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WO2021225135A1 - Therapeutic agent for nakajo-nishimura syndrome - Google Patents

Therapeutic agent for nakajo-nishimura syndrome Download PDF

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WO2021225135A1
WO2021225135A1 PCT/JP2021/017367 JP2021017367W WO2021225135A1 WO 2021225135 A1 WO2021225135 A1 WO 2021225135A1 JP 2021017367 W JP2021017367 W JP 2021017367W WO 2021225135 A1 WO2021225135 A1 WO 2021225135A1
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mcp
mls
production
nns
cudc
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潤 齋藤
直也 加瀬
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Kyoto University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a therapeutic agent for Nakajo-Nishimura syndrome.
  • This application claims priority based on US Patent Application No. 63 / 021,112, which was provisionally filed in the United States on May 7, 2020, the contents of which are incorporated herein by reference.
  • Nakajo-Nishimura syndrome is one of the proteasome-related autoinflammatory syndromes (PRAAS) and is caused by homozygous mutations in the proteasome subunit beta 8 (PSMB8) / LMP-7 gene. NNS begins with a chilblain-like rash in early infancy, develops regular high fever, and ultimately results in fat / muscle atrophy and joint contracture, primarily in the upper body.
  • PRAAS proteasome-related autoinflammatory syndromes
  • PSMB8 proteasome subunit beta 8
  • All NNS patients are homozygous for the PSMB8 gene. It has a 602G> T mutation (a guanine to thymine mutation at the 602nd nucleotide residue), which is a G201V amino acid mutation at the ⁇ 5i subunit (a glycine to valine mutation at the 201st amino acid residue). Mutation).
  • NNS patients are thought to cause immune proteasome dysfunction, which induces cell stress and thus inflammation.
  • MCP-1 monocyte chemotaxis protein-1
  • IP-10 interferon gamma-induced protein 10
  • Oral steroids are used as a treatment method for NNS, but they are not effective for fat atrophy and joint contracture, and have serious side effects especially for infants, so the development of new therapeutic agents is eagerly desired.
  • the inventors have previously prepared NNS disease model cells using human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) (see Non-Patent Document 1). Specifically, wild-type iPSCs (WT-iPS) were prepared by recovering mutations from mutant iPSCs (MT-iPS) established from NNS patients using the genome editing technology CRISPR / Cas9 system. In addition, a mutant type (MT-ES) in which mutations were introduced into the human embryonic stem cell line KhES-1 (WT-ES) was prepared in the same manner.
  • WT-iPS wild-type iPSCs
  • MT-iPS mutant iPSCs
  • KhES-1 human embryonic stem cell line
  • MLs monocyte phenotype monocytic cell lines
  • WT-iPS-MLs monocyte phenotype monocytic cell lines
  • MT-iPS-MLs monocyte phenotype monocytic cell lines
  • WT-ES-MLs WT-ES-MLs
  • MT-ES-MLs monocyte phenotype monocytic cell lines
  • Non-Patent Document 1 also describes antioxidants and Janus Kinase (JAK) inhibitors as candidates for therapeutic agents for NNS.
  • JK Janus Kinase
  • an object of the present invention is to provide a therapeutic agent for NNS.
  • a therapeutic agent for Nakajo-Nishimura syndrome which contains a histone deacetylase inhibitor.
  • the histone deacetylase inhibitor is at least one compound selected from the group consisting of CUDC-907, JNJ-26481585, LAQ824, romidepsin and tricostatin A, or a pharmacologically acceptable salt thereof or
  • the therapeutic agent for Nakajo-Nishimura syndrome according to [1] which is a solvate thereof.
  • FIG. 1 is a schematic diagram showing an experimental schedule of Experimental Example 1.
  • FIG. 2 is a graph showing the production inhibition rates of the compounds MCP-1 and IP-10 in the primary screening of Experimental Example 1.
  • FIG. 3 is a graph showing the production inhibition rates of the compounds MCP-1 and IP-10 in the secondary screening of Experimental Example 1.
  • FIG. 4 is a graph showing the production inhibition rates of the compounds MCP-1 and IP-10 in MT-ES-MLs of Experimental Example 1.
  • FIG. 5 is a graph showing the results of cytotoxicity evaluation in Experimental Example 1.
  • FIG. 6 is a schematic diagram showing the results of high-throughput screening in Experimental Example 1.
  • 7 (a) to 7 (c) are dose-response curves for the production of MCP-1 of each compound measured in Experimental Example 2.
  • FIG. 7 (d) to 7 (f) are dose-response curves for the production of IP-10 of each compound measured in Experimental Example 2.
  • FIG. 8A is a graph showing the results of measuring intracellular reduced nicotinamide adenine dinucleotide (NADH) in Experimental Example 2.
  • FIG. 8B is a graph showing the results of measuring extracellular lactate dehydrogenase (LDH) activity in Experimental Example 2.
  • FIG. 9 is a graph showing the expression level of MCP-1 by each cell measured in Experimental Example 3.
  • FIG. 10 is a graph showing the dose-dependent effect of inhibiting the production of MCP-1 of CUDC-907 in fibroblasts derived from NNS patients, which was measured in Experimental Example 3.
  • FIG. 11A is a graph showing the results of measuring intracellular NADH in Experimental Example 3.
  • FIG. 11B is a graph showing the results of measuring the extracellular LDH activity in Experimental Example 3.
  • FIG. 12A is a graph showing the results of comparing the production amount of MCP-1 in each cell in Experimental Example 4.
  • FIG. 12B is a graph showing the results of comparing the amount of IP-10 produced in each cell in Experimental Example 4.
  • FIG. 13 is a graph showing the amount of MCP-1 produced in a fibroblast cell line derived from a healthy subject and a fibroblast cell derived from an NNS patient, which was measured in Experimental Example 4.
  • FIG. 14A is a graph showing the results of measuring the expression level of the CCL2 gene in Experimental Example 5.
  • FIG. 14A is a graph showing the results of measuring the expression level of the CCL2 gene in Experimental Example 5.
  • FIG. 14B is a graph showing the results of measuring the expression level of the CXCL10 gene in Experimental Example 5.
  • FIG. 15 is an image showing the results of typical Western blotting in Experimental Example 5.
  • FIG. 16 (a) is a dose-response curve for the production of MCP-1 of each compound measured in Experimental Example 6.
  • FIG. 16 (b) is a dose-response curve for the production of IP-10 of each compound measured in Experimental Example 6.
  • the present invention provides a therapeutic agent for Nakajo-Nishimura syndrome, which comprises a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • HDAC inhibitors suppress the overproduction of MCP-1 and IP-10 in NNS disease model cells and reduce them to healthy levels. Therefore, HDAC inhibitors can be used as therapeutic agents for Nakajo-Nishimura syndrome.
  • histone deacetylase inhibitors examples include CUDC-907 (CAS number: 1339928-25-4), JNJ-26481585 (CAS number: 875320-29-9), LAQ824 (CAS number: 404951-53-7), and the like.
  • examples thereof include romidepsin (CAS number: 128517-07-7), tricostatin A (CAS number: 58880-19-6), pharmacologically acceptable salts thereof, and solvates thereof.
  • Trichostatin A The chemical formula of Trichostatin A is shown in the following formula (5).
  • pharmaceutically acceptable salts include, for example, inorganic acid salts, alkali metal salts, alkaline earth metal salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like.
  • inorganic acid salts such as hydrochlorides, sulfates, hydrobromates, nitrates, phosphates; acetates, mesylates, succinates, maleates, fumarates, etc.
  • Organic acid salts such as citrate and tartrate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; metal salts such as aluminum salt and zinc salt; ammonium salt and tetra Ammonium salts such as methylammonium salt; organic amine addition salts such as morpholin and piperidine; amino acid addition salts such as glycine, phenylalanine, lysine, aspartic acid and glutamic acid can be mentioned.
  • Examples of pharmaceutically acceptable solvates include hydrates and organic solvates.
  • the therapeutic agent of the present embodiment is preferably formulated as a pharmaceutical composition containing the above-mentioned compound and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be administered orally in the form of, for example, liquids, powders, granules, tablets, capsules, etc., or parenterally in the form of injections, suppositories, external skin preparations, etc. .. More specific examples of the external skin preparation include dosage forms such as ointments and patches.
  • binders such as gelatin, cornstarch, tragant gum, and gum arabic
  • excipients such as starch and crystalline cellulose
  • swelling agents such as alginic acid
  • solvents for injections such as water, ethanol, and glycerin
  • adhesives such as rubber-based adhesives and silicone-based adhesives.
  • the pharmaceutical composition may contain additives.
  • Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and martitol; flavors such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid. Buffering agents such as salts and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants such as ascorbic acid; preservatives such as paraoxybenzoic acid esters (paraben), benzalconium chloride, chlorobutanol, cresol, etc. Agents and the like can be mentioned.
  • the dose of the pharmaceutical composition varies depending on the subject's symptoms, body weight, age, gender, etc. and cannot be unconditionally determined, but in the case of oral administration, for example, 0.1 to 100 mg / kg body weight per administration unit form.
  • the active ingredient (the above-mentioned compound) may be administered once a day or in 2 to 4 divided doses. In the case of an injection, for example, 0.01 to 50 mg of the active ingredient may be administered per administration unit form.
  • the present invention provides a method for treating Nakajo-Nishimura syndrome, which comprises administering an effective amount of a histone deacetylase inhibitor to a subject in need of treatment.
  • the histone deacetylase inhibitor is the same as described above.
  • the "effective amount" of the compound refers to the amount of the compound required to bring about a therapeutic effect on the treated subject, and more specifically, at least one or more related to Nakajo-Nishimura syndrome. The amount may be sufficient to prevent, delay, or minimize the symptoms of.
  • the "effective amount" of a compound is the prevention, delay, or minimization of one or more symptoms associated with Nakajo-Nishimura syndrome, either alone or in combination with other compounds or other treatments. It may be sufficient to do so. Effective amounts of compounds may vary depending on the severity of symptoms, route of administration, use of excipients, and combination with other therapeutic treatments, as will be appreciated by those skilled in the art.
  • the present invention provides a histone deacetylase inhibitor for use in the treatment of Nakajo-Nishimura syndrome.
  • the histone deacetylase inhibitor is the same as described above.
  • the present invention provides the use of histone deacetylase inhibitors for the production of therapeutic agents for Nakajo-Nishimura syndrome.
  • the histone deacetylase inhibitor is the same as described above.
  • Cell culture Fibroblasts collected from 2 healthy subjects (healthy subjects # 1 and # 2) and 2 NNS patients (NNS # 1 and # 2) were used. As cells, 10% fetal bovine serum (catalog number "F0926", Sigma-Aldrich) was added to Dulbecco's modified Eagle's medium (catalog number "08459-64", Nacalai Tesque). Cells were dissociated and subcultured into single cells using 0.25% trypsin EDTA solution (Cat. No. "25200-072", Thermo Fisher Scientific).
  • the fibroblasts used in the experiment were 10 ng / mL TNF- ⁇ (catalog number "210-TA”, R & D Systems) and 10 ng / mL IFN- ⁇ (catalog number "285-IF") to induce immune proteasomes. R & D Systems) stimulated for 72 hours.
  • MT-iPS-MLs is a cell line obtained by inducing differentiation of iPS cells (iPSCs) established from fibroblasts (NNS # 1) into cells having a homozygous phenotype (MLs), and is a PSMB8 gene.
  • iPSCs iPS cells
  • NNS # 1 fibroblasts
  • MLs a homozygous phenotype
  • WT-iPS-MLs are wild-type cell lines that are genetically homogeneous with MT-iPS-MLs.
  • MT-iPS-MLs a homozygous mutation of the PSMB8 gene similar to MT-iPS-MLs was introduced into KhES1, which is a human embryonic stem cell (ESCs) strain, by genome editing, and this was introduced into a cell line having a monocyte phenotype. Differentiation was induced into MLs) to obtain MT-ES-MLs.
  • WT-ES-MLs are wild-type cell lines that are genetically homogeneous with MT-ES-MLs.
  • WT-iPS-MLs, MT-iPS-MLs, WT-ES-MLs, MT-ES-MLs were added to StemPro TM- 34 SFM medium (catalog number "10639-011", Thermofisher Scientific) at 2 mM L. -Glutamine (catalog number "25030-081", Thermofisher Scientific), 50 ng / mL macrophage Colony Stimulating Factor (M-CSF, catalog number "216-MC”, R & D Systems) and 50 ng / mL granulocyte-macrosis It was cultured in a medium supplemented with factoror (GM-CSF, catalog number "215-GM”, R & D Systems).
  • M-CSF macrophage Colony Stimulating Factor
  • High-throughput screening 5,821 compounds, which have already been reported to have bioactive effects, were screened by high-throughput screening (HTS). Screening was performed by chemokine measurement based on homogeneous time-resolved fluorescence (HTRF). Table 1 below shows the compound library used for screening.
  • MT-iPS-MLs were seeded in 384-well plates at 5 ⁇ 10 3 per well and compounds were added at 1 ⁇ M (primary screening) or 100 nM (secondary screening). After incubation for 3 hours, 50 ng / mL lipopolysaccharide (Lipopolysaccharide (LPS), catalog number "tlll-peklps", in vivogen) for induction of MCP-1 or 100 ng / for induction of IP-10. mL IFN- ⁇ was added.
  • LPS lipopolysaccharide
  • ELISA Enzyme-linked immunosorbent assay
  • MT-ES-MLs were 5 ⁇ 10 4 (cell viability) or 1 ⁇ 10 4 (cytotoxic), NNS # One fibroblast was seeded at a cell density of 5 ⁇ 10 3 (cell viability) or 2 ⁇ 10 3 (cytotoxic), and the compound was added at an arbitrary concentration. After incubation for 24 hours, a commercially available kit (product name "Cell Counting Kit-8", catalog number "CK04", Cytotoxicity Research Institute) was used to measure cell viability, and a commercially available kit (product) was used to measure cytotoxicity.
  • RNA extraction and quantitative RT-PCR Total RNA was extracted using the RNeasy Mini kit (catalog number “74106", Qiagen) and treated with RNase-free DNase (catalog number "79254", Qiagen). The purified RNA was reverse transcribed using a commercially available kit (product name "PrimeScript TM RT Master Mix”, catalog number "RR037A”, Takara). qPCR was performed using a commercially available kit (product name "StepOnePlus TM ", Applied Biosystems, product name "TB Green Premix Ex Taq II", catalog number "RR820A”, Takara). The base sequence of the primers used is shown in Table 2 below.
  • the total amount of protein in the cytolyte was measured using a commercially available kit (product name "DC TM Protein Assay", catalog number “500-0116JA”, Biorad) and EnVision Multilabel Plate Readers (PerkinElmer), and each was measured. The protein concentration during cytolysis was adjusted to be the same.
  • cytolytic solution was boiled for 5 minutes in 4 ⁇ Remley sample buffer (catalog number “161-0747”, Bio-Rad) containing 2-mercaptoethanol (catalog number “21418-42”, Nacalai Tesque). Subsequently, the protein was separated by SDS polyacrylamide gel electrophoresis and transferred to Immobilon-P membrane (catalog number "IPVH000010], Merck).
  • the membrane is Tris-buffered saline supplemented with 10% skim milk (catalog number "190-12865", Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.1% Tween 20 (catalog number "9005-64-5", Sigma-Aldrich). It was blocked with water and reacted with the antibody.
  • Anti-GAPDH monoclonal antibody catalog number "2118", cell signaling technology
  • anti-MCP-1 polyclonal antibody catalog number "ab9669", abcam
  • anti-IP-10 polyclonal antibody catalog number "ab8098", abcam
  • HRP labeling Anti-rabbit IgG antibody catalog number "7074", cell signaling technology
  • HRP-labeled anti-mouse IgG antibody catalog number "7076", cell signaling technology
  • HTS histone deacetylase inhibitors as potential therapeutic agents for NNS
  • HTS identified potential therapeutic agents for NNS.
  • Compounds were evaluated by the rate of inhibition of production of MCP-1 and IP-10, both of which are pro-inflammatory chemokines that are particularly elevated in NNS patients.
  • IL-6 is also particularly elevated in NNS patients, but blocking the IL-6 receptor by administration of the anti-IL-6 receptor antibody tocilizumab has a limited therapeutic effect on NNS, and thus MCP- We focused on compounds that inhibit the production of both 1 and IP-10.
  • the compound library was composed of 5,821 compounds and contained approved agents, kinase inhibitors, and bioactive compounds.
  • FIG. 1 shows the experiment schedule. After treating the cells with the compound for 3 hours, LPS or IFN- ⁇ treatment induced the production of MCP-1 or MP-10. MT-iPS-MLs were used in the primary and secondary screenings.
  • FIG. 2 is a graph showing the production inhibition rates of MCP-1 and IP-10.
  • 642 compounds having an MCP-1 production inhibition rate of more than 60%, an IP-10 production inhibition rate of more than 50%, or both production inhibition rates of more than 30% are defined as hit compounds. bottom.
  • the compound existing in the region surrounded by the square is a hit compound. The reproducibility of the hit compounds was confirmed and those suspected of having strong cytotoxicity were removed, and 108 of the 642 compounds were evaluated by the secondary screening.
  • FIG. 3 is a graph showing the production inhibition rates of MCP-1 and IP-10. As shown in FIG. 3, 26 compounds in which the production inhibition rate of MCP-1 and IP-10 was more than 80% with respect to the compound concentration of 100 nM were designated as hit compounds. In FIG. 3, the compound existing in the region surrounded by the square is a hit compound.
  • FIG. 4 is a graph showing the production inhibition rates of MCP-1 and IP-10. As a result, as shown in FIG. 4, 13 compounds having a production inhibition rate of MCP-1 and IP-10 of more than 80% were designated as hit compounds. In FIG. 4, the compound existing in the region surrounded by the square is a hit compound.
  • FIG. 5 is a graph showing the results of cytotoxicity evaluation.
  • the vertical axis shows the amount of free lactate dehydrogenase (LDH).
  • the dotted line indicates the mean value + 1 standard deviation (SD).
  • “United” shows the result of the negative control in which DMSO was added instead of the compound
  • “Lysis” shows the result of the cytolytic solution used as the positive control.
  • the four compounds were considered non-cytotoxic because the amount of free LDH was less than the mean of the untreated control + 1 standard deviation (SD).
  • FIG. 6 is a schematic diagram showing the results of HTS. As shown in FIG. 6, since 3 of the 4 hit compounds were histone deacetylase (HDAC) inhibitors, a more detailed study was conducted on these 3 compounds.
  • HDAC histone deacetylase
  • Table 3 below shows the 50% inhibitory concentration (IC 50 ) of each compound calculated based on FIGS. 7 (a) to 7 (f).
  • CUDC-907 was found to exhibit the lowest an IC 50 value for the production of MCP-1 and IP-10.
  • HDAC inhibitors are known to arrest the cell cycle. Therefore, in order to confirm the safety of CUDC-907, the effect on cell viability and cytotoxicity on MT-iPS-MLs was examined.
  • CUDC-907 was treated at a maximum concentration of 100 nM, and the cell viability and cytotoxicity with respect to the DMSO-treated group were examined.
  • Cell viability was assessed by detecting intracellular reduced nicotinamide adenine dinucleotide (NADH).
  • Cytotoxicity was assessed by detecting extracellular lactate dehydrogenase (LDH) activity.
  • DMSO indicates the result of adding DMSO instead of the compound
  • Lysite indicates the result of cytolysis.
  • CUDC-907 showed almost the same cell viability and cytotoxicity as the DMSO-treated group at any concentration. From this result, it was clarified that CUDC-907 is a compound showing an effect of inhibiting the production of MCP-1 and IP-10 with extremely low toxicity to MT-iPS-MLs.
  • the expression level of immune proteasome in fibroblasts is lower than the expression level in immune cells. Therefore, the immunoproteasome was induced by treatment with TNF- ⁇ and IFN- ⁇ for a longer period of time (72 hours) to sufficiently show the phenotype of the NNS patient, and then the amount of cytokine produced was measured.
  • "UT” indicates the result of fibroblasts not treated with TNF- ⁇ and IFN- ⁇ .
  • "*”, "**”, and "***” have significant differences in p ⁇ 0.05, p ⁇ 0.01, and p ⁇ 0.005 as a result of Student's T-test, respectively. Show that.
  • FIG. 10 is a graph showing the dose-dependent effect of inhibiting the production of MCP-1 of CUDC-907 in NNS # 1, which is a fibroblast derived from an NNS patient.
  • IC 50 of the production inhibition of MCP-1 by CUDC-907 in fibroblasts was found to be comparable with the values in the MT-iPS-MLs.
  • DMSO indicates the result of adding DMSO instead of CUDC-907
  • Lysite indicates the result of cytolysis.
  • CUDC-907 shows almost no cytotoxicity at an effective concentration. From the above results, it is clear that CUDC-907 inhibits the production of MCP-1 by fibroblasts derived from NNS patients at the same concentration as that in MT-iPS-MLs without showing cytotoxicity. It became.
  • "**", "***”, and "*****” are the results of one-way analysis of variance (ANOVA) and Dunnett's multiple comparison, respectively, and p ⁇ 0. 0.01, p ⁇ 0.005, p ⁇ 0.001 indicate that there is a significant difference, and "NS" indicates that there is no significant difference.
  • “**” indicates that there is a significant difference at p ⁇ 0.01 as a result of Student's T-test, and “NS” indicates that there is no significant difference.
  • WT-iPS-MLs cells were treated with 10 nM CUDC-907 for 3 hours and then stimulated with 100 ng / mL TNF- ⁇ and 100 ng / mL IFN- ⁇ for 3, 9, 15 and 21 hours for quantitative RT-PCR. went.
  • the expression level of each gene was standardized by the expression level of the GAPDH gene.
  • FIG. 14A is a graph showing the results of measuring the expression level of the CCL2 gene. Further, FIG. 14 (b) is a graph showing the results of measuring the expression level of the CXCL10 gene. In FIGS. 14A and 14B, the vertical axis of the graph indicates the expression level (relative value).
  • CUDC-907 increases the expression level of these genes, unlike the result of ELISA. This result indicates that CUDC-907 inhibits the production of MCP-1 and IP-10 after transcription.
  • MCP-1 and IP-10 were examined at the protein level.
  • the extracellular release of these cytokines was inhibited by Brefeldin A treatment.
  • Breferdin A is known to inhibit protein transport from the endoplasmic reticulum to the Golgi apparatus.
  • WT-iPS-MLs cells were treated with 10 nM CUDC-907 for 30 minutes and then stimulated with 100 ng / mL TNF- ⁇ and 100 ng / mL IFN- ⁇ for 6 hours. Breferdin A was added 1 hour prior to cell recovery.
  • FIG. 15 is an image showing a typical Western blotting result. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as a loading control. In FIG. 15, “-" indicates that it was not added, and “+” indicates that it was added.
  • Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase
  • CUDC-907 reduced intracellular levels of MCP-1 and IP-10. This result indicates that CUDC-907 inhibits post-transcriptional production of MCP-1 and IP-10.
  • FIG. 16 (a) is a dose-response curve for the production of MCP-1 of each compound.
  • FIG. 16 (b) is a dose-response curve for IP-10 production of each compound.
  • Table 4 below shows the 50% inhibitory concentration (IC 50 ) of each compound calculated based on FIGS. 16 (a) and 16 (b).

