Pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorder, hereditary contralateral pigmentary disorder model mouse, and screening method for compounds for the treatment of hereditary contralateral pigmentary disorder

Abstract

This invention provides a pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders, a mouse model for hereditary contralateral pigmentary disorders, and a screening method for compounds for the treatment of hereditary contralateral pigmentary disorders. [Solution] A heterozygous Adar1 knockout mouse that has lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout the body and exhibits a phenotype of mottled skin and / or fur color.

Description

Technical Field

[0001] The disclosure of the present application relates to a pharmaceutical composition for treating dyschromatosis symmetrica hereditaria, a model mouse for dyschromatosis symmetrica hereditaria, and a method for screening a compound for treating dyschromatosis symmetrica hereditaria.

Background Art

[0002] Dyschromatosis symmetrica hereditaria (hereinafter sometimes referred to as "DSH") is a pigmentary disease in which pigmented macules and leukoderma appear as "macules" on both dorsa of the hands and both dorsa of the feet. In humans, a gene encoding double-stranded RNA-specific adenosine deaminase 1 (Adenosine Deaminase Acting on RNA1, hereinafter sometimes referred to as "ADAR1" or "ADAR". Note that the double-stranded RNA-specific adenosine deaminase 1 of mice is referred to as "Adar1". Also, regarding model cells, it may be referred to as "Adar1" regardless of the origin.) is the causative gene. It is known that by converting adenosine in RNA to inosine, a protein different from the DNA information is generated. It has been clarified that having a heterozygous mutation in the ADAR1 gene causes DSH (see Non-Patent Document 1).

[0003] DSH rarely has neurological complications and does not cause life-threatening serious symptoms. However, since it develops from childhood and the symptoms appear on the exposed parts, there is a problem that the patient suffers a heavy mental burden due to appearance problems. Therefore, the establishment of therapeutic drugs and treatment methods is required, but currently there are no effective therapeutic drugs and treatment methods.

[0004] By the way, in the research of disease treatment, it is generally carried out to prepare a model animal such as a mouse having the disease, examine the pathological condition of the disease using the model animal, administer a test compound to the model animal, and screen a therapeutic compound.

Prior Art Documents

[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 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] However, simply knocking out Adar1 does not produce the phenotype seen in DSH patients (hereinafter sometimes referred to as the "DSH phenotype") in mice, and therefore the mice cannot be used for DSH research.

[0007] The disclosure in this application was made to solve the above-mentioned problems, and through diligent research, we have newly discovered that (1) DSH can be treated by using a JAK inhibitor or a STAT inhibitor as the active ingredient, (2) when a predetermined substance is administered to heterozygous Adar1 knockout mice that have lost Adar1 function throughout the body, or when the gene that produces it is expressed, they exhibit the DSH phenotype, (3) homozygous Adar1 knockout mice that have lost Adar1 function specifically in melanocytes exhibit the DSH phenotype, and (4) a screening of DSH therapeutic compounds can be performed using the created knockout mice (hereditary contralateral pigment disorder model mice) or cells with reduced or lost Adar1 function (model cells).

[0008] In other words, the purpose of the disclosure of this application is to provide a pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders, a model mouse for hereditary contralateral pigmentary disorders, and a screening method for compounds for the treatment of hereditary contralateral pigmentary disorders. [Means for solving the problem]

[0009] The disclosures of this application relate to a pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders, a model mouse for hereditary contralateral pigmentary disorders, and a screening method for compounds for the treatment of hereditary contralateral pigmentary disorders, as described below.

