WO2021193224A1 - Pharmaceutical composition for treating dyschromatosis symmetrica hereditaria, dyschromatosis symmetrica hereditaria model mouse, and screening method for compound for treating dyschromatosis symmetrica hereditaria - Google Patents

Pharmaceutical composition for treating dyschromatosis symmetrica hereditaria, dyschromatosis symmetrica hereditaria model mouse, and screening method for compound for treating dyschromatosis symmetrica hereditaria Download PDF

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WO2021193224A1
WO2021193224A1 PCT/JP2021/010546 JP2021010546W WO2021193224A1 WO 2021193224 A1 WO2021193224 A1 WO 2021193224A1 JP 2021010546 W JP2021010546 W JP 2021010546W WO 2021193224 A1 WO2021193224 A1 WO 2021193224A1
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adar1
mouse
cells
dsh
dyschromatosis
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PCT/JP2021/010546
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French (fr)
Japanese (ja)
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通浩 河野
真志 秋山
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国立大学法人東海国立大学機構
国立大学法人秋田大学
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Publication of WO2021193224A1 publication Critical patent/WO2021193224A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
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    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
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    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the disclosure of this application relates to a pharmaceutical composition for treating hereditary contralateral dyschromatosis, a mouse model for hereditary contralateral dyschromatosis, and a method for screening a compound for treating hereditary contralateral dyschromatosis.
  • RNA-specific adenosine deaminase 1 Adenosine Deaminase Acting on RNA1, which is one of the RNA editing enzymes, hereinafter may be referred to as “ADAR1” or “ADAR”.
  • ADAR1 RNA-specific adenosine deaminase 1
  • the gene encoding heavy-chain RNA-specific adenosine deaminase 1 is described as "Adar1", and the model cell may be described as "Adar1" regardless of its origin). be. It is known that converting RNA adenosine to inosine produces a protein different from DNA information. It has been clarified that having a heterozygous mutation in the ADAR1 gene causes DSH (see Non-Patent Document 1).
  • DSH rarely has neurological symptoms, but does not cause serious life-threatening symptoms. However, since it develops from childhood and the symptoms appear in the exposed part, there is a problem that a heavy mental burden is caused to the patient due to the appearance problem. Therefore, it is required to establish a therapeutic agent and a therapeutic method, but there is currently no effective therapeutic agent and therapeutic method.
  • a model animal such as a mouse having the disease is prepared, the pathological condition of the disease is investigated using the model animal, or a test compound is administered to the model animal to obtain a therapeutic compound. Screening is common practice.
  • DSH phenotype the phenotype indicated by the DSH patient (hereinafter, may be referred to as "DSH phenotype") does not appear in the mouse, and there is a problem that it cannot be used for DSH research. ..
  • DSH can be treated by using a JAK inhibitor or a STAT inhibitor as an active ingredient.
  • Heterozygous Adar1 knockout mice that have lost the function of Adar1 systemically show DSH phenotype when a predetermined substance is administered or a gene that produces them is expressed (3) Melanosite-specifically.
  • the homozygous Adar1 knockout mouse that lost the function of Adar1 showed a DSH phenotype, and (4) the prepared knockout mouse (hereditary contralateral dyschromatosis model mouse) and the function of Adar1 were reduced or lost.
  • screening of DSH therapeutic compounds can be performed by using cells (model cells).
  • the purpose of the disclosure of the present application is to screen a pharmaceutical composition for treating hereditary contralateral dyschromatosis, a mouse model for hereditary contralateral dyschromatosis, and a compound for treating hereditary contralateral dyschromatosis.
  • the method is to provide.
  • the disclosure of this application is the screening of pharmaceutical compositions for the treatment of hereditary contralateral dyschromatosis, hereditary contralateral dyschromatosis model mice, and compounds for the treatment of hereditary contralateral dyschromatosis, as shown below. Regarding the method.
  • a pharmaceutical composition for treating hereditary contralateral dyschromatosis which comprises 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, Itacitinib, Solcitinib, AZD1480, Ruxolitinib, JAK3-IN-6, Curcumol, Peficitinib, Cerdulatinib, FM-381, Filgotinib, and Delgocitinib.
  • the STAT inhibitor Selected from Napabucasin, Ochromycine, Fludarabine, Nifuroxazide, C188-9, and AS1517499, The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to (1) above.
  • the JAK inhibitor is Delgocitinib.
  • the JAK inhibitor is Ruxolitinib.
  • the JAK inhibitor is Tofacitinib.
  • the JAK inhibitor is Peficitinib.
  • the JAK inhibitor is Cerdulatinib.
  • the p150 of Adar1 was knocked out.
  • Adar1 KO Dct-LacZ Tg mouse obtained by mating a Dct-LacZ transgenic mouse with an Adar1 heterozygous knockout mouse that has lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout the body. (11) The p150 of Adar1 was knocked out.
  • the Adar1 KO Dct-LacZ Tg mouse according to (10) above.
  • Adar1 KO Dct-LacZ Tg: K14-Scf Tg obtained by mating a K14-Scf Tg mouse with the Adar1 KO: Dct-LacZ Tg mouse according to (10) or (11) above. mouse.
  • Adar1 homozygous knockout mouse in which the function of Adar1 (Adenosine Deaminase Acting on RNA1) was specifically lost in a melanocyte manner.
  • (14) Shows the phenotype that the color of the hair is mottled, The Adar1 homozygous knockout mouse according to (13) above.
  • (15) The step of administering the test compound to the knockout mouse according to any one of (8) to (14) above, and A test compound selection step of selecting a test compound that suppresses depigmentation spots, A method for screening a compound for treating hereditary contralateral dyschromatosis, including.
  • the step of comparing the number of viable cells and the step of comparing the number of viable cells after the above A test compound selection step of selecting a test compound from the comparison result of the survival cell number comparison step and a test compound selection step.
  • a method for screening a compound for treating hereditary contralateral dyschromatosis including.
  • the pharmaceutical composition for treating hereditary contralateral dyschromatosis disclosed in this application can treat hereditary contralateral dyschromatosis.
  • the hereditary contralateral dyschromatosis model mouse and model cells disclosed in this application it is possible to screen a compound for treating hereditary contralateral dyschromatosis.
  • FIG. 1 is a drawing-substituting photograph, which is a photograph of an Adar1 heterozygous knockout mouse produced in Example 1.
  • FIG. 2 is a drawing substitute photograph.
  • FIG. 2A is a photograph of the Adar1 heterozygous knockout mouse prepared in Example 2
  • FIG. 2B is a photograph of a wild-type mouse with a C57BL / 6 background for comparison.
  • FIG. 3 is a drawing-substituting photograph, which is a photograph of the Adar1 heterozygous knockout mouse produced in Example 3.
  • FIG. 4 is a drawing-substituting photograph, which is a photograph of the Adar1 homozygous knockout mouse produced in Example 4.
  • FIG. 5 is a drawing substitute photograph. Column A in FIG.
  • FIG. 5 is a photograph of the control cells and DSH model cells prepared in Example 5. Rows B and C of FIG. 5 are photographs of control cells and DSH model cells when Ruxolitinib and Tofacitinib were administered in Example 6.
  • FIG. 6 is a drawing substitute photograph.
  • FIG. 6A is a photograph of Tofacitinib transdermally administered to a DSH model mouse in Example 7
  • FIG. 6B is a photograph of DMSO transdermally administered to a DSH model mouse in Comparative Example 1.
  • FIG. 7 is a drawing substitute photograph.
  • FIG. 7A is a photograph of Tofacitinib orally administered to a DSH model mouse in Example 8, and FIG.
  • FIG. 7B is a photograph of DMSO orally administered to a DSH model mouse in Comparative Example 2.
  • FIG. 8 is a drawing substitute photograph.
  • Column A in FIG. 8 is a photograph of the control cells and DSH model cells prepared in Example 5.
  • Column B of FIG. 8 is a photograph of control cells and DSH model cells when Delgocitinib was administered in Example 10.
  • FIG. 9 is a drawing substitute photograph.
  • FIG. 9B is a photograph of Delgocitinib transdermally administered to a DSH model mouse in Example 11
  • FIG. 9A is a photograph of DMSO transdermally administered to a DSH model mouse in Comparative Example 3.
  • the screening method for the DSH therapeutic pharmaceutical composition disclosed in this application the hereditary contralateral dyschromatosis model mouse (Adar1 knockout mouse), and the DSH therapeutic compound will be described in detail below.
  • the Adar1 knockout mice disclosed in this application include Adar1 heterozygous knockout mice and Adar1 homozygous knockout mice.
  • the Adar1 heterozygous knockout mouse and the Adar1 homozygous knockout mouse will be described in more detail below.
  • the Adar1 heterozygous knockout mouse according to the first embodiment first prepares a heterozygous knockout mouse in which the function of Adar1 is lost systemically.
  • the heterozygous knockout mouse that has lost the function of Adar1 is not limited in the production method as long as the function of Adar1 is lost. For example, it may be produced by the following procedure.
  • A Clone all or part of the knockout gene (Adar1) on the mouse genome, or purchase and obtain the already cloned genomic DNA, and obtain the gene (or part of the gene). And create a base sequence that includes the peripheral part. Mutations are introduced there or parts are deleted to partially modify the gene so that it is inactivated. It then incorporates marker genes that result in observable differences (color, fluorescence, antibiotic resistance genes, etc.) as well as inactivation changes such as deletions.
  • the gene information of mouse Adar1 is known, and for example, the gene information can be obtained from GenBank Gene ID: 56417 and the like.
  • the knockout region of Adar1 is not particularly limited.
  • Adar1 has isoforms such as p110 localized in the nucleus and p150 localized in the cytoplasm, even if the p150 isoform is specifically deleted. good.
  • B Isolate embryonic stem cells from mouse blastocysts (early mouse embryos, spherical undifferentiated cells surrounded by extraembryonic cells). As the embryonic stem cells, for example, brown mouse embryonic stem cells can be used for cell culture in vitro.
  • C The nucleotide sequence prepared in (a) above is gene-introduced into the embryonic stem cells obtained in (b) above by means such as electroporation.
  • embryonic stem cells (heterozygous type) that have actually undergone recombination to a new nucleotide sequence are isolated.
  • the homologous-recombinant embryonic stem cells separated in (c) above are injected into, for example, a black mouse blastocyst, and the blastocyst is injected into the uterus of a female mouse to produce a pup.
  • the pups become a chimera containing two cells, part of the body derived from the original blastocyst and the other part derived from genetically engineered embryonic stem cells. Therefore, the coat color becomes brown and black spots.
  • mice only germ cells (eggs or sperms) derived from genetically engineered cells are used. Specifically, among newly born mice obtained by mating chimeric mice with black mice, mice having a brown body are derived from embryonic stem cells, and the modified genes among them are confirmed to be heterozygous by PCR or the like. Then, a heterozygous Adar1 knockout mouse is obtained. Further, this Adar1 heterozygous knockout mouse is backcrossed to obtain a black mouse.
  • germ cells eggs or sperms
  • the Adar1 heterozygous knockout mouse may use the above procedure or one obtained from an institution such as Mutant Mouse Regional Resource Centers (MMRRC) (Stock No. 034620-JAX).
  • MMRRC Mutant Mouse Regional Resource Centers
  • the hair of the Adar1 heterozygous knockout mouse remains black and does not show the DSH phenotype simply by losing the function of Adar1.
  • the Adar1 heterozygous knockout mouse according to the first embodiment has been subjected to diligent studies. ) It was newly found that the administration of the ligand causes the hair color to become mottled and shows a DSH phenotype.
  • the color of the hair of the Adar1 heterozygous knockout mouse according to the first embodiment is "spot", which means that the hair is white in the entire lesion as compared with the black hair in the surrounding non-lesion. It means that there is, or a mixture of white and black hair.
  • Interferon is a protein secreted by cells in response to the invasion of foreign substances such as pathogens (especially viruses) and tumor cells in the animal body, and is a substance for suppressing the growth of viruses and cells.
  • Toll-like receptors are receptor proteins on the cell surface of animals and have the function of detecting various pathogens and activating innate immunity.
  • Adar1 heterozygous mice are thought to exhibit the DSH phenotype when they are hyperimmunized or hyperinflammatory with reduced or lost ADAR1 function.
  • Interferon is not particularly limited as long as the Adar1 heterozygous mouse exhibits the DSH phenotype.
  • m-Ifn ⁇ , m-Ifn ⁇ , m-Ifn ⁇ , m-Ifn ⁇ , m-Ifn ⁇ , m-Ifn ⁇ , m-Ifn ⁇ and the like can be mentioned.
  • homologues corresponding to type 1 interferon (IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ , IFN ⁇ ), type 2 interferon (IFN ⁇ ), and type 3 interferon (IFN ⁇ ) in humans can also be mentioned.
  • the Toll-like receptor ligand is not particularly limited as long as the Adar1 heterozygous mouse shows the DSH phenotype.
  • Pam3CSK4 synthetic tripalmitylated lipopeptide
  • Histone Histone
  • Zymosan yeast-derived cell wall component
  • MARP-2 MARP-2
  • Poly (I: C) Polyynosinic-polycyticidylic acid sodium salt
  • E E.
  • coli membrane component lipo paclitaxel
  • Lipid a flagellin (Salmonella typhimurium-derived protein), Gardiquimod, imidazoquinoline amine analog Imiquimod / R-837 (guanosine), Imidazoquinoline compounds of Imidazoquinoline Resiquimod / R-848 (low molecular weight), Loxoribine, CpG ODN 2006 , CpG ODN 1668, Profilin, and other commercially available Toll-like receptor ligands may be used.
  • the method of administration of these substances may be either topical administration to the skin or systemic administration by oral administration or injection.
  • the skin of adult wild-type mice does not have pigment cells (melanocytes) that produce brown to black pigment (melanin), but the hair follicles of body hair have pigment cells. Therefore, when screening a DSH therapeutic compound using the Adar1 heterozygous mouse according to the first embodiment, changes in hair may be observed by administration of the test compound.
  • DSH shows autosomal dominant inheritance and has a mutation in only one allele of Adar1 (heterozygous type).
  • the knockout mouse according to the first embodiment also has the effect of exhibiting a phenotype under the same genetic conditions as human DSH.
  • the Adar1 heterozygous knockout mouse according to the second embodiment is the first embodiment in that the Adar1 heterozygous knockout mouse is mated with a transgenic mouse expressing LacZ instead of administration of an interferon or a Toll-like receptor ligand.
  • the other points are the same as the Adar1 heterozygous knockout mouse according to the first embodiment.
  • the transgenic mouse expressing LacZ may be systemically expressed at the LacZ expression site, or may be limited to the skin or a part of skin cells.
  • the Dct-LacZ transgenic mouse shown in the following examples is a mouse that expresses LacZ with the Dct promoter, that is, expresses LacZ specifically in a pigment cell.
  • ROSA26-lacZ Soriano P. Nat Genet. 1999 Jan; 21 (1): 70-1
  • CAG-lacZ C57BL / 6-Tg (Cag-LacZ) 11Miya, Kumamoto University Life Resources Development Research and Support Center Animal Resources Development Research Division CARD ID 1455.
  • 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 show the DSH phenotype prepared in the same procedure as the first embodiment. Obtained by doing.
  • the obtained knockout mouse according to the second embodiment (hereinafter, may be referred to as “Adar1 KO: Dct-LacZ Tg mouse”) is different from the first embodiment in that it is an interferon or Toll-like receptor ligand. Shows the DSH phenotype without administration of.
  • LacZ is known as one of the reporter genes and produces ⁇ -galactosidase, which decomposes lactose into glucose and galactose.
  • Adar1 KO: Dct-LacZ Tg mice the mechanism of action in Adar1 KO: Dct-LacZ Tg mice is not clear, the DSH phenotype is also exhibited by a mechanism of action different from the hyperimmune or hyperinflammatory state of the first embodiment.
  • the DSH phenotype "spot" that appears in the Adar1 knockout mouse according to the second embodiment means that white body hair and black body hair are mixed as in the Adar1 knockout mouse according to the first embodiment. means.
  • the DSH phenotype is produced only in the portion to which the interferon or Toll-like receptor ligand is administered, whereas the Adar1 heterozygous type according to the second embodiment is produced.
  • Type knockout mice have different effects in that a wide range of DSH phenotypes occur.
  • the DSH phenotype appears without administration of interferon or Toll-like receptor ligand. Therefore, since it is not necessary to externally administer a substance for making the Adar1 knockout mouse highly immune, it is easy to handle and the DSH therapeutic compound screening method described later can be carried out at low cost.
  • the Adar1 heterozygous knockout mouse according to the third embodiment is obtained by mating a K14-Scf Tg mouse with the Adar1 heterozygous knockout mouse (Adar1 KO: Dct-LacZ Tg mouse) according to the second embodiment.
  • the obtained mouse may be described as "Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mouse").
  • the Adar1 KO: Dct-LacZ Tg mouse according to the third embodiment is the skin of the Adar1 KO: Dct-LacZ Tg mouse according to the second embodiment by mating the K14-Scf Tg mouse. Pigment cells can be introduced into the mouse. Therefore, in the Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mouse according to the third embodiment, in addition to the phenotype according to the second embodiment, the skin also becomes white and black (brown) "spots". Indicates the phenotype.
