Therapeutic agent for Nakajo-Nishimura syndrome

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

Technical Field

[0001] The present invention relates to a therapeutic agent for Nakajo-Nishimura syndrome. This application claims priority based on U.S. Patent Application No. 63 / 021,112, which was filed in the United States on May 7, 2020, and the content thereof is incorporated herein by reference.

Background Art

[0002] Nakajo-Nishimura syndrome (NNS) is one of the proteasome-related autoinflammatory syndromes (PRAAS) and is caused by a homozygous mutation in the proteasome subunit beta 8 (PSMB8) / LMP-7 gene. NNS begins in early infancy with a rash resembling heat rash, develops periodic high fevers, and ultimately results in fat and muscle atrophy and joint contractures mainly in the upper body.

[0003] All NNS patients have a homozygous c.602G>T mutation (a mutation from guanine to thymine at the 602nd nucleotide residue) in the PSMB8 gene, which results in a G201V amino acid mutation (a mutation from glycine to valine at the 201st amino acid residue) in the β5i subunit.

[0004] The mutation of the PSMB8 gene in NNS patients is thought to cause immune proteasome dysfunction, which induces inflammation by inducing cellular stress. However, the detailed mechanism of inflammation induced by immune proteasome dysfunction remains largely unclear. NNS patients are known to overproduce the inflammatory chemokines monocyte chemotactic protein-1 (MCP-1) and interferon gamma-induced protein 10 (IP-10), which are considered to be related to the inflammatory symptoms.

[0005] As a treatment for NNS, oral administration of steroids is performed, but it has no effect on lipoatrophy or joint contracture, and especially the side effects on infants are serious, so the development of new therapeutic drugs is eagerly desired.

[0006] Previously, the inventors 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) with restored mutations were prepared from mutant iPSCs (MT-iPS) established from NNS patients using the genome editing technology CRISPR / Cas9 system. In addition, mutant types (MT-ES) with mutations introduced in the same way were prepared for the human embryonic stem cell line KhES-1 (WT-ES).

[0007] Furthermore, in order to confirm the reproduction of NNS diseases in inflammation, these cells were differentiated and established into a monocytic cell line (MLs) with the phenotype of monocytes (WT-iPS-MLs, MT-iPS-MLs, WT-ES-MLs, MT-ES-MLs).

[0008] Subsequently, when comparing the production amounts of cytokines and chemokines induced by TNF-α and IFN-γ in the prepared MLs, it was confirmed that the production of MCP-1, IP-10, and interleukin-6 (IL-6), which are specifically overproduced in NNS, increased in MT-iPS-MLs and MT-ES-MLs.

[0009] Non-Patent Document 1 also describes antioxidants and Janus Kinase (JAK) inhibitors as candidates for NNS therapeutic drugs.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] However, antioxidants and JAK inhibitors are insufficient to reduce the production of excessive MCP-1 and IP-10 in NNS disease model cells to normal levels. Therefore, an object of the present invention is to provide a therapeutic agent for NNS.

Means for Solving the Problems

[0012] The present invention includes the following aspects. [1] A therapeutic agent for Nakajo-Nishimura syndrome containing a histone deacetylase inhibitor. [2] The therapeutic agent for Nakajo-Nishimura syndrome according to [1], wherein the histone deacetylase inhibitor is at least one compound selected from the group consisting of CUDC-907, JNJ-26481585, LAQ824, romidepsin and trichostatin A, or a pharmacologically acceptable salt thereof or a solvate thereof.

Effects of the Invention

[0013] According to the present invention, a therapeutic agent for Nakajo-Nishimura syndrome can be provided.

Brief Description of the Drawings

[0014]

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Mode for Carrying Out the Invention

[0015] [Therapeutic Agent for Nakajima-Nishimura Syndrome] In one embodiment, the present invention provides a therapeutic agent for Nakajima-Nishimura syndrome, comprising a histone deacetylase (HDAC) inhibitor.