[0010] (1) A pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders, comprising as an active ingredient at least one compound selected from the group consisting of JAK inhibitors and STAT inhibitors. (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, Selected from Napabucasin, Ochromycinone, Fludarabine, Nifuroxazide, C188-9, and AS1517499, A pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders as described in (1) above. (3) The JAK inhibitor is Delgocitinib, A pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders as described in (2) above. (4) The JAK inhibitor is Ruxolitinib, A pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders as described in (2) above. (5) The JAK inhibitor is Tofacitinib, A pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders as described in (2) above. (6) The JAK inhibitor is peficitinib, A pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders as described in (2) above. (7) The JAK inhibitor is Cerdulatinib, A pharmaceutical composition for the treatment of hereditary contralateral pigmentary disorders as described in (2) above. (8) A heterozygous Adar1 knockout mouse in which the function of Adar1 (Adenosine Deaminase Acting on RNA1) is lost throughout the body, The phenotype is that the skin and / or hair color is patchy. Adar1 heterozygous knockout mouse. (9) The p150 of Adar1 was knocked out, Adar1 heterozygous knockout mouse as described in (8) above. (10) Adar1 KO:Dct-LacZ Tg mouse obtained by mating an Adar1 heterozygous knockout mouse, which has lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout its body, with a Dct-LacZ transgenic mouse. (11) The p150 of Adar1 was knocked out, The Adar1 KO:Dct-LacZ Tg mouse described in (10) above. (12) An Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse obtained by crossing an Adar1 KO:Dct-LacZ Tg mouse described in (10) or (11) above with a K14-Scf Tg mouse. (13) Adar1 homozygous knockout mouse in which melanocytes have specifically lost the function of Adar1 (Adenosine Deaminase Acting on RNA1). (14) Exhibiting a phenotype in which the color of the body hair is mottled, Adar1 homozygous knockout mouse as described in (13) above. (15) A step of administering the test compound to a knockout mouse described in any one of (8) to (14) above, A test compound selection step of selecting a test compound that suppresses depigmented spots, A screening method for a compound for treating hereditary symmetrical dyschromatosis, which includes (16) A DSH model cell preparation step of preparing cells with inactivated Adar1, A control cell preparation step of preparing control cells without inactivated Adar1, A step of administering a test compound, interferon, and / or a Toll-like receptor ligand to the DSH model cells, A step of administering a test compound, interferon, and / or a Toll-like receptor ligand to the control cells, A viable cell number comparison step of comparing the number of viable cells after performing the step of administering a test compound, interferon, and / or a Toll-like receptor ligand to the DSH model cells with the number of viable cells after performing the step of administering a test compound, interferon, and / or a Toll-like receptor ligand to the control cells, A test compound selection step of selecting a test compound from the comparison result of the viable cell number comparison step, A screening method for a compound for treating hereditary symmetrical dyschromatosis, which includes

Advantages of the Invention

[0011] The pharmaceutical composition for treating hereditary symmetrical dyschromatosis disclosed in the present application can treat hereditary symmetrical dyschromatosis. In addition, by using the hereditary symmetrical dyschromatosis model mice and model cells disclosed in the present application, a compound for treating hereditary symmetrical dyschromatosis can be screened.

Brief Description of the Drawings

[0012] [Figure 1] Figure 1 is a photograph substituting for a drawing, and is a photograph of an Adar1 heterozygous knockout mouse prepared in Example 1. [Figure 2] Figure 2 is a photograph substituting for a drawing. Figure 2A is a photograph of an Adar1 heterozygous knockout mouse prepared in Example 2, and Figure 2B is a photograph of a wild-type mouse with a C57BL / 6 background for comparison. [Figure 3] Figure 3 is a photograph used as a substitute for a drawing, and is a photograph of an Adar1 heterozygous knockout mouse prepared in Example 3. [Figure 4] Figure 4 is a photograph used as a substitute for a drawing, and shows an Adar1 homozygous knockout mouse prepared in Example 4. [Figure 5] Figure 5 is a photograph used as a substitute for a drawing. Column A of Figure 5 shows photographs of control cells and DSH model cells prepared in Example 5. Columns B and C of Figure 5 show photographs of control cells and DSH model cells after administration of Ruxolitinib and Tofacitinib in Example 6. [Figure 6] Figure 6 is a photograph used as a substitute for a drawing. Figure 6A is a photograph of transdermal administration of tofacitinib to DSH model mice in Example 7, and Figure 6B is a photograph of transdermal administration of DMSO to DSH model mice in Comparative Example 1. [Figure 7] Figure 7 is a photograph used as a substitute for a drawing. Figure 7A is a photograph of orally administered tofacitinib to DSH model mice in Example 8, and Figure 7B is a photograph of orally administered DMSO to DSH model mice in Comparative Example 2. [Figure 8] Figure 8 is a photograph used as a substitute for a drawing. Column A of Figure 8 shows photographs of control cells and DSH model cells prepared in Example 5. Column B of Figure 8 shows photographs of control cells and DSH model cells after administration of Delgocitinib in Example 10. [Figure 9] Figure 9 is a photograph used as a substitute for a drawing. Figure 9B is a photograph of transdermal administration of Delgocitinib to DSH model mice in Example 11, and Figure 9A is a photograph of transdermal administration of DMSO to DSH model mice in Comparative Example 3. [Modes for carrying out the invention]

[0013] The following describes in detail the pharmaceutical composition for DSH treatment, the hereditary contralateral pigment disorder model mouse (Adar1 knockout mouse), and the screening method for DSH treatment compounds disclosed in this application.

[0014] First, let's describe the Adar1 knockout mice. The Adar1 knockout mice disclosed in this application include Adar1 heterozygous knockout mice and Adar1 homozygous knockout mice. The Adar1 heterozygous knockout mice and Adar1 homozygous knockout mice will be described in more detail below.