  • the DSH phenotype of the body hair of the mouse according to the third embodiment is a phenotype in which the white hair and the black hair shown by the mouse of the second embodiment are finely mixed (salt and pepper hair like). It shows a mottled DSH phenotype in which the white hair part and the black hair part are more clearly separated (polka dots, polka dot). Therefore, since large white spots are formed even when evaluated by coat color, it becomes easy to understand the effect determination of the DSH therapeutic compound.
  • the Adar1 homozygous knockout mouse can be produced by using the Adar1 conditional knockout mouse using the Cre-lox system, if the site is limited, such as knockout of only pigment cells. If the genetic background of the mouse at that time is a black mouse, it becomes possible to determine whether or not the DSH phenotype appears.
  • Conditional knockout is a method also called conditional gene disruption that can be performed by crossing flox mice and Cre-expressing mice.
  • a mouse flox mouse or floxed mouse
  • a target gene region is sandwiched between Cre recombinase target sequence loxP is prepared.
  • Cre-expressing mice deletion of the target gene occurs only in specific target cells.
  • a homozygous knockout mouse in which the melanocyte-specific Adar1 function is lost can be produced by targeting the melanocyte Adar1 as a target gene region.
  • the target is not particularly limited as long as it can specifically lose the function of Adar1 of melanocytes, and examples thereof include p110 and / or p150.
  • a known method may be used as a procedure for producing a homozygous knockout mouse using the Cre-lox system.
  • Adar1 homozygous knockout mice die during the fetal period and are not born.
  • Adar1 homozygous knockout mouse of the present embodiment since Adar1 is knocked out specifically in a melanocyte, an adult mouse can be obtained.
  • Adar1 homozygous knockout mice exhibit a phenotype in which black and white hairs are finely mixed to form (salt and pepper-like) spots due to melanocyte knockout.
  • the region of Adar1 to be knocked out in melanocytes may include, for example, p110 and / or p150, but may be other regions.
  • the Adar1 homozygous knockout mouse tends to have stronger symptoms (more white hair) than the Adar1 heterozygous knockout mouse according to the second embodiment. Therefore, when several kinds of test compounds that can be expected to have a high therapeutic effect are found, the therapeutic effect of the test compound can be evaluated at a high level by using the Adar1 homozygous knockout mouse.
  • the Adar1 heterozygous knockout mouse according to the second embodiment has the effect of being useful for the initial screening of the test compound.
  • the method for screening a compound for treating DSH can be carried out using the above-mentioned Adar1 knockout mouse (hereinafter, may be referred to as “DSH model mouse”) or cells such as pigment cells.
  • An embodiment of a method for screening a compound for treating DSH using a DSH model mouse is described as an embodiment. -The step of administering the test compound to the DSH model mouse and -A test compound selection step of selecting a test compound that suppresses depigmentation spots (white spots, white hair), and including.
  • Test compounds include, for example, 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, and fermentation. Examples thereof include microbial products, marine organism extracts, and plant extracts.
  • the administration of the test compound to the 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, attachment to the skin, or internal administration by injection or the like. Then, depending on the administered test compound, suppression of depigmentation spots (white spots, white hair) in DSH model mice (hair color changes from white to black (brown) in the case of body hair, and less white spots in the case of skin (pigment). A compound that suppresses depigmentation spots may be selected by determining the presence or absence of)).
  • the step of comparing the number of viable cells and the step of comparing the number of viable cells after the step is carried out, -The test compound selection step of selecting the test compound from the comparison result of the survival
  • Examples of cells include mouse or human-derived pigment cells.
  • Adar1 is also expressed in cells other than pigment cells, cells expressing Adar1 may be used instead of pigment cells.
  • Examples of cells expressing Adar1 include cos7 cells, HeLa cells, melanoma cells, HaCaT cells and the like.
  • As the cells cells of primary culture or cells that can be passaged may be used.
  • the inactivation of Adar1 in cells is not particularly limited as long as it can specifically inactivate Adar1 in cells. For example, a known method such as introducing siRNA having a sequence that inactivates Adar1 into a cell can be mentioned.
  • cells may be primary cultured from Adar1 heterozygous knockout mice and wild-type mice, respectively.
  • human-derived cells into which mutation-introduced ADAR1 and wild-type ADAR1 have been gene-introduced and mouse-derived cells in which mutation-introduced Adar1 and wild-type Adar1 have been gene-introduced may be used.
  • Interferon and / or Toll-like receptor ligands are added to cells in which Adar1 is inactivated (hereinafter, may be referred to as "DSH model cells") and control cells in which control siRNA that does not inactivate Adar1 is introduced into cells.
  • DSH model cells Interferon and / or Toll-like receptor ligands are added to cells in which Adar1 is inactivated
  • control siRNA that does not inactivate Adar1 is introduced into cells.
  • the number of viable cells may be compared. It is considered that the closer the ratio of the viable cells in the groups (1) and (2) was, the higher the cell viability was due to the test compound.
  • the pharmaceutical composition for treating DSH is not particularly limited as long as it contains an ingredient effective for treating DSH.
  • JAK inhibitors and STAT inhibitors were confirmed in various commercially available types, as shown in Examples described later. Therefore, JAK inhibitors and STAT inhibitors, as well as similar compounds, can be used in DSH therapeutic pharmaceutical compositions.
  • Dosage forms of DSH therapeutic pharmaceutical compositions include, for example, tablets, pills, powders, rosenge, sachets, cashiers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid).
  • a medium ointments, gelatin soft and hard capsules, syrups, sterilized injection solutions, sterilized encapsulants and the like.
  • the pharmaceutical composition for DSH treatment may contain a carrier, a mold release agent, and a diluent.
  • a carrier for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacant, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, phosphate buffered saline (PBS). , Syrup, methylcellulose, methyl-and propyl oxybenzoate, talc, magnesium stearate and mineral oil.
  • vitamins, lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners or flavoring agents may be added.
  • the pharmaceutical composition for DSH treatment may be combined with a known drug.
  • a p150 Adar1 heterozygous knockout mouse (C57BL / 6 background) was produced by the following procedure.
  • a BAC clone containing the Adar1 gene was purchased from the BACPAC Resources Center (BPRC) (https://bacpacresources.org), and a p150-specific exon of the Adar1 gene was cut out from the genomic DNA clone with a restriction enzyme.
  • Genomic DNA clones were translocated into pBluescript vectors, linearized and gene-introduced into embryonic stem cells. Embryonic stem cells in which the introduced gene and the endogenous gene were recombined were selected with antibiotics.
  • Embryonic stem cells undergoing gene recombination were driven into blastocysts and returned to the uterus of mice to obtain chimeric mice.
  • the chimeric mouse and a wild mouse were crossed to select a mouse born from embryonic stem cell-derived sperm by hair, and the genotype was confirmed by PCR to obtain a p150 Adar1 heterozygous knockout mouse. Further, this mouse was backcrossed to obtain a C57BL / 6 background.
  • the newborns of p150 Adar1 heterozygous knockout mice (C57BL / 6 background) on the second day after birth were subcutaneously injected with 3000 U of m-Ifn ⁇ (manufactured by PBL) once a day.
  • FIG. 1 is a photograph of an Adar1 heterozygous knockout mouse prepared in Example 1. Wild-type mice with a C57BL / 6 background have black hair as usual, but the Adar1 heterozygous knockout mouse prepared in Example 1 has a C57BL / 6 background, but the hair that grows after subcutaneous injection turns white. It was confirmed that (see the elliptical part in FIG. 1).
  • LPS subcutaneously injected at 50 ⁇ L on the same schedule using LPS (manufactured by wako), which is a Toll-like receptor ligand.
  • LPS manufactured by wako
  • Toll-like receptor ligand poly I: C
  • a model mouse showing a DSH phenotype can be produced by administering an interferon or Toll-like receptor ligand to an Adar1 heterozygous knockout mouse.
  • Example 2 Dct-LacZ transgenic (Tg) mice (Kumamoto University Life Resources Research and Support Center, ID: 782) are bred to Adar1 heterozygous knockout mice to which the interferon and Toll-like receptor ligands of Example 1 have not been administered.
  • Adar1 KO Dct-LacZ Tg mice were prepared.
  • FIG. 2A is a photograph of the mouse produced in Example 2.
  • a photograph of a wild-type mouse with a C57BL / 6 background is shown in FIG. 2B. As shown in FIG. 2A, it was confirmed that the mouse had white hair mixed with normal black hair.
  • mice prepared in Example 2 exhibited the DSH phenotype without administration of interferon and Toll-like receptor ligands.
  • Example 3 By mating the Adar1 KO: Dct-LacZ Tg mouse prepared in Example 2 with a K14-Scf Tg mouse (Kunisada T, et al., J Exp Med. 1998; 187 (10): 1565-73.). , Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mice were produced.
  • FIG. 3 is a photograph of the mouse produced in Example 3. In Example 3, by mating K14-Scf Tg mice, pigment cells were allowed to be present in the skin of the Adar1 KO: Dct-LacZ Tg mice prepared in Example 2.
  • Example 4 Adar1 flox mice (Adar tm1a (EUCOMM) Wtsi / tm1a (EUCOMM) Wtsi (MBLD; EPD0087_1_D11), Sanger Institute, UK) and Dct-Cre Tg mice (Prof. , Guyonneau L, et al., Pigment cell res, 15: 305-309, 2002).
  • MBLD has a genetic modification in Adar1 in a homozygous state, and was prepared by mating Dct-Cre Tg mice to prepare the mouse of Example 4 (Adar tm1a (EUCOMM) Wtsi / tm1a (EUCOMM) Wtsi ; Dct-Cre_Tg. ), It became an Adar1 homozygous knockout mouse in which Adar1 was knocked out only in melanosite.
  • FIG. 4 is a photograph of the mouse produced in Example 4. As shown in FIG. 4, the mouse produced in Example 4 showed a phenotype in which the hair became spots in white and black areas.
  • Example 5 [Preparation of DSH model cells] ⁇ Example 5> In Example 1, the DSH phenotype was exhibited by administering interferon or Toll-like receptor ligand to the Adar1 heterozygous knockout mouse. Therefore, DSH model cells using pigment cells were prepared by the following procedure. ..
  • mice melanocytes, melan5 cells were added to a 12-well plate at 0.5x10. 5 cell / well was sprinkled and 1 mL of medium (see Oka S, et al., Pigment Cell Res. 14 (4): 268-74, 2001) was added.
  • Adar1 siRNA (Adar1 (1), 5'-AGAAGGGUUUCUUUCA) (SEQ ID NO: 1) and control siRNA (Allstar negative siRNA, manufactured by Qiagen) are used, and attached to HiPerfect (Qiagen).
  • the gene was introduced into cultured cells according to the method.
  • the upper row of row A is a photograph of control cells (control siRNA + m-Ifn) into which control siRNA has been introduced
  • the lower row of row A is a photograph of DSH model cells (Adar1 siRNA + m-Ifn) into which Adar1 siRNA has been introduced.
  • the mortality rate of DSH model cells was higher than that of control cells.
  • Each cell was peeled off with trypsin, and the number of viable cells was measured with a TC20 fully automatic cell counter (Bio-Rad), and the ratio was taken as the survival rate.
  • the cell viability of DSH model cells relative to control cells was 10.5%.
  • the prepared DSH model cells show the properties of the pigment cells of the DSH patient, because the survival rate of the pigment cells in which the expression of Adar1 was decreased decreased.
  • Example 6 [Experiment using DSH model cells] ⁇ Example 6> Experiments were carried out in the same procedure as in Example 5 except that in addition to mouse interferon ⁇ , Ruxolitinib was added to [day 3] in Example 5 so that the final concentration of Ruxolitinib was 0.5 ⁇ M and the final concentration of Tofacitinib was 5 ⁇ M. Was done. The administration concentration of the drug was determined based on the cytotoxicity evaluation in the control cells and the DSH model cells. In addition, DMSO was used to adjust the drug concentration. The upper part of the B column of FIG.
  • FIG. 5 is a photograph when Ruxolitinib is added to the control cells, and the lower part of the B column is a photograph when Ruxolitinib is added to the DSH model cells.
  • the upper part of row C in FIG. 5 is a photograph when Tofacitinib is added to control cells, and the lower part of row C is a photograph when Tofacitinib is added to DSH model cells.
  • the survival rate of DSH model cells is improved when either the immunosuppressive substance Ruxolitinib or Tofacitinib is added, and in particular, in Tofacitinib, the cells are improved.
  • the survival rate was 98.8% (the number of DSH model cells administered with Tofacitinib / the number of control cells administered with Tofacitinib). This means that the DSH model cells in which the function of Adar1 was reduced or lost were restored to the survival rate by the addition of Ruxolitinib and Tofacitinib. Therefore, the function of Adar1 was reduced or lost. It suggests that it is useful in the treatment of diseases.
  • Example 6 [Experiment using DSH model mouse] In Example 6, the following experiments were carried out using Tofacitinib, which had a high cell viability.
  • Example 7 Transdermal administration> The neonates of the p150 Adar1 heterozygous knockout mouse described in Example 1 were subcutaneously injected with 3000 U of m-Ifn ⁇ (manufactured by PBL) once a day. Injection of m-Ifn ⁇ was started on the 7th day after birth and continued for 10 days until the 16th day after birth. Tofacitinib was subcutaneously injected at 50 mg / kg / day for 10 days from day 7 to day 16 after birth.
  • FIG. 6A is a photograph of the 22nd day after birth.
  • FIG. 6B is a photograph of the 22nd day after birth.
  • Example 8 Oral administration> The neonates of the p150 Adar1 heterozygous knockout mouse described in Example 1 were subcutaneously injected with 3000 U of m-Ifn ⁇ (manufactured by PBL) once a day. Injection of m-Ifn ⁇ was started on the 7th day after birth and continued daily until the 16th day. Tofacitinib was orally administered at 50 mg / kg / day daily for 10 days from the 7th day to the 16th day after birth.
  • FIG. 7A is a photograph of the 22nd day after birth.
  • FIG. 7B is a photograph of the 22nd day after birth.
  • Example 9 [Screening of DSH therapeutic compounds] As described above, since it was confirmed that the DSH model cells and the DSH model mice can be used for the screening of the DSH therapeutic compound, various compounds were screened using the DSH model cells. Table 1 below shows the compounds, concentrations, known uses of the compounds, and cell viability that were screened in the same procedure as in Example 6. The compounds confirmed in Example 6 are also shown in Table 1 for comparison. The exemplified compounds were obtained from Sigma Aldrich for Tofacitinib, Tokyo Chemical Industry for Curcumol, Cayman chemical for Celdulatinib, and all others from Selleck.
  • JAK inhibitors and STAT inhibitors showed therapeutic effects in various commercially available types. Among them, JAK inhibitors were confirmed to be preferable as pharmaceutical compositions for the treatment of DSH because the cell viability of most types was 50% or more.
  • Example 10 [Confirmation of usefulness of other JAK inhibitors] ⁇ Example 10> The experiment was carried out in the same procedure as in Example 6 except that Delgocitinib (manufactured by ChemScene), which is a JAK inhibitor, was added in place of Ruxolitinib and Tofacitinib in Example 6 so as to have a final concentration of 1 ⁇ M.
  • the upper part of row B in FIG. 8 is a photograph when Delgocitinib was added to the control cells, and the lower part of row B is a photograph when Delgocitinib was added to the DSH model cells.
  • Example 11 A transdermal administration experiment was performed in the same procedure as in Example 7 except that Delgocitinib was used instead of Tofacitinib. Specifically, 3000 U of m-Ifn ⁇ (manufactured by PBL) was subcutaneously injected into the neonates of the p150 Adar1 heterozygous knockout mouse described in Example 1 once a day. Injection of m-Ifn ⁇ was started on the 2nd day after birth and was performed 10 times until the 14th day after birth. Delgocitinib was subcutaneously injected 10 times a day at 15 mg / kg / day from the 2nd day to the 14th day after birth. FIG. 9B is a photograph of the 15th day after birth.
  • FIG. 9A is a photograph of the 15th day after birth.
  • Delgocitinib which is a JAK inhibitor, is useful as a compound for treating DSH.
  • DSH model mouse and model cells disclosed in this application it is possible to elucidate the pathophysiology of DSH and screen compounds for the treatment of DSH.
  • JAK inhibitors and STAT inhibitors can be used as pharmaceutical compositions for the treatment of DSH. Therefore, it is useful for research and development of DSH treatment in universities, medical institutions, pharmaceutical companies, etc.

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

遺伝性対側性色素異常症治療用医薬組成物、遺伝性対側性色素異常症モデルマウス、および、遺伝性対側性色素異常症治療用化合物のスクリーニング方法Screening method for a pharmaceutical composition for treating hereditary contralateral dyschromatosis, a mouse model for hereditary contralateral dyschromatosis, and a compound for treating hereditary contralateral dyschromatosis.
　本出願の開示は、遺伝性対側性色素異常症治療用医薬組成物、遺伝性対側性色素異常症モデルマウス、および、遺伝性対側性色素異常症治療用化合物のスクリーニング方法に関する。 The disclosure of this application relates to a pharmaceutical composition for treating hereditary contralateral dyschromatosis, a mouse model for hereditary contralateral dyschromatosis, and a method for screening a compound for treating hereditary contralateral dyschromatosis.
