] Conditional knockout, also known as conditional gene disruption, is achieved by crossing floxed mice with Cre-expressing mice. First, mice (floxed or floxed mice) are created in which the target gene region is flanked by the Cre recombinase target sequence loxP. Next, by crossing the floxed mice with Cre-expressing mice, the target gene is deleted only in specific target cells.

[0033] In an embodiment of an Adar1 homozygous knockout mouse, Adar1 in melanocytes is targeted as the gene region, thereby enabling the generation of a homozygous knockout mouse in which Adar1 function is specifically lost in melanocytes. The target may be any gene that specifically loses Adar1 function in melanocytes, and examples of the target include p110 and / or p150. Furthermore, known methods may be used to generate homozygous knockout mice using the Cre-lox system.

[0034] Homozygous knockout mice die during the fetal stage and are not born. In the Adar1 homozygous knockout mouse of this embodiment, Adar1 is knocked out specifically in melanocytes, making it possible to obtain adult mice. Due to the knockout in melanocytes, the Adar1 homozygous knockout mouse exhibits a phenotype in which black and white body hair are intermingled (resembling a salt-and-pepper head). Examples of the Adar1 region knocked out in melanocytes include p110 and / or p150, but other regions may also be used.

[0035] Adar1 homozygous knockout mice tend to show more severe symptoms (more white hair) than Adar1 heterozygous knockout mice according to the second embodiment. Therefore, when several test compounds that are expected to have a high therapeutic effect are found, the use of Adar1 homozygous knockout mice provides the advantage of enabling a high level of evaluation of the therapeutic effect of the test compounds. On the other hand, the Adar1 heterozygous knockout mice according to the second embodiment provide the advantage of being useful for the initial screening of test compounds.

[0036] Next, a method for screening for a compound for treating DSH will be described. The method for screening for a compound for treating DSH can be performed using the above-mentioned Adar1 knockout mouse (hereinafter sometimes referred to as a "DSH model mouse") or cells such as pigment cells.

[0037] (Embodiment of screening method using DSH model mouse) An embodiment of the method for screening a compound for treating DSH using a DSH model mouse includes: administering a test compound to a DSH model mouse; a test compound selection step of selecting a test compound that suppresses depigmentation (vitiligo, white hair); Includes:

[0038] Test compounds include, for example, single compounds such as natural compounds, organic compounds, inorganic compounds, proteins, antibodies, and peptides, as well as compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, and plant extracts.

[0039] The method of administering a test compound to a DSH model mouse is not particularly limited, as long as it is taken into the body of the DSH model mouse, such as oral administration, application to the skin, or internal administration by injection, etc. Then, by determining whether or not the administered test compound suppresses depigmentation (vitiligo, white hair) in the DSH model mouse (in the case of body hair, the hair color changes from white to black (brown), and in the case of skin, the number of white spots decreases (pigment becomes darker)), a compound that suppresses depigmentation can be selected.

[0040] (Embodiment of a screening method using cells) An embodiment of the cell-based method for screening compounds for treating DSH comprises: A DSH model cell preparation step of preparing cells in which Adar1 is inactivated; a control cell preparation step of preparing control cells in which Adar1 is not inactivated; administering a test compound and interferon and / or a Toll-like receptor ligand to the DSH model cells; administering a test compound and interferon and / or a Toll-like receptor ligand to the control cells; a step of comparing the number of viable cells after administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells with the number of viable cells after administering the test compound and interferon and / or Toll-like receptor ligand to the control cells; a test compound selection step of selecting a test compound based on the comparison result of the viable cell number comparison step; Includes:

[0041] Examples of cells include mouse or human pigment cells. Since Adar1 is also expressed in cells other than pigment cells, Adar1-expressing cells may be used instead of pigment cells. Examples of Adar1-expressing cells include cos7 cells, HeLa cells, melanoma cells, and HaCaT cells. Primary cultured cells or subcultured cells may be used. Inactivation of Adar1 in cells is not particularly limited as long as it can specifically inactivate Adar1 in the cells. For example, known methods include introducing siRNA with a sequence that inactivates Adar1 into cells. Alternatively, cells may be primary cultured from Adar1 heterozygous knockout mice and wild-type mice. Human-derived cells transfected with a mutant ADAR1 and a wild-type ADAR1, or mouse-derived cells transfected with a mutant Adar1 and a wild-type Adar1 may also be used. When interferon and / or Toll-like receptor ligands are administered to cells in which Adar1 has been inactivated (hereinafter sometimes referred to as "DSH model cells") and control cells transfected with control siRNA that does not inactivate Adar1, the viability of the Adar1-inactivated DSH model cells is reduced compared to the control cells in which Adar1 has not been inactivated. Therefore, the number of surviving cells can be compared between (1) a group in which a test compound and interferon and / or Toll-like receptor ligands are administered to DSH model cells, and (2) a group in which a test compound and interferon and / or Toll-like receptor ligands are administered to control cells. The closer the ratio of surviving cells between groups (1) and (2), the greater the increase in cell viability due to the test compound is considered to be.