[0015] (First embodiment of Adar1 heterozygous knockout mouse) In the first embodiment, a heterozygous knockout mouse of Adar1 is first created in which the function of Adar1 is lost throughout the body. There are no restrictions on the method of creating a heterozygous knockout mouse that has lost the function of Adar1, as long as the function of Adar1 is lost. For example, it can be created by following the procedure below.

[0016] (a) Cloning all or part of the knockout gene (Adar1) from the mouse genome, or obtaining already cloned genomic DNA, and creating a base sequence including that gene (or part of the gene) and surrounding regions. Introducing mutations or deleting parts of it to partially modify the gene so that it becomes inactive. At that time, in addition to modifications that inactivate the gene, such as deletions, marker genes that produce observable differences (such as color, fluorescence, or antibiotic resistance genes) are incorporated. The genetic information for mouse Adar1 is publicly available and can be obtained, for example, from GenBank Gene ID: 56417. There are no particular restrictions on the region of Adar1 to knock out, but 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) Isolate embryonic stem cells derived from mouse blastocysts (early mouse embryos, in which spherical undifferentiated cells are surrounded by extraembryonic cells). Embryonic stem cells can be cultured in vitro, for example, using embryonic stem cells from brown mice. (c) The embryonic stem cells obtained in (b) above are genetically modified using the base sequence prepared in (a) above by means of electroporation or other methods. Next, embryonic stem cells (heterozygous) that have undergone recombination to the new base sequence are isolated using the marker gene incorporated in (a) above. (d) The homologous recombination-induced embryonic stem cells isolated in (c) above are injected, for example, into a black mouse blastocyst, which is then injected into the uterus of a female mouse, resulting in the birth of a pup. This pup is a chimera, with part of its body derived from the original blastocyst and the other part from the genetically modified embryonic stem cells. As a result, its fur color is a mix of brown and black. (e) Of the chimeric mice, only those whose germ cells (eggs or sperm) are derived from genetically modified cells are used. Specifically, chimeric mice are crossed with black mice, and among the newly born mice, those that are entirely brown are derived from embryonic stem cells. Heterozygous genes are then confirmed by PCR or other methods to obtain heterozygous Adar1 knockout mice. These Adar1 heterozygous knockout mice are then backcrossed to produce black mice.

[0017] Adar1 heterozygous knockout mice can be obtained using the procedure described above, or from institutions such as Mutant Mouse Regional Resource Centers (MMRRC) (Stock No. 034620-JAX).

[0018] As shown in the examples described later, simply losing the function of Adar1 does not result in the fur of Adar1 heterozygous knockout mice remaining black, and they do not exhibit the DSH phenotype. On the other hand, after diligent study, it was newly discovered that administering mouse interferon (m-Ifn) or a Toll-like receptor ligand to Adar1 heterozygous knockout mice prepared using the above procedure resulted in mottled fur coloration and the exhibiting of the DSH phenotype. In the case of Adar1 heterozygous knockout mice according to the first embodiment, "mottled" fur coloration means that in the lesional area, the fur is white overall compared to the black fur of the surrounding non-lesional area, or that white and black fur are mixed together.

[0019] Interferons are proteins secreted by cells in animals in response to the invasion of foreign substances such as pathogens (especially viruses) and tumor cells, and are substances that suppress the proliferation of viruses and cells. Toll-like receptors are receptor proteins on the surface of animal cells that sense various pathogens and activate innate immunity. It is thought that when Adar1 function is reduced or lost, and a state of high immunity or high inflammation is created, Adar1 heterozygous mice exhibit the DSH phenotype, either through the interaction between loss of Adar1 function and externally administered immune or inflammatory substances, or through the interaction between decreased or lost Adar1 function and the internal expression of immune or inflammatory substances induced by ligand administration. In other words, it is thought that Adar1 heterozygous mice exhibit the DSH phenotype when Adar1 function is reduced or lost and a state of high immunity or high inflammation is created.

[0020] The interferon used is not particularly limited as long as the Adar1 heterozygous mouse exhibits the DSH phenotype. Examples include m-Ifnα, m-Ifnβ, m-Ifnω, m-Ifnε, m-Ifnκ, m-Ifnζ, m-Ifnγ, and m-Ifnλ. Homologs corresponding to human type 1 interferons (IFNα, IFNβ, IFNω, IFNε, IFNκ), type 2 interferons (IFNγ), and type 3 interferons (IFNλ) are also acceptable.