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Abstract

This therapeutic agent for Nakajo-Nishimura syndrome comprises a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor may be: at least one compound selected from the group consisting of CUDC-907, JNJ-26481585, LAQ824, romidepsin, and trichostatin A; a pharmaceutically acceptable salt thereof; or solvates thereof.

Description

中條・西村症候群の治療剤A therapeutic agent for Nakajo-Nishimura syndrome

　本発明は、中條・西村症候群の治療剤に関する。本願は、２０２０年５月７日に米国に仮出願された米国特許出願第６３／０２１，１１２号明細書に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a therapeutic agent for Nakajo-Nishimura syndrome. This application claims priority based on US Patent Application No. 63 / 021,112, which was provisionally filed in the United States on May 7, 2020, the contents of which are incorporated herein by reference.

　中條・西村症候群（ＮＮＳ）は、プロテアソーム関連自己炎症性症候群（ＰＲＡＡＳ）の１つであり、ｐｒｏｔｅａｓｏｍｅ　ｓｕｂｕｎｉｔ　ｂｅｔａ　８（ＰＳＭＢ８）／ＬＭＰ－７遺伝子におけるホモ接合型の変異により生じる。ＮＮＳは、乳児期初期に、しもやけのような発疹を示すことから始まり、定期的な高熱を発症し、最終的には、主に上半身における脂肪・筋萎縮及び関節拘縮を生じる。 Nakajo-Nishimura syndrome (NNS) is one of the proteasome-related autoinflammatory syndromes (PRAAS) and is caused by homozygous mutations in the proteasome subunit beta 8 (PSMB8) / LMP-7 gene. NNS begins with a chilblain-like rash in early infancy, develops regular high fever, and ultimately results in fat / muscle atrophy and joint contracture, primarily in the upper body.