[0016] As will be described later in the Examples, the inventors have clarified that the HDAC inhibitor suppresses the overproduction of MCP-1 and IP-10 in NNS disease model cells and reduces them to normal levels. Therefore, the HDAC inhibitor can be used as a therapeutic agent for Nakajima-Nishimura syndrome.

[0017] 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), romidepsin (CAS number: 128517-07-7), trichostatin A (CAS number: 58880-19-6), their pharmacologically acceptable salts, their solvates, and the like.

[0018] The chemical formula of CUDC-907 is shown in the following formula (1).

Chemical formula

[0019] The chemical formula of JNJ-26481585 is shown in the following formula (2).

Chemical formula

[0020] The chemical formula of LAQ824 is shown in the following formula (3).

Chemical formula

[0021] The chemical formula of romidepsin is shown in the following formula (4).

Chemical formula

[0022] The chemical formula of trichostatin A is shown in the following formula (5).

Chemical formula

[0023] In the therapeutic agent of the present embodiment, examples of pharmaceutically acceptable salts include 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. More specifically, for example, inorganic acid salts such as hydrochloride, sulfate, hydrobromide, nitrate, and phosphate; organic acid salts such as acetate, mesylate, succinate, maleate, fumarate, 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 salts such as ammonium salt and tetramethylammonium salt; organic amine addition salts such as morpholine and piperidine; amino acid addition salts such as glycine, phenylalanine, lysine, aspartic acid, and glutamic acid, and the like. Further, examples of pharmaceutically acceptable solvates include hydrates, organic solvent solvates, and the like.

[0024] The therapeutic agent of the present embodiment is preferably formulated as a pharmaceutical composition containing the above-described compound and a pharmaceutically acceptable carrier. The pharmaceutical composition can be administered orally, for example, in the form of a solution, powder, granule, tablet, capsule, etc., or parenterally, in the form of an injection, suppository, external preparation for skin, etc. More specifically, examples of external preparations for skin include dosage forms such as ointments and patches.

[0025] As the pharmaceutically acceptable carrier, those usually used in the formulation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth 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, and the like.

[0026] The pharmaceutical composition may contain additives. Examples of additives include lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin, and maltitol; flavoring agents such as peppermint and perilla oil; stabilizers such as benzyl alcohol and phenol; buffers such as phosphates and sodium acetate; solubilizing agents such as benzyl benzoate and benzyl alcohol; antioxidants such as ascorbic acid; preservatives such as parabens, benzalkonium chloride, chlorobutanol, and cresol, etc.

[0027] The dosage of the pharmaceutical composition varies depending on the symptoms, weight, age, gender, etc. of the subject and cannot be determined generally. In the case of oral administration, for example, an effective ingredient (the above-mentioned compound) at 0.1 - 100 mg / kg body weight per dosage unit form may be administered once a day or divided into about 2 - 4 times a day. In the case of an injection, for example, an effective ingredient at 0.01 - 50 mg per dosage unit form may be administered.

[0028] [Other Embodiments] In one embodiment, the present invention provides a method for treating Nakajo - Nishimura syndrome, which includes administering an effective amount of a histone deacetylase inhibitor to a subject in need of treatment. The histone deacetylase inhibitor is the same as those described above. Here, the "effective amount" of a compound refers to the amount of the compound required to bring about a therapeutic effect in the treated subject, and more specifically, it may be an amount sufficient to prevent, delay, or minimize at least one or more symptoms associated with Nakajo - Nishimura syndrome. In one aspect, the "effective amount" of a compound may be an amount sufficient to prevent, delay, or minimize one or more symptoms associated with Nakajo - Nishimura syndrome, either alone or in combination with other compounds or other treatments. As recognized by those skilled in the art, the effective amount of a compound can vary depending on the degree of symptoms, route of administration, use of excipients, and combination with other therapeutic treatments.

[0029] 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.

[0030] In one embodiment, the present invention provides the use of a histone deacetylase inhibitor for the manufacture of a therapeutic agent for Nakajo-Nishimura syndrome. The histone deacetylase inhibitor is the same as described above.