[0021] Furthermore, there are no particular restrictions on the Toll-like receptor ligand, as long as the Adar1 heterozygous mouse exhibits the 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) from the membrane of E. coli, paclitaxel, Lipid A, Flagellin (a protein derived from Salmonella typhimurium), Gardiquimod, Imiquimod / R-837 (an imidazoquinoline amine analog of guanosine), Imidazoquinoline Resiquimod / R-848 (a low molecular weight imidazoquinoline compound), Loxoribine, CpG ODN 2006, CpG ODN 1668, and Profilin can be used.

[0022] These substances can be administered either topically to the skin or systemically by oral or injectable means.

[0023] Wild-type adult mice do not have melanocytes (pigment cells that produce brown or black pigment, or melanin) in their skin, but they do have pigment cells in their hair follicles. Therefore, when screening DSH therapeutic compounds using Adar1 heterozygous mice according to the first embodiment, it is sufficient to observe changes in body hair after administering the test compound. Furthermore, DSH is inherited in an autosomal dominant manner, and heterozygotes have a mutation in only one allele of Adar1. The knockout mice according to the first embodiment also have the effect of exhibiting the same phenotype as human DSH under the same genetic conditions.

[0024] (Second embodiment of Adar1 heterozygous knockout mouse) Next, a second embodiment of the 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 Toll-like receptor ligand, the Adar1 heterozygous knockout mouse is crossed with a transgenic mouse expressing LacZ, while in other respects it is the same as the Adar1 heterozygous knockout mouse according to the first embodiment. Therefore, the second embodiment will be described mainly in terms of the differences from the first embodiment, and repetitive explanations of matters already explained in the first embodiment will be omitted. Thus, it goes without saying that even if not explicitly explained in the second embodiment, matters already explained in the first embodiment can be adopted in the second embodiment.

[0025] Transgenic mice expressing LacZ may express LacZ throughout the body, or its expression may be limited to the skin or a portion of skin cells. The Dct-LacZ transgenic mice shown in the following examples express LacZ using the Dct promoter, that is, they express LacZ specifically in pigment cells. Examples of transgenic mice that express 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, Kumamoto University Bioresource Development Research and Support Center, Animal Resource Development Research Division CARD ID 1455).

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

[0027] LacZ is known as a reporter gene and produces β-galactosidase, which breaks down lactose into glucose and galactose. The mechanism of action in Adar1 KO:Dct-LacZ Tg mice is not clear, but the DSH phenotype is shown through a mechanism different from the hyperimmune or hyperinflammatory state of the first embodiment. The "spotted" DSH phenotype that appears in the Adar1 knockout mouse according to the second embodiment means that white and black body hairs are mixed together, similar to the Adar1 knockout mouse according to the first embodiment. However, while the Adar1 heterozygous knockout mouse according to the first embodiment produces the DSH phenotype only in the parts to which interferon or Toll-like receptor ligand was administered, the Adar1 heterozygous knockout mouse according to the second embodiment exhibits a different effect in that the DSH phenotype is produced over a wide area.

[0028] The Adar1 KO:Dct-LacZ Tg mice according to the second embodiment exhibit the DSH phenotype without the administration of interferon or Toll-like receptor ligands. Therefore, since external administration of substances to induce a highly immune state in Adar1 knockout mice is unnecessary, the method is easy to handle and allows for the low-cost screening of DSH therapeutic compounds described later.

[0029] (Third embodiment of Adar1 heterozygous knockout mouse) Next, a third embodiment of the Adar1 heterozygous knockout mouse will be described. The Adar1 heterozygous knockout mouse according to the third embodiment is obtained by mating 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 described as "Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse"). By mating the Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse according to the third embodiment with a K14-Scf Tg mouse, pigment cells can be introduced 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 becomes "spotted" with white and black (brown) 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, the effect of being able to screen for DSH therapeutic compounds by observing changes in skin color is also achieved. Furthermore, the DSH phenotype of the body hair of the mouse according to the third embodiment shows a spotted DSH phenotype in which the white and black hairs are more clearly separated (polka dot pattern) than the phenotype shown by the mouse of the second embodiment, in which the white and black hairs are finely mixed (salt and pepper hair-like). Therefore, when evaluating by hair color, large white spots are formed, making it easier to determine the effect of the DSH therapeutic compound.

[0031] (Embodiment of Adar1 homozygous knockout mouse) Adar1 homozygous knockout mice can be created using the Cre-lox system to produce conditional Adar1 knockout mice, provided that the knockout site is limited, such as by knocking out only pigment cells. If the genetic background of these mice is set to black, it becomes possible to determine whether or not the DSH phenotype will appear.

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

[0033] In the embodiment of the Adar1 homozygous knockout mouse, the Adar1 gene region in melanocytes is targeted, thereby enabling the creation of homozygous knockout mice in which the function of Adar1 is specifically lost in melanocytes. The target is not particularly limited as long as it specifically eliminates the function of Adar1 in melanocytes, and examples include p110 and / or p150. Furthermore, the procedure for creating homozygous knockout mice using the Cre-lox system can be carried out using known methods.