　遺伝性対側性色素異常症（Ｄｙｓｃｈｒｏｍａｔｏｓｉｓ　Ｓｙｍｍｅｔｒｉｃａ　Ｈｅｒｅｄｉｔａｒｉａ、以下「ＤＳＨ」と記載することがある。）は、両手背と両足背に色素斑と白斑が「斑」に出現する色素性疾患である。ヒトでは、ＲＮＡ編集酵素の一つである二重鎖ＲＮＡ特異的アデノシン脱アミノ化酵素１（Ａｄｅｎｏｓｉｎｅ　Ｄｅａｍｉｎａｓｅ　Ａｃｔｉｎｇ　ｏｎ　ＲＮＡ１、以下「ＡＤＡＲ１」もしくは「ＡＤＡＲ」と記載することがある。なお、マウスの二重鎖ＲＮＡ特異的アデノシン脱アミノ化酵素１に関しては「Ａｄａｒ１」と記載する。また、モデル細胞に関しては、由来を問わず「Ａｄａｒ１」と記載する場合がある。）をコードする遺伝子が病因遺伝子である。ＲＮＡのアデノシンをイノシンに変換することでＤＮＡ情報とは異なるタンパク質を生成してしまうことが知られている。ＡＤＡＲ１遺伝子にヘテロ接合性の変異を持つとＤＳＨを発症することが明らかになっている（非特許文献１参照）。 Hereditary contralateral dyschromatosis (dyschromatosis symmetryca hereditaria, hereinafter sometimes referred to as "DSH") is a pigmented disease in which pigmented spots and white spots appear on the backs of both hands and feet. In humans, double-stranded RNA-specific adenosine deaminase 1 (Adenosine Deaminase Acting on RNA1, which is one of the RNA editing enzymes, hereinafter may be referred to as “ADAR1” or “ADAR”. The gene encoding heavy-chain RNA-specific adenosine deaminase 1 is described as "Adar1", and the model cell may be described as "Adar1" regardless of its origin). be. It is known that converting RNA adenosine to inosine produces a protein different from DNA information. It has been clarified that having a heterozygous mutation in the ADAR1 gene causes DSH (see Non-Patent Document 1).
　ＤＳＨは、神経症状の合併が稀にあるが、生命にかかわる重篤な症状は引き起こさない。しかしながら、小児期から発症し、症状は露出部に現れるため、見た目の問題から患者に重い精神的な負担が生じるという問題がある。そのため、治療薬および治療方法の確立が求められるが、現在のところ有効な治療薬および治療方法はない。 DSH rarely has neurological symptoms, but does not cause serious life-threatening symptoms. However, since it develops from childhood and the symptoms appear in the exposed part, there is a problem that a heavy mental burden is caused to the patient due to the appearance problem. Therefore, it is required to establish a therapeutic agent and a therapeutic method, but there is currently no effective therapeutic agent and therapeutic method.
　ところで、疾患治療の研究には、当該疾患を有したマウス等のモデル動物を作製し、当該モデル動物を用いて疾患の病態を調べたり、当該モデル動物に被験化合物を投与し、治療用化合物をスクリーニングすることが、一般的に行われている。 By the way, in research on disease treatment, a model animal such as a mouse having the disease is prepared, the pathological condition of the disease is investigated using the model animal, or a test compound is administered to the model animal to obtain a therapeutic compound. Screening is common practice.
　しかしながら、単にＡｄａｒ１をノックアウトしても、マウスにはＤＳＨ患者が示す表現型（以下、「ＤＳＨ表現型」と記載することがある。）は現れず、ＤＳＨの研究には使用できないという問題がある。 However, even if Adar1 is simply knocked out, the phenotype indicated by the DSH patient (hereinafter, may be referred to as "DSH phenotype") does not appear in the mouse, and there is a problem that it cannot be used for DSH research. ..
　本出願の開示は、上記問題点を解決するためになされたものであり、鋭意研究を行ったところ、（１）ＪＡＫ阻害剤またはＳＴＡＴ阻害剤を有効成分とすることで、ＤＳＨの治療ができること、（２）全身でＡｄａｒ１の機能を喪失したヘテロ接合型Ａｄａｒ１ノックアウトマウスに、所定の物質を投与、或いは、生産する遺伝子を発現させると、ＤＳＨ表現型を示すこと、（３）メラノサイト特異的にＡｄａｒ１の機能を喪失したホモ接合型のＡｄａｒ１ノックアウトマウスは、ＤＳＨ表現型を示すこと、（４）作製したノックアウトマウス（遺伝性対側性色素異常症モデルマウス）やＡｄａｒ１の機能を低下もしくは喪失した細胞（モデル細胞）を用いることで、ＤＳＨ治療用化合物のスクリーニングを実施できること、を新たに見出した。 The disclosure of this application was made in order to solve the above-mentioned problems, and as a result of diligent research, (1) DSH can be treated by using a JAK inhibitor or a STAT inhibitor as an active ingredient. , (2) Heterozygous Adar1 knockout mice that have lost the function of Adar1 systemically show DSH phenotype when a predetermined substance is administered or a gene that produces them is expressed, (3) Melanosite-specifically. The homozygous Adar1 knockout mouse that lost the function of Adar1 showed a DSH phenotype, and (4) the prepared knockout mouse (hereditary contralateral dyschromatosis model mouse) and the function of Adar1 were reduced or lost. We have newly found that screening of DSH therapeutic compounds can be performed by using cells (model cells).
　すなわち、本出願の開示の目的は、遺伝性対側性色素異常症治療用医薬組成物、遺伝性対側性色素異常症モデルマウス、および、遺伝性対側性色素異常症治療用化合物のスクリーニング方法、を提供することである。 That is, the purpose of the disclosure of the present application is to screen a pharmaceutical composition for treating hereditary contralateral dyschromatosis, a mouse model for hereditary contralateral dyschromatosis, and a compound for treating hereditary contralateral dyschromatosis. The method is to provide.
　本出願の開示は、以下に示す、遺伝性対側性色素異常症治療用医薬組成物、遺伝性対側性色素異常症モデルマウス、および、遺伝性対側性色素異常症治療用化合物のスクリーニング方法、に関する。 The disclosure of this application is the screening of pharmaceutical compositions for the treatment of hereditary contralateral dyschromatosis, hereditary contralateral dyschromatosis model mice, and compounds for the treatment of hereditary contralateral dyschromatosis, as shown below. Regarding the method.
（１）ＪＡＫ阻害剤、および、ＳＴＡＴ阻害剤から成る群から選択した少なくとも一種の化合物を有効成分として含む、遺伝性対側性色素異常症治療用医薬組成物。
（２）前記ＪＡＫ阻害剤が、
　　Ｔｏｆａｃｉｔｉｎｉｂ、Ｉｔａｃｉｎｉｔｉｂ、Ｓｏｌｃｉｔｉｎｉｂ、ＡＺＤ１４８０、Ｒｕｘｏｌｉｔｉｎｉｂ、ＪＡＫ３－ＩＮ－６、Ｃｕｒｃｕｍｏｌ、Ｐｅｆｉｃｉｔｉｎｉｂ、Ｃｅｒｄｕｌａｔｉｎｉｂ、ＦＭ－３８１、Ｆｉｌｇｏｔｉｎｉｂ、および、Ｄｅｌｇｏｃｉｔｉｎｉｂから選択され、
　前記ＳＴＡＴ阻害剤が、
　　Ｎａｐａｂｕｃａｓｉｎ、Ｏｃｈｒｏｍｙｃｉｎｏｎｅ、Ｆｌｕｄａｒａｂｉｎｅ、Ｎｉｆｕｒｏｘａｚｉｄｅ、Ｃ１８８－９、および、ＡＳ１５１７４９９から選択される、
上記（１）に記載の遺伝性対側性色素異常症治療用医薬組成物。
（３）前記ＪＡＫ阻害剤がＤｅｌｇｏｃｉｔｉｎｉｂである、
上記（２）に記載の遺伝性対側性色素異常症治療用医薬組成物。
（４）前記ＪＡＫ阻害剤がＲｕｘｏｌｉｔｉｎｉｂである、
上記（２）に記載の遺伝性対側性色素異常症治療用医薬組成物。
（５）前記ＪＡＫ阻害剤がＴｏｆａｃｉｔｉｎｉｂである、
上記（２）に記載の遺伝性対側性色素異常症治療用医薬組成物。
（６）前記ＪＡＫ阻害剤がＰｅｆｉｃｉｔｉｎｉｂである、
上記（２）に記載の遺伝性対側性色素異常症治療用医薬組成物。
（７）前記ＪＡＫ阻害剤がＣｅｒｄｕｌａｔｉｎｉｂである、
上記（２）に記載の遺伝性対側性色素異常症治療用医薬組成物。
（８）全身でＡｄａｒ１（Ａｄｅｎｏｓｉｎｅ　Ｄｅａｍｉｎａｓｅ　Ａｃｔｉｎｇ　ｏｎ　ＲＮＡ１）の機能を喪失したヘテロ接合型Ａｄａｒ１ノックアウトマウスであって、
　皮膚及び／又は体毛の色が斑であるとの表現型を示す、
Ａｄａｒ１ヘテロ接合型ノックアウトマウス。
（９）前記Ａｄａｒ１のｐ１５０をノックアウトした、
上記（８）に記載のＡｄａｒ１ヘテロ接合型ノックアウトマウス。
（１０）全身でＡｄａｒ１（Ａｄｅｎｏｓｉｎｅ　Ｄｅａｍｉｎａｓｅ　Ａｃｔｉｎｇ　ｏｎ　ＲＮＡ１）の機能を喪失したＡｄａｒ１ヘテロ接合型ノックアウトマウスに、Ｄｃｔ－ＬａｃＺトランスジェニックマウスを交配することで得られた、Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウス。
（１１）前記Ａｄａｒ１のｐ１５０をノックアウトした、
上記（１０）に記載のＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウス。
（１２）上記（１０）または（１１）に記載のＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウスに、Ｋ１４－Ｓｃｆ　Ｔｇマウスを交配することで得られた、Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇ：Ｋ１４－Ｓｃｆ　Ｔｇマウス。
（１３）メラノサイト特異的にＡｄａｒ１（Ａｄｅｎｏｓｉｎｅ　Ｄｅａｍｉｎａｓｅ　Ａｃｔｉｎｇ　ｏｎ　ＲＮＡ１）の機能を喪失した、Ａｄａｒ１ホモ接合型ノックアウトマウス。
（１４）体毛の色が斑であるとの表現型を示す、
上記（１３）に記載のＡｄａｒ１ホモ接合型ノックアウトマウス。
（１５）上記（８）～（１４）の何れか一つに記載のノックアウトマウスに被験化合物を投与する工程と、
　脱色素斑を抑制する被験化合物を選択する被験化合物選択工程と、
を含む、遺伝性対側性色素異常症治療用化合物のスクリーニング方法。
（１６）Ａｄａｒ１を不活性化した細胞を準備するＤＳＨモデル細胞準備工程と、
　Ａｄａｒ１を不活性化していないコントロール細胞を準備するコントロール細胞準備工程と、
　前記ＤＳＨモデル細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程と、
　前記コントロール細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程と、
　前記ＤＳＨモデル細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程を実施後の生存細胞数と、前記コントロール細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程を実施後の生存細胞数とを対比する、生存細胞数対比工程と、
　前記生存細胞数対比工程の対比結果から、被験化合物を選択する被験化合物選択工程と、
を含む、遺伝性対側性色素異常症治療用化合物のスクリーニング方法。 (1) A pharmaceutical composition for treating hereditary contralateral dyschromatosis, which comprises at least one compound selected from the group consisting of a JAK inhibitor and a STAT inhibitor as an active ingredient.
(2) The JAK inhibitor
Selected from Tofacitinib, Itacitinib, Solcitinib, AZD1480, Ruxolitinib, JAK3-IN-6, Curcumol, Peficitinib, Cerdulatinib, FM-381, Filgotinib, and Delgocitinib.
The STAT inhibitor
Selected from Napabucasin, Ochromycine, Fludarabine, Nifuroxazide, C188-9, and AS1517499,
The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to (1) above.
(3) The JAK inhibitor is Delgocitinib.
The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to (2) above.
(4) The JAK inhibitor is Ruxolitinib.
The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to (2) above.
(5) The JAK inhibitor is Tofacitinib.
The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to (2) above.
(6) The JAK inhibitor is Peficitinib.
The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to (2) above.
(7) The JAK inhibitor is Cerdulatinib.
The pharmaceutical composition for treating hereditary contralateral dyschromatosis 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.
Indicates a phenotype that the color of the skin and / or hair is mottled,
Adar1 heterozygous knockout mouse.
(9) The p150 of Adar1 was knocked out.
The Adar1 heterozygous knockout mouse according to (8) above.
(10) Adar1 KO: Dct-LacZ Tg mouse obtained by mating a Dct-LacZ transgenic mouse with an Adar1 heterozygous knockout mouse that has lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout the body.
(11) The p150 of Adar1 was knocked out.
The Adar1 KO: Dct-LacZ Tg mouse according to (10) above.
(12) Adar1 KO: Dct-LacZ Tg: K14-Scf Tg obtained by mating a K14-Scf Tg mouse with the Adar1 KO: Dct-LacZ Tg mouse according to (10) or (11) above. mouse.
(13) Adar1 homozygous knockout mouse in which the function of Adar1 (Adenosine Deaminase Acting on RNA1) was specifically lost in a melanocyte manner.
(14) Shows the phenotype that the color of the hair is mottled,
The Adar1 homozygous knockout mouse according to (13) above.
(15) The step of administering the test compound to the knockout mouse according to any one of (8) to (14) above, and
A test compound selection step of selecting a test compound that suppresses depigmentation spots,
A method for screening a compound for treating hereditary contralateral dyschromatosis, including.
(16) A DSH model cell preparation step for preparing cells in which Adar1 is inactivated, and
A control cell preparation step for preparing control cells that have not inactivated Adar1 and
The step of administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells, and
The step of administering the test compound and interferon and / or Toll-like receptor ligand to the control cells, and
The number of surviving cells after performing the step of administering the test compound and the interferon and / or Toll-like receptor ligand to the DSH model cells, and the step of administering the test compound and the interferon and / or Toll-like receptor ligand to the control cells. The step of comparing the number of viable cells and the step of comparing the number of viable cells after the above
A test compound selection step of selecting a test compound from the comparison result of the survival cell number comparison step and a test compound selection step.
A method for screening a compound for treating hereditary contralateral dyschromatosis, including.
　本出願で開示する遺伝性対側性色素異常症治療用医薬組成物により、遺伝性対側性色素異常症の治療ができる。また、本出願で開示する遺伝性対側性色素異常症モデルマウスおよびモデル細胞を用いることで、遺伝性対側性色素異常症治療用化合物のスクリーニングができる。 The pharmaceutical composition for treating hereditary contralateral dyschromatosis disclosed in this application can treat hereditary contralateral dyschromatosis. In addition, by using the hereditary contralateral dyschromatosis model mouse and model cells disclosed in this application, it is possible to screen a compound for treating hereditary contralateral dyschromatosis.
図１は図面代用写真で、実施例１で作製した、Ａｄａｒ１ヘテロ接合型ノックアウトマウスの写真である。FIG. 1 is a drawing-substituting photograph, which is a photograph of an Adar1 heterozygous knockout mouse produced in Example 1. 図２は図面代用写真である。図２Ａは実施例２で作製したＡｄａｒ１ヘテロ接合型ノックアウトマウスの写真、図２Ｂは、比較としてのＣ５７ＢＬ／６背景の野生型マウスの写真である。FIG. 2 is a drawing substitute photograph. FIG. 2A is a photograph of the Adar1 heterozygous knockout mouse prepared in Example 2, and FIG. 2B is a photograph of a wild-type mouse with a C57BL / 6 background for comparison. 図３は図面代用写真で、実施例３で作製した、Ａｄａｒ１ヘテロ接合型ノックアウトマウスの写真である。FIG. 3 is a drawing-substituting photograph, which is a photograph of the Adar1 heterozygous knockout mouse produced in Example 3. 図４は図面代用写真で、実施例４で作製した、Ａｄａｒ１ホモ接合型ノックアウトマウスの写真である。FIG. 4 is a drawing-substituting photograph, which is a photograph of the Adar1 homozygous knockout mouse produced in Example 4. 図５は図面代用写真である。図５のＡ列は、実施例５で作製したコントロール細胞およびＤＳＨモデル細胞の写真である。図５のＢ列およびＣ列は、実施例６において、ＲｕｘｏｌｉｔｉｎｉｂおよびＴｏｆａｃｉｔｉｎｉｂを投与した時の、コントロール細胞およびＤＳＨモデル細胞の写真である。FIG. 5 is a drawing substitute photograph. Column A in FIG. 5 is a photograph of the control cells and DSH model cells prepared in Example 5. Rows B and C of FIG. 5 are photographs of control cells and DSH model cells when Ruxolitinib and Tofacitinib were administered in Example 6. 図６は図面代用写真である。図６Ａは実施例７において、ＴｏｆａｃｉｔｉｎｉｂをＤＳＨモデルマウスに経皮投与した写真、図６Ｂは比較例１において、ＤＭＳＯをＤＳＨモデルマウスに経皮投与した写真である。FIG. 6 is a drawing substitute photograph. FIG. 6A is a photograph of Tofacitinib transdermally administered to a DSH model mouse in Example 7, and FIG. 6B is a photograph of DMSO transdermally administered to a DSH model mouse in Comparative Example 1. 図７は図面代用写真である。図７Ａは実施例８において、ＴｏｆａｃｉｔｉｎｉｂをＤＳＨモデルマウスに経口投与した写真、図７Ｂは比較例２において、ＤＭＳＯをＤＳＨモデルマウスに経口投与した写真である。FIG. 7 is a drawing substitute photograph. FIG. 7A is a photograph of Tofacitinib orally administered to a DSH model mouse in Example 8, and FIG. 7B is a photograph of DMSO orally administered to a DSH model mouse in Comparative Example 2. 図８は図面代用写真である。図８のＡ列は、実施例５で作製したコントロール細胞およびＤＳＨモデル細胞の写真である。図８のＢ列は、実施例１０において、Ｄｅｌｇｏｃｉｔｉｎｉｂを投与した時の、コントロール細胞およびＤＳＨモデル細胞の写真である。FIG. 8 is a drawing substitute photograph. Column A in FIG. 8 is a photograph of the control cells and DSH model cells prepared in Example 5. Column B of FIG. 8 is a photograph of control cells and DSH model cells when Delgocitinib was administered in Example 10. 図９は図面代用写真である。図９Ｂは実施例１１において、ＤｅｌｇｏｃｉｔｉｎｉｂをＤＳＨモデルマウスに経皮投与した写真、図９Ａは比較例３において、ＤＭＳＯをＤＳＨモデルマウスに経皮投与した写真である。FIG. 9 is a drawing substitute photograph. FIG. 9B is a photograph of Delgocitinib transdermally administered to a DSH model mouse in Example 11, and FIG. 9A is a photograph of DMSO transdermally administered to a DSH model mouse in Comparative Example 3.