　全てのＮＮＳ患者は、ＰＳＭＢ８遺伝子に、ホモ接合型のｃ．６０２Ｇ＞Ｔ変異（第６０２番目のヌクレオチド残基におけるグアニンからチミンへの変異）を有しており、この変異はβ５ｉサブユニットにおけるＧ２０１Ｖアミノ酸変異（第２０１番目のアミノ酸残基におけるグリシンからバリンへの変異）をもたらす。 All NNS patients are homozygous for the PSMB8 gene. It has a 602G> T mutation (a guanine to thymine mutation at the 602nd nucleotide residue), which is a G201V amino acid mutation at the β5i subunit (a glycine to valine mutation at the 201st amino acid residue). Mutation).

　ＮＮＳ患者におけるＰＳＭＢ８遺伝子の変異は、免疫プロテアソームの機能不全を引き起こし、これが細胞ストレスを誘導することで炎症を引き起こすと考えられている。しかしながら、免疫プロテアソームの機能不全により誘導される炎症の詳細なメカニズムは依然として不明な点が多い。ＮＮＳ患者は炎症性ケモカインのｍｏｎｏｃｙｔｅ　ｃｈｅｍｏｔａｃｔｉｃ　ｐｒｏｔｅｉｎ－１（ＭＣＰ－１）及びｉｎｔｅｒｆｅｒｏｎ　ｇａｍｍａ－ｉｎｄｕｃｅｄ　ｐｒｏｔｅｉｎ　１０（ＩＰ－１０）を過剰産生することが知られており、これが炎症症状に関連しているとされている。 Mutations in the PSMB8 gene in NNS patients are thought to cause immune proteasome dysfunction, which induces cell stress and thus inflammation. However, the detailed mechanism of inflammation induced by immune proteasome dysfunction remains unclear. NNS patients are known to overproduce the inflammatory chemokines monocyte chemotaxis protein-1 (MCP-1) and interferon gamma-induced protein 10 (IP-10), which have been implicated in inflammatory symptoms. ing.

　ＮＮＳの治療法としてはステロイド内服が行われているが、脂肪萎縮や関節拘縮には効果がなく、特に乳幼児に対する副作用も重篤なことから、新規治療薬の開発が切望されている。 Oral steroids are used as a treatment method for NNS, but they are not effective for fat atrophy and joint contracture, and have serious side effects especially for infants, so the development of new therapeutic agents is eagerly desired.

　発明者らは、以前に、ヒト人工多能性幹細胞（ｉＰＳＣｓ）及びヒト胚性幹細胞（ＥＳＣｓ）を用いてＮＮＳ疾患モデル細胞を作製した（非特許文献１を参照。）。具体的には、ＮＮＳの患者より樹立した変異型ｉＰＳＣｓ（ＭＴ－ｉＰＳ）に対し、ゲノム編集技術ＣＲＩＳＰＲ／Ｃａｓ９システムを用いて変異を回復させた野生型ｉＰＳＣｓ（ＷＴ－ｉＰＳ）を作製した。また、ヒト胚性幹細胞株のＫｈＥＳ－１（ＷＴ－ＥＳ）に対しても同様の方法で変異を導入した変異型（ＭＴ－ＥＳ）を作製した。 The inventors have previously prepared NNS disease model cells using human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) (see Non-Patent Document 1). Specifically, wild-type iPSCs (WT-iPS) were prepared by recovering mutations from mutant iPSCs (MT-iPS) established from NNS patients using the genome editing technology CRISPR / Cas9 system. In addition, a mutant type (MT-ES) in which mutations were introduced into the human embryonic stem cell line KhES-1 (WT-ES) was prepared in the same manner.

　更に、炎症におけるＮＮＳの疾患の再現を確認するため、これらの細胞を単球の表現型を持つｍｏｎｏｃｙｔｉｃ　ｃｅｌｌ　ｌｉｎｅ（ＭＬｓ）に分化及び株化させた（ＷＴ－ｉＰＳ－ＭＬｓ、ＭＴ－ｉＰＳ－ＭＬｓ、ＷＴ－ＥＳ－ＭＬｓ、ＭＴ－ＥＳ－ＭＬｓ）。 Furthermore, in order to confirm the reproduction of NNS disease in inflammation, these cells were differentiated and established into monocyte phenotype monocytic cell lines (MLs) (WT-iPS-MLs, MT-iPS-MLs). , WT-ES-MLs, MT-ES-MLs).

　続いて、作製したＭＬｓに対し、ＴＮＦ－α及びＩＦＮ－γにより誘導されるサイトカインやケモカインの産生量を比較したところ、ＭＴ－ｉＰＳ－ＭＬｓ、ＭＴ－ＥＳ－ＭＬｓにおいて、ＮＮＳ特異的に過剰産生される、ＭＣＰ－１、ＩＰ－１０及びｉｎｔｅｒｌｅｕｋｉｎ－６（ＩＬ－６）の産生が増加していることが確認された。 Subsequently, when the production amounts of cytokines and chemokines induced by TNF-α and IFN-γ were compared with the produced MLs, overproduction was specifically overproduced in MT-iPS-MLs and MT-ES-MLs. It was confirmed that the production of MCP-1, IP-10 and interleukin-6 (IL-6) was increased.

　非特許文献１にはまた、ＮＮＳの治療薬の候補として、抗酸化剤やＪａｎｕｓ　Ｋｉｎａｓｅ（ＪＡＫ）阻害剤が記載されている。 Non-Patent Document 1 also describes antioxidants and Janus Kinase (JAK) inhibitors as candidates for therapeutic agents for NNS.

Honda-Ozaki and Terashima et al., Pluripotent Stem Cell Model of Nakajo-Nishimura Syndrome Untangles Proinflammatory Pathways Mediated by Oxidative Stress, Stem Cell Reports, 10 (6), 1835-1850, 2018.Honda-Ozaki and Terashima et al., Pluripotent Stem Cell Model of Nakajo-Nishimura Syndrome Untangles Proinflammatory Pathways Mediated by Oxidative Stress, Stem Cell Reports, 10 (6), 1835-1850, 2018.

　しかしながら、抗酸化剤やＪＡＫ阻害剤は、ＮＮＳ疾患モデル細胞における過剰なＭＣＰ－１及びＩＰ－１０の産生を健常レベルまで低下させるのに不十分である。そこで、本発明は、ＮＮＳの治療剤を提供することを目的とする。 However, antioxidants and JAK inhibitors are inadequate to reduce excess MCP-1 and IP-10 production in NNS disease model cells to healthy levels. Therefore, an object of the present invention is to provide a therapeutic agent for NNS.

　本発明は以下の態様を含む。
［１］ヒストン脱アセチル化酵素阻害剤を含む、中條・西村症候群の治療剤。
［２］前記ヒストン脱アセチル化酵素阻害剤が、ＣＵＤＣ－９０７、ＪＮＪ－２６４８１５８５、ＬＡＱ８２４、ロミデプシン及びトリコスタチンＡからなる群より選択される少なくとも１つの化合物又はその薬理学的に許容される塩若しくはそれらの溶媒和物である、［１］に記載の中條・西村症候群の治療剤。 The present invention includes the following aspects.
[1] A therapeutic agent for Nakajo-Nishimura syndrome, which contains a histone deacetylase inhibitor.
[2] The histone deacetylase inhibitor is at least one compound selected from the group consisting of CUDC-907, JNJ-26481585, LAQ824, romidepsin and tricostatin A, or a pharmacologically acceptable salt thereof or The therapeutic agent for Nakajo-Nishimura syndrome according to [1], which is a solvate thereof.

　本発明によれば、中條・西村症候群の治療剤を提供することができる。 According to the present invention, it is possible to provide a therapeutic agent for Nakajo-Nishimura syndrome.

図１は、実験例１の実験スケジュールを示す模式図である。FIG. 1 is a schematic diagram showing an experimental schedule of Experimental Example 1. 図２は、実験例１の１次スクリーニングにおける、化合物のＭＣＰ－１及びＩＰ－１０の産生阻害率を示すグラフである。FIG. 2 is a graph showing the production inhibition rates of the compounds MCP-1 and IP-10 in the primary screening of Experimental Example 1. 図３は、実験例１の２次スクリーニングにおける、化合物のＭＣＰ－１及びＩＰ－１０の産生阻害率を示すグラフである。FIG. 3 is a graph showing the production inhibition rates of the compounds MCP-1 and IP-10 in the secondary screening of Experimental Example 1. 図４は、実験例１のＭＴ－ＥＳ－ＭＬｓにおける、化合物のＭＣＰ－１及びＩＰ－１０の産生阻害率を示すグラフである。FIG. 4 is a graph showing the production inhibition rates of the compounds MCP-1 and IP-10 in MT-ES-MLs of Experimental Example 1. 図５は、実験例１における細胞毒性評価の結果を示すグラフである。FIG. 5 is a graph showing the results of cytotoxicity evaluation in Experimental Example 1. 図６は、実験例１におけるハイスループットスクリーニングの結果を示す模式図である。FIG. 6 is a schematic diagram showing the results of high-throughput screening in Experimental Example 1. 図７（ａ）～（ｃ）は、実験例２において測定した、各化合物のＭＣＰ－１の産生に対する用量反応曲線である。図７（ｄ）～（ｆ）は、実験例２において測定した、各化合物のＩＰ－１０の産生に対する用量反応曲線である。7 (a) to 7 (c) are dose-response curves for the production of MCP-1 of each compound measured in Experimental Example 2. 7 (d) to 7 (f) are dose-response curves for the production of IP-10 of each compound measured in Experimental Example 2. 図８（ａ）は、実験例２において、細胞内還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）を測定した結果を示すグラフである。図８（ｂ）は、実験例２において、細胞外乳酸脱水素酵素（ＬＤＨ）活性を測定した結果を示すグラフである。FIG. 8A is a graph showing the results of measuring intracellular reduced nicotinamide adenine dinucleotide (NADH) in Experimental Example 2. FIG. 8B is a graph showing the results of measuring extracellular lactate dehydrogenase (LDH) activity in Experimental Example 2. 図９は、実験例３において測定した、各細胞によるＭＣＰ－１の発現量を示すグラフである。FIG. 9 is a graph showing the expression level of MCP-1 by each cell measured in Experimental Example 3. 図１０は、実験例３において測定した、ＮＮＳ患者由来の線維芽細胞における、ＣＵＤＣ－９０７の用量依存的なＭＣＰ－１の産生阻害効果を示すグラフである。FIG. 10 is a graph showing the dose-dependent effect of inhibiting the production of MCP-1 of CUDC-907 in fibroblasts derived from NNS patients, which was measured in Experimental Example 3. 図１１（ａ）は、実験例３において、細胞内ＮＡＤＨを測定した結果を示すグラフである。図１１（ｂ）は、実験例３において細胞外ＬＤＨ活性を測定した結果を示すグラフである。FIG. 11A is a graph showing the results of measuring intracellular NADH in Experimental Example 3. FIG. 11B is a graph showing the results of measuring the extracellular LDH activity in Experimental Example 3. 図１２（ａ）は、実験例４において、各細胞におけるＭＣＰ－１の産生量を比較した結果を示すグラフである。図１２（ｂ）は、実験例４において、各細胞におけるＩＰ－１０の産生量を比較した結果を示すグラフである。FIG. 12A is a graph showing the results of comparing the production amount of MCP-1 in each cell in Experimental Example 4. FIG. 12B is a graph showing the results of comparing the amount of IP-10 produced in each cell in Experimental Example 4. 図１３は、実験例４において測定した、健常者由来の線維芽細胞株及びＮＮＳ患者由来の線維芽細胞におけるＭＣＰ－１の産生量を示すグラフである。FIG. 13 is a graph showing the amount of MCP-1 produced in a fibroblast cell line derived from a healthy subject and a fibroblast cell derived from an NNS patient, which was measured in Experimental Example 4. 図１４（ａ）は、実験例５において、ＣＣＬ２遺伝子の発現量を測定した結果を示すグラフである。図１４（ｂ）は、実験例５において、ＣＸＣＬ１０遺伝子の発現量を測定した結果を示すグラフである。FIG. 14A is a graph showing the results of measuring the expression level of the CCL2 gene in Experimental Example 5. FIG. 14B is a graph showing the results of measuring the expression level of the CXCL10 gene in Experimental Example 5. 図１５は、実験例５における代表的なウエスタンブロッティングの結果を示す画像である。FIG. 15 is an image showing the results of typical Western blotting in Experimental Example 5. 図１６（ａ）は、実験例６において測定した、各化合物のＭＣＰ－１の産生に対する用量反応曲線である。図１６（ｂ）は、実験例６において測定した、各化合物のＩＰ－１０の産生に対する用量反応曲線である。FIG. 16 (a) is a dose-response curve for the production of MCP-1 of each compound measured in Experimental Example 6. FIG. 16 (b) is a dose-response curve for the production of IP-10 of each compound measured in Experimental Example 6.