[0034] Homozygous knockout mice die during the fetal stage and are not born. In this embodiment, since Adar1 homozygous knockout mice have Adar1 specifically knocked out in melanocytes, adult mice can be obtained. Adar1 homozygous knockout mice exhibit a phenotype in which black and white body hairs are finely interspersed (resembling salt and pepper) due to the knockout of melanocytes. The Adar1 region to be knocked out in melanocytes may be, for example, p110 and / or p150, but other regions may also be used.

[0035] Adar1 homozygous knockout mice tend to exhibit more severe symptoms (more white hair) than Adar1 heterozygous knockout mice according to the second embodiment. Therefore, when several test compounds with high therapeutic efficacy are found, using Adar1 homozygous knockout mice allows for a high-level evaluation of the therapeutic effect of the test compounds. On the other hand, Adar1 heterozygous knockout mice according to the second embodiment are useful for initial screening of test compounds.

[0036] Next, we will describe a screening method for DSH therapeutic compounds. The screening method for DSH therapeutic compounds can be performed using the Adar1 knockout mice (hereinafter sometimes referred to as "DSH model mice") or cells such as pigment cells.

[0037] (Embodiment of a screening method using DSH model mice) An example of a method for screening DSH therapeutic compounds using a DSH model mouse is: The process involves administering the test compound to a DSH model mouse, • A test compound selection step in which a test compound is selected to suppress depigmented spots (vitiligo, white hair), Includes.

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

[0039] There are no particular restrictions on the method of administering the test compound to DSH model mice, as long as it is taken into the DSH model mouse's body, such as oral administration, skin application, or internal administration by injection. Then, by determining whether or not the administered test compound suppresses depigmentation spots (white spots, white hair) in DSH model mice (in the case of body hair, the hair color changes from white to black (brown), and in the case of skin, the white spots decrease (pigmentation darkens)), a compound that suppresses depigmentation spots should be selected.

[0040] (Embodiment of a screening method using cells) An example of a cell-based screening method for DSH therapeutic compounds is: • A DSH model cell preparation step to prepare cells with Adar1 inactivated, • A control cell preparation step to prepare control cells that have not had Adar1 inactivated, The process involves administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells. The steps include administering the test compound and interferon and / or Toll-like receptor ligand to the control cells, A step of comparing the number of surviving cells after administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells, and the number of surviving cells after administering the test compound and interferon and / or Toll-like receptor ligand to the control cells, • A test compound selection step in which a test compound is selected based on the comparison results of the viable cell number comparison step, Includes.

[0041] Examples of cells include mouse or human-derived pigment cells. Since Adar1 is also expressed in cells other than pigment cells, cells expressing Adar1 may be used instead of pigment cells. Examples of Adar1-expressing cells include cos7 cells, HeLa cells, melanoma cells, and HaCaT cells. The cells may be primary cultured cells or passaged cells. Inactivation of Adar1 in cells is not particularly limited as long as it specifically inactivates Adar1 in the cells. For example, known methods such as introducing siRNA containing an Adar1-inactivating sequence into cells can be used. Alternatively, cells may be primary cultured from Adar1 heterozygous knockout mice and wild-type mice, respectively. Furthermore, human-derived cells with the mutated ADAR1 and wild-type ADAR1 gene transfers, or mouse-derived cells with the mutated Adar1 and wild-type Adar1 gene transfers, may also be used. When interferon and / or Toll-like receptor ligands are administered to cells with Adar1 inactivation (hereinafter sometimes referred to as "DSH model cells") and control cells into which a control siRNA that does not inactivate Adar1 has been introduced, the survival rate of the Adar1-inactivated DSH model cells decreases compared to the control cells that do not have Adar1 inactivation. Therefore, it is sufficient to compare the number of surviving cells in (1) the group of DSH model cells administered with the test compound and interferon and / or Toll-like receptor ligands, and (2) the group of control cells administered with the test compound and interferon and / or Toll-like receptor ligands. The closer the ratio of surviving cells in groups (1) and (2), the more likely it is that the test compound increased cell viability.

[0042] (Embodiment of a pharmaceutical composition for treating DSH) The pharmaceutical composition for treating DSH is not particularly limited as long as it contains an ingredient effective in treating DSH. The inventors conducted experiments with various known pharmaceuticals and confirmed the therapeutic effects of various commercially available types of JAK inhibitors and STAT inhibitors, as shown in the examples described later. Therefore, JAK inhibitors, STAT inhibitors, and similar compounds can be used in pharmaceutical compositions for treating DSH.