　以下に、本出願で開示するＤＳＨ治療用医薬組成物、遺伝性対側性色素異常症モデルマウス（Ａｄａｒ１ノックアウトマウス）、および、ＤＳＨ治療用化合物のスクリーニング方法について、詳しく説明する。 The screening method for the DSH therapeutic pharmaceutical composition disclosed in this application, the hereditary contralateral dyschromatosis model mouse (Adar1 knockout mouse), and the DSH therapeutic compound will be described in detail below.
　先ず、Ａｄａｒ１ノックアウトマウスについて説明する。本出願で開示するＡｄａｒ１ノックアウトマウスには、Ａｄａｒ１ヘテロ接合型ノックアウトマウスおよびＡｄａｒ１ホモ接合型ノックアウトマウスが含まれる。以下に、Ａｄａｒ１ヘテロ接合型ノックアウトマウスおよびＡｄａｒ１ホモ接合型ノックアウトマウスについて、より具体的に説明する。 First, the Adar1 knockout mouse will be described. The Adar1 knockout mice disclosed in this application include Adar1 heterozygous knockout mice and Adar1 homozygous knockout mice. The Adar1 heterozygous knockout mouse and the Adar1 homozygous knockout mouse will be described in more detail below.
（Ａｄａｒ１ヘテロ接合型ノックアウトマウスの第１の実施形態）
　第１の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスは、先ず、全身でＡｄａｒ１の機能を喪失したヘテロ接合型のノックアウトマウスを作製する。Ａｄａｒ１の機能を喪失したヘテロ接合型のノックアウトマウスは、Ａｄａｒ１の機能を喪失すれば作製方法に制限はなく、例えば、以下の手順で作製すればよい。 (First Embodiment of Adar1 Heterozygous Knockout Mouse)
The Adar1 heterozygous knockout mouse according to the first embodiment first prepares a heterozygous knockout mouse in which the function of Adar1 is lost systemically. The heterozygous knockout mouse that has lost the function of Adar1 is not limited in the production method as long as the function of Adar1 is lost. For example, it may be produced by the following procedure.
（ａ）マウスのゲノム上のノックアウトする遺伝子（Ａｄａｒ１）の全体もしくは一部をクローン化してくる、もしくはすでにクローン化したゲノムＤＮＡを購入したりして入手し、その遺伝子（もしくは遺伝子の一部）と周辺部分を含む塩基配列を創り出す。そこに変異を導入したり、一部を欠失させたりして、その遺伝子が不活性化するように一部変更する。その時に欠失などの不活性化の変更だけでなく、観察可能な差異（色や蛍光、抗生物質耐性遺伝子など）をもたらすマーカー遺伝子を組み込む。
　なお、マウスＡｄａｒ１の遺伝子情報は公知であり、例えば、ＧｅｎＢａｎｋ　Ｇｅｎｅ　ＩＤ：５６４１７等から遺伝子情報を入手することができる。Ａｄａｒ１のノックアウトする領域は特に制限はないが、例えば、Ａｄａｒ１には核に局在するｐ１１０、細胞質に局在するｐ１５０等のアイソフォームがあるため、ｐ１５０アイソフォームを特異的に欠失させてもよい。
（ｂ）マウスの胚盤胞（初期のマウスの胚であり、球状の未分化細胞が胚体外細胞に囲まれている）由来の胚性幹細胞を分離する。胚性幹細胞は、例えば、茶色マウスの胚性幹細胞を用い、ｉｎ　ｖｉｔｒｏで細胞培養ができる。
（ｃ）上記（ｂ）で得られた胚性幹細胞に、上記（ａ）で作製した塩基配列を、電気穿孔法等の手段を用いて遺伝子導入する。次に、上記（ａ）で組み入れたマーカー遺伝子を利用し、実際に新しい塩基配列へ組換えを起こした胚性幹細胞（ヘテロ接合型）を分離する。
（ｄ）上記（ｃ）で分離した相同組換えを起こした胚性幹細胞を、例えば、黒色のマウスの胚盤胞に注入し、当該胚盤胞は雌マウスの子宮に注入され、仔マウスが出産される。この仔マウスは、体の一部がオリジナルの胚盤胞に由来し、他の部分は遺伝子操作された胚性幹細胞に由来する２つの細胞を含んだキメラになる。そのため、毛色は茶と黒の斑になる。
（ｅ）キメラマウスのうち、生殖細胞（卵子もしくは***）が、遺伝子操作された細胞由来のものだけが利用される。具体的には、キメラマウスを黒色のマウスを交配させて新たに生まれたマウスのうち、全身が茶色のマウスが胚性幹細胞由来となり、それらのうち変更した遺伝子をＰＣＲなどでヘテロ接合性を確認し、ヘテロ接合型のＡｄａｒ１ノックアウトマウスが得られる。さらにこのＡｄａｒ１ヘテロ接合型ノックアウトマウスを戻し交配で、黒色マウスとする。 (A) Clone all or part of the knockout gene (Adar1) on the mouse genome, or purchase and obtain the already cloned genomic DNA, and obtain the gene (or part of the gene). And create a base sequence that includes the peripheral part. Mutations are introduced there or parts are deleted to partially modify the gene so that it is inactivated. It then incorporates marker genes that result in observable differences (color, fluorescence, antibiotic resistance genes, etc.) as well as inactivation changes such as deletions.
The gene information of mouse Adar1 is known, and for example, the gene information can be obtained from GenBank Gene ID: 56417 and the like. The knockout region of Adar1 is not particularly limited. For example, since Adar1 has isoforms such as p110 localized in the nucleus and p150 localized in the cytoplasm, even if the p150 isoform is specifically deleted. good.
(B) Isolate embryonic stem cells from mouse blastocysts (early mouse embryos, spherical undifferentiated cells surrounded by extraembryonic cells). As the embryonic stem cells, for example, brown mouse embryonic stem cells can be used for cell culture in vitro.
(C) The nucleotide sequence prepared in (a) above is gene-introduced into the embryonic stem cells obtained in (b) above by means such as electroporation. Next, using the marker gene incorporated in (a) above, embryonic stem cells (heterozygous type) that have actually undergone recombination to a new nucleotide sequence are isolated.
(D) The homologous-recombinant embryonic stem cells separated in (c) above are injected into, for example, a black mouse blastocyst, and the blastocyst is injected into the uterus of a female mouse to produce a pup. Give birth. The pups become a chimera containing two cells, part of the body derived from the original blastocyst and the other part derived from genetically engineered embryonic stem cells. Therefore, the coat color becomes brown and black spots.
(E) Among chimeric mice, only germ cells (eggs or sperms) derived from genetically engineered cells are used. Specifically, among newly born mice obtained by mating chimeric mice with black mice, mice having a brown body are derived from embryonic stem cells, and the modified genes among them are confirmed to be heterozygous by PCR or the like. Then, a heterozygous Adar1 knockout mouse is obtained. Further, this Adar1 heterozygous knockout mouse is backcrossed to obtain a black mouse.
　Ａｄａｒ１ヘテロ接合型ノックアウトマウスは、上記の手順や、例えば、Ｍｕｔａｎｔ　Ｍｏｕｓｅ　Ｒｅｇｉｏｎａｌ　Ｒｅｓｏｕｒｃｅ　Ｃｅｎｔｅｒｓ　（ＭＭＲＲＣ）（Ｓｔｏｃｋ　Ｎｏ．０３４６２０－ＪＡＸ）等の機関から入手したものを用いてもよい。 The Adar1 heterozygous knockout mouse may use the above procedure or one obtained from an institution such as Mutant Mouse Regional Resource Centers (MMRRC) (Stock No. 034620-JAX).
　後述する実施例に示すとおり、単にＡｄａｒ１の機能を喪失したのみでは、Ａｄａｒ１ヘテロ接合型ノックアウトマウスの体毛は黒色のままであり、ＤＳＨ表現型を示さない。一方、第１の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスは、鋭意検討の結果、上記手順で作製したＡｄａｒ１ヘテロ接合型マウスにマウスインターフェロン（ｍ－Iｆｎ）やＴｏｌｌ様受容体（Ｔｏｌｌ－ｌｉｋｅ　ｒｅｃｅｐｔｏｒ）リガンドを投与することで、体毛の色が斑になり、ＤＳＨ表現型を示すことを新たに見出した。なお、第１の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスの体毛の色が「斑」とは、病変部では周囲の非病変部の黒い体毛と比較して、病変部全体で体毛が白である、もしくは白い体毛と黒い体毛が入り交じっていることを意味する。 As shown in the examples described later, the hair of the Adar1 heterozygous knockout mouse remains black and does not show the DSH phenotype simply by losing the function of Adar1. On the other hand, the Adar1 heterozygous knockout mouse according to the first embodiment has been subjected to diligent studies. ) It was newly found that the administration of the ligand causes the hair color to become mottled and shows a DSH phenotype. The color of the hair of the Adar1 heterozygous knockout mouse according to the first embodiment is "spot", which means that the hair is white in the entire lesion as compared with the black hair in the surrounding non-lesion. It means that there is, or a mixture of white and black hair.
　インターフェロンは、動物体内で病原体（特にウイルス）や腫瘍細胞などの異物の侵入に反応して細胞が分泌するタンパク質で、ウイルスや細胞の増殖を抑制するための物質である。また、Ｔｏｌｌ様受容体（Ｔｏｌｌ－ｌｉｋｅ　ｒｅｃｅｐｔｏｒ）は、動物の細胞表面にある受容体タンパク質で、種々の病原体を感知して自然免疫を作動させる機能がある。Ａｄａｒ１機能の喪失と外的投与された免疫もしくは炎症を生じる物質との相互作用、或いは、Ａｄａｒ１機能の低下もしくは喪失とリガンド投与による免疫もしくは炎症を生じる物質の内的発現との相互作用、換言すると、ＡＤＡＲ１機能を低下もしくは喪失した状態で高免疫もしくは高炎症状態にすると、Ａｄａｒ１ヘテロ接合型マウスがＤＳＨ表現型を示すと考えられる。 Interferon is a protein secreted by cells in response to the invasion of foreign substances such as pathogens (especially viruses) and tumor cells in the animal body, and is a substance for suppressing the growth of viruses and cells. In addition, Toll-like receptors are receptor proteins on the cell surface of animals and have the function of detecting various pathogens and activating innate immunity. The interaction between the loss of ADAR1 function and an externally administered substance that causes immunity or inflammation, or the interaction between the decrease or loss of ADAR1 function and the internal expression of a substance that causes immunity or inflammation due to ligand administration, in other words. Adar1 heterozygous mice are thought to exhibit the DSH phenotype when they are hyperimmunized or hyperinflammatory with reduced or lost ADAR1 function.
　インターフェロンは、Ａｄａｒ１ヘテロ接合型マウスがＤＳＨ表現型を示せば特に制限はない。例えば、ｍ－Ｉｆｎα、ｍ－Ｉｆｎβ、ｍ－Ｉｆｎω、ｍ－Ｉｆｎε、ｍ－Ｉｆｎκ、ｍ－Ｉｆｎζ、ｍ－Ｉｆｎγ、ｍ－Ｉｆｎλ等が挙げられる。また、ヒトにおける１型インターフェロン（ＩＦＮα、ＩＦＮβ、ＩＦＮω、ＩＦＮε、ＩＦＮκ）、２型インターフェロン（ＩＦＮγ）、３型インターフェロン（ＩＦＮλ）に相当するホモログ等も挙げられる。 Interferon is not particularly limited as long as the Adar1 heterozygous mouse exhibits the DSH phenotype. For example, m-Ifnα, m-Ifnβ, m-Ifnω, m-Ifnε, m-Ifnκ, m-Ifnζ, m-Ifnγ, m-Ifnλ and the like can be mentioned. In addition, homologues corresponding to type 1 interferon (IFNα, IFNβ, IFNω, IFNε, IFNκ), type 2 interferon (IFNγ), and type 3 interferon (IFNλ) in humans can also be mentioned.
　また、Ｔｏｌｌ様受容体リガンドも、Ａｄａｒ１ヘテロ接合型マウスがＤＳＨ表現型を示せば特に制限はない。例えば、Ｐａｍ３ＣＳＫ４（合成トリパルミトイル化リポペプチド）、Ｈｉｓｔｏｎｅ、Ｚｙｍｏｓａｎ（酵母由来の細胞壁構成成分）、ＭＡＬＰ－２、Ｐｏｌｙ（Ｉ：Ｃ）（Ｐｏｌｙｉｎｏｓｉｎｉｃ－ｐｏｌｙｃｙｔｉｄｙｌｉｃ　ａｃｉｄ　ｓｏｄｉｕｍ　ｓａｌｔ）や大腸菌膜成分のｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ（ＬＰＳ）、パクリタクセル、Ｌｉｐｉｄ　Ａ、Ｆｌａｇｅｌｌｉｎ（Ｓａｌｍｏｎｅｌｌａ　ｔｙｐｈｉｍｕｒｉｕｍ由来タンパク）、Ｇａｒｄｉｑｕｉｍｏｄ、Ｉｍｉｑｕｉｍｏｄ／Ｒ－８３７（グアノシンのｉｍｉｄａｚｏｑｕｉｎｏｌｉｎｅ　ａｍｉｎｅアナログ）、Ｉｍｉｄａｚｏｑｕｉｎｏｌｉｎｅ　Ｒｅｓｉｑｕｉｍｏｄ／Ｒ－８４８（低分子量のｉｍｉｄａｚｏｑｕｉｎｏｌｉｎｅ系化合物）、Ｌｏｘｏｒｉｂｉｎｅ、ＣｐＧ　ＯＤＮ　２００６、ＣｐＧ　ＯＤＮ　１６６８、Ｐｒｏｆｉｌｉｎなど、市販されているＴｏｌｌ様受容体リガンドを用いればよい。 Further, the Toll-like receptor ligand is not particularly limited as long as the Adar1 heterozygous mouse shows the DSH phenotype. For example, Pam3CSK4 (synthetic tripalmitylated lipopeptide), Histone, Zymosan (yeast-derived cell wall component), MARP-2, Poly (I: C) (Polyynosinic-polycyticidylic acid sodium salt) and E. coli membrane component lipo ), paclitaxel, Lipid a, flagellin (Salmonella typhimurium-derived protein), Gardiquimod, imidazoquinoline amine analog Imiquimod / R-837 (guanosine), Imidazoquinoline compounds of Imidazoquinoline Resiquimod / R-848 (low molecular weight), Loxoribine, CpG ODN 2006 , CpG ODN 1668, Profilin, and other commercially available Toll-like receptor ligands may be used.
　それらの物質の投与方法は皮膚への局所投与でも経口や注射での全身的な投与のいずれでもかまわない。 The method of administration of these substances may be either topical administration to the skin or systemic administration by oral administration or injection.
　野生型のマウス成体の皮膚には、褐色ないし黒色の色素（メラニン）を生成する色素細胞（メラノサイト）はないが、体毛の毛包には色素細胞がある。したがって、第１の実施形態に係るＡｄａｒ１ヘテロ接合型マウスを用いてＤＳＨ治療用化合物をスクリーニングする際には、被験化合物の投与により、体毛の変化を観察すればよい。また、ＤＳＨは常染色体優性遺伝を示し、Ａｄａｒ１の一方のアリルにのみ変異を持っている（ヘテロ接合型）。第１の実施形態に係るノックアウトマウスは、ヒトのＤＳＨと同じ遺伝子条件で表現型を示しているとの効果も奏する。 The skin of adult wild-type mice does not have pigment cells (melanocytes) that produce brown to black pigment (melanin), but the hair follicles of body hair have pigment cells. Therefore, when screening a DSH therapeutic compound using the Adar1 heterozygous mouse according to the first embodiment, changes in hair may be observed by administration of the test compound. In addition, DSH shows autosomal dominant inheritance and has a mutation in only one allele of Adar1 (heterozygous type). The knockout mouse according to the first embodiment also has the effect of exhibiting a phenotype under the same genetic conditions as human DSH.