Examples

[0031] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0032] [Materials and Methods] The following experiments were approved by the ethics committees of Kyoto University (R0091 / G0259) and Wakayama Medical University. Informed consent in writing was obtained from the patients or their guardians in accordance with the Declaration of Helsinki. The use of human embryonic stem cells (ESCs) was approved by the Ministry of Education, Culture, Sports, Science and Technology.

[0033] (Cell Culture) Fibroblasts were obtained from two healthy individuals (Healthy #1 and #2) and two NNS patients (NNS #1 and #2). The cells were used in Dulbecco's modified Eagle's medium (Catalog number "08459-64", Nacalai Tesque) supplemented with 10% fetal bovine serum (Catalog number "F0926", Sigma-Aldrich). The cells were dissociated into single cells using 0.25% trypsin-EDTA solution (Catalog number "25200-072", Thermo Fisher Scientific) and subcultured. The fibroblasts used in the experiment were stimulated with 10 ng / mL TNF-α (Catalog number "210-TA", R&D Systems) and 10 ng / mL IFN-γ (Catalog number "285-IF", R&D Systems) for 72 hours to induce immunoproteasome.

[0034] 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 the differentiation of iPS cells (iPSCs) established from fibroblasts (NNS#1) into cells (MLs) with a monocyte phenotype, and has a homozygous mutation in the PSMB8 gene. WT-iPS-MLs is a wild-type cell line genetically homogeneous with MT-iPS-MLs.

[0035] In addition, a homozygous mutation in the PSMB8 gene similar to that of MT-iPS-MLs was introduced into the human embryonic stem cell (ESC) line KhES1 by genome editing, and this was induced to differentiate into a cell line (MLs) with a monocyte phenotype to obtain MT-ES-MLs. WT-ES-MLs is a wild-type cell line genetically homogeneous with MT-ES-MLs.

[0036] WT-iPS-MLs, MT-iPS-MLs, WT-ES-MLs, and MT-ES-MLs were cultured in StemPro TM -34 SFM medium (catalog number "10639-011", Thermo Fisher Scientific) supplemented with 2 mM L-glutamine (catalog number "25030-081", Thermo Fisher Scientific), 50 ng / mL macrophage Colony Stimulating Factor (M-CSF, catalog number "216-MC", R&D Systems), and 50 ng / mL granulocyte macrophage colony-stimulating factor (GM-CSF, catalog number "215-GM", R&D Systems).

[0037] (High-throughput screening) 5,821 compounds that have already been reported to have bioactive effects were screened by high-throughput screening (HTS). The screening was performed by chemokine measurement based on homogeneous time-resolved fluorescence (HTRF). Table 1 below shows the compound library used for the screening.

[0038]

Table 1

[0039] In the primary and secondary screenings, MT-iPS-MLs were seeded at 5×10 3 cells per well in a 384-well plate, and the compound was added at 1 μM (primary screening) or 100 nM (secondary screening). After incubating for 3 hours, 50 ng / mL lipopolysaccharide (Lipopolysaccharid (LPS), catalog number "tlrl-peklps", InvivoGen) was added for the induction of MCP-1, or 100 ng / mL IFN-γ was added for the induction of IP-10. Subsequently, after incubating for 21 hours, the supernatant was transferred to another 384-well plate, and a Human CCL2 (MCP-1) Kit (catalog number "62HCCL2PEG", Cisbio) or a 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 compounds were verified by the same procedure as in the secondary screening.

[0040] (Enzyme-linked immunosorbent assay (ELISA)) The measurement of the concentrations of MCP-1 and IP-10 in the culture supernatants of MLs or fibroblasts was performed using a commercially available kit (product name "LEGENDplex TM Human Adipokine Panel, BioLegend). For the collection of the supernatant, the cells were seeded at 1×10 per well in a 96-well plate4 cells (MLs) or 5 × 10 3 cells were seeded at a cell density of (fibroblasts), and a compound dissolved in dimethyl sulfoxide (DMSO, catalog number "D2650", Sigma-Aldrich) was added at any concentration.