[0043] Dosage forms of pharmaceutical compositions for the treatment of DSH include, for example, tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid or in a liquid medium), ointments, gelatin soft and hard capsules, suppositories, sterile injection solutions, and sterile encapsulated powders.

[0044] Furthermore, the DSH therapeutic pharmaceutical composition may contain carriers, excipients, and 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 oxybenzoates, talc, magnesium stearate, and mineral oil. In addition, vitamins, lubricants, humectants, emulsifiers and suspending agents, preservatives, sweeteners, or flavorings may be added. The DSH therapeutic pharmaceutical composition may also be combined with known drugs.

[0045] The embodiments disclosed in this application are described below in detail by illustrating the embodiments, but these embodiments are solely for illustrative purposes and are not intended to limit or restrict the scope of the invention disclosed in this application. [Examples]

[0046] [Creation of DSH model mice] <Example 1> p150 Adar1 heterozygous knockout mice (C57BL / 6 background) were prepared using the following procedure. We purchased a BAC clone containing the Adar1 gene from the BACPAC Resources Center (BPRC) (https: / / bacpacresources.org), and extracted the p150-specific exon of the Adar1 gene from the genomic DNA clone using restriction enzymes. We then transferred the genomic DNA clone into a pBluescript vector, linearized it, and introduced it into embryonic stem cells. Embryonic stem cells in which recombination between the introduced gene and the endogenous gene had occurred were selected using antibiotics. Embryonic stem cells with recombination were injected into blastocysts, which were then returned to the uterus of mice to obtain chimeric mice. These chimeric mice were crossed with wild mice, and mice born from sperm derived from embryonic stem cells were selected by body hair. By confirming the genotype using PCR, we obtained p150 Adar1 heterozygous knockout mice. These mice were then backcrossed to create a C57BL / 6 background. Newborn p150 Adar1 heterozygous knockout mice (C57BL / 6 background) on day 2 of life were subcutaneously injected with 3000 U of m-Ifnβ (PBL Corporation) once daily. Subcutaneous injection of m-Ifnβ was continued for 10 days. Figure 1 is a photograph of the Adar1 heterozygous knockout mice prepared in Example 1. Wild-type mice on a C57BL / 6 background have normal black fur, but the Adar1 heterozygous knockout mice prepared in Example 1, also on a C57BL / 6 background, had white fur that grew after subcutaneous injection (see the oval area in Figure 1).

[0047] Also, instead of m-Ifnβ, • When 0.5 μg / μL of LPS (manufactured by Wako), a Toll-like receptor ligand, was subcutaneously injected at a dose of 50 μL over the same period, • When poly(I:C) (manufactured by GE Healthcare), a Toll-like receptor ligand, was administered via subcutaneous injection of 50 μL at a dose of 2 μg / μL of poly(I:C) over the same period, We also confirmed that the body hair turned white, similar to Figure 1.

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

[0049] Based on these results, we confirmed that a model mouse exhibiting the DSH phenotype can be created by administering interferon or a Toll-like receptor ligand to Adar1 heterozygous knockout mice.

[0050] <Example 2> Adar1 KO:Dct-LacZ Tg mice were created by mating Adar1 heterozygous knockout mice, which had not been administered interferon and Toll-like receptor ligand as in Example 1, with Dct-LacZ transgenic (Tg) mice (Kumamoto University Bioresource Research and Support Center, ID:782). Figure 2A is a photograph of the mouse created 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, it was confirmed that the mouse had white hairs mixed in with the normal black hairs. This phenotype better represents the clinical appearance of DSH, which is a mixture of pigmented and depigmented spots. Furthermore, it was confirmed that the mouse created in Example 2 exhibited the DSH phenotype even without administration of interferon and Toll-like receptor ligand.

[0051] <Example 3> Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mice were created by mating the Adar1 KO:Dct-LacZ Tg mice prepared in Example 2 with K14-Scf Tg mice (Kunisada T, et al., J Exp Med. 1998; 187(10): 1565-73.). Figure 3 shows a photograph of the mouse prepared in Example 3. In Example 3, pigment cells were introduced into the skin of the Adar1 KO:Dct-LacZ Tg mice prepared in Example 2 by mating with K14-Scf Tg mice. As a result, the mice prepared in Example 3 showed large circular white spots (polka dots) compared to the "salt and pepper" phenotype of Example 2, where white and black hairs were finely mixed, and were confirmed to be closer to the human DSH phenotype.