（Ａｄａｒ１ヘテロ接合型ノックアウトマウスの第２の実施形態）
　次に、Ａｄａｒ１ヘテロ接合型ノックアウトマウスの第２の実施形態について説明する。第２の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスは、インターフェロンやＴｏｌｌ様受容体リガンドの投与に代え、Ａｄａｒ１ヘテロ接合型ノックアウトマウスにＬａｃＺを発現するトランスジェニックマウスを交配する点で第１の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスと異なり、その他の点は第１の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスと同じである。したがって、第２の実施形態では、第１の実施形態と異なる点を中心に説明し、第１の実施形態において説明済みの事項についての繰り返しとなる説明は省略する。よって、第２の実施形態において明示的に説明されなかったとしても、第２の実施形態において、第１の実施形態で説明済みの事項を採用可能であることは言うまでもない。 (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 is the first embodiment in that the Adar1 heterozygous knockout mouse is mated with a transgenic mouse expressing LacZ instead of administration of an interferon or a Toll-like receptor ligand. Unlike the Adar1 heterozygous knockout mouse according to the morphology, the other points are the same as the Adar1 heterozygous knockout mouse according to the first embodiment. Therefore, in the second embodiment, the points different from those in the first embodiment will be mainly described, and the repetitive description of the matters explained in the first embodiment will be omitted. Therefore, it goes without saying that the matters explained in the first embodiment can be adopted in the second embodiment even if they are not explicitly explained in the second embodiment.
　ＬａｃＺを発現するトランスジェニックマウスは、ＬａｃＺの発現部位について全身に発現してもよいし、皮膚、もしくは皮膚の細胞の一部に限定してもよい。以下の実施例で示すＤｃｔ－ＬａｃＺトランスジェニックマウスは、ＤｃｔプロモーターでＬａｃＺを発現させる、つまり、色素細胞特異的にＬａｃＺを発現させるマウスである。全身で発現させる場合は、ＲＯＳＡ２６－ｌａｃＺ（Ｓｏｒｉａｎｏ　Ｐ．　Ｎａｔ　Ｇｅｎｅｔ．　１９９９　Ｊａｎ；２１（１）：７０－１）やＣＡＧ－ｌａｃＺ（Ｃ５７ＢＬ／６－Ｔｇ（Ｃａｇ－ＬａｃＺ）１１Ｍｉｙａ、熊本大学生命資源開発研究・支援センター　動物資源開発研究部門ＣＡＲＤ　ＩＤ　１４５５）などがある。 The transgenic mouse expressing LacZ may be systemically expressed at the LacZ expression site, or may be limited to the skin or a part of skin cells. The Dct-LacZ transgenic mouse shown in the following examples is a mouse that expresses LacZ with the Dct promoter, that is, expresses LacZ specifically in a pigment cell. For systemic expression, ROSA26-lacZ (Soriano P. Nat Genet. 1999 Jan; 21 (1): 70-1), CAG-lacZ (C57BL / 6-Tg (Cag-LacZ) 11Miya, Kumamoto University Life Resources Development Research and Support Center Animal Resources Development Research Division CARD ID 1455).
　第２の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスは、第１の実施形態と同様の手順で作製したＤＳＨ表現型を示していないＡｄａｒ１ヘテロ接合型ノックアウトマウスに、Ｄｃｔ－ＬａｃＺトランスジェニックマウスを交配することで得られる。得られた第２の実施形態に係るノックアウトマウス（以下、「Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウス」と記載することがある。）は、第１の実施形態と異なり、インターフェロンやＴｏｌｌ様受領体リガンドを投与することなく、ＤＳＨ表現型を示す。 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 show the DSH phenotype prepared in the same procedure as the first embodiment. Obtained by doing. The obtained knockout mouse according to the second embodiment (hereinafter, may be referred to as “Adar1 KO: Dct-LacZ Tg mouse”) is different from the first embodiment in that it is an interferon or Toll-like receptor ligand. Shows the DSH phenotype without administration of.
　ＬａｃＺは、レポーター遺伝子の一つとして知られており、ラクトースをグルコースとガラクトースに分解するβガラクトシダーゼを生成する。Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウス内における作用機序は明らかではないが、第１の実施形態の高免疫もしくは高炎症状態とは異なる作用機序でもＤＳＨ表現型が示される。なお、第２の実施形態に係るＡｄａｒ１ノックアウトマウスに現れるＤＳＨ表現型である「斑」とは、第１の実施形態に係るＡｄａｒ１ノックアウトマウスと同様、白い体毛と黒い体毛が入り交じっていることを意味する。ただし、第１の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスでは、インターフェロンやＴｏｌｌ様受容体リガンドを投与した部分のみでＤＳＨ表現型を生じるのに対して、第２の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスは、広範囲にＤＳＨ表現型が生じる点で異なる効果を奏する。 LacZ is known as one of the reporter genes and produces β-galactosidase, which decomposes lactose into glucose and galactose. Although the mechanism of action in Adar1 KO: Dct-LacZ Tg mice is not clear, the DSH phenotype is also exhibited by a mechanism of action different from the hyperimmune or hyperinflammatory state of the first embodiment. The DSH phenotype "spot" that appears in the Adar1 knockout mouse according to the second embodiment means that white body hair and black body hair are mixed as in the Adar1 knockout mouse according to the first embodiment. means. However, in the Adar1 heterozygous knockout mouse according to the first embodiment, the DSH phenotype is produced only in the portion to which the interferon or Toll-like receptor ligand is administered, whereas the Adar1 heterozygous type according to the second embodiment is produced. Type knockout mice have different effects in that a wide range of DSH phenotypes occur.
　第２の実施形態に係るＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウスは、インターフェロンやＴｏｌｌ様受容体リガンドを投与することなく、ＤＳＨ表現型が現れる。したがって、Ａｄａｒ１ノックアウトマウスを高免疫状態にするための物質の外的投与が不要であることから、取り扱いが容易で、低コストで後述するＤＳＨ治療用化合物のスクリーニング方法が実施できるという効果を奏する。 In the Adar1 KO: Dct-LacZ Tg mouse according to the second embodiment, the DSH phenotype appears without administration of interferon or Toll-like receptor ligand. Therefore, since it is not necessary to externally administer a substance for making the Adar1 knockout mouse highly immune, it is easy to handle and the DSH therapeutic compound screening method described later can be carried out at low cost.
（Ａｄａｒ１ヘテロ接合型ノックアウトマウスの第３の実施形態）
　次に、Ａｄａｒ１ヘテロ接合型ノックアウトマウスの第３の実施形態について説明する。第３の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスは、第２の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウス（Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウス）に、Ｋ１４－Ｓｃｆ　Ｔｇマウスを交配することで得られる（得られたマウスを、「Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇ：Ｋ１４－Ｓｃｆ　Ｔｇマウス」と記載することがある。）。第３の実施形態に係るＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇ：Ｋ１４－Ｓｃｆ　Ｔｇマウスは、Ｋ１４－Ｓｃｆ　Ｔｇマウスを交配することで、第２の実施形態に係るＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウスの皮膚に色素細胞を導入できる。したがって、第３の実施形態に係るＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇ：Ｋ１４－Ｓｃｆ　Ｔｇマウスは、第２の実施形態に係る表現型に加え、皮膚も白と黒（褐色）の「斑」になるという表現型を示す。 (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 a K14-Scf Tg mouse with the Adar1 heterozygous knockout mouse (Adar1 KO: Dct-LacZ Tg mouse) according to the second embodiment. (The obtained mouse may be described as "Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mouse"). The Adar1 KO: Dct-LacZ Tg mouse according to the third embodiment is the skin of the Adar1 KO: Dct-LacZ Tg mouse according to the second embodiment by mating the K14-Scf Tg mouse. Pigment cells can be introduced into the mouse. Therefore, in the Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mouse according to the third embodiment, in addition to the phenotype according to the second embodiment, the skin also becomes white and black (brown) "spots". Indicates the phenotype.
　第３の実施形態に係るＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇ：Ｋ１４－Ｓｃｆ　Ｔｇマウスを用いてスクリーニング方法を実施する場合、第１および第２の実施形態に記載の効果に加え、皮膚の色の変化を観察することでも、ＤＳＨ治療用化合物のスクリーニングができるという効果を奏する。また、第３の実施形態に係るマウスの体毛のＤＳＨ表現型は、第２の実施形態のマウスが示す白い毛と黒い毛が細かくまじりあった（ごま塩頭状、ｓａｌｔ　ａｎｄ　ｐｅｐｐｅｒ　ｈａｉｒ　ｌｉｋｅ）表現型より、白い毛の部分と黒い毛の部分がより明確に分かれた（水玉模様、ｐｏｌｋａ　ｄｏｔ）斑状のＤＳＨ表現型を示す。したがって、毛色で評価する際も大きな白い斑点ができるため、ＤＳＨ治療用化合物の効果判定が分かりやすくなる。 When the screening method is carried out using Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mice according to the third embodiment, in addition to the effects described in the first and second embodiments, changes in skin color. Also, the effect of being able to screen for DSH therapeutic compounds is obtained. In addition, the DSH phenotype of the body hair of the mouse according to the third embodiment is a phenotype in which the white hair and the black hair shown by the mouse of the second embodiment are finely mixed (salt and pepper hair like). It shows a mottled DSH phenotype in which the white hair part and the black hair part are more clearly separated (polka dots, polka dot). Therefore, since large white spots are formed even when evaluated by coat color, it becomes easy to understand the effect determination of the DSH therapeutic compound.
（Ａｄａｒ１ホモ接合型ノックアウトマウスの実施形態）
　Ａｄａｒ１ホモ接合型ノックアウトマウスは、色素細胞のみのノックアウトなど、部位を限定すれば、Ｃｒｅ－ｌｏｘシステムを用いたＡｄａｒ１のコンディショナルノックアウトマウスを用いて作製することができる。その際のマウスの遺伝的背景を黒色マウスとすれば、ＤＳＨの表現型が現れるかどうか判断可能になる。 (Embodiment of Adar1 homozygous knockout mouse)
The Adar1 homozygous knockout mouse can be produced by using the Adar1 conditional knockout mouse using the Cre-lox system, if the site is limited, such as knockout of only pigment cells. If the genetic background of the mouse at that time is a black mouse, it becomes possible to determine whether or not the DSH phenotype appears.
　コンディショナルノックアウトとは、ｆｌｏｘマウスとＣｒｅ発現マウスをかけ合わせることでできる、条件付き遺伝子破壊とも呼ばれる方法である。先ず、標的となる遺伝子領域をＣｒｅリコンビナーゼ標的配列ｌｏｘＰで挟んだマウス（ｆｌｏｘマウスまたはｆｌｏｘｅｄマウス）を作製する。次に、ｆｌｏｘマウスをＣｒｅ発現マウスと交配することで、特定の標的細胞のみで標的遺伝子の欠失が起きる。 Conditional knockout is a method also called conditional gene disruption that can be performed by crossing flox mice and Cre-expressing mice. First, a mouse (flox mouse or floxed mouse) in which a target gene region is sandwiched between Cre recombinase target sequence loxP is prepared. Next, by mating flox mice with Cre-expressing mice, deletion of the target gene occurs only in specific target cells.
　Ａｄａｒ１ホモ接合型ノックアウトマウスの実施形態では、メラノサイトのＡｄａｒ１を標的となる遺伝子領域とすることで、メラノサイト特異的にＡｄａｒ１の機能を喪失したホモ接合型のノックアウトマウスを作製することができる。標的は、メラノサイトのＡｄａｒ１の機能を特異的に喪失できれば特に制限はなく、ｐ１１０及び／又はｐ１５０が挙げられる。また、Ｃｒｅ－ｌｏｘシステムを用いたホモ接合型ノックアウトマウスの作製手順は、公知の方法を用いればよい。 In the embodiment of the Adar1 homozygous knockout mouse, a homozygous knockout mouse in which the melanocyte-specific Adar1 function is lost can be produced by targeting the melanocyte Adar1 as a target gene region. The target is not particularly limited as long as it can specifically lose the function of Adar1 of melanocytes, and examples thereof include p110 and / or p150. Further, as a procedure for producing a homozygous knockout mouse using the Cre-lox system, a known method may be used.
　ホモ接合型のノックアウトマウスは胎児期に死亡して生まれてこない。本実施形態の、Ａｄａｒ１ホモ接合型ノックアウトマウスでは、メラノサイト特異的にＡｄａｒ１をノックアウトしていることから、マウス成獣を得ることができる。Ａｄａｒ１ホモ接合型ノックアウトマウスは、メラノサイトのノックアウトにより、黒い体毛と白い体毛が細かく入り交じって（ごま塩頭状の）斑になるという表現型を示す。メラノサイトにおいてノックアウトするＡｄａｒ１の領域は、例えば、ｐ１１０及び／又はｐ１５０が挙げられ得るが、その他の領域であってもよい。 Homozygous knockout mice die during the fetal period and are not born. In the Adar1 homozygous knockout mouse of the present embodiment, since Adar1 is knocked out specifically in a melanocyte, an adult mouse can be obtained. Adar1 homozygous knockout mice exhibit a phenotype in which black and white hairs are finely mixed to form (salt and pepper-like) spots due to melanocyte knockout. The region of Adar1 to be knocked out in melanocytes may include, for example, p110 and / or p150, but may be other regions.
　Ａｄａｒ１ホモ接合型ノックアウトマウスは、第２の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスよりも症状が強く出る（白毛が多い）傾向がある。そのため、高い治療効果が期待できる被験化合物が数種類みつかった場合に、Ａｄａｒ１ホモ接合型ノックアウトマウスを用いることで、被験化合物の治療効果を高レベルで評価できるという効果を奏する。一方、第２の実施形態に係るＡｄａｒ１ヘテロ接合型ノックアウトマウスは、被験化合物の初期のスクリーニングに有用であるという効果を奏する。 The Adar1 homozygous knockout mouse tends to have stronger symptoms (more white hair) than the Adar1 heterozygous knockout mouse according to the second embodiment. Therefore, when several kinds of test compounds that can be expected to have a high therapeutic effect are found, the therapeutic effect of the test compound can be evaluated at a high level by using the Adar1 homozygous knockout mouse. On the other hand, the Adar1 heterozygous knockout mouse according to the second embodiment has the effect of being useful for the initial screening of the test compound.
　次に、ＤＳＨ治療用化合物のスクリーニング方法について説明する。ＤＳＨ治療用化合物のスクリーニング方法は、上記のＡｄａｒ１ノックアウトマウス（以下において、「ＤＳＨモデルマウス」と記載することがある。）、または、色素細胞などの細胞を用いて実施できる。 Next, a screening method for DSH therapeutic compounds will be described. The method for screening a compound for treating DSH can be carried out using the above-mentioned Adar1 knockout mouse (hereinafter, may be referred to as “DSH model mouse”) or cells such as pigment cells.
（ＤＳＨモデルマウスを用いたスクリーニング方法の実施形態）
　ＤＳＨモデルマウスを用いたＤＳＨ治療用化合物のスクリーニング方法の実施形態は、
・ＤＳＨモデルマウスに被験化合物を投与する工程と、
・脱色素斑（白斑、白毛）を抑制する被験化合物を選択する被験化合物選択工程と、
を含む。 (Embodiment of Screening Method Using DSH Model Mouse)
An embodiment of a method for screening a compound for treating DSH using a DSH model mouse is described as an embodiment.
-The step of administering the test compound to the DSH model mouse and
-A test compound selection step of selecting a test compound that suppresses depigmentation spots (white spots, white hair), and
including.
　被験化合物としては、例えば、天然化合物、有機化合物、無機化合物、タンパク質、抗体、ペプチドなどの単一化合物、並びに、化合物ライブラリー、遺伝子ライブラリーの発現産物、細胞抽出物、細胞培養上清、発酵微生物産生物、海洋生物抽出物、植物抽出物等を挙げることができる。 Test compounds include, for example, 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, and fermentation. Examples thereof include microbial products, marine organism extracts, and plant extracts.
　ＤＳＨモデルマウスへの被験化合物の投与は、経口投与、皮膚への貼着、又は注射等による体内投与等、ＤＳＨモデルマウスの体内に取り込まれる方法であれば特に制限は無い。そして、投与した被験化合物により、ＤＳＨモデルマウス脱色素斑（白斑、白毛）の抑制（体毛の場合は毛の色が白から黒（褐色）に変化、皮膚の場合は白斑が少なくなる（色素が濃くなる））の有無を判定することで、脱色素斑を抑制する化合物を選択すればよい。 The administration of the test compound to the 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, attachment to the skin, or internal administration by injection or the like. Then, depending on the administered test compound, suppression of depigmentation spots (white spots, white hair) in DSH model mice (hair color changes from white to black (brown) in the case of body hair, and less white spots in the case of skin (pigment). A compound that suppresses depigmentation spots may be selected by determining the presence or absence of)).