[0041] Subsequently, after incubating for 3 hours, TNF-α and IFN-γ at 100 ng / mL (MLs) or 10 ng / mL (fibroblasts) were added and the cells were stimulated. After incubating for 21 hours, the supernatant was collected.

[0042] (Measurement of cell viability and cytotoxicity) For the evaluation of cell viability and cytotoxicity, cells were seeded in 96-well plates at a cell density of 5 × 10 cells per well for MT-ES-MLs (cell viability) or 1 × 10 cells per well (cytotoxicity), and 5 × 10 cells per well for NNS#1 fibroblasts (cell viability) or 2 × 10 cells per well (cytotoxicity), and a compound was added at any concentration. After incubating for 24 hours, for the measurement of cell viability, a commercially available kit (product name "Cell Counting Kit-8", catalog number "CK04", Dojindo Laboratories), and for the measurement of cytotoxicity, a commercially available kit (product name " 4 cells (cell viability) or 1 × 10 4 cells (cytotoxicity), NNS#1 fibroblasts were seeded at a cell density of 5 × 10 3 cells (cell viability) or 2 × 10 3 cells (cytotoxicity), and a compound was added at any concentration. After incubating for 24 hours, for the measurement of cell viability, a commercially available kit (product name "Cell Counting Kit-8", catalog number "CK04", Dojindo Laboratories), and for the measurement of cytotoxicity, a commercially available kit (product name "Cytotoxicity Detection Kit PLUS ", catalog number "4744934001", Merck) was added, and the measurement was performed using a 2104 EnVision Multilabel Plate Reader (PerkinElmer). As a positive control (100% cell death) for the evaluation of cell viability and cytotoxicity, a lysate of the same number of cells was used.

[0043] (RNA extraction and quantitative RT-PCR (qPCR)) Total RNA was extracted using an 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 TMReverse transcription was performed using the "RT Master Mix", catalog number "RR037A", Takara). qPCR was performed using a commercially available kit (product name "StepOnePlus" TM ", Applied Biosystems, and product name "TB Green Premix Ex Taq II", catalog number "RR820A", Takara). The nucleotide sequences of the primers used are shown in Table 2 below.

[0044]

Table 2

[0045] (Western blotting) 3×10 5 cells were allowed to stand on ice for 30 minutes and lysed in RIPA buffer (catalog number "188-02453", Fujifilm Wako Pure Chemical Industries) supplemented with a protease inhibitor cocktail (catalog number "04080-11", Nacalai Tesque). Subsequently, after centrifugation at 150,000×g for 5 minutes at 4°C, the supernatant was collected.

[0046] The total protein amount in the cell lysate was measured using a commercially available kit (product name "DC TM Protein Assay", catalog number "500-0116JA", Bio-Rad) and an EnVision Multilabel Plate Reader (PerkinElmer), and the protein concentration in each cell lysate was adjusted to be the same.

[0047] The cell lysate was boiled in 4× Laemmli sample buffer (catalog number "161-0747", Bio-Rad) containing 2-mercaptoethanol (catalog number "21418-42", Nacalai Tesque) for 5 minutes. Subsequently, the proteins were separated by SDS polyacrylamide gel electrophoresis and transferred to an Immobilon-P membrane (catalog number "IPVH00010", Merck).

[0048] The membrane was blocked with Tris-buffered saline supplemented with 10% skim milk (catalog number "190-12865", FUJIFILM Wako Pure Chemical Corporation) and 0.1% Tween 20 (catalog number "9005-64-5", Sigma-Aldrich) and reacted with the antibodies.

[0049] 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-labeled anti-rabbit IgG antibody (catalog number "7074", Cell Signaling Technology), and HRP-labeled anti-mouse IgG antibody (catalog number "7076", Cell Signaling Technology).

[0050] For chemiluminescence, a commercially available kit (product name "SuperSignal TM West Femto Maximum Sensitivity Substrate", catalog number "34095", Thermo Fisher Scientific) was used. Images were acquired using ImageQuant LAS 4000 (GE Healthcare).