[0052] <Example 4> Adar1 Flox Mouse (Adar tm1a(EUCOMM)Wtsi / tm1a(EUCOMM)Wtsi (MBLD;EPD0087_1_D11), Sanger Institute, UK) was 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. MBLD has a gene modification in Adar1 in a homozygous state, and by crossing it with Dct-Cre Tg mice, the mouse of Example 4 (Adar) was produced. tm1a(EUCOMM)Wtsi / tm1a(EUCOMM)Wtsi In the Dct-Cre_Tg model, Adar1 was knocked out only in melanocytes, resulting in Adar1 homozygous knockout mice. Figure 4 is a photograph of the mouse produced in Example 4. As shown in Figure 4, the mouse produced in Example 4 exhibited a phenotype in which the body hair was patchy with white and black areas.

[0053] [Creation of DSH model cells] <Example 5> In Example 1, administering interferon or Toll-like receptor ligand to Adar1 heterozygous knockout mice resulted in the DSH phenotype. Therefore, DSH model cells using pigment cells were created using the following procedure.

[0054] [Day 1] Place 0.5x10⁶ mouse melanocytes (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) into a 12-well plate. 5 Cells were seeded in a 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) (Sequence ID: 1) and control siRNA (Allstar negative siRNA, Qiagen) were introduced into cultured cells according to the instructions provided with HiPerfect (Qiagen). [Day 3] Mouse interferon β (PBL) was added at a rate of 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 control cells (control siRNA + m-Ifn) transfected with control siRNA, and the lower row of column A shows DSH model cells (Adar1 siRNA + m-Ifn) transfected with Adar1 siRNA. As is clear from the images 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 measured using a TC20 fully automated cell counter (Bio-Rad). The ratio of viability was defined as the cell viability of DSH model cells compared to control cells.

[0055] Based on these results, the survival rate of pigment cells with reduced Adar1 expression decreased, suggesting that the DSH model cells created exhibit the characteristics of pigment cells from DSH patients.

[0056] [Confirmation of the usefulness of DSH model cells and DSH model mice] Next, we confirmed the usefulness of the DSH model mice created in Example 1 and the DSH model cells created in Example 5. As mentioned above, the DSH model mice created in Example 1 did not show the DSH phenotype in Adar1 heterozygous knockout mice. However, after diligent research, we found that when Adar1 function was lost and a state of high immunity or high inflammation was induced, Adar1 heterozygous mice showed the DSH phenotype. Therefore, we considered that drugs with immunosuppressive or anti-inflammatory functions would be useful in treating Adar1, and after conducting various studies on drugs with immunosuppressive functions, 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 conducted in the same manner as in Example 5, except that, in addition to mouse interferon-β, Ruxolitinib was added to achieve a final concentration of 0.5 μM and Tofacitinib to achieve a final concentration of 5 μM on day 3 of Example 5. The drug 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 shows images of control cells after Ruxolitinib was added, and the lower row of column B shows images of DSH model cells after Ruxolitinib was added. The upper row of column C in Figure 5 shows images of control cells after Tofacitinib was added, and the lower row of column C shows images of DSH model cells after Tofacitinib was added.

[0058] As is evident from the photographs in columns B and C of Figure 5, the survival rate of DSH model cells improved when either the immunosuppressant Ruxolitinib or Tofacitinib was added. In particular, with Tofacitinib, the cell survival rate was 98.8% (number of DSH model cells treated with Tofacitinib / number of control cells treated with Tofacitinib). This means that DSH model cells with reduced or lost Adar1 function recovered their survival rate upon the addition of Ruxolitinib and Tofacitinib, suggesting their usefulness in treating diseases characterized by reduced or lost Adar1 function.

[0059] [Experiments using DSH model mice] In Example 6, the following experiment was performed using tofacitinib, which showed a high cell viability rate.

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

[0061] <Comparative Example 1: Transdermal Administration> The experiment was carried out using the same procedure as in Example 7, except that DMSO, the solvent for tofacitinib, was injected instead of tofacitinib. Figure 6B is a photograph taken at 22 days postpartum.

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

[0063] <Comparative Example 2: Oral Administration> The experiment was conducted using the same procedure as in Example 8, except that DMSO, the solvent for tofacitinib, was administered orally instead of tofacitinib. Figure 7B is a photograph taken at 22 days postpartum.

[0064] As is evident from the photographs, DSH model mice that received tofacitinib via transdermal injection (Example 7: Figure 6A) or oral administration (Example 8: Figure 7A) showed a change in fur color from white to black. On the other hand, DSH model mice that did not receive tofacitinib via transdermal injection (Comparative Example 1: Figure 6B) or oral administration (Comparative Example 2: Figure 7B) retained their white fur.