（細胞を用いたスクリーニング方法の実施形態）
　細胞を用いたＤＳＨ治療用化合物のスクリーニング方法の実施形態は、
・Ａｄａｒ１を不活性化した細胞を準備するＤＳＨモデル細胞準備工程と、
・Ａｄａｒ１を不活性化していないコントロール細胞を準備するコントロール細胞準備工程と、
・前記ＤＳＨモデル細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程と、
・前記コントロール細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程と、
・前記ＤＳＨモデル細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程を実施後の生存細胞数と、前記コントロール細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程を実施後の生存細胞数とを対比する、生存細胞数対比工程と、
・前記生存細胞数対比工程の対比結果から、被験化合物を選択する被験化合物選択工程と、
を含む。 (Embodiment of screening method using cells)
An embodiment of a method for screening a compound for DSH treatment using cells is described as an embodiment.
・ DSH model cell preparation step to prepare cells inactivated Adar1 and
-Control cell preparation step to prepare control cells that have not inactivated Adar1 and
-A step of administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells, and
-A step of administering the test compound and interferon and / or Toll-like receptor ligand to the control cells, and
-The number of surviving cells after the step of administering the test compound and the interferon and / or Toll-like receptor ligand to the DSH model cells, and the test compound and the interferon and / or Toll-like receptor ligand are administered to the control cells. The step of comparing the number of viable cells and the step of comparing the number of viable cells after the step is carried out,
-The test compound selection step of selecting the test compound from the comparison result of the survival cell number comparison step and the test compound selection step.
including.
　細胞の例としては、マウス或いはヒト由来の色素細胞が挙げられる。また、Ａｄａｒ１は色素細胞以外の細胞でも発現していることから、色素細胞に代え、Ａｄａｒ１を発現している細胞を用いてもよい。Ａｄａｒ１を発現している細胞としては、例えば、ｃｏｓ７細胞、ＨｅＬａ細胞、メラノーマ細胞、ＨａＣａＴ細胞等が挙げられる。細胞は、初代培養の細胞を用いても継代可能な細胞を用いてもよい。細胞のＡｄａｒ１の不活性化は、細胞のＡｄａｒ１を特異的に不活性化できれば特に制限はない。例えば、Ａｄａｒ１を不活性化する配列のｓｉＲＮＡを細胞へ導入する等の公知の方法が挙げられる。また、Ａｄａｒ１ヘテロ接合型ノックアウトマウスと野生型のマウスからそれぞれ細胞を初代培養してもよい。また、変異を導入したＡＤＡＲ１と野生型のＡＤＡＲ１を遺伝子導入したヒト由来細胞、変異を導入したＡｄａｒ１と野生型のＡｄａｒ１を遺伝子導入したマウス由来細胞を用いてもよい。Ａｄａｒ１を不活性化した細胞（以下、「ＤＳＨモデル細胞」と記載することがある。）とＡｄａｒ１を不活性化しないコントロールｓｉＲＮＡを細胞に導入したコントロール細胞にインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与すると、Ａｄａｒ１を不活性化していないコントロール細胞と比較して、Ａｄａｒ１を不活性化したＤＳＨモデル細胞の生存率は低下する。したがって、（１）ＤＳＨモデル細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与した群、（２）コントロール細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与した群、の生存細胞数を対比すればよい。（１）と（２）の群の生存細胞の比率が近い程、被験化合物により細胞生存率が高まったと考えられる。 Examples of cells include mouse or human-derived pigment cells. Moreover, since Adar1 is also expressed in cells other than pigment cells, cells expressing Adar1 may be used instead of pigment cells. Examples of cells expressing Adar1 include cos7 cells, HeLa cells, melanoma cells, HaCaT cells and the like. As the cells, cells of primary culture or cells that can be passaged may be used. The inactivation of Adar1 in cells is not particularly limited as long as it can specifically inactivate Adar1 in cells. For example, a known method such as introducing siRNA having a sequence that inactivates Adar1 into a cell can be mentioned. In addition, cells may be primary cultured from Adar1 heterozygous knockout mice and wild-type mice, respectively. In addition, human-derived cells into which mutation-introduced ADAR1 and wild-type ADAR1 have been gene-introduced, and mouse-derived cells in which mutation-introduced Adar1 and wild-type Adar1 have been gene-introduced may be used. Interferon and / or Toll-like receptor ligands are added to cells in which Adar1 is inactivated (hereinafter, may be referred to as "DSH model cells") and control cells in which control siRNA that does not inactivate Adar1 is introduced into cells. When administered, the viability of Adar1 inactivated DSH model cells is reduced compared to control cells in which Adar1 is not inactivated. Therefore, (1) a group in which the DSH model cells were administered with the test compound and interferon and / or Toll-like receptor ligand, and (2) a group in which the control cells were administered with the test compound and interferon and / or Toll-like receptor ligand. The number of viable cells may be compared. It is considered that the closer the ratio of the viable cells in the groups (1) and (2) was, the higher the cell viability was due to the test compound.
（ＤＳＨ治療用医薬組成物の実施形態）
　ＤＳＨ治療用医薬組成物は、ＤＳＨの治療に有効となる成分を含んでいれば特に制限はない。本発明者らは、公知の様々な医薬品で実験を行ったところ、後述する実施例に示すとおり、ＪＡＫ阻害剤およびＳＴＡＴ阻害剤は、市販されている様々な種類で治療効果を確認した。したがって、ＪＡＫ阻害剤およびＳＴＡＴ阻害剤、並びに、類似した化合物は、ＤＳＨ治療用医薬組成物に用いることができる。 (Embodiment of Pharmaceutical Composition for DSH Treatment)
The pharmaceutical composition for treating DSH is not particularly limited as long as it contains an ingredient effective for treating DSH. When the present inventors conducted experiments with various known pharmaceuticals, the therapeutic effects of JAK inhibitors and STAT inhibitors were confirmed in various commercially available types, as shown in Examples described later. Therefore, JAK inhibitors and STAT inhibitors, as well as similar compounds, can be used in DSH therapeutic pharmaceutical compositions.
　ＤＳＨ治療用医薬組成物の剤型としては、例えば、錠剤、丸剤、粉剤、ロゼンジ、サシェ剤、カシェ剤、エリキシル剤、懸濁剤、乳剤、溶液剤、シロップ、エアロゾル剤(固形としてまたは液体媒質中)、軟膏剤、ゼラチン軟および硬カプセル剤、座剤、滅菌注射用溶液および滅菌封入粉剤等が挙げられる。 Dosage forms of DSH therapeutic pharmaceutical compositions include, for example, tablets, pills, powders, rosenge, sachets, cashiers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid). In a medium), ointments, gelatin soft and hard capsules, syrups, sterilized injection solutions, sterilized encapsulants and the like.
　また、ＤＳＨ治療用医薬組成物は、担体、賦型剤、希釈剤を含んでいてもよい。例えば、ラクトース、デキストロース、シュクロース、ソルビトール、マンニトール、デンプン、ガムアカシア、リン酸カルシウム、アルギネート、トラガカント、ゼラチン、ケイ酸カルシウム、微結晶セルロース、ポリビニルピロリドン、セルロース、水、リン酸緩衝生理食塩水（ＰＢＳ）、シロップ、メチルセルロース、オキシ安息香酸メチル－およびプロピル、タルク、ステアリン酸マグネシウムおよび鉱油が挙げられる。更に、ビタミン、潤滑剤、湿潤剤、乳化剤および懸濁化剤、保存剤、甘味剤または着香剤を加えてもよい。また、ＤＳＨ治療用医薬組成物を公知の薬剤と組み合わせてもよい。 Further, the pharmaceutical composition for DSH treatment may contain a carrier, a mold release agent, and a diluent. For example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, tragacant, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, phosphate buffered saline (PBS). , Syrup, methylcellulose, methyl-and propyl oxybenzoate, talc, magnesium stearate and mineral oil. In addition, vitamins, lubricants, wetting agents, emulsifiers and suspending agents, preservatives, sweeteners or flavoring agents may be added. In addition, the pharmaceutical composition for DSH treatment may be combined with a known drug.
　以下に実施例を掲げ、本出願で開示する実施形態を具体的に説明するが、この実施例は単に実施形態の説明のためのものである。本出願で開示する発明の範囲を限定したり、あるいは制限することを表すものではない。 Examples are given below to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments. It does not represent limiting or limiting the scope of the invention disclosed in this application.
［ＤＳＨモデルマウスの作製］
＜実施例１＞
　ｐ１５０　Ａｄａｒ１　ヘテロ接合型ノックアウトマウス（Ｃ５７ＢＬ／６背景）を、次の手順で作製した。
　ＢＡＣＰＡＣ　Ｒｅｓｏｕｒｃｅｓ　Ｃｅｎｔｅｒ（ＢＰＲＣ）（ｈｔｔｐｓ：／／ｂａｃｐａｃｒｅｓｏｕｒｃｅｓ．ｏｒｇ）よりＡｄａｒ１遺伝子を含んだＢＡＣクローンを購入し、Ａｄａｒ１遺伝子のｐ１５０特異的なエクソンをそのゲノムＤＮＡクローンから制限酵素で切り出した。ｐＢｌｕｅｓｃｒｉｐｔベクターにゲノムＤＮＡクローンを載せ替えて、直線化して、胚性幹細胞に遺伝子導入した。導入した遺伝子と内因性の遺伝子の組換えが起こっている胚性幹細胞を抗生剤で選択した。遺伝子の組換えが起こっている胚性幹細胞を胚盤胞に打ち込み、それをマウスの子宮に戻して、キメラマウスを得た。そのキメラマウスと野生マウスを交配して、体毛によって胚性幹細胞由来の***から誕生したマウスを選択し、ＰＣＲで遺伝子型を確認することでｐ１５０　Ａｄａｒ１　ヘテロ接合型ノックアウトマウスを得た。さらにこのマウスを戻し交配によりＣ５７ＢＬ／６背景とした。生後２日目のｐ１５０　Ａｄａｒ１　ヘテロ接合型ノックアウトマウス（Ｃ５７ＢＬ／６背景）の新生仔に、３０００Ｕのｍ－Ｉｆｎβ（ＰＢＬ社製）を１日１回皮下注射した。ｍ－Ｉｆｎβの皮下注射は１０日間継続した。図１は実施例１で作製した、Ａｄａｒ１ヘテロ接合型ノックアウトマウスの写真である。Ｃ５７ＢＬ／６背景の野生型マウスは通常どおり黒色の体毛であるが、実施例１で作製したＡｄａｒ１ヘテロ接合型ノックアウトマウスは、Ｃ５７ＢＬ／６背景であるが皮下注射後に生えてきた体毛が白色になることを確認した（図１の楕円部分参照）。 [Preparation of DSH model mouse]
<Example 1>
A p150 Adar1 heterozygous knockout mouse (C57BL / 6 background) was produced by the following procedure.
A BAC clone containing the Adar1 gene was purchased from the BACPAC Resources Center (BPRC) (https://bacpacresources.org), and a p150-specific exon of the Adar1 gene was cut out from the genomic DNA clone with a restriction enzyme. Genomic DNA clones were translocated into pBluescript vectors, linearized and gene-introduced into embryonic stem cells. Embryonic stem cells in which the introduced gene and the endogenous gene were recombined were selected with antibiotics. Embryonic stem cells undergoing gene recombination were driven into blastocysts and returned to the uterus of mice to obtain chimeric mice. The chimeric mouse and a wild mouse were crossed to select a mouse born from embryonic stem cell-derived sperm by hair, and the genotype was confirmed by PCR to obtain a p150 Adar1 heterozygous knockout mouse. Further, this mouse was backcrossed to obtain a C57BL / 6 background. The newborns of p150 Adar1 heterozygous knockout mice (C57BL / 6 background) on the second day after birth were subcutaneously injected with 3000 U of m-Ifnβ (manufactured by PBL) once a day. Subcutaneous injection of m-Ifnβ continued for 10 days. FIG. 1 is a photograph of an Adar1 heterozygous knockout mouse prepared in Example 1. Wild-type mice with a C57BL / 6 background have black hair as usual, but the Adar1 heterozygous knockout mouse prepared in Example 1 has a C57BL / 6 background, but the hair that grows after subcutaneous injection turns white. It was confirmed that (see the elliptical part in FIG. 1).
　また、ｍ－Ｉｆｎβに代え、
・Ｔｏｌｌ様受容体リガンドであるＬＰＳ（ｗａｋｏ社製）を用い、０．５μｇ／μＬのＬＰＳを、同様の日程で５０μＬ皮下注射した場合、
・Ｔｏｌｌ様受容体リガンドであるｐｏｌｙ（Ｉ：Ｃ）（ＧＥヘルスケア社製）を用い、２μｇ／μＬのｐｏｌｙ（Ｉ：Ｃ）を、同様の日程で５０μＬ皮下注射した場合、
にも、図１と同様に、体毛が白色になることを確認した。 Also, instead of m-Ifnβ
-When 0.5 μg / μL LPS is subcutaneously injected at 50 μL on the same schedule using LPS (manufactured by wako), which is a Toll-like receptor ligand.
-When 50 μL of poly (I: C) of 2 μg / μL is subcutaneously injected on the same schedule using Toll-like receptor ligand poly (I: C) (manufactured by GE Healthcare).
Also, as in FIG. 1, it was confirmed that the body hair became white.
　しかしながら、ｍ－ＩｆｎまたはＴｏｌｌ様受容体リガンドを皮下注射しなかったＡｄａｒ１ヘテロ接合型ノックアウトマウスは、体毛が黒のままであった。 However, Adar1 heterozygous knockout mice that were not subcutaneously injected with m-Ifn or Toll-like receptor ligands remained black in hair.
　以上の結果より、Ａｄａｒ１ヘテロ接合型ノックアウトマウスにインターフェロンまたはＴｏｌｌ様受容体リガンドを投与することで、ＤＳＨ表現型を示すモデルマウスを作製できることを確認した。 From the above results, it was confirmed that a model mouse showing a DSH phenotype can be produced by administering an interferon or Toll-like receptor ligand to an Adar1 heterozygous knockout mouse.
＜実施例２＞
　実施例１のインターフェロンおよびＴｏｌｌ様受容体リガンドを投与していないＡｄａｒ１ヘテロ接合型ノックアウトマウスに、Ｄｃｔ－ＬａｃＺトランスジェニック（Ｔｇ）マウス（熊本大学生命資源研究・支援センター、ＩＤ：７８２）を交配することで、Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウスを作製した。図２Ａは、実施例２で作製したマウスの写真である。また、比較として、Ｃ５７ＢＬ／６背景の野生型マウスの写真を図２Ｂに示す。図２Ａに示すように、マウスに通常の黒い毛に混じって白い毛が生えたことを確認した。この表現型は色素斑と脱色素斑が混じっているＤＳＨの臨床像をより良く示している。また、実施例２で作製したマウスは、インターフェロンおよびＴｏｌｌ様受容体リガンドを投与しなくても、ＤＳＨ表現型を示すことを確認した。 <Example 2>
Dct-LacZ transgenic (Tg) mice (Kumamoto University Life Resources Research and Support Center, ID: 782) are bred to Adar1 heterozygous knockout mice to which the interferon and Toll-like receptor ligands of Example 1 have not been administered. As a result, Adar1 KO: Dct-LacZ Tg mice were prepared. FIG. 2A is a photograph of the mouse produced in Example 2. For comparison, a photograph of a wild-type mouse with a C57BL / 6 background is shown in FIG. 2B. As shown in FIG. 2A, it was confirmed that the mouse had white hair mixed with normal black hair. This phenotype better illustrates the clinical picture of DSH with a mixture of pigmented and depigmented spots. It was also confirmed that the mice prepared in Example 2 exhibited the DSH phenotype without administration of interferon and Toll-like receptor ligands.
＜実施例３＞
　実施例２で作製したＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウスに、Ｋ１４－Ｓｃｆ　Ｔｇマウス（Ｋｕｎｉｓａｄａ　Ｔ，　ｅｔ　ａｌ．，　Ｊ　Ｅｘｐ　Ｍｅｄ．１９９８；１８７（１０）：１５６５－７３．）を交配することで、Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇ：Ｋ１４－Ｓｃｆ　Ｔｇマウスを作製した。図３は実施例３で作製したマウスの写真である。実施例３においては、Ｋ１４－Ｓｃｆ　Ｔｇマウスを交配することで、実施例２で作製したＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウスの皮膚に色素細胞が存在するようにした。その結果、実施例３で作製したマウスは、実施例２の表現型「ごま塩頭」状に白い毛と黒い毛が細かく混じり合った状態と比べて、大きな円形の白いまだら（水玉模様）が出現して、よりヒトのＤＳＨ表現型に近くなったことを確認した。 <Example 3>
By mating the Adar1 KO: Dct-LacZ Tg mouse prepared in Example 2 with a K14-Scf Tg mouse (Kunisada T, et al., J Exp Med. 1998; 187 (10): 1565-73.). , Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mice were produced. FIG. 3 is a photograph of the mouse produced in Example 3. In Example 3, by mating K14-Scf Tg mice, pigment cells were allowed to be present in the skin of the Adar1 KO: Dct-LacZ Tg mice prepared in Example 2. As a result, in the mouse produced in Example 3, large circular white mottles (polka dots) appeared as compared with the state in which white hair and black hair were finely mixed in the phenotype "salt and pepper head" of Example 2. Then, it was confirmed that it became closer to the human DSH phenotype.