［中條・西村症候群の治療剤］
　１実施形態において、本発明は、ヒストン脱アセチル化酵素（ＨＤＡＣ）阻害剤を含む、中條・西村症候群の治療剤を提供する。 [Therapeutic agent for Nakajo-Nishimura syndrome]
In one embodiment, the present invention provides a therapeutic agent for Nakajo-Nishimura syndrome, which comprises a histone deacetylase (HDAC) inhibitor.

　実施例において後述するように、発明者らは、ＨＤＡＣ阻害剤がＮＮＳ疾患モデル細胞におけるＭＣＰ－１及びＩＰ－１０の過剰産生を抑制し、健常レベルまで低下させることを明らかにした。したがって、ＨＤＡＣ阻害剤を中條・西村症候群の治療剤として用いることができる。 As will be described later in the Examples, the inventors have shown that HDAC inhibitors suppress the overproduction of MCP-1 and IP-10 in NNS disease model cells and reduce them to healthy levels. Therefore, HDAC inhibitors can be used as therapeutic agents for Nakajo-Nishimura syndrome.

　ヒストン脱アセチル化酵素阻害剤としては、ＣＵＤＣ－９０７（ＣＡＳ番号：１３３９９２８－２５－４）、ＪＮＪ－２６４８１５８５（ＣＡＳ番号：８７５３２０－２９－９）、ＬＡＱ８２４（ＣＡＳ番号：４０４９５１－５３－７）、ロミデプシン（ＣＡＳ番号：１２８５１７－０７－７）、トリコスタチンＡ（ＣＡＳ番号：５８８８０－１９－６）、これらの薬理学的に許容される塩、これらの溶媒和物等が挙げられる。 Examples of histone deacetylase inhibitors include CUDC-907 (CAS number: 1339928-25-4), JNJ-26481585 (CAS number: 875320-29-9), LAQ824 (CAS number: 404951-53-7), and the like. Examples thereof include romidepsin (CAS number: 128517-07-7), tricostatin A (CAS number: 58880-19-6), pharmacologically acceptable salts thereof, and solvates thereof.

　ＣＵＤＣ－９０７の化学式を下記式（１）に示す。

Figure JPOXMLDOC01-appb-C000001
The chemical formula of CUDC-907 is shown in the following formula (1).
Figure JPOXMLDOC01-appb-C000001

　ＪＮＪ－２６４８１５８５の化学式を下記式（２）に示す。

Figure JPOXMLDOC01-appb-C000002
The chemical formula of JNJ-26481585 is shown in the following formula (2).
Figure JPOXMLDOC01-appb-C000002

　ＬＡＱ８２４の化学式を下記式（３）に示す。

Figure JPOXMLDOC01-appb-C000003
The chemical formula of LAQ824 is shown in the following formula (3).
Figure JPOXMLDOC01-appb-C000003

　ロミデプシンの化学式を下記式（４）に示す。

Figure JPOXMLDOC01-appb-C000004
The chemical formula of romidepsin is shown in the following formula (4).
Figure JPOXMLDOC01-appb-C000004

　トリコスタチンＡの化学式を下記式（５）に示す。

Figure JPOXMLDOC01-appb-C000005
The chemical formula of Trichostatin A is shown in the following formula (5).
Figure JPOXMLDOC01-appb-C000005

　本実施形態の治療剤において、薬学的に許容される塩としては、例えば、無機酸塩、アルカリ金属塩、アルカリ土類金属塩、金属塩、アンモニウム塩、有機アミン付加塩、アミノ酸付加塩等が挙げられる。より具体的には、例えば、塩酸塩、硫酸塩、臭化水素酸塩、硝酸塩、リン酸塩等の無機酸塩；酢酸塩、メシル酸塩、コハク酸塩、マレイン酸塩、フマル酸塩、クエン酸塩、酒石酸塩等の有機酸塩；ナトリウム塩、カリウム塩等のアルカリ金属塩；マグネシウム塩、カルシウム塩等のアルカリ土類金属塩；アルミニウム塩、亜鉛塩等の金属塩；アンモニウム塩、テトラメチルアンモニウム塩等のアンモニウム塩；モルホリン、ピペリジン等の有機アミン付加塩；グリシン、フェニルアラニン、リジン、アスパラギン酸、グルタミン酸等のアミノ酸付加塩等が挙げられる。また、薬学的に許容される溶媒和物としては、例えば、水和物、有機溶媒和物等が挙げられる。 In the therapeutic agent of the present embodiment, pharmaceutically acceptable salts include, for example, inorganic acid salts, alkali metal salts, alkaline earth metal salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like. Can be mentioned. More specifically, for example, inorganic acid salts such as hydrochlorides, sulfates, hydrobromates, nitrates, phosphates; acetates, mesylates, succinates, maleates, fumarates, etc. Organic acid salts such as citrate and tartrate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; metal salts such as aluminum salt and zinc salt; ammonium salt and tetra Ammonium salts such as methylammonium salt; organic amine addition salts such as morpholin and piperidine; amino acid addition salts such as glycine, phenylalanine, lysine, aspartic acid and glutamic acid can be mentioned. Examples of pharmaceutically acceptable solvates include hydrates and organic solvates.

　本実施形態の治療剤は、上述した化合物と、薬学的に許容される担体とを含む医薬組成物として製剤化されていることが好ましい。医薬組成物は、例えば、液剤、散剤、顆粒剤、錠剤、カプセル剤等の形態で経口的に、あるいは、注射剤、坐剤、皮膚外用剤等の形態で非経口的に投与することができる。皮膚外用剤としては、より具体的には、軟膏剤、貼付剤等の剤型が挙げられる。 The therapeutic agent of the present embodiment is preferably formulated as a pharmaceutical composition containing the above-mentioned compound and a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered orally in the form of, for example, liquids, powders, granules, tablets, capsules, etc., or parenterally in the form of injections, suppositories, external skin preparations, etc. .. More specific examples of the external skin preparation include dosage forms such as ointments and patches.

　薬学的に許容される担体としては、通常医薬組成物の製剤に用いられるものを特に制限なく用いることができる。より具体的には、例えば、ゼラチン、コーンスターチ、トラガントガム、アラビアゴム等の結合剤；デンプン、結晶性セルロース等の賦形剤；アルギン酸等の膨化剤；水、エタノール、グリセリン等の注射剤用溶剤；ゴム系粘着剤、シリコーン系粘着剤等の粘着剤等が挙げられる。 As the pharmaceutically acceptable carrier, those usually used for the preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, cornstarch, tragant gum, and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injections such as water, ethanol, and glycerin; Examples thereof include adhesives such as rubber-based adhesives and silicone-based adhesives.

　医薬組成物は添加剤を含んでいてもよい。添加剤としては、ステアリン酸カルシウム、ステアリン酸マグネシウム等の潤滑剤；ショ糖、乳糖、サッカリン、マルチトール等の甘味剤；ペパーミント、アカモノ油等の香味剤；ベンジルアルコール、フェノール等の安定剤；リン酸塩、酢酸ナトリウム等の緩衝剤；安息香酸ベンジル、ベンジルアルコール等の溶解補助剤；アスコルビン酸等の酸化防止剤；パラオキシ安息香酸エステル類（パラベン）、塩化ベンザルコニウム、クロロブタノール、クレゾール等の防腐剤等が挙げられる。 The pharmaceutical composition may contain additives. Additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and martitol; flavors such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid. Buffering agents such as salts and sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants such as ascorbic acid; preservatives such as paraoxybenzoic acid esters (paraben), benzalconium chloride, chlorobutanol, cresol, etc. Agents and the like can be mentioned.

　医薬組成物の投与量は、対象の症状、体重、年齢、性別等によって異なり、一概には決定できないが、経口投与の場合には、例えば、投与単位形態あたり０．１～１００ｍｇ／ｋｇ体重の有効成分（上述した化合物）を１日１回又は２～４回程度に分けて投与すればよい。また、注射剤の場合には、例えば、投与単位形態あたり０．０１～５０ｍｇの有効成分を投与すればよい。 The dose of the pharmaceutical composition varies depending on the subject's symptoms, body weight, age, gender, etc. and cannot be unconditionally determined, but in the case of oral administration, for example, 0.1 to 100 mg / kg body weight per administration unit form. The active ingredient (the above-mentioned compound) may be administered once a day or in 2 to 4 divided doses. In the case of an injection, for example, 0.01 to 50 mg of the active ingredient may be administered per administration unit form.

［その他の実施形態］
　１実施形態において、本発明は、ヒストン脱アセチル化酵素阻害剤の有効量を、治療を必要とする対象に投与することを含む、中條・西村症候群の治療方法を提供する。ヒストン脱アセチル化酵素阻害剤については上述したものと同様である。ここで、化合物の「有効量」とは、処置された対象に治療効果をもたらすのに必要とされる化合物の量を指し、より具体的には、中條・西村症候群に関連する少なくとも１つ以上の症状を阻止、遅延、又は最小限にするために十分な量であってよい。ある態様において、化合物の「有効量」とは、化合物単独で、又は他の化合物ないしは他の処置を組み合わせて、中條・西村症候群に関連する１つ以上の症状を阻止、遅延、又は最小限にするために十分な量であってよい。化合物の有効量は、当業者によって認識されるように、症状の程度、投与経路、賦形剤の使用、及び他の治療処置との併用に応じて変化し得る。 [Other Embodiments]
In one embodiment, the present invention provides a method for treating Nakajo-Nishimura syndrome, which comprises administering an effective amount of a histone deacetylase inhibitor to a subject in need of treatment. The histone deacetylase inhibitor is the same as described above. Here, the "effective amount" of the compound refers to the amount of the compound required to bring about a therapeutic effect on the treated subject, and more specifically, at least one or more related to Nakajo-Nishimura syndrome. The amount may be sufficient to prevent, delay, or minimize the symptoms of. In some embodiments, the "effective amount" of a compound is the prevention, delay, or minimization of one or more symptoms associated with Nakajo-Nishimura syndrome, either alone or in combination with other compounds or other treatments. It may be sufficient to do so. Effective amounts of compounds may vary depending on the severity of symptoms, route of administration, use of excipients, and combination with other therapeutic treatments, as will be appreciated by those skilled in the art.

　１実施形態において、本発明は、中條・西村症候群の治療における使用のための、ヒストン脱アセチル化酵素阻害剤を提供する。ヒストン脱アセチル化酵素阻害剤については上述したものと同様である。 In one embodiment, the present invention provides a histone deacetylase inhibitor for use in the treatment of Nakajo-Nishimura syndrome. The histone deacetylase inhibitor is the same as described above.

　１実施形態において、本発明は、中條・西村症候群の治療剤の製造のための、ヒストン脱アセチル化酵素阻害剤の使用を提供する。ヒストン脱アセチル化酵素阻害剤については上述したものと同様である。 In one embodiment, the present invention provides the use of histone deacetylase inhibitors for the production of therapeutic agents for Nakajo-Nishimura syndrome. The histone deacetylase inhibitor is the same as described above.

　次に実施例を示して本発明を更に詳細に説明するが、本発明は以下の実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

［材料及び方法］
　以下の実験は、京都大学（Ｒ００９１／Ｇ０２５９）と和歌山県立医科大学の倫理委員会によって承認された。ヘルシンキ宣言にしたがって、患者又はその保護者から書面によるインフォームドコンセントを得た。ヒト胚性幹細胞（ＥＳＣｓ）の使用は、文部科学省によって承認された。 [Materials and methods]
The following experiments were approved by the Institutional Review Board of Kyoto University (R0091 / G0259) and Wakayama Medical University. Written informed consent was obtained from the patient or his or her guardian in accordance with the Declaration of Helsinki. The use of human embryonic stem cells (ESCs) has been approved by the Ministry of Education, Culture, Sports, Science and Technology.