[0065] From these results, it was confirmed that DSH model mice that were not administered tofacitinib continued to exhibit the DSH phenotype, but that the DSH phenotype was resolved upon administration of tofacitinib. In other words, it was confirmed 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 with DSH model cells and DSH model mice, confirming that DSH model cells and DSH model mice can be used for screening DSH therapeutic compounds.

[0066] <Example 9> [Screening of compounds for the treatment of DSH] As described above, since we confirmed that DSH model cells and DSH model mice can be used for screening DSH therapeutic compounds, we screened various compounds using DSH model cells. Table 1 below shows the compounds screened using the same procedure as in Example 6, their concentrations, known uses, and cell viability. Compounds already identified in Example 6 are also listed in Table 1 for comparison. The example compounds were obtained from Sigma Aldrich (Tofacitinib), Tokyo Chemical Industry (Curcumol), Cayman Chemical (Cerdulatinib), and Selleck (all others).

[0067] [Table 1]

[0068] As shown in Table 1, JAK inhibitors and STAT inhibitors demonstrated therapeutic efficacy in various commercially available types. In particular, JAK inhibitors showed cell viability of 50% or more in most types, confirming their suitability as pharmaceutical compositions for DSH treatment.

[0069] [Confirmation of the usefulness of other JAK inhibitors] <Example 10> The experiment was carried out in the same manner as in Example 6, except that Delgocitinib (ChemScene), a JAK inhibitor, was added to a final concentration of 1 μM instead of Ruxolitinib and Tofacitinib as in Example 6. The upper row of column B in Figure 8 shows photographs of control cells after Delgocitinib was added, and the lower row of column B shows photographs of DSH model cells after Delgocitinib was added.

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

[0071] <Example 11> Transdermal administration experiments were performed using a procedure similar to that of Example 7, except that Delgocitinib was used instead of Tofacitinib. Specifically, 3000 U of m-Ifnβ (manufactured by PBL) was subcutaneously injected once daily into neonate p150 Adar1 heterozygous knockout mice described in Example 1. Injections of m-Ifnβ were started on postnatal day 2 and continued for 10 doses until postnatal day 14. Delgocitinib was administered subcutaneously at a dose of 15 mg / kg / day once daily for 10 doses from postnatal day 2 to postnatal day 14. Figure 9B is a photograph taken on postnatal day 15.

[0072] <Comparative Example 3> The experiment was carried out using the same procedure as in Example 11, except that DMSO, the solvent for Delgocitinib, was injected instead of Delgocitinib. Figure 9A is a photograph taken at 15 days postpartum.

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

[0074] Based on these results, we confirmed that Delgocitinib, a JAK inhibitor, is a useful compound for treating DSH in both DSH model cell and DSH model mouse experiments. [Industrial applicability]

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

Claims

1. A heterozygous Adar1 knockout mouse in which the function of Adar1 (Adenosine Deaminase Acting on RNA1) is lost throughout the body, The phenotype is that the skin and / or hair color is patchy. Adar1 heterozygous knockout mouse.

2. The p150 of Adar1 was knocked out. The Adar1 heterozygous knockout mouse according to claim 1.

3. Adar1 KO:Dct-LacZ Tg mouse was obtained by mating an Adar1 heterozygous knockout mouse, which had lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout its body, with a Dct-LacZ transgenic mouse.

4. The p150 of Adar1 was knocked out. Adar1 KO:Dct-LacZ Tg mouse as described in claim 3.

5. An Adar1 KO:Dct-LacZ Tg:K14-Scf Tg mouse obtained by crossing an Adar1 KO:Dct-LacZ Tg mouse described in claim 3 or 4 with a K14-Scf Tg mouse.

6. Adar1 homozygous knockout mouse in which the function of Adar1 (Adenosine Deaminase Acting on RNA1) is specifically lost in melanocytes.

7. The phenotype is that the body hair color is mottled. Adar1 homozygous knockout mouse according to claim 6.

8. A step of administering a test compound to a knockout mouse according to any one of claims 1 to 7, A test compound selection step in which a test compound is selected to suppress depigmentation, A screening method for compounds for the treatment of hereditary contralateral pigmentary disorders, including those mentioned above.

9. A DSH model cell preparation step to prepare cells in which Adar1 has been inactivated, A control cell preparation step to prepare control cells in which Adar1 has not been inactivated, The process involves administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells, The process involves administering the test compound and interferon and / or Toll-like receptor ligand to the control cells, A step of comparing the number of surviving cells after administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells, and the number of surviving cells after administering the test compound and interferon and / or Toll-like receptor ligand to the control cells, Based on the comparison results of the aforementioned viable cell number comparison step, a test compound selection step is performed to select the test compound, A screening method for compounds for the treatment of hereditary contralateral pigmentary disorders, including those mentioned above.

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