＜実施例４＞
　Ａｄａｒ１のｆｌｏｘマウス（Ａｄａｒtm1a(EUCOMM)Wtsi/tm1a(EUCOMM)Wtsi（ＭＢＬＤ；ＥＰＤ００８７＿１＿Ｄ１１），Ｓａｎｇｅｒ　Ｉｎｓｔｉｔｕｔｅ，ＵＫ）と、メラノサイト特異的にＣｒｅを発現するＤｃｔ－Ｃｒｅ　Ｔｇマウス（Ｐｒｏｆ．Ｂｅｅｒｍａｎｎ　Ｆより提供、Ｇｕｙｏｎｎｅａｕ　Ｌ，　ｅｔ　ａｌ．，　Ｐｉｇｍｅｎｔ　ｃｅｌｌ　ｒｅｓ，１５：３０５－３０９，２００２）を交配させた。ＭＢＬＤはホモの状態でＡｄａｒ１に遺伝子改変が入っており、Ｄｃｔ－Ｃｒｅ　Ｔｇマウスを交配させることで、作製した実施例４のマウス（Ａｄａｒtm1a(EUCOMM)Wtsi/tm1a(EUCOMM)Wtsi；Ｄｃｔ－Ｃｒｅ＿Ｔｇ）では、メラノサイトでのみＡｄａｒ１がノックアウトされたＡｄａｒ１ホモ接合型ノックアウトマウスとなった。図４は実施例４で作製したマウスの写真である。図４に示すように、実施例４で作製したマウスは、体毛が白い領域と黒い領域の斑になるという表現型を示した。 <Example 4>
Adar1 flox mice (Adar tm1a (EUCOMM) Wtsi / tm1a (EUCOMM) Wtsi (MBLD; EPD0087_1_D11), Sanger Institute, UK) and Dct-Cre Tg mice (Prof. , Guyonneau L, et al., Pigment cell res, 15: 305-309, 2002). MBLD has a genetic modification in Adar1 in a homozygous state, and was prepared by mating Dct-Cre Tg mice to prepare the mouse of Example 4 (Adar tm1a (EUCOMM) Wtsi / tm1a (EUCOMM) Wtsi ; Dct-Cre_Tg. ), It became an Adar1 homozygous knockout mouse in which Adar1 was knocked out only in melanosite. FIG. 4 is a photograph of the mouse produced in Example 4. As shown in FIG. 4, the mouse produced in Example 4 showed a phenotype in which the hair became spots in white and black areas.
［ＤＳＨモデル細胞の作製］
＜実施例５＞
　実施例１において、Ａｄａｒ１ヘテロ接合型ノックアウトマウスにインターフェロンやＴｏｌｌ様受容体リガンドを投与することで、ＤＳＨ表現型を示したことから、以下の手順により、色素細胞を用いたＤＳＨモデル細胞を作製した。 [Preparation of DSH model cells]
<Example 5>
In Example 1, the DSH phenotype was exhibited by administering interferon or Toll-like receptor ligand to the Adar1 heterozygous knockout mouse. Therefore, DSH model cells using pigment cells were prepared by the following procedure. ..
［ｄａｙ１］　マウスメラノサイトであるｍｅｌａｎ５細胞（聖マリアンナ医科大学　溝口昌子博士より供与、Ｏｏｋａ　Ｓ，　ｅｔ　ａｌ．，　Ｐｉｇｍｅｎｔ　Ｃｅｌｌ　Ｒｅｓ．　１４（４）：２６８－７４，２００１）を１２　ｗｅｌｌ　ｐｌａｔｅに０．５ｘ１０5　ｃｅｌｌ／ｗｅｌｌで撒いて１ｍＬのｍｅｄｉｕｍ（Ｏｏｋａ　Ｓ，　ｅｔ　ａｌ．，　Ｐｉｇｍｅｎｔ　Ｃｅｌｌ　Ｒｅｓ．　１４（４）：２６８－７４，２００１参照）を入れた。
［ｄａｙ２］　０．３μＬ　Ａｄａｒ１　ｓｉＲＮＡ（Ａｄａｒ１（１），５’－ＡＧＡＡＧＡＣＧＧＵＵＵＣＵＵＵＵＣＡ）（配列番号：１）とコントロールｓｉＲＮＡ（Ａｌｌｓｔａｒ　ｎｅｇａｔｉｖｅ　ｓｉＲＮＡ，Ｑｉａｇｅｎ社製））を、ＨｉＰｅｒｆｅｃｔ（Ｑｉａｇｅｎ社製）の添付の使用法に従って、培養細胞に遺伝子導入した。
［ｄａｙ３］　マウスインターフェロンβ（ＰＢＬ社）を、３３０Ｕ／ｗｅｌｌ添加した。
［ｄａｙ５］　Ａｄａｒ１　ｓｉＲＮＡと、コントロールｓｉＲＮＡがそれぞれトランスフェクションされた細胞を観察した。図５のＡ列の上段はコントロールｓｉＲＮＡを導入したコントロール細胞（ｃｏｎｔｒｏｌ　ｓｉＲＮＡ＋ｍ－Ｉｆｎ）の写真、Ａ列の下段はＡｄａｒ１　ｓｉＲＮＡを導入したＤＳＨモデル細胞（Ａｄａｒ１　ｓｉＲＮＡ＋ｍ－Ｉｆｎ）の写真である。Ａ列の写真から明らかなように、コントロール細胞に比べて、ＤＳＨモデル細胞の死亡率が高いことが分かった。なお、それぞれの細胞をトリプシンで剥がし、ＴＣ２０全自動セルカウンター（Ｂｉｏ－Ｒａｄ社）で生細胞数を測定し、その比を生存率とした。コントロール細胞に対するＤＳＨモデル細胞の細胞生存率は１０．５％であった。 [Day1] mouse melanocytes, melan5 cells (donated by Dr. Masako Mizonokuchi, St. Marianna University School of Medicine, Oka S, et al., Pigment Cell Res. 14 (4): 268-74, 2001) were added to a 12-well plate at 0.5x10. 5 cell / well was sprinkled and 1 mL of medium (see Oka S, et al., Pigment Cell Res. 14 (4): 268-74, 2001) was added.
[Day2] 0.3 μL Adar1 siRNA (Adar1 (1), 5'-AGAAGGGUUUCUUUCA) (SEQ ID NO: 1) and control siRNA (Allstar negative siRNA, manufactured by Qiagen) are used, and attached to HiPerfect (Qiagen). The gene was introduced into cultured cells according to the method.
[Day3] Mouse interferon β (PBL) was added at 330 U / well.
[Day5] Adar1 siRNA and control siRNA-transfected cells were observed. In FIG. 5, the upper row of row A is a photograph of control cells (control siRNA + m-Ifn) into which control siRNA has been introduced, and the lower row of row A is a photograph of DSH model cells (Adar1 siRNA + m-Ifn) into which Adar1 siRNA has been introduced. As is clear from the photographs in column A, it was found that the mortality rate of DSH model cells was higher than that of control cells. Each cell was peeled off with trypsin, and the number of viable cells was measured with a TC20 fully automatic cell counter (Bio-Rad), and the ratio was taken as the survival rate. The cell viability of DSH model cells relative to control cells was 10.5%.
　以上の結果より、Ａｄａｒ１の発現が低下した色素細胞の生存率が下がったことから、作製したＤＳＨモデル細胞は、ＤＳＨ患者の色素細胞の性質を示していると言える。 From the above results, it can be said that the prepared DSH model cells show the properties of the pigment cells of the DSH patient, because the survival rate of the pigment cells in which the expression of Adar1 was decreased decreased.
［ＤＳＨモデル細胞およびＤＳＨモデルマウスの有用性の確認］
　次に、実施例１で作製したＤＳＨモデルマウス、および、実施例５で作製したＤＳＨモデル細胞の有用性の確認を行った。実施例１で作製したＤＳＨモデルマウスは、上記のとおり、Ａｄａｒ１ヘテロ接合型ノックアウトマウスではＤＳＨ表現型を示さなかったが、鋭意研究の結果、Ａｄａｒ１機能を喪失した状態で高免疫もしくは高炎症状態にすると、Ａｄａｒ１ヘテロ接合型マウスがＤＳＨ表現型を示した。したがって、免疫もしくは炎症抑制機能を有する薬剤がＡｄａｒ１の治療に有用であると考え、免疫抑制機能を有する薬剤の中から種々検討を重ね、先ず、Ｒｕｘｏｌｉｔｉｎｉｂ（Ｓｅｌｌｅｃｋ社製）、Ｔｏｆａｃｉｔｉｎｉｂ（Ｓｉｇｍａ　Ａｌｄｒｉｃｈ社製）について、ＤＳＨモデル細胞を用いた実験を行った。 [Confirmation of usefulness of DSH model cells and DSH model mice]
Next, the usefulness of the DSH model mouse prepared in Example 1 and the DSH model cell prepared in Example 5 was confirmed. As described above, the DSH model mouse prepared in Example 1 did not show the DSH phenotype in the Adar1 heterozygous knockout mouse, but as a result of diligent research, it became highly immune or highly inflammatory with the Adar1 function lost. Then, the Adar1 heterozygous mouse showed the DSH phenotype. Therefore, it is considered that a drug having an immunosuppressive function or an anti-inflammatory function is useful for the treatment of Adar1, and various studies are repeated from among the drugs having an immunosuppressive function. First, Ruxolitinib (manufactured by Selleck) and Tofacitinib (manufactured by Sigma Aldrich) ), An experiment was conducted using DSH model cells.
［ＤＳＨモデル細胞を用いた実験］
＜実施例６＞
　実施例５の［ｄａｙ３］に、マウスインターフェロンβに加え、Ｒｕｘｏｌｉｔｉｎｉｂの最終濃度が０．５μＭ、Ｔｏｆａｃｉｔｉｎｉｂの最終濃度が５μＭとなるように、それぞれ添加した以外は、実施例５と同様の手順で実験を行った。なお、薬剤の投与濃度は、コントロール細胞およびＤＳＨモデル細胞における細胞毒性評価を基に決定した。また、薬剤濃度の調整には、ＤＭＳＯを用いた。図５のＢ列の上段はコントロール細胞にＲｕｘｏｌｉｔｉｎｉｂを添加した時の写真、Ｂ列の下段はＤＳＨモデル細胞にＲｕｘｏｌｉｔｉｎｉｂを添加した時の写真である。図５のＣ列の上段はコントロール細胞にＴｏｆａｃｉｔｉｎｉｂを添加した時の写真、Ｃ列の下段はＤＳＨモデル細胞にＴｏｆａｃｉｔｉｎｉｂを添加した時の写真である。 [Experiment using DSH model cells]
<Example 6>
Experiments were carried out in the same procedure as in Example 5 except that in addition to mouse interferon β, Ruxolitinib was added to [day 3] in Example 5 so that the final concentration of Ruxolitinib was 0.5 μM and the final concentration of Tofacitinib was 5 μM. Was done. The administration concentration of the drug was determined based on the cytotoxicity evaluation in the control cells and the DSH model cells. In addition, DMSO was used to adjust the drug concentration. The upper part of the B column of FIG. 5 is a photograph when Ruxolitinib is added to the control cells, and the lower part of the B column is a photograph when Ruxolitinib is added to the DSH model cells. The upper part of row C in FIG. 5 is a photograph when Tofacitinib is added to control cells, and the lower part of row C is a photograph when Tofacitinib is added to DSH model cells.
　図５のＢ列およびＣ列の写真から明らかなように、免疫抑制物質であるＲｕｘｏｌｉｔｉｎｉｂ、および、Ｔｏｆａｃｉｔｉｎｉｂの何れを添加した場合にも、ＤＳＨモデル細胞の生存率が向上し、特に、Ｔｏｆａｃｉｔｉｎｉｂでは細胞生存率は９８．８％（Ｔｏｆａｃｉｔｉｎｉｂを投与したＤＳＨモデル細胞数／Ｔｏｆａｃｉｔｉｎｉｂを投与したコントロール細胞数）であった。このことは、Ａｄａｒ１の機能が低下もしく喪失したＤＳＨモデル細胞が、Ｒｕｘｏｌｉｔｉｎｉｂ、および、Ｔｏｆａｃｉｔｉｎｉｂの添加により、生存率が回復したことを意味していることから、Ａｄａｒ１の機能が低下もしく喪失した疾患の治療に有用であることを示唆している。 As is clear from the photographs of columns B and C in FIG. 5, the survival rate of DSH model cells is improved when either the immunosuppressive substance Ruxolitinib or Tofacitinib is added, and in particular, in Tofacitinib, the cells are improved. The survival rate was 98.8% (the number of DSH model cells administered with Tofacitinib / the number of control cells administered with Tofacitinib). This means that the DSH model cells in which the function of Adar1 was reduced or lost were restored to the survival rate by the addition of Ruxolitinib and Tofacitinib. Therefore, the function of Adar1 was reduced or lost. It suggests that it is useful in the treatment of diseases.
［ＤＳＨモデルマウスを用いた実験］
　実施例６において、細胞生存率が高かったＴｏｆａｃｉｔｉｎｉｂを用い、以下の実験を行った。 [Experiment using DSH model mouse]
In Example 6, the following experiments were carried out using Tofacitinib, which had a high cell viability.
＜実施例７：経皮投与＞
　実施例１に記載のｐ１５０　Ａｄａｒ１　ヘテロ接合型ノックアウトマウスの新生仔に、３０００Ｕのｍ－Ｉｆｎβ（ＰＢＬ社製）を１日１回皮下注射した。ｍ－Ｉｆｎβの注射は、生後７日目から開始し、生後１６日目まで１０日間行った。Ｔｏｆａｃｉｔｉｎｉｂは、生後７日目から１６日目まで１０日間、５０ｍｇ／ｋｇ／ｄａｙで皮下注射した。図６Ａは、生後２２日目の写真である。 <Example 7: Transdermal administration>
The neonates of the p150 Adar1 heterozygous knockout mouse described in Example 1 were subcutaneously injected with 3000 U of m-Ifnβ (manufactured by PBL) once a day. Injection of m-Ifnβ was started on the 7th day after birth and continued for 10 days until the 16th day after birth. Tofacitinib was subcutaneously injected at 50 mg / kg / day for 10 days from day 7 to day 16 after birth. FIG. 6A is a photograph of the 22nd day after birth.
＜比較例１：経皮投与＞
　Ｔｏｆａｃｉｔｉｎｉｂの代わりに、Ｔｏｆａｃｉｔｉｎｉｂの溶媒であるＤＭＳＯを注射した以外は、実施例７と同様の手順で実験を行った。図６Ｂは、生後２２日目の写真である。 <Comparative example 1: Transdermal administration>
The experiment was carried out in the same procedure as in Example 7 except that DMSO, which is a solvent of Tofacitinib, was injected instead of Tofacitinib. FIG. 6B is a photograph of the 22nd day after birth.
＜実施例８：経口投与＞
　実施例１に記載のｐ１５０　Ａｄａｒ１　ヘテロ接合型ノックアウトマウスの新生仔に、３０００Ｕのｍ－Ｉｆｎβ（ＰＢＬ社製）を１日１回皮下注射した。ｍ－Ｉｆｎβの注射は、生後７日目から開始し、１６日目まで毎日行った。Ｔｏｆａｃｉｔｉｎｉｂは、生後７日目から１６日目までの１０日間の毎日、５０ｍｇ／ｋｇ／ｄａｙで経口投与した。図７Ａは、生後２２日目の写真である。 <Example 8: Oral administration>
The neonates of the p150 Adar1 heterozygous knockout mouse described in Example 1 were subcutaneously injected with 3000 U of m-Ifnβ (manufactured by PBL) once a day. Injection of m-Ifnβ was started on the 7th day after birth and continued daily until the 16th day. Tofacitinib was orally administered at 50 mg / kg / day daily for 10 days from the 7th day to the 16th day after birth. FIG. 7A is a photograph of the 22nd day after birth.
＜比較例２：経口投与＞
　Ｔｏｆａｃｉｔｉｎｉｂの代わりに、Ｔｏｆａｃｉｔｉｎｉｂの溶媒であるＤＭＳＯを経口投与した以外は、実施例８と同様の手順で実験を行った。図７Ｂは、生後２２日目の写真である。 <Comparative example 2: Oral administration>
The experiment was carried out in the same procedure as in Example 8 except that DMSO, which is a solvent of Tofacitinib, was orally administered instead of Tofacitinib. FIG. 7B is a photograph of the 22nd day after birth.
　Ｔｏｆａｃｉｔｉｎｉｂを経皮注射（実施例７：図６Ａ）、または、経口投与（実施例８：図７Ａ）したＤＳＨモデルマウスは、写真から明らかなように、体毛が白色から黒色に変化した。一方、Ｔｏｆａｃｉｔｉｎｉｂを経皮注射（比較例１：図６Ｂ）、または、経口投与（比較例２：図７Ｂ）しなかったＤＳＨモデルマウスは、体毛が白いままであった。 In the DSH model mice to which Tofacitinib was injected transdermally (Example 7: FIG. 6A) or orally administered (Example 8: FIG. 7A), the body hair changed from white to black, as is clear from the photograph. On the other hand, the DSH model mice to which Tofacitinib was not transdermally injected (Comparative Example 1: FIG. 6B) or orally administered (Comparative Example 2: FIG. 7B) had white hair.