（細胞培養）
　線維芽細胞は、健常者２名（健常者＃１及び＃２）及びＮＮＳ患者２名（ＮＮＳ＃１及び＃２）より採取したものを用いた。細胞は、ダルベッコ変法イーグル培地（カタログ番号「０８４５９－６４」、ナカライテスク）に１０％ウシ胎児血清（カタログ番号「Ｆ０９２６」、シグマ－アルドリッチ）を加えたものを用いた。細胞は０．２５％トリプシンＥＤＴＡ溶液（カタログ番号「２５２００－０７２」、サーモフィッシャーサイエンティフィック）を用いて単一細胞に解離して継代した。実験に用いた線維芽細胞は、免疫プロテアソームを誘導するため、１０ｎｇ／ｍＬ　ＴＮＦ－α（カタログ番号「２１０－ＴＡ」、Ｒ＆Ｄシステムズ）及び１０ｎｇ／ｍＬ　ＩＦＮ－γ（カタログ番号「２８５－ＩＦ」、Ｒ＆Ｄシステムズ）で７２時間刺激した。 (Cell culture)
Fibroblasts collected from 2 healthy subjects (healthy subjects # 1 and # 2) and 2 NNS patients (NNS # 1 and # 2) were used. As cells, 10% fetal bovine serum (catalog number "F0926", Sigma-Aldrich) was added to Dulbecco's modified Eagle's medium (catalog number "08459-64", Nacalai Tesque). Cells were dissociated and subcultured into single cells using 0.25% trypsin EDTA solution (Cat. No. "25200-072", Thermo Fisher Scientific). The fibroblasts used in the experiment were 10 ng / mL TNF-α (catalog number "210-TA", R & D Systems) and 10 ng / mL IFN-γ (catalog number "285-IF") to induce immune proteasomes. R & D Systems) stimulated for 72 hours.

　非特許文献１に記載された、ＷＴ－ｉＰＳ－ＭＬｓ、ＭＴ－ｉＰＳ－ＭＬｓ、ＷＴ－ＥＳ－ＭＬｓ、ＭＴ－ＥＳ－ＭＬｓ細胞を使用した。ＭＴ－ｉＰＳ－ＭＬｓは、線維芽細胞（ＮＮＳ＃１）から樹立したｉＰＳ細胞（ｉＰＳＣｓ）を、単球の表現型を持つ細胞（ＭＬｓ）に分化誘導して得た細胞株であり、ＰＳＭＢ８遺伝子にホモ接合型の変異を有する。ＷＴ－ｉＰＳ－ＭＬｓは、ＭＴ－ｉＰＳ－ＭＬｓと遺伝的に同質の野生型細胞株である。 The WT-iPS-MLs, MT-iPS-MLs, WT-ES-MLs, and MT-ES-MLs cells described in Non-Patent Document 1 were used. MT-iPS-MLs is a cell line obtained by inducing differentiation of iPS cells (iPSCs) established from fibroblasts (NNS # 1) into cells having a homozygous phenotype (MLs), and is a PSMB8 gene. Has a homozygous mutation in. WT-iPS-MLs are wild-type cell lines that are genetically homogeneous with MT-iPS-MLs.

　また、ヒト胚性幹細胞（ＥＳＣｓ）株であるＫｈＥＳ１に、ゲノム編集によりＭＴ－ｉＰＳ－ＭＬｓと同様のＰＳＭＢ８遺伝子のホモ接合型の変異を導入し、これを単球の表現型を持つ細胞株（ＭＬｓ）に分化誘導してＭＴ－ＥＳ－ＭＬｓを得た。ＷＴ－ＥＳ－ＭＬｓは、ＭＴ－ＥＳ－ＭＬｓと遺伝的に同質の野生型細胞株である。 In addition, a homozygous mutation of the PSMB8 gene similar to MT-iPS-MLs was introduced into KhES1, which is a human embryonic stem cell (ESCs) strain, by genome editing, and this was introduced into a cell line having a monocyte phenotype. Differentiation was induced into MLs) to obtain MT-ES-MLs. WT-ES-MLs are wild-type cell lines that are genetically homogeneous with MT-ES-MLs.

　ＷＴ－ｉＰＳ－ＭＬｓ、ＭＴ－ｉＰＳ－ＭＬｓ、ＷＴ－ＥＳ－ＭＬｓ、ＭＴ－ＥＳ－ＭＬｓは、ＳｔｅｍＰｒｏＴＭ－３４　ＳＦＭ培地（カタログ番号「１０６３９－０１１」、サーモフィッシャーサイエンティフィック）に、２ｍＭ　Ｌ－グルタミン（カタログ番号「２５０３０－０８１」、サーモフィッシャーサイエンティフィック）、５０ｎｇ／ｍＬ　ｍａｃｒｏｐｈａｇｅ　Ｃｏｌｏｎｙ　Ｓｔｉｍｕｌａｔｉｎｇ　Ｆａｃｔｏｒ（Ｍ－ＣＳＦ、カタログ番号「２１６－ＭＣ」、Ｒ＆Ｄシステムズ）及び５０ｎｇ／ｍＬ　ｇｒａｎｕｌｏｃｙｔｅ　ｍａｃｒｏｐｈａｇｅ　ｃｏｌｏｎｙ－ｓｔｉｍｕｌａｔｉｎｇ　ｆａｃｔｏｒ（ＧＭ－ＣＳＦ、カタログ番号「２１５－ＧＭ」、Ｒ＆Ｄシステムズ）を添加した培地で培養した。 WT-iPS-MLs, MT-iPS-MLs, WT-ES-MLs, MT-ES-MLs were added to StemPro TM- 34 SFM medium (catalog number "10639-011", Thermofisher Scientific) at 2 mM L. -Glutamine (catalog number "25030-081", Thermofisher Scientific), 50 ng / mL macrophage Colony Stimulating Factor (M-CSF, catalog number "216-MC", R & D Systems) and 50 ng / mL granulocyte-macrosis It was cultured in a medium supplemented with factoror (GM-CSF, catalog number "215-GM", R & D Systems).

（ハイスループットスクリーニング）
　既に生理活性作用が報告されている５，８２１化合物をハイスループットスクリーニング（ＨＴＳ）によりスクリーニングした。スクリーニングは、均一系時間分解蛍光（ｈｏｍｏｇｅｎｅｏｕｓ　ｔｉｍｅ－ｒｅｓｏｌｖｅｄ　ｆｌｕｏｒｅｓｃｅｎｃｅ、ＨＴＲＦ）に基づくケモカイン測定により行った。下記表１に、スクリーニングに使用した化合物ライブラリを示す。 (High-throughput screening)
5,821 compounds, which have already been reported to have bioactive effects, were screened by high-throughput screening (HTS). Screening was performed by chemokine measurement based on homogeneous time-resolved fluorescence (HTRF). Table 1 below shows the compound library used for screening.

Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006

　１次及び２次スクリーニングでは、３８４ウェルプレートにＭＴ－ｉＰＳ－ＭＬｓを１ウェルあたり５×１０３個で播種し、化合物を１μＭ（１次スクリーニング）又は１００ｎＭ（２次スクリーニング）添加した。３時間インキュベートした後、ＭＣＰ－１の誘導のために５０ｎｇ／ｍＬリポ多糖（Ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄ（ＬＰＳ）、カタログ番号「ｔｌｒｌ－ｐｅｋｌｐｓ」、インビボジェン）を、又は、ＩＰ－１０の誘導のために１００ｎｇ／ｍＬ　ＩＦＮ－γを添加した。続いて、２１時間インキュベートした後、上清を別の３８４ウェルプレートに移し、Ｈｕｍａｎ　ＣＣＬ２（ＭＣＰ－１）Ｋｉｔ（カタログ番号「６２ＨＣＣＬ２ＰＥＧ」、シスバイオ）又はＨｕｍａｎ　ＣＸＣＬ１０（ＩＰ－１０）Ｋｉｔ（カタログ番号「６２ＨＣＸ１０ＰＥＧ、シスバイオ）を加え、ＰｏｗｅｒＳｃａｎ４（ＤＳファーマバイオメディカル）によりＭＣＰ－１又はＩＰ－１０の濃度を測定した。続いて、ＭＴ－ＥＳ－ＭＬｓを用いて、２次スクリーニングと同様の手順によりヒット化合物の検証を行った。 In the primary and secondary screenings, MT-iPS-MLs were seeded in 384-well plates at 5 × 10 3 per well and compounds were added at 1 μM (primary screening) or 100 nM (secondary screening). After incubation for 3 hours, 50 ng / mL lipopolysaccharide (Lipopolysaccharide (LPS), catalog number "tlll-peklps", in vivogen) for induction of MCP-1 or 100 ng / for induction of IP-10. mL IFN-γ was added. Subsequently, after incubating for 21 hours, the supernatant was transferred to another 384-well plate and transferred to Human CCL2 (MCP-1) Kit (catalog number "62HCCL2PEG", cisbio) or Human CXCL10 (IP-10) Kit (catalog number "". 62HCX10PEG, cisbio) was added and the concentration of MCP-1 or IP-10 was measured by PowerScan4 (DS Pharma Biomedical). Subsequently, using MT-ES-MLs, the hit compound was subjected to the same procedure as in the secondary screening. Was verified.

（酵素結合免疫吸着検定法（ＥＬＩＳＡ））
　ＭＬｓ又は線維芽細胞の培養上清中のＭＣＰ－１及びＩＰ－１０の濃度の測定は、市販のキット（製品名「ＬＥＧＥＮＤｐｌｅｘＴＭ　Ｈｕｍａｎ　Ａｄｉｐｏｋｉｎｅ　Ｐａｎｅｌ、バイオレジェンド）を用いて行なった。上清採取のために、細胞を９６ウェルプレートに１ウェルあたり１×１０４個（ＭＬｓ）又は５×１０３個（線維芽細胞）の細胞密度で播種し、ジメチルスルホキシド（ＤＭＳＯ、カタログ番号「Ｄ２６５０」、シグマアルドリッチ）に溶解した化合物を任意の濃度で添加した。 (Enzyme-linked immunosorbent assay (ELISA))
The concentrations of MCP-1 and IP-10 in the culture supernatant of MLs or fibroblasts were measured using a commercially available kit (product name "LEGENDplex TM Human Adipokine Panel, BioLegend"). To allow cells to be seeded in 96-well plates at a cell density of 1 x 10 4 (MLs) or 5 x 10 3 (fibroblasts) per well, dimethylsulfoxide (DMSO, catalog number "D2650", sigma). The compound dissolved in Aldrich) was added at an arbitrary concentration.

　続いて、３時間インキュベートした後、１００ｎｇ／ｍＬ（ＭＬｓ）又は１０ｎｇ／ｍＬ（線維芽細胞）のＴＮＦ－α及びＩＦＮ－γを添加し刺激した。２１時間インキュベートした後、上清を採取した。 Subsequently, after incubating for 3 hours, 100 ng / mL (MLs) or 10 ng / mL (fibroblasts) of TNF-α and IFN-γ were added and stimulated. After incubating for 21 hours, the supernatant was collected.

（細胞生存率及び細胞毒性の測定）
　細胞生存率及び細胞毒性評価のために、細胞を９６ウェルプレートに１ウェルあたり、ＭＴ－ＥＳ－ＭＬｓは５×１０４個（細胞生存率）又は１×１０４個（細胞毒性）、ＮＮＳ＃１線維芽細胞は５×１０３個（細胞生存率）又は２×１０３個（細胞毒性）の細胞密度で播種し、化合物を任意の濃度で添加した。２４時間インキュベートした後、細胞生存率の測定では、市販のキット（製品名「Ｃｅｌｌ　Ｃｏｕｎｔｉｎｇ　Ｋｉｔ－８」、カタログ番号「ＣＫ０４」、同仁化学研究所）、細胞毒性の測定では、市販のキット（製品名「Ｃｙｔｏｔｏｘｉｃｉｔｙ　Ｄｅｔｅｃｔｉｏｎ　ＫｉｔＰＬＵＳ」、カタログ番号「４７４４９３４００１」、メルク）を加え、２１０４　ＥｎＶｉｓｉｏｎ　Ｍｕｌｔｉｌａｂｅｌ　Ｐｌａｔｅ　Ｒｅａｄｅｒｓ（パーキンエルマー）で測定した。細胞生存率・細胞毒性評価の陽性コントロール（１００％細胞死）として、同数の細胞の溶解液を使用した。 (Measurement of cell viability and cytotoxicity)
For cell viability and cytotoxicity assessment, per well of cells in a 96-well plate, MT-ES-MLs were 5 × 10 4 (cell viability) or 1 × 10 4 (cytotoxic), NNS # One fibroblast was seeded at a cell density of 5 × 10 3 (cell viability) or 2 × 10 3 (cytotoxic), and the compound was added at an arbitrary concentration. After incubation for 24 hours, a commercially available kit (product name "Cell Counting Kit-8", catalog number "CK04", Cytotoxicity Research Institute) was used to measure cell viability, and a commercially available kit (product) was used to measure cytotoxicity. The name "Cytotoxicity Detection Kit PLUS ", catalog number "4744934001", Merck) was added, and the measurement was performed with 2104 EnVision Multilabel Plate Readers (PerkinElmer). As a positive control (100% cell death) for cell viability / cytotoxicity evaluation, the same number of cell lysates were used.