[0042] (Embodiments of pharmaceutical compositions for treating DSH) There are no particular limitations on the pharmaceutical composition for treating DSH, as long as it contains an ingredient that is effective in treating DSH. The inventors conducted experiments using various known pharmaceuticals and, as shown in the Examples below, confirmed the therapeutic effects of various commercially available JAK inhibitors and STAT inhibitors. Therefore, JAK inhibitors, STAT inhibitors, and similar compounds can be used in pharmaceutical compositions for treating DSH.

[0043] Dosage forms of pharmaceutical compositions for treating DSH include, for example, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile sealed powders.

[0044] The pharmaceutical composition for treating DSH may also contain carriers, excipients, or diluents. Examples include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, phosphate-buffered saline (PBS), syrup, methylcellulose, methyl and propyl hydroxybenzoates, talc, magnesium stearate, and mineral oil. Vitamins, lubricants, wetting agents, emulsifying and suspending agents, preservatives, sweeteners, or flavoring agents may also be added. The pharmaceutical composition for treating DSH may also be combined with known drugs.

[0045] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application. [Example]

[0046] [Generation of DSH model mice] Example 1 p150 Adar1 heterozygous knockout mice (C57BL / 6 background) were generated by the following procedure. A BAC clone containing the Adar1 gene was purchased from the BACPAC Resources Center (BPRC) (https: / / bacpacresources.org), and the p150-specific exon of the Adar1 gene was excised from the genomic DNA clone using restriction enzymes. The genomic DNA clone was then transferred into the pBluescript vector, linearized, and transfected into embryonic stem cells. Embryonic stem cells showing recombination between the transgene and the endogenous gene were selected with antibiotics. Embryonic stem cells showing recombination were then injected into blastocysts and transferred to mouse uteruses to generate chimeric mice. These chimeric mice were then mated with wild-type mice, and mice born from embryonic stem cell-derived sperm were selected based on their body hair. p150 Adar1 heterozygous knockout mice were obtained by PCR genotyping. These mice were then backcrossed to a C57BL / 6 background. Two-day-old newborn p150 Adar1 heterozygous knockout mice (C57BL / 6 background) were subcutaneously injected with 3000 U of m-Ifnβ (PBL) once daily. Subcutaneous injection of m-Ifnβ was continued for 10 days. Figure 1 shows photographs of the Adar1 heterozygous knockout mice generated in Example 1. While wild-type mice on the C57BL / 6 background have normal black fur, the Adar1 heterozygous knockout mice generated in Example 1 were confirmed to have white fur after subcutaneous injection, despite being on the C57BL / 6 background (see the oval in Figure 1).

[0047] In addition, instead of m-Ifnβ, When 50 μL of 0.5 μg / μL of LPS (manufactured by Wako), a Toll-like receptor ligand, was subcutaneously injected on the same schedule, When 50 μL of 2 μg / μL poly(I:C) (GE Healthcare), a Toll-like receptor ligand, was subcutaneously injected on the same schedule, As shown in Figure 1, we confirmed that the body hair turned white.

[0048] However, Adar1 heterozygous knockout mice that were not subcutaneously injected with m-Ifn or Toll-like receptor ligands retained their black fur.

[0049] These results demonstrate that administration of interferon or Toll-like receptor ligands to Adar1 heterozygous knockout mice can produce model mice exhibiting the DSH phenotype.