　以上の結果から、Ｔｏｆａｃｉｔｉｎｉｂを投与しなかったＤＳＨモデルマウスは、ＤＳＨ表現型を示したままであったが、Ｔｏｆａｃｉｔｉｎｉｂを投与することで、ＤＳＨ表現型が解消することを確認した。つまり、Ｔｏｆａｃｉｔｉｎｉｂが、経皮あるいは経口を問わず、ＤＳＨ治療用化合物として有用であることを確認した。また、実施例５、実施例７および実施例８に示すとおり、ＤＳＨモデル細胞およびＤＳＨモデルマウスで同じ結果が得られたことから、ＤＳＨモデル細胞およびＤＳＨモデルマウスは、ＤＳＨ治療用化合物のスクリーニングに使用できることを確認した。 From the above results, it was confirmed that the DSH model mice to which Tofacitinib was not administered remained showing the DSH phenotype, but the DSH phenotype was eliminated by administering Tofacitinib. That is, it was confirmed that Tofacitinib is useful as a DSH therapeutic compound regardless of whether it is transdermal or oral. Further, as shown in Examples 5, 7 and 8, the same results were obtained in the DSH model cells and the DSH model mice. Therefore, the DSH model cells and the DSH model mice were used for screening the DSH therapeutic compound. Confirmed that it can be used.
＜実施例９＞
［ＤＳＨ治療用化合物のスクリーニング］
　上記のとおり、ＤＳＨモデル細胞およびＤＳＨモデルマウスが、ＤＳＨ治療用化合物のスクリーニングに使用できることを確認したことから、ＤＳＨモデル細胞を用いて、種々の化合物のスクリーニングを行った。以下の表１に、実施例６と同様の手順でスクリーニングを行った化合物、濃度、化合物の公知の用途、細胞生存率を示す。なお、実施例６で確認済みの化合物についても、対比のため、表１に記載した。例示した化合物は、ＴｏｆａｃｉｔｉｎｉｂはＳｉｇｍａ　Ａｌｄｒｉｃｈ社、Ｃｕｒｃｕｍｏｌは東京化成工業、ＣｅｒｄｕｌａｔｉｎｉｂはＣａｙｍａｎ　ｃｈｅｍｉｃａｌから入手し、そのほかは全てＳｅｌｌｅｃｋ社から入手した。 <Example 9>
[Screening of DSH therapeutic compounds]
As described above, since it was confirmed that the DSH model cells and the DSH model mice can be used for the screening of the DSH therapeutic compound, various compounds were screened using the DSH model cells. Table 1 below shows the compounds, concentrations, known uses of the compounds, and cell viability that were screened in the same procedure as in Example 6. The compounds confirmed in Example 6 are also shown in Table 1 for comparison. The exemplified compounds were obtained from Sigma Aldrich for Tofacitinib, Tokyo Chemical Industry for Curcumol, Cayman chemical for Celdulatinib, and all others from Selleck.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１に示すとおり、ＪＡＫ阻害剤およびＳＴＡＴ阻害剤は、市販されている様々な種類で治療効果を示した。中でもＪＡＫ阻害剤は、殆どの種類で細胞生存率が５０％以上であったことから、ＤＳＨ治療用医薬組成物として好ましいことを確認した。 As shown in Table 1, JAK inhibitors and STAT inhibitors showed therapeutic effects in various commercially available types. Among them, JAK inhibitors were confirmed to be preferable as pharmaceutical compositions for the treatment of DSH because the cell viability of most types was 50% or more.
［その他のＪＡＫ阻害剤の有用性の確認］
＜実施例１０＞
　実施例６のＲｕｘｏｌｉｔｉｎｉｂおよびＴｏｆａｃｉｔｉｎｉｂに代え、ＪＡＫ阻害剤であるＤｅｌｇｏｃｉｔｉｎｉｂ（ＣｈｅｍＳｃｅｎｅ社製）の最終濃度が１μＭとなるように添加した以外は、実施例６と同様の手順で実験を行った。図８のＢ列の上段はコントロール細胞にＤｅｌｇｏｃｉｔｉｎｉｂを添加した時の写真、Ｂ列の下段はＤＳＨモデル細胞にＤｅｌｇｏｃｉｔｉｎｉｂを添加した時の写真である。 [Confirmation of usefulness of other JAK inhibitors]
<Example 10>
The experiment was carried out in the same procedure as in Example 6 except that Delgocitinib (manufactured by ChemScene), which is a JAK inhibitor, was added in place of Ruxolitinib and Tofacitinib in Example 6 so as to have a final concentration of 1 μM. The upper part of row B in FIG. 8 is a photograph when Delgocitinib was added to the control cells, and the lower part of row B is a photograph when Delgocitinib was added to the DSH model cells.
　図８の写真から明らかなように、Ｄｅｌｇｏｃｉｔｉｎｉｂを添加した場合には、添加しなかった場合と比較して、ＤＳＨモデル細胞の生存率が向上することを確認した。 As is clear from the photograph of FIG. 8, it was confirmed that when Delgocitinib was added, the survival rate of the DSH model cells was improved as compared with the case where it was not added.
＜実施例１１＞
　Ｔｏｆａｃｉｔｉｎｉｂに代え、Ｄｅｌｇｏｃｉｔｉｎｉｂを用いた以外は、実施例７と類似の手順で経皮投与実験を行った。具体的には、実施例１に記載のｐ１５０　Ａｄａｒ１　ヘテロ接合型ノックアウトマウスの新生仔に、３０００Ｕのｍ－Ｉｆｎβ（ＰＢＬ社製）を１日１回皮下注射した。ｍ－Ｉｆｎβの注射は、生後２日目から開始し、生後１４日目まで１０回行った。Ｄｅｌｇｏｃｉｔｉｎｉｂは、生後２日目から１４日目までに１日１回１５ｍｇ／ｋｇ／ｄａｙで１０回皮下注射した。図９Ｂは、生後１５日目の写真である。 <Example 11>
A transdermal administration experiment was performed in the same procedure as in Example 7 except that Delgocitinib was used instead of Tofacitinib. Specifically, 3000 U of m-Ifnβ (manufactured by PBL) was subcutaneously injected into the neonates of the p150 Adar1 heterozygous knockout mouse described in Example 1 once a day. Injection of m-Ifnβ was started on the 2nd day after birth and was performed 10 times until the 14th day after birth. Delgocitinib was subcutaneously injected 10 times a day at 15 mg / kg / day from the 2nd day to the 14th day after birth. FIG. 9B is a photograph of the 15th day after birth.
＜比較例３＞
　Ｄｅｌｇｏｃｉｔｉｎｉｂの代わりに、Ｄｅｌｇｏｃｉｔｉｎｉｂの溶媒であるＤＭＳＯを注射した以外は、実施例１１と同様の手順で実験を行った。図９Ａは、生後１５日目の写真である。 <Comparative example 3>
The experiment was carried out in the same procedure as in Example 11 except that DMSO, which is a solvent of Delgocitinib, was injected instead of Delgocitinib. FIG. 9A is a photograph of the 15th day after birth.
　Ｄｅｌｇｏｃｉｔｉｎｉｂを経皮注射したＤＳＨモデルマウス（実施例１１：図９Ｂ）は、写真から明らかなように、体毛が白色から黒色に変化した。一方、Ｄｅｌｇｏｃｉｔｉｎｉｂを経皮注射しなかったＤＳＨモデルマウス（比較例３：図９Ａ）は、体毛が白いままであった。 In the DSH model mouse (Example 11: FIG. 9B) in which Delgocitinib was transdermally injected, the hair changed from white to black, as is clear from the photograph. On the other hand, the DSH model mouse (Comparative Example 3: FIG. 9A) in which Delgocitinib was not injected transdermally had white hair.
　以上の結果から、ＪＡＫ阻害剤であるＤｅｌｇｏｃｉｔｉｎｉｂは、ＤＳＨ治療用化合物として有用であることを、ＤＳＨモデル細胞およびＤＳＨモデルマウスの両方の実験で確認した。 From the above results, it was confirmed in both experiments of DSH model cells and DSH model mice that Delgocitinib, which is a JAK inhibitor, is useful as a compound for treating DSH.
　本出願で開示するＤＳＨモデルマウスおよびモデル細胞を用いることで、ＤＳＨの病態の解明やＤＳＨ治療用の化合物のスクリーニングができる。また、ＪＡＫ阻害剤およびＳＴＡＴ阻害剤はＤＳＨ治療用医薬組成物として用いることができる。したがって、大学、医療機関、製薬会社等におけるＤＳＨ治療の研究・開発に有用である。 By using the DSH model mouse and model cells disclosed in this application, it is possible to elucidate the pathophysiology of DSH and screen compounds for the treatment of DSH. In addition, JAK inhibitors and STAT inhibitors can be used as pharmaceutical compositions for the treatment of DSH. Therefore, it is useful for research and development of DSH treatment in universities, medical institutions, pharmaceutical companies, etc.

Claims (16)

  1. 　ＪＡＫ阻害剤、および、ＳＴＡＴ阻害剤から成る群から選択した少なくとも一種の化合物を有効成分として含む、遺伝性対側性色素異常症治療用医薬組成物。 A pharmaceutical composition for treating hereditary contralateral dyschromatosis, which comprises at least one compound selected from the group consisting of a JAK inhibitor and a STAT inhibitor as an active ingredient.
  2. 　前記ＪＡＫ阻害剤が、
    　　Ｔｏｆａｃｉｔｉｎｉｂ、Ｉｔａｃｉｎｉｔｉｂ、Ｓｏｌｃｉｔｉｎｉｂ、ＡＺＤ１４８０、Ｒｕｘｏｌｉｔｉｎｉｂ、ＪＡＫ３－ＩＮ－６、Ｃｕｒｃｕｍｏｌ、Ｐｅｆｉｃｉｔｉｎｉｂ、Ｃｅｒｄｕｌａｔｉｎｉｂ、ＦＭ－３８１、Ｆｉｌｇｏｔｉｎｉｂ、および、Ｄｅｌｇｏｃｉｔｉｎｉｂから選択され、
    　前記ＳＴＡＴ阻害剤が、
    　　Ｎａｐａｂｕｃａｓｉｎ、Ｏｃｈｒｏｍｙｃｉｎｏｎｅ、Ｆｌｕｄａｒａｂｉｎｅ、Ｎｉｆｕｒｏｘａｚｉｄｅ、Ｃ１８８－９、および、ＡＳ１５１７４９９から選択される、
    請求項１に記載の遺伝性対側性色素異常症治療用医薬組成物。     The JAK inhibitor
    Selected from Tofacitinib, Itacitinib, Solcitinib, AZD1480, Ruxolitinib, JAK3-IN-6, Curcumol, Peficitinib, Cerdulatinib, FM-381, Filgotinib, and Delgocitinib.
    The STAT inhibitor
    Selected from Napabucasin, Ochromycine, Fludarabine, Nifuroxazide, C188-9, and AS1517499,
    The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to claim 1.
  3. 　前記ＪＡＫ阻害剤がＤｅｌｇｏｃｉｔｉｎｉｂである、
    請求項２に記載の遺伝性対側性色素異常症治療用医薬組成物。     The JAK inhibitor is Delgocitinib,
    The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to claim 2.
  4. 　前記ＪＡＫ阻害剤がＲｕｘｏｌｉｔｉｎｉｂである、
    請求項２に記載の遺伝性対側性色素異常症治療用医薬組成物。     The JAK inhibitor is Ruxolitinib,
    The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to claim 2.
  5. 　前記ＪＡＫ阻害剤がＴｏｆａｃｉｔｉｎｉｂである、
    請求項２に記載の遺伝性対側性色素異常症治療用医薬組成物。     The JAK inhibitor is Tofacitinib.
    The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to claim 2.
  6. 　前記ＪＡＫ阻害剤がＰｅｆｉｃｉｔｉｎｉｂである、
    請求項２に記載の遺伝性対側性色素異常症治療用医薬組成物。     The JAK inhibitor is Peficitinib,
    The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to claim 2.
  7. 　前記ＪＡＫ阻害剤がＣｅｒｄｕｌａｔｉｎｉｂである、
    請求項２に記載の遺伝性対側性色素異常症治療用医薬組成物。     The JAK inhibitor is Cerdulatinib,
    The pharmaceutical composition for treating hereditary contralateral dyschromatosis according to claim 2.
  8. 　全身でＡｄａｒ１（Ａｄｅｎｏｓｉｎｅ　Ｄｅａｍｉｎａｓｅ　Ａｃｔｉｎｇ　ｏｎ　ＲＮＡ１）の機能を喪失したヘテロ接合型Ａｄａｒ１ノックアウトマウスであって、
    　皮膚及び／又は体毛の色が斑であるとの表現型を示す、
    Ａｄａｒ１ヘテロ接合型ノックアウトマウス。     A heterozygous Adar1 knockout mouse that has lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout the body.
    Indicates a phenotype that the color of the skin and / or hair is mottled,
    Adar1 heterozygous knockout mouse.
  9. 　前記Ａｄａｒ１のｐ１５０をノックアウトした、
    請求項８に記載のＡｄａｒ１ヘテロ接合型ノックアウトマウス。     The p150 of Adar1 was knocked out.
    The Adar1 heterozygous knockout mouse according to claim 8.
  10. 　全身でＡｄａｒ１（Ａｄｅｎｏｓｉｎｅ　Ｄｅａｍｉｎａｓｅ　Ａｃｔｉｎｇ　ｏｎ　ＲＮＡ１）の機能を喪失したＡｄａｒ１ヘテロ接合型ノックアウトマウスに、Ｄｃｔ－ＬａｃＺトランスジェニックマウスを交配することで得られた、Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウス。 Adar1 KO: Dct-LacZ Tg mouse obtained by mating a Dct-LacZ transgenic mouse with an Adar1 heterozygous knockout mouse that has lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) throughout the body.
  11. 　前記Ａｄａｒ１のｐ１５０をノックアウトした、
    請求項１０に記載のＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウス。     The p150 of Adar1 was knocked out.
    The Adar1 KO: Dct-LacZ Tg mouse according to claim 10.
  12. 　請求項１０または１１に記載のＡｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇマウスに、Ｋ１４－Ｓｃｆ　Ｔｇマウスを交配することで得られた、Ａｄａｒ１　ＫＯ：Ｄｃｔ－ＬａｃＺ　Ｔｇ：Ｋ１４－Ｓｃｆ　Ｔｇマウス。 Adar1 KO: Dct-LacZ Tg: K14-Scf Tg mouse obtained by mating a K14-Scf Tg mouse with the Adar1 KO: Dct-LacZ Tg mouse according to claim 10 or 11.
  13. 　メラノサイト特異的にＡｄａｒ１（Ａｄｅｎｏｓｉｎｅ　Ｄｅａｍｉｎａｓｅ　Ａｃｔｉｎｇ　ｏｎ　ＲＮＡ１）の機能を喪失した、Ａｄａｒ１ホモ接合型ノックアウトマウス。 Adar1 homozygous knockout mouse that has lost the function of Adar1 (Adenosine Deaminase Acting on RNA1) specifically for melanocytes.
  14. 　体毛の色が斑であるとの表現型を示す、
    請求項１３に記載のＡｄａｒ１ホモ接合型ノックアウトマウス。     Shows the phenotype that the color of the hair is mottled,
    The Adar1 homozygous knockout mouse according to claim 13.
  15. 　請求項８～１４の何れか一項に記載のノックアウトマウスに被験化合物を投与する工程と、
    　脱色素斑を抑制する被験化合物を選択する被験化合物選択工程と、
    を含む、遺伝性対側性色素異常症治療用化合物のスクリーニング方法。     The step of administering the test compound to the knockout mouse according to any one of claims 8 to 14.
    A test compound selection step of selecting a test compound that suppresses depigmentation spots,
    A method for screening a compound for treating hereditary contralateral dyschromatosis, including.
  16. 　Ａｄａｒ１を不活性化した細胞を準備するＤＳＨモデル細胞準備工程と、
    　Ａｄａｒ１を不活性化していないコントロール細胞を準備するコントロール細胞準備工程と、
    　前記ＤＳＨモデル細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程と、
    　前記コントロール細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程と、
    　前記ＤＳＨモデル細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程を実施後の生存細胞数と、前記コントロール細胞に被験化合物とインターフェロン及び／又はＴｏｌｌ様受容体リガンドを投与する工程を実施後の生存細胞数とを対比する、生存細胞数対比工程と、
    　前記生存細胞数対比工程の対比結果から、被験化合物を選択する被験化合物選択工程と、
    を含む、遺伝性対側性色素異常症治療用化合物のスクリーニング方法。
          DSH model cell preparation step to prepare cells inactivated Adar1 and
    A control cell preparation step for preparing control cells that have not inactivated Adar1 and
    The step of administering the test compound and interferon and / or Toll-like receptor ligand to the DSH model cells, and
    The step of administering the test compound and interferon and / or Toll-like receptor ligand to the control cells, and
    The number of surviving cells after performing the step of administering the test compound and the interferon and / or Toll-like receptor ligand to the DSH model cells, and the step of administering the test compound and the interferon and / or Toll-like receptor ligand to the control cells. The step of comparing the number of viable cells and the step of comparing the number of viable cells after the above
    A test compound selection step of selecting a test compound from the comparison result of the survival cell number comparison step and a test compound selection step.
    A method for screening a compound for treating hereditary contralateral dyschromatosis, including.
PCT/JP2021/010546 2020-03-24 2021-03-16 Pharmaceutical composition for treating dyschromatosis symmetrica hereditaria, dyschromatosis symmetrica hereditaria model mouse, and screening method for compound for treating dyschromatosis symmetrica hereditaria WO2021193224A1 (en)

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