（ＲＮＡ抽出及び定量的ＲＴ－ＰＣＲ（ｑＰＣＲ））
　ＲＮｅａｓｙ　Ｍｉｎｉ　ｋｉｔ（カタログ番号「７４１０６」、キアゲン）を用いて全ＲＮＡを抽出し、ＲＮａｓｅ－ｆｒｅｅ　ＤＮａｓｅ（カタログ番号「７９２５４」、キアゲン）で処理した。精製したＲＮＡは市販のキット（製品名「ＰｒｉｍｅＳｃｒｉｐｔＴＭ　ＲＴ　Ｍａｓｔｅｒ　Ｍｉｘ」、カタログ番号「ＲＲ０３７Ａ」、タカラ）を用いて逆転写した。ｑＰＣＲは市販のキット（製品名「ＳｔｅｐＯｎｅＰｌｕｓＴＭ」、アプライドバイオシステムズ、及び、製品名「ＴＢ　Ｇｒｅｅｎ　Ｐｒｅｍｉｘ　Ｅｘ　Ｔａｑ　ＩＩ」、カタログ番号「ＲＲ８２０Ａ」、タカラ）を用いて行った。使用したプライマーの塩基配列を下記表２に示す。 (RNA extraction and quantitative RT-PCR (qPCR))
Total RNA was extracted using the RNeasy Mini kit (catalog number "74106", Qiagen) and treated with RNase-free DNase (catalog number "79254", Qiagen). The purified RNA was reverse transcribed using a commercially available kit (product name "PrimeScript TM RT Master Mix", catalog number "RR037A", Takara). qPCR was performed using a commercially available kit (product name "StepOnePlus TM ", Applied Biosystems, product name "TB Green Premix Ex Taq II", catalog number "RR820A", Takara). The base sequence of the primers used is shown in Table 2 below.

Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007

（ウエスタンブロッティング）
　３×１０５個の細胞を、プロテアーゼ阻害剤カクテル（カタログ番号「０４０８０－１１」、ナカライテスク）を添加したＲＩＰＡバッファー（カタログ番号「１８８－０２４５３」、富士フイルム和光純薬）中、氷上で３０分静置して溶解した。続いて、１５０，０００×ｇ、４℃で５分間遠心した後、上清を回収した。 (Western blotting)
30 x 10 5 cells on ice in a RIPA buffer (catalog number "188-02453", Fujifilm Wako Pure Chemical Industries) supplemented with a protease inhibitor cocktail (catalog number "04080-11", Nacalai Tesque). It was allowed to stand for a minute and dissolved. Subsequently, after centrifuging at 150,000 × g at 4 ° C. for 5 minutes, the supernatant was collected.

　細胞溶解液中の全タンパク質量を、市販のキット（製品名「ＤＣＴＭ　Ｐｒｏｔｅｉｎ　Ａｓｓａｙ」、カタログ番号「５００－０１１６ＪＡ」、バイオラッド）及びＥｎＶｉｓｉｏｎ　Ｍｕｌｔｉｌａｂｅｌ　Ｐｌａｔｅ　Ｒｅａｄｅｒｓ（パーキンエルマー）を用いて測定し、各細胞溶解中のタンパク質濃度が同一になるよう調整した。 The total amount of protein in the cytolyte was measured using a commercially available kit (product name "DC TM Protein Assay", catalog number "500-0116JA", Biorad) and EnVision Multilabel Plate Readers (PerkinElmer), and each was measured. The protein concentration during cytolysis was adjusted to be the same.

　細胞溶解液を、２－メルカプトエタノール（カタログ番号「２１４１８－４２」、ナカライテスク）を含む４×レムリーサンプルバッファー（カタログ番号「１６１－０７４７」、バイオラッド）中で５分間煮沸した。続いて、タンパク質をＳＤＳポリアクリルアミドゲル電気泳動により分離し、Ｉｍｍｏｂｉｌｏｎ－Ｐ　ｍｅｍｂｒａｎｅ（カタログ番号「ＩＰＶＨ０００１０］、メルク）に転写した。 The cytolytic solution was boiled for 5 minutes in 4 × Remley sample buffer (catalog number “161-0747”, Bio-Rad) containing 2-mercaptoethanol (catalog number “21418-42”, Nacalai Tesque). Subsequently, the protein was separated by SDS polyacrylamide gel electrophoresis and transferred to Immobilon-P membrane (catalog number "IPVH000010], Merck).

　膜は、１０％脱脂乳（カタログ番号「１９０－１２８６５」、富士フイルム和光純薬）及び０．１％Ｔｗｅｅｎ２０（カタログ番号「９００５－６４－５」、シグマ－アルドリッチ）を添加したトリス緩衝生理食塩水でブロッキングし、抗体と反応させた。 The membrane is Tris-buffered saline supplemented with 10% skim milk (catalog number "190-12865", Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.1% Tween 20 (catalog number "9005-64-5", Sigma-Aldrich). It was blocked with water and reacted with the antibody.

　使用した抗体は以下の通りであった。抗ＧＡＰＤＨモノクローナル抗体（カタログ番号「２１１８」、セルシグナリングテクノロジー）、抗ＭＣＰ－１ポリクローナル抗体（カタログ番号「ａｂ９６６９」、アブカム）、抗ＩＰ－１０ポリクローナル抗体（カタログ番号「ａｂ８０９８」、アブカム）、ＨＲＰ標識抗ウサギＩｇＧ抗体（カタログ番号「７０７４」、セルシグナリングテクノロジー）及びＨＲＰ標識抗マウスＩｇＧ抗体（カタログ番号「７０７６」、セルシグナリングテクノロジー）。 The antibodies used were as follows. Anti-GAPDH monoclonal antibody (catalog number "2118", cell signaling technology), anti-MCP-1 polyclonal antibody (catalog number "ab9669", abcam), anti-IP-10 polyclonal antibody (catalog number "ab8098", abcam), HRP labeling Anti-rabbit IgG antibody (catalog number "7074", cell signaling technology) and HRP-labeled anti-mouse IgG antibody (catalog number "7076", cell signaling technology).

　化学発光には市販のキット（製品名「ＳｕｐｅｒＳｉｇｎａｌＴＭ　Ｗｅｓｔ　Ｆｅｍｔｏ　Ｍａｘｉｍｕｍ　Ｓｅｎｓｉｔｉｖｉｔｙ　Ｓｕｂｓｔｒａｔｅ」、カタログ番号「３４０９５」、サーモフィッシャーサイエンティフィック）を使用した。イメージは　ＩｍａｇｅＱｕａｎｔ　ＬＡＳ　４０００（ＧＥヘルスケア）を使用して取得した。 A commercially available kit (product name "SuperSignal TM West Femto Maximum Sensitivity Substrate", catalog number "34095", Thermo Fisher Scientific) was used for chemiluminescence. Images were acquired using ImageQuant LAS 4000 (GE Healthcare).

（統計解析）
　全ての統計解析及びＩＣ５０値の算出はＧｒａｐｈＰａｄ　Ｐｒｉｓｍソフトウエア（ＧｒａｐｈＰａｄ　Ｓｏｆｔｗａｒｅ）を使用して行った。 (Statistical analysis)
Calculated for all statistical analyzes and IC 50 values were performed using GraphPad Prism software (GraphPad Software).

［実験例１］
（ＨＴＳにより、ヒストン脱アセチル化酵素阻害剤がＮＮＳの治療剤の候補であることが同定された）
　ＨＴＳにより、ＮＮＳの治療剤の候補を同定した。いずれもＮＮＳ患者で特に上昇する炎症誘発性ケモカインである、ＭＣＰ－１及びＩＰ－１０の産生阻害率で化合物を評価した。ＩＬ－６もＮＮＳ患者で特に上昇するが、抗ＩＬ－６受容体抗体であるトシリズマブの投与によりＩＬ－６受容体をブロックしても、ＮＮＳの治療効果が限定的であることから、ＭＣＰ－１及びＩＰ－１０の双方の産生を阻害する化合物に着目した。 [Experimental Example 1]
(HTS identified histone deacetylase inhibitors as potential therapeutic agents for NNS)
HTS identified potential therapeutic agents for NNS. Compounds were evaluated by the rate of inhibition of production of MCP-1 and IP-10, both of which are pro-inflammatory chemokines that are particularly elevated in NNS patients. IL-6 is also particularly elevated in NNS patients, but blocking the IL-6 receptor by administration of the anti-IL-6 receptor antibody tocilizumab has a limited therapeutic effect on NNS, and thus MCP- We focused on compounds that inhibit the production of both 1 and IP-10.

　上記表１に示すように、化合物ライブラリは５，８２１化合物から構成され、承認薬、キナーゼ阻害薬、生理活性化合物を含んでいた。図１に実験スケジュールを示す。細胞を化合物で３時間処理した後、ＬＰＳ又はＩＦＮ－γ処理によりＭＣＰ－１又はＭＰ－１０の産生を誘導した。１次及び２次スクリーニングでは、ＭＴ－ｉＰＳ－ＭＬｓを使用した。 As shown in Table 1 above, the compound library was composed of 5,821 compounds and contained approved agents, kinase inhibitors, and bioactive compounds. FIG. 1 shows the experiment schedule. After treating the cells with the compound for 3 hours, LPS or IFN-γ treatment induced the production of MCP-1 or MP-10. MT-iPS-MLs were used in the primary and secondary screenings.

　まず、５，８２１化合物のＭＴ－ｉＰＳ－ＭＬｓに対する効果を１μＭの濃度で評価した。図２は、ＭＣＰ－１及びＩＰ－１０の産生阻害率を示すグラフである。図２に示すように、ＭＣＰ－１の産生阻害率が６０％超、ＩＰ－１０の産生阻害率が５０％超、又は、両者の産生阻害率が３０％超である６４２化合物をヒット化合物とした。図２中、四角で囲んだ領域に存在する化合物がヒット化合物である。ヒット化合物に対し、再現性の確認及び強い細胞毒性が疑われるものの除去を行い、化合物６４２種のうち１０８種を２次スクリーニングで評価した。 First, the effect of 5,821 compounds on MT-iPS-MLs was evaluated at a concentration of 1 μM. FIG. 2 is a graph showing the production inhibition rates of MCP-1 and IP-10. As shown in FIG. 2, 642 compounds having an MCP-1 production inhibition rate of more than 60%, an IP-10 production inhibition rate of more than 50%, or both production inhibition rates of more than 30% are defined as hit compounds. bottom. In FIG. 2, the compound existing in the region surrounded by the square is a hit compound. The reproducibility of the hit compounds was confirmed and those suspected of having strong cytotoxicity were removed, and 108 of the 642 compounds were evaluated by the secondary screening.

　２次スクリーニングも同様にＭＣＰ－１及びＩＰ－１０の産生阻害率で評価を行った。図３は、ＭＣＰ－１及びＩＰ－１０の産生阻害率を示すグラフである。図３に示すように、化合物の濃度１００ｎＭに対し、ＭＣＰ－１及びＩＰ－１０の産生阻害率が８０％超である２６化合物をヒット化合物とした。図３中、四角で囲んだ領域に存在する化合物がヒット化合物である。 The secondary screening was also evaluated based on the production inhibition rate of MCP-1 and IP-10. FIG. 3 is a graph showing the production inhibition rates of MCP-1 and IP-10. As shown in FIG. 3, 26 compounds in which the production inhibition rate of MCP-1 and IP-10 was more than 80% with respect to the compound concentration of 100 nM were designated as hit compounds. In FIG. 3, the compound existing in the region surrounded by the square is a hit compound.

　次に、これらヒット化合物のＭＣＰ－１及びＩＰ－１０産生阻害効果がクローン特異的である可能性を排除するため、ＭＴ－ＥＳ－ＭＬｓを用い２次スクリーニングと同様の条件で２６化合物を評価した。図４は、ＭＣＰ－１及びＩＰ－１０の産生阻害率を示すグラフである。その結果、図４に示すように、ＭＣＰ－１及びＩＰ－１０の産生阻害率が８０％超である１３化合物をヒット化合物とした。図４中、四角で囲んだ領域に存在する化合物がヒット化合物である。 Next, in order to eliminate the possibility that the MCP-1 and IP-10 production inhibitory effects of these hit compounds were clone-specific, 26 compounds were evaluated using MT-ES-MLs under the same conditions as in the secondary screening. .. FIG. 4 is a graph showing the production inhibition rates of MCP-1 and IP-10. As a result, as shown in FIG. 4, 13 compounds having a production inhibition rate of MCP-1 and IP-10 of more than 80% were designated as hit compounds. In FIG. 4, the compound existing in the region surrounded by the square is a hit compound.