[0051] (Statistical analysis) All statistical analyses and IC 50 value calculations were performed using GraphPad Prism software (GraphPad Software).

[0052] [Experimental Example 1] (It was identified by HTS that histone deacetylase inhibitors are candidates for therapeutic agents for NNS) Using HTS, candidates for therapeutic agents for NNS were identified. All compounds were evaluated based on the inhibition rates of the production of MCP-1 and IP-10, which are inflammatory chemokines that are particularly elevated in NNS patients. Although IL-6 is also particularly elevated in NNS patients, since the therapeutic effect of NNS is limited even when the IL-6 receptor is blocked by administration of tocilizumab, an anti-IL-6 receptor antibody, compounds that inhibit the production of both MCP-1 and IP-10 were focused on.

[0053] As shown in Table 1 above, the compound library consisted of 5,821 compounds and included approved drugs, kinase inhibitors, and bioactive compounds. The experimental schedule is shown in Figure 1. After treating the cells with the compounds for 3 hours, the production of MCP-1 or MP-10 was induced by LPS or IFN-γ treatment. MT-iPS-MLs were used for the primary and secondary screenings.

[0054] First, the effects of the 5,821 compounds on MT-iPS-MLs were evaluated at a concentration of 1 μM. Figure 2 is a graph showing the inhibition rates of the production of MCP-1 and IP-10. As shown in Figure 2, 642 compounds with an MCP-1 production inhibition rate of over 60%, an IP-10 production inhibition rate of over 50%, or both production inhibition rates of over 30% were designated as hit compounds. In Figure 2, the compounds present in the region surrounded by squares are the hit compounds. For the hit compounds, reproducibility was confirmed and those suspected of having strong cytotoxicity were removed, and 108 out of the 642 compounds were evaluated in the secondary screening.

[0055] The secondary screening was also evaluated in the same manner based on the inhibition rates of the production of MCP-1 and IP-10. Figure 3 is a graph showing the inhibition rates of the production of MCP-1 and IP-10. As shown in Figure 3, 26 compounds with an MCP-1 and IP-10 production inhibition rate of over 80% at a compound concentration of 100 nM were designated as hit compounds. In Figure 3, the compounds present in the region surrounded by squares are the hit compounds.

[0056] Next, to rule out the possibility that the inhibitory effects of these hit compounds on MCP-1 and IP-10 production are clone-specific, 26 compounds were evaluated using MT-ES-MLs under the same conditions as the secondary screening. Figure 4 is a graph showing the inhibition rates of MCP-1 and IP-10 production. As a result, as shown in Figure 4, 13 compounds with inhibition rates of MCP-1 and IP-10 production exceeding 80% were designated as hit compounds. In Figure 4, the compounds present in the region surrounded by squares are the hit compounds.

[0057] Finally, the toxicity of these 13 compounds against MT-ES-MLs at a concentration of 100 nM was evaluated. Figure 5 is a graph showing the results of the cytotoxicity evaluation. The vertical axis indicates the amount of released lactate dehydrogenase (LDH). The dotted line indicates the mean value + 1 standard deviation (SD). In Figure 5, "Untreated" shows the results of the negative control with DMSO added instead of the compound, and "Lysis" shows the results of the cell lysate used as the positive control. As shown in Figure 5, 4 compounds were considered to be non-cytotoxic because the amount of released LDH was less than the mean value + 1 standard deviation (SD) of the untreated control.

[0058] Figure 6 is a schematic diagram showing the results of the HTS. As shown in Figure 6, since 3 out of the 4 hit compounds were histone deacetylase (HDAC) inhibitors, more detailed examinations were conducted on these 3 compounds.

[0059] [Experimental Example 2] (CUDC-907 inhibited the production of MCP-1 and IP-10 at low concentrations) Regarding CUDC-907, JNJ-26481585, and LAQ824, which are the three HDAC inhibitors identified in Experimental Example 1, the inhibitory effects on the production of MCP-1 and IP-10 by MT-iPS-MLs were examined.