[0050] <Example 2> Adar1 KO:Dct-LacZ Tg mice were generated by crossing the Adar1 heterozygous knockout mice (not administered interferon or Toll-like receptor ligands) of Example 1 with Dct-LacZ transgenic (Tg) mice (Kumamoto University, Bioresources Research and Development Center, ID: 782). Figure 2A shows a photograph of the mice generated in Example 2. For comparison, Figure 2B shows a photograph of a wild-type mouse on a C57BL / 6 background. As shown in Figure 2A, the mice were confirmed to have white hair mixed with their normal black hair. This phenotype better reflects the clinical picture of DSH, which is a mixture of pigmented and depigmented spots. Furthermore, it was confirmed that the mice generated in Example 2 exhibited the DSH phenotype even without administration of interferon or Toll-like receptor ligands.

[0051] Example 3 Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mice were generated by crossing the Adar1 KO:Dct-LacZ Tg mice generated in Example 2 with K14-Scf Tg mice (Kunisada T, et al., J Exp Med. 1998;187(10):1565-73). Figure 3 shows photographs of the mice generated in Example 3. In Example 3, the Adar1 KO:Dct-LacZ Tg mice generated in Example 2 were crossed with K14-Scf Tg mice to allow the presence of pigment cells in the skin. As a result, the mice generated in Example 3 displayed large, circular white spots (polka dots) compared to the "salt-and-pepper" phenotype of Example 2, which had a fine mixture of white and black hair. This confirmed that the mice generated in Example 3 more closely resembled the human DSH phenotype.

[0052] Example 4 Adar1 floxed mice (Adar tm1a(EUCOMM)Wtsi / tm1a(EUCOMM)Wtsi The mice (MBLD; EPD0087_1_D11, Sanger Institute, UK) were crossed with Dct-Cre Tg mice (provided by Prof. Beermann F, Guyonneau L, et al., Pigment cell res, 15:305-309, 2002) that express Cre specifically in melanocytes. The MBLD mice were homozygous for Adar1 gene modification, and the mice (Adar1) of Example 4 were generated by crossing with Dct-Cre Tg mice. tm1a(EUCOMM)Wtsi / tm1a(EUCOMM)Wtsi ;Dct-Cre_Tg) resulted in Adar1 homozygous knockout mice in which Adar1 was knocked out only in melanocytes. Figure 4 shows photographs of the mice produced in Example 4. As shown in Figure 4, the mice produced in Example 4 exhibited a phenotype in which their body hair was patchy with white and black areas.

[0053] [Creation of DSH model cells] <Example 5> In Example 1, Adar1 heterozygous knockout mice were administered interferon or a Toll-like receptor ligand and exhibited a DSH phenotype. Therefore, DSH model cells were prepared using pigment cells by the following procedure.

[0054] [Day 1] Mouse melanocyte melan5 cells (provided by Dr. Masako Mizoguchi, St. Marianna University School of Medicine; Ooka S, et al., Pigment Cell Res. 14(4):268-74, 2001) were cultured at 0.5x10 cells / well in a 12-well plate. 5 The cells were plated at 1 cell / well and 1 mL of medium (see Ooka S, et al., Pigment Cell Res. 14(4):268-74, 2001) was added. [Day 2] 0.3 μL of Adar1 siRNA (Adar1(1), 5'-AGAAGACGGUUUCUUUUCA) (SEQ ID NO: 1) and control siRNA (Allstar negative siRNA, manufactured by Qiagen) were transfected into the cultured cells according to the instructions attached to HiPerfect (manufactured by Qiagen). [Day 3] Mouse interferon β (PBL) was added at 330 U / well. [Day 5] Cells transfected with Adar1 siRNA and control siRNA were observed. The upper row of column A in Figure 5 shows photographs of control cells transfected with control siRNA (control siRNA+m-Ifn), and the lower row of column A shows photographs of DSH model cells transfected with Adar1 siRNA (Adar1 siRNA+m-Ifn). As is clear from the photographs in column A, the mortality rate of DSH model cells was higher than that of control cells. Each cell was detached with trypsin, and the number of viable cells was counted using a TC20 automated cell counter (Bio-Rad). The ratio of viable cells to control cells was 10.5%.

[0055] These results suggest that the survival rate of pigment cells with reduced Adar1 expression was reduced, suggesting that the DSH model cells we created exhibit the properties of pigment cells in DSH patients.