　最後に、これらの１３化合物の濃度１００ｎＭにおけるＭＴ－ＥＳ－ＭＬｓに対する毒性を評価した。図５は、細胞毒性評価の結果を示すグラフである。縦軸は遊離した乳酸脱水素酵素（ＬＤＨ）の量を示す。点線は、平均値＋１標準偏差（ＳＤ）を示す。図５中、「Ｕｎｔｒｅａｔｅｄ」は化合物の代わりにＤＭＳＯを添加したネガティブコントロールの結果を示し、「Ｌｙｓｉｓ」はポジティブコントロールとして用いた細胞溶解液の結果を示す。図５に示すように、４化合物は、遊離したＬＤＨの量が未処理コントロールの平均値＋１標準偏差（ＳＤ）未満であったことから、細胞毒性がないと考えられた。 Finally, the toxicity of these 13 compounds to MT-ES-MLs at a concentration of 100 nM was evaluated. FIG. 5 is a graph showing the results of cytotoxicity evaluation. The vertical axis shows the amount of free lactate dehydrogenase (LDH). The dotted line indicates the mean value + 1 standard deviation (SD). In FIG. 5, "United" shows the result of the negative control in which DMSO was added instead of the compound, and "Lysis" shows the result of the cytolytic solution used as the positive control. As shown in FIG. 5, the four compounds were considered non-cytotoxic because the amount of free LDH was less than the mean of the untreated control + 1 standard deviation (SD).

　図６は、ＨＴＳの結果を示す模式図である。図６に示すように、ヒットした４化合物のうち３化合物がヒストン脱アセチル化酵素（ＨＤＡＣ）阻害剤であったことから、これらの３化合物についてより詳細な検討を行った。 FIG. 6 is a schematic diagram showing the results of HTS. As shown in FIG. 6, since 3 of the 4 hit compounds were histone deacetylase (HDAC) inhibitors, a more detailed study was conducted on these 3 compounds.

［実験例２］
（ＣＵＤＣ－９０７は低濃度でＭＣＰ－１及びＩＰ－１０の産生を阻害した）
　実験例１で同定された３種のＨＤＡＣ阻害剤である、ＣＵＤＣ－９０７、ＪＮＪ－２６４８１５８５及びＬＡＱ８２４について、ＭＴ－ｉＰＳ－ＭＬｓによるＭＣＰ－１及びＩＰ－１０の産生阻害効果を検討した。 [Experimental Example 2]
(CUDC-907 inhibited the production of MCP-1 and IP-10 at low concentrations)
For the three HDAC inhibitors identified in Experimental Example 1, CUDC-907, JNJ-26481585 and LAQ824, the effect of MT-iPS-MLs on the production of MCP-1 and IP-10 was examined.

　図７（ａ）～（ｃ）は各化合物のＭＣＰ－１の産生に対する用量反応曲線である（ｎ＝３）。図７（ｄ）～（ｆ）は各化合物のＩＰ－１０の産生に対する用量反応曲線である（ｎ＝３）。下記表３に、図７（ａ）～（ｆ）に基づいて算出した各化合物の５０％阻害濃度（ＩＣ５０）を示す。 7 (a) to 7 (c) are dose-response curves for the production of MCP-1 of each compound (n = 3). 7 (d) to 7 (f) are dose-response curves for the production of IP-10 of each compound (n = 3). Table 3 below shows the 50% inhibitory concentration (IC 50 ) of each compound calculated based on FIGS. 7 (a) to 7 (f).

Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008

　その結果、３化合物のうち、ＣＵＤＣ－９０７がＭＣＰ－１及びＩＰ－１０の産生に対する最も低いＩＣ５０値を示すことが明らかとなった。 As a result, among the three compounds, CUDC-907 was found to exhibit the lowest an IC 50 value for the production of MCP-1 and IP-10.

　ＨＤＡＣ阻害剤は細胞周期を停止させることが知られている。そこで、ＣＵＤＣ－９０７の安全性を確認するため、ＭＴ－ｉＰＳ－ＭＬｓに対する細胞生存率及び細胞毒性への影響を検討した。 HDAC inhibitors are known to arrest the cell cycle. Therefore, in order to confirm the safety of CUDC-907, the effect on cell viability and cytotoxicity on MT-iPS-MLs was examined.

　ＣＵＤＣ－９０７を最大１００ｎＭの濃度で処理し、ＤＭＳＯ処理群に対する細胞生存率及び細胞毒性を検討した。細胞生存率は、細胞内還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）を検出することにより評価した。細胞毒性は、細胞外乳酸脱水素酵素（ＬＤＨ）活性を検出することにより評価した。 CUDC-907 was treated at a maximum concentration of 100 nM, and the cell viability and cytotoxicity with respect to the DMSO-treated group were examined. Cell viability was assessed by detecting intracellular reduced nicotinamide adenine dinucleotide (NADH). Cytotoxicity was assessed by detecting extracellular lactate dehydrogenase (LDH) activity.

　図８（ａ）は、細胞内ＮＡＤＨを測定した結果を示すグラフである（ｎ＝３）。図８（ｂ）は、細胞外ＬＤＨ活性を測定した結果を示すグラフである（ｎ＝３）。図８（ａ）及び（ｂ）中、「ＤＭＳＯ」は化合物の代わりにＤＭＳＯを添加した結果であることを示し、「Ｌｙｓａｔｅ」は細胞溶解液の結果であることを示す。 FIG. 8A is a graph showing the results of measuring intracellular NADH (n = 3). FIG. 8B is a graph showing the results of measuring extracellular LDH activity (n = 3). In FIGS. 8A and 8B, "DMSO" indicates the result of adding DMSO instead of the compound, and "Lysite" indicates the result of cytolysis.

　その結果、ＣＵＤＣ－９０７は、いずれの濃度においてもＤＭＳＯ処理群とほぼ同等の細胞生存率及び細胞毒性を示した。この結果から、ＣＵＤＣ－９０７が、ＭＴ－ｉＰＳ－ＭＬｓに対して、極めて低い毒性でＭＣＰ－１及びＩＰ－１０の産生阻害効果を示す化合物であることが明らかとなった。 As a result, CUDC-907 showed almost the same cell viability and cytotoxicity as the DMSO-treated group at any concentration. From this result, it was clarified that CUDC-907 is a compound showing an effect of inhibiting the production of MCP-1 and IP-10 with extremely low toxicity to MT-iPS-MLs.

［実験例３］
（ＣＵＤＣ－９０７はＮＮＳ患者由来の線維芽細胞におけるＭＣＰ－１及びＩＰ－１０の産生を効果的に阻害した）
　ＣＵＤＣ－９０７の効果がＭＴ－ｉＰＳ－ＭＬｓ特異的でないことを確認するため、ＮＮＳ患者由来線維芽細胞を用いてその効果を確認した。 [Experimental Example 3]
(CUDC-907 effectively inhibited the production of MCP-1 and IP-10 in fibroblasts from NNS patients)
In order to confirm that the effect of CUDC-907 is not specific to MT-iPS-MLs, the effect was confirmed using fibroblasts derived from NNS patients.

　線維芽細胞における免疫プロテアソームの発現量は免疫細胞における発現量よりも低い。そこで、ＴＮＦ－α及びＩＦＮ－γで、より長時間（７２時間）処理して免疫プロテアソームを誘導し、ＮＮＳ患者の表現型を十分に示す状態にした後にサイトカインの産生量を測定した。 The expression level of immune proteasome in fibroblasts is lower than the expression level in immune cells. Therefore, the immunoproteasome was induced by treatment with TNF-α and IFN-γ for a longer period of time (72 hours) to sufficiently show the phenotype of the NNS patient, and then the amount of cytokine produced was measured.

　線維芽細胞からのＩＰ－１０の発現量は非常に少なかったため、健常者由来の線維芽細胞とＮＮＳ患者由来の線維芽細胞を比較することはできなかった。 Since the expression level of IP-10 from fibroblasts was very low, it was not possible to compare fibroblasts derived from healthy subjects with fibroblasts derived from NNS patients.

　図９は、健常者由来の線維芽細胞である、Ｈｅａｌｔｈｙ＃１、Ｈｅａｌｔｈｙ＃２、及び、ＮＮＳ患者由来の線維芽細胞である、ＮＮＳ＃１、ＮＮＳ＃２におけるＭＣＰ－１の発現量を示すグラフである（ｎ＝３）。図９中、「ＵＴ」はＴＮＦ－α及びＩＦＮ－γで処理していない線維芽細胞の結果であることを示す。また、「＊」、「＊＊」、「＊＊＊」は、それぞれ、スチューデントのＴ検定の結果、ｐ＜０．０５、ｐ＜０．０１、ｐ＜０．００５で有意差が存在することを示す。 FIG. 9 shows the expression levels of MCP-1 in Health # 1, Health # 2, and NNS patient-derived fibroblasts, NNS # 1 and NNS # 2, which are fibroblasts derived from healthy subjects. It is a graph (n = 3). In FIG. 9, "UT" indicates the result of fibroblasts not treated with TNF-α and IFN-γ. In addition, "*", "**", and "***" have significant differences in p <0.05, p <0.01, and p <0.005 as a result of Student's T-test, respectively. Show that.

　図１０は、ＮＮＳ患者由来の線維芽細胞であるＮＮＳ＃１における、ＣＵＤＣ－９０７の用量依存的なＭＣＰ－１の産生阻害効果を示すグラフである。その結果、線維芽細胞におけるＣＵＤＣ－９０７によるＭＣＰ－１の産生阻害のＩＣ５０はＭＴ－ｉＰＳ－ＭＬｓにおける値と同程度であることが明らかとなった。 FIG. 10 is a graph showing the dose-dependent effect of inhibiting the production of MCP-1 of CUDC-907 in NNS # 1, which is a fibroblast derived from an NNS patient. As a result, IC 50 of the production inhibition of MCP-1 by CUDC-907 in fibroblasts was found to be comparable with the values in the MT-iPS-MLs.

　続いて、ＮＮＳ患者由来線維芽細胞であるＮＮＳ＃１に対するＣＵＤＣ－９０７の細胞生存率及び細胞毒性への影響を検討した。図１１（ａ）は、細胞内ＮＡＤＨを測定した結果を示すグラフである（ｎ＝３）。図１１（ｂ）は、細胞外ＬＤＨ活性を測定した結果を示すグラフである（ｎ＝３）。図１１（ａ）及び（ｂ）中、「ＤＭＳＯ」はＣＵＤＣ－９０７の代わりにＤＭＳＯを添加した結果であることを示し、「Ｌｙｓａｔｅ」は細胞溶解液の結果であることを示す。 Subsequently, the effects of CUDC-907 on cell viability and cytotoxicity on NNS # 1, which is a fibroblast derived from NNS patients, were examined. FIG. 11A is a graph showing the results of measuring intracellular NADH (n = 3). FIG. 11B is a graph showing the results of measuring extracellular LDH activity (n = 3). In FIGS. 11 (a) and 11 (b), "DMSO" indicates the result of adding DMSO instead of CUDC-907, and "Lysite" indicates the result of cytolysis.

　その結果、ＣＵＤＣ－９０７は、有効濃度においてほとんど細胞毒性を示さないことが明らかとなった。以上の結果から、ＣＵＤＣ－９０７は、ＮＮＳ患者由来の線維芽細胞によるＭＣＰ－１の産生を、ＭＴ－ｉＰＳ－ＭＬｓにおけるものと同程度の濃度で、細胞毒性を示さずに阻害することが明らかとなった。 As a result, it was clarified that CUDC-907 shows almost no cytotoxicity at an effective concentration. From the above results, it is clear that CUDC-907 inhibits the production of MCP-1 by fibroblasts derived from NNS patients at the same concentration as that in MT-iPS-MLs without showing cytotoxicity. It became.

［実験例４］
（ＣＵＤＣ－９０７によるＭＣＰ－１及びＩＰ－１０の産生阻害効果の検討）
　ＣＵＤＣ－９０７のＮＮＳ疾患モデルに対するＭＣＰ－１及びＩＰ－１０の産生阻害効果が、野生型コントロールの産生量と比較して十分であるかを確認した。 [Experimental Example 4]
(Examination of the effect of CUDC-907 on inhibiting the production of MCP-1 and IP-10)
It was confirmed whether the production inhibitory effect of MCP-1 and IP-10 on the NNS disease model of CUDC-907 was sufficient as compared with the production amount of the wild-type control.

　図１２（ａ）は、ＣＵＤＣ－９０７の存在下及び非存在下において、ＷＴ－ｉＰＳ－ＭＬｓ及びＭＴ－ｉＰＳ－ＭＬｓのＭＣＰ－１の産生量を比較した結果を示すグラフである（ｎ＝３）。図１２（ｂ）は、ＣＵＤＣ－９０７の存在下及び非存在下において、ＷＴ－ｉＰＳ－ＭＬｓ及びＭＴ－ｉＰＳ－ＭＬｓのＩＰ－１０の産生量を比較した結果を示すグラフである（ｎ＝３）。図１２（ａ）及び（ｂ）中、「＊＊」、「＊＊＊」、「＊＊＊＊」は、それぞれ、一元配置分散分析（ＡＮＯＶＡ）及びダネットの多重比較の結果、ｐ＜０．０１、ｐ＜０．００５、ｐ＜０．００１で有意差が存在することを示し、「Ｎ．Ｓ．」は有意差がないことを示す。 FIG. 12A is a graph showing the results of comparing the production amounts of MCP-1 of WT-iPS-MLs and MT-iPS-MLs in the presence and absence of CUDC-907 (n = 3). ). FIG. 12B is a graph showing the results of comparing the production amounts of IP-10 of WT-iPS-MLs and MT-iPS-MLs in the presence and absence of CUDC-907 (n = 3). ). In FIGS. 12A and 12B, "**", "***", and "*****" are the results of one-way analysis of variance (ANOVA) and Dunnett's multiple comparison, respectively, and p <0. 0.01, p <0.005, p <0.001 indicate that there is a significant difference, and "NS" indicates that there is no significant difference.

　その結果、１０ｎＭのＣＵＤＣ－９０７をＭＴ－ｉＰＳ－ＭＬｓに処理したところ、ＭＣＰ－１及びＩＰ－１０の産生量はＷＴ－ｉＰＳ－ＭＬｓと同等かそれ以下まで減少することが明らかとなった。 As a result, when 10 nM CUDC-907 was treated into MT-iPS-MLs, it was clarified that the production amounts of MCP-1 and IP-10 were reduced to the same level as or less than WT-iPS-MLs.

　また、図１３は、健常者由来の線維芽細胞株である、Ｈｅａｌｔｈｙ＃１、Ｈｅａｌｔｈｙ＃２におけるＭＣＰ－１の産生量、及び、ＣＵＤＣ－９０７の存在下及び非存在下において、ＮＮＳ患者由来の線維芽細胞である、ＮＮＳ＃１、ＮＮＳ＃２におけるＭＣＰ－１の産生量を示すグラフである（ｎ＝３）。図１３中、「＊＊」は、スチューデントのＴ検定の結果、ｐ＜０．０１で有意差が存在することを示し、「Ｎ．Ｓ．」は有意差がないことを示す。 In addition, FIG. 13 shows the amount of MCP-1 produced in Health # 1 and Health # 2, which are fibroblast cell lines derived from healthy subjects, and derived from NNS patients in the presence and absence of CUDC-907. It is a graph which shows the production amount of MCP-1 in NNS # 1 and NNS # 2, which are fibroblasts (n = 3). In FIG. 13, “**” indicates that there is a significant difference at p <0.01 as a result of Student's T-test, and “NS” indicates that there is no significant difference.

　その結果、Ｈｅａｌｔｈｙ＃１、Ｈｅａｌｔｈｙ＃２に対しＮＮＳ＃１、＃２でＭＣＰ－１産生の増加が確認された。更に、３０ｎＭのＣＵＤＣ－９０７をＮＮＳ＃１、＃２に処理したところ、ＭＣＰ－１の産生量はＨｅａｌｔｈｙ＃１、Ｈｅａｌｔｈｙ＃２と同等かそれ以下まで減少した。 As a result, an increase in MCP-1 production was confirmed in NNS # 1 and # 2 with respect to Health # 1 and Health # 2. Furthermore, when 30 nM CUDC-907 was treated with NNS # 1 and # 2, the amount of MCP-1 produced decreased to the same level as or less than that of Health # 1 and Health # 2.

　以上の結果から、ＣＵＤＣ－９０７の効果は、ＮＮＳによる過剰なＭＣＰ－１及びＩＰ－１０の産生を健常レベルまで減少させるのに十分であることが明らかとなった。 From the above results, it was clarified that the effect of CUDC-907 is sufficient to reduce the excessive production of MCP-1 and IP-10 by NNS to a healthy level.

［実験例５］
（ＣＵＤＣ－９０７は転写後にＭＣＰ－１及びＩＰ－１０の産生を阻害した）
　ＣＵＤＣ－９０７によるＭＣＰ－１及びＩＰ－１０の産生阻害のメカニズムを明らかにするために、ＭＣＰ－１をコードするＣＣＬ２遺伝子及びＩＰ－１０をコードするＣＸＣＬ１０遺伝子の発現レベルを定量した。 [Experimental Example 5]
(CUDC-907 inhibited the production of MCP-1 and IP-10 after transcription)
To elucidate the mechanism by which CUDC-907 inhibits the production of MCP-1 and IP-10, the expression levels of the CCL2 gene encoding MCP-1 and the CXCL10 gene encoding IP-10 were quantified.

　ＷＴ－ｉＰＳ－ＭＬｓ細胞を１０ｎＭのＣＵＤＣ－９０７で３時間処理した後、１００ｎｇ／ｍＬ　ＴＮＦ－α及び１００ｎｇ／ｍＬ　ＩＦＮ－γで３、９、１５、２１時間刺激し、定量的ＲＴ－ＰＣＲを行った。各遺伝子の発現量はＧＡＰＤＨ遺伝子の発現量で標準化した。 WT-iPS-MLs cells were treated with 10 nM CUDC-907 for 3 hours and then stimulated with 100 ng / mL TNF-α and 100 ng / mL IFN-γ for 3, 9, 15 and 21 hours for quantitative RT-PCR. went. The expression level of each gene was standardized by the expression level of the GAPDH gene.

　図１４（ａ）はＣＣＬ２遺伝子の発現量を測定した結果を示すグラフである。また、図１４（ｂ）はＣＸＣＬ１０遺伝子の発現量を測定した結果を示すグラフである。図１４（ａ）及び（ｂ）中、グラフの縦軸は、発現量（相対値）を示す。 FIG. 14A is a graph showing the results of measuring the expression level of the CCL2 gene. Further, FIG. 14 (b) is a graph showing the results of measuring the expression level of the CXCL10 gene. In FIGS. 14A and 14B, the vertical axis of the graph indicates the expression level (relative value).

　その結果、ＥＬＩＳＡの結果と異なり、ＣＵＤＣ－９０７はこれらの遺伝子の発現レベルを上昇させることが明らかとなった。この結果は、ＣＵＤＣ－９０７が転写後にＭＣＰ－１及びＩＰ－１０の産生を阻害することを示す。 As a result, it was clarified that CUDC-907 increases the expression level of these genes, unlike the result of ELISA. This result indicates that CUDC-907 inhibits the production of MCP-1 and IP-10 after transcription.

　続いて、ＭＣＰ－１及びＩＰ－１０の発現量をタンパク質レベルで検討した。ウエスタンブロッティングにより細胞内におけるＭＣＰ－１及びＩＰ－１０の発現量を検出するため、これらのサイトカインの細胞外への放出をブレフェルジンＡ（Ｂｒｅｆｅｌｄｉｎ　Ａ）処理により阻害した。ブレフェルジンＡは、小胞体からゴルジ体へのタンパク質輸送を阻害することが知られている。 Subsequently, the expression levels of MCP-1 and IP-10 were examined at the protein level. In order to detect the intracellular expression levels of MCP-1 and IP-10 by Western blotting, the extracellular release of these cytokines was inhibited by Brefeldin A treatment. Breferdin A is known to inhibit protein transport from the endoplasmic reticulum to the Golgi apparatus.

　ＷＴ－ｉＰＳ－ＭＬｓ細胞を１０ｎＭのＣＵＤＣ－９０７で３０分間処理した後、１００ｎｇ／ｍＬ　ＴＮＦ－α及び１００ｎｇ／ｍＬ　ＩＦＮ－γで６時間刺激した。ブレフェルジンＡは細胞回収の１時間前に添加した。 WT-iPS-MLs cells were treated with 10 nM CUDC-907 for 30 minutes and then stimulated with 100 ng / mL TNF-α and 100 ng / mL IFN-γ for 6 hours. Breferdin A was added 1 hour prior to cell recovery.

　図１５は代表的なウエスタンブロッティングの結果を示す画像である。グリセルアルデヒド３リン酸脱水素酵素（ＧＡＰＤＨ）をローディングコントロールとして検出した。図１５中、「－」は添加しなかったことを示し、「＋」は添加したことを示す。 FIG. 15 is an image showing a typical Western blotting result. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as a loading control. In FIG. 15, "-" indicates that it was not added, and "+" indicates that it was added.

　その結果、ブレフェルジンＡの存在下では、ＭＣＰ－１及びＩＰ－１０の細胞内レベルが上昇した。この結果は、ブレフェルジンＡ処理がＭＣＰ－１及びＩＰ－１０の細胞外への放出を阻害したことを示す。 As a result, in the presence of Breferdin A, the intracellular levels of MCP-1 and IP-10 increased. This result indicates that Breferdin A treatment inhibited the extracellular release of MCP-1 and IP-10.

　また、ブレフェルジンＡの存在下において、ＣＵＤＣ－９０７は、ＭＣＰ－１及びＩＰ－１０の細胞内レベルを減少させた。この結果は、ＣＵＤＣ－９０７が、ＭＣＰ－１及びＩＰ－１０の産生を転写後に阻害することを示す。 Also, in the presence of Breferdin A, CUDC-907 reduced intracellular levels of MCP-1 and IP-10. This result indicates that CUDC-907 inhibits post-transcriptional production of MCP-1 and IP-10.

［実験例６］
（ＨＤＡＣ阻害剤によるＭＣＰ－１及びＩＰ－１０の産生阻害）
　ＨＤＡＣ阻害剤である、ＣＵＤＣ－９０７、ＪＮＪ－２６４８１５８５、ＬＡＱ８２４、ロミデプシン及びトリコスタチンＡについて、ＭＴ－ｉＰＳ－ＭＬｓによるＭＣＰ－１及びＩＰ－１０の産生阻害効果を検討した。 [Experimental Example 6]
(Inhibition of MCP-1 and IP-10 production by HDAC inhibitors)
The effects of MT-iPS-MLs on the production of MCP-1 and IP-10 were investigated for the HDAC inhibitors CUDC-907, JNJ-26481585, LAQ824, romidepsin and tricostatin A.

　図１６（ａ）は各化合物のＭＣＰ－１の産生に対する用量反応曲線である。図１６（ｂ）は各化合物のＩＰ－１０の産生に対する用量反応曲線である。下記表４に、図１６（ａ）及び（ｂ）に基づいて算出した各化合物の５０％阻害濃度（ＩＣ５０）を示す。 FIG. 16 (a) is a dose-response curve for the production of MCP-1 of each compound. FIG. 16 (b) is a dose-response curve for IP-10 production of each compound. Table 4 below shows the 50% inhibitory concentration (IC 50 ) of each compound calculated based on FIGS. 16 (a) and 16 (b).

Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009

　本発明によれば、中條・西村症候群の治療剤を提供することができる。 According to the present invention, it is possible to provide a therapeutic agent for Nakajo-Nishimura syndrome.

Claims (2)

　ヒストン脱アセチル化酵素阻害剤を含む、中條・西村症候群の治療剤。 A therapeutic agent for Nakajo-Nishimura syndrome, including a histone deacetylase inhibitor. 　前記ヒストン脱アセチル化酵素阻害剤が、ＣＵＤＣ－９０７、ＪＮＪ－２６４８１５８５、ＬＡＱ８２４、ロミデプシン及びトリコスタチンＡからなる群より選択される少なくとも１つの化合物又はその薬理学的に許容される塩若しくはそれらの溶媒和物である、請求項１に記載の中條・西村症候群の治療剤。 The histone deacetylase inhibitor is at least one compound selected from the group consisting of CUDC-907, JNJ-26481585, LAQ824, romidepsin and tricostatin A, or a pharmacologically acceptable salt thereof or a solvent thereof. The therapeutic agent for Nakajo-Nishimura syndrome according to claim 1, which is a Japanese product.
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
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BREHM, A. ET AL. ET AL.: "Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 125, no. 11, 2015, pages 4196 - 4211, XP055873092 *
COWARD, W. ET AL. ET AL.: "Repression of IP-10 by Interactions between Histone Deacetylation and Hypermethylation in Idiopathic Pulmonary Fibrosis", MOLECULAR AND CELLULAR BIOLOGY, vol. 30, no. 12, 2010, pages 2874 - 2886, XP055873095 *
HONDA-OZAKI, F. ET AL. ET AL.: "Pluripotent Stem Cell Model of Nakajo-Nishimura Syndrome Untangles Proinflammatory Pathways Mediated by Oxidative Stress", STEM CELL REPORTS, vol. 10, 2018, pages 1835 - 1850, XP055873090 *
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