[0056] [Confirming the usefulness of DSH model cells and DSH model mice] Next, the usefulness of the DSH model mice prepared in Example 1 and the DSH model cells prepared in Example 5 was confirmed. As described above, the DSH model mice prepared in Example 1 did not exhibit a DSH phenotype in Adar1 heterozygous knockout mice. However, as a result of extensive research, Adar1 heterozygous mice exhibited a DSH phenotype when placed in a hyperimmune or hyperinflammatory state with loss of Adar1 function. Therefore, we considered that drugs with immune or inflammatory suppressive functions would be useful for treating Adar1. After extensive investigation, we first conducted experiments using DSH model cells with Ruxolitinib (Selleck) and Tofacitinib (Sigma-Aldrich).

[0057] [Experiments using DSH model cells] Example 6 The experiment was performed in the same manner as in Example 5, except that on [day 3] in Example 5, in addition to mouse interferon β, Ruxolitinib was added to a final concentration of 0.5 μM, and Tofacitinib was added to a final concentration of 5 μM. The drug administration concentrations were determined based on cytotoxicity evaluations in control cells and DSH model cells. DMSO was used to adjust the drug concentrations. The upper row of column B in Figure 5 is a photograph of control cells with Ruxolitinib added, and the lower row of column B is a photograph of DSH model cells with Ruxolitinib added. The upper row of column C in Figure 5 is a photograph of control cells with Tofacitinib added, and the lower row of column C is a photograph of DSH model cells with Tofacitinib added.

[0058] As is clear from the photographs in columns B and C of Figure 5, the addition of either the immunosuppressants ruxolitinib or tofacitinib improved the viability of DSH model cells. In particular, the cell viability was 98.8% with tofacitinib (number of DSH model cells treated with tofacitinib / number of control cells treated with tofacitinib). This means that the addition of ruxolitinib and tofacitinib restored the viability of DSH model cells in which Adar1 function was reduced or lost, suggesting that these drugs may be useful in treating diseases in which Adar1 function is reduced or lost.

[0059] [Experiments using DSH model mice] The following experiment was carried out using Tofacitinib, which showed a high cell viability in Example 6.

[0060] Example 7: Transdermal administration Neonatal p150 Adar1 heterozygous knockout mice described in Example 1 were subcutaneously injected with 3000 U of m-Ifnβ (PBL) once daily. m-Ifnβ injections were initiated on postnatal day 7 and continued for 10 days until postnatal day 16. Tofacitinib was subcutaneously injected at 50 mg / kg / day for 10 days from postnatal day 7 to postnatal day 16. Figure 6A shows a photograph of a mouse on postnatal day 22.

[0061] Comparative Example 1: Transdermal Administration Except for injecting DMSO, a solvent for tofacitinib, instead of tofacitinib, the experiment was carried out in the same manner as in Example 7. Figure 6B is a photograph of the mouse on day 22 after birth.

[0062] Example 8: Oral administration Neonatal p150 Adar1 heterozygous knockout mice described in Example 1 were subcutaneously injected with 3000 U of m-Ifnβ (PBL) once daily. m-Ifnβ injections were initiated on postnatal day 7 and continued daily until postnatal day 16. Tofacitinib was orally administered at 50 mg / kg / day for 10 days from postnatal day 7 to postnatal day 16. Figure 7A shows a photograph of the mice on postnatal day 22.

[0063] Comparative Example 2: Oral Administration Except for orally administering DMSO, a solvent for tofacitinib, instead of tofacitinib, the experiment was carried out in the same manner as in Example 8. Figure 7B is a photograph of the mouse on day 22 after birth.

[0064] As is clear from the photographs, the body hair of DSH model mice that received tofacitinib by subcutaneous injection (Example 7: Figure 6A) or oral administration (Example 8: Figure 7A) changed from white to black. On the other hand, the body hair of DSH model mice that did not receive tofacitinib by subcutaneous injection (Comparative Example 1: Figure 6B) or oral administration (Comparative Example 2: Figure 7B) remained white.

[0065] These results confirmed that DSH model mice that were not administered tofacitinib still exhibited the DSH phenotype, but that the administration of tofacitinib resolved the DSH phenotype. This confirms that tofacitinib is useful as a DSH therapeutic compound, whether administered transdermally or orally. Furthermore, as shown in Examples 5, 7, and 8, the same results were obtained in the DSH model cells and DSH model mice, confirming that the DSH model cells and DSH model mice can be used to screen for DSH therapeutic compounds.

[0066] Example 9 [Screening of compounds for treating DSH] As described above, we confirmed that DSH model cells and DSH model mice can be used to screen compounds for DSH treatment. Therefore, we used DSH model cells to screen various compounds. Table 1 below shows the compounds screened using the same procedure as in Example 6, their concentrations, known uses, and cell viability. For comparison, the compounds already identified in Example 6 are also listed in Table 1. Of the exemplified compounds, tofacitinib was obtained from Sigma-Aldrich, curcumol from Tokyo Chemical Industry Co., Ltd., and cerdulatinib from Cayman Chemical; all others were obtained from Selleck.

[0067] [Table 1]

[0068] As shown in Table 1, various commercially available JAK inhibitors and STAT inhibitors showed therapeutic effects. Most of the JAK inhibitors showed cell viability rates of 50% or higher, confirming their suitability as pharmaceutical compositions for treating DSH.

[0069] [Confirm the usefulness of other JAK inhibitors] Example 10 An experiment was performed in the same manner as in Example 6, except that the JAK inhibitor Delgocitinib (ChemScene) was added to a final concentration of 1 μM instead of Ruxolitinib and Tofacitinib in Example 6. The upper row of column B in Figure 8 is a photograph of control cells to which Delgocitinib was added, and the lower row of column B is a photograph of DSH model cells to which Delgocitinib was added.

[0070] As is clear from the photograph in FIG. 8, it was confirmed that the survival rate of DSH model cells was improved when delgocitinib was added compared to when it was not added.

[0071] Example 11 A transdermal administration experiment was performed using a procedure similar to that of Example 7, except that delgocitinib was used instead of tofacitinib. Specifically, 3000 U of m-Ifnβ (PBL) was subcutaneously injected once daily into newborn p150 Adar1 heterozygous knockout mice described in Example 1. m-Ifnβ injections were initiated on postnatal day 2 and continued 10 times until postnatal day 14. Delgocitinib was subcutaneously injected 10 times at 15 mg / kg / day from postnatal day 2 to postnatal day 14. Figure 9B shows a photograph of a mouse on day 15 after birth.

[0072] <Comparative Example 3> Except for injecting DMSO, a solvent for delgocitinib, instead of delgocitinib, the experiment was carried out in the same manner as in Example 11. Figure 9A is a photograph taken on the 15th day after birth.

[0073] As is clear from the photograph, the body hair of the DSH model mouse that received a transdermal injection of delgocitinib (Example 11: FIG. 9B) changed from white to black. On the other hand, the body hair of the DSH model mouse that did not receive a transdermal injection of delgocitinib (Comparative Example 3: FIG. 9A) remained white.

[0074] These results demonstrate that the JAK inhibitor delgocitinib is a useful compound for treating DSH, as confirmed by experiments using both DSH model cells and DSH model mice. [Industrial Applicability]

[0075] The DSH model mice and model cells disclosed in this application can be used to elucidate the pathology of DSH and to screen for compounds for treating DSH. Furthermore, JAK inhibitors and STAT inhibitors can be used as pharmaceutical compositions for treating DSH. Therefore, they are useful for research and development of DSH treatments at universities, medical institutions, pharmaceutical companies, and the like.

Claims

1. The present invention relates to a method for treating rheumatoid arthritis, the method comprising administering to a patient a therapeutically effective amount of at least one compound selected from the group consisting of a JAK inhibitor and a STAT inhibitor as an active ingredient, The JAK inhibitor selected from Tofacitinib, Itacinitib, Solcitinib, AZD1480, Ruxolitinib, JAK3-IN-6, Curcumol, Pericitinib, Cerdulatinib, FM-381, Filgotinib, and Delgocitinib; The STAT inhibitor is A pharmaceutical composition for treating hereditary symmetrical pigmentary disorders, which is selected from napabucasin, ochromycinone, fludarabine, nifuroxazide, C188-9, and AS1517499.

2. the JAK inhibitor is Delgocitinib; The pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to claim 1.

3. the JAK inhibitor is Ruxolitinib; The pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to claim 1.

4. the JAK inhibitor is tofacitinib; The pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to claim 1.

5. The JAK inhibitor is pificitinib. The pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to claim 1.

6. the JAK inhibitor is cerdulatinib; The pharmaceutical composition for treating hereditary symmetric pigmentation disorder according to claim 1.

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