[0060] Figures 7(a)-(c) are dose-response curves for the production of MCP-1 by each compound (n = 3). Figures 7(d)-(f) are dose-response curves for the production of IP-10 by each compound (n = 3). Table 3 below shows the 50% inhibitory concentration (IC 50 ) of each compound calculated based on Figures 7(a)-(f).

[0061]

Table 3

[0062] As a result, among the three compounds, it was revealed that CUDC-907 showed the lowest IC 50 value for the production of MCP-1 and IP-10.

[0063] HDAC inhibitors are known to arrest the cell cycle. Therefore, to confirm the safety of CUDC-907, the effects on cell viability and cytotoxicity against MT-iPS-MLs were examined.

[0064] CUDC-907 was treated at a concentration of up to 100 nM, and the cell viability and cytotoxicity against the DMSO-treated group were examined. Cell viability was evaluated by detecting intracellular reduced nicotinamide adenine dinucleotide (NADH). Cytotoxicity was evaluated by detecting extracellular lactate dehydrogenase (LDH) activity.

[0065] Figure 8(a) is a graph showing the results of measuring intracellular NADH (n = 3). Figure 8(b) is a graph showing the results of measuring extracellular LDH activity (n = 3). In Figures 8(a) and (b), "DMSO" indicates the result of adding DMSO instead of the compound, and "Lysate" indicates the result of the cell lysate.

[0066] As a result, CUDC-907 showed almost the same cell viability and cytotoxicity as the DMSO-treated group at any concentration. From these results, it was revealed that CUDC-907 is a compound that exhibits an inhibitory effect on the production of MCP-1 and IP-10 with extremely low toxicity against MT-iPS-MLs.

[0067] [Experimental Example 3] (CUDC-907 effectively inhibited the production of MCP-1 and IP-10 in fibroblasts derived from NNS patients) To confirm that the effect of CUDC-907 is not specific to MT-iPS-MLs, its effect was confirmed using fibroblasts derived from NNS patients.

[0068] The expression level of immunoproteasome in fibroblasts is lower than that in immune cells. Therefore, TNF-α and IFN-γ were used for a longer time (72 hours) to induce immunoproteasome, and after bringing the phenotype of NNS patients into a state that sufficiently shows it, the production amount of cytokines was measured.

[0069] Since the expression level of IP-10 from fibroblasts was very low, it was not possible to compare fibroblasts derived from healthy individuals with those derived from NNS patients.

[0070] Figure 9 is a graph showing the expression levels of MCP-1 in Healthy#1 and Healthy#2, which are fibroblasts derived from healthy individuals, and NNS#1 and NNS#2, which are fibroblasts derived from NNS patients (n = 3). In Figure 9, "UT" indicates the results of fibroblasts not treated with TNF-α and IFN-γ. Also, "*", "**", "***" indicate that there are significant differences in the results of Student's t-test, p < 0.05, p < 0.01, p < 0.005, respectively.

[0071] Figure 10 is a graph showing the dose-dependent inhibitory effect of CUDC-907 on the production of MCP-1 in NNS#1, which is a fibroblast derived from an NNS patient. As a result, the IC of the inhibition of MCP-1 production by CUDC-907 in fibroblasts50 It was revealed that it was comparable to the value in MT-iPS-MLs.

[0072] Subsequently, the effects of CUDC-907 on the cell viability and cytotoxicity against NNS#1, which is a fibroblast derived from an NNS patient, were examined. Figure 11(a) is a graph showing the results of measuring intracellular NADH (n = 3). Figure 11(b) is a graph showing the results of measuring extracellular LDH activity (n = 3). In Figures 11(a) and (b), "DMSO" indicates the result of adding DMSO instead of CUDC-907, and "Lysate" indicates the result of cell lysate.

[0073] As a result, it was revealed that CUDC-907 showed almost no cytotoxicity at the effective concentration. From the above results, it was revealed that CUDC-907 inhibits the production of MCP-1 by fibroblasts derived from NNS patients at a concentration comparable to that in MT-iPS-MLs without showing cytotoxicity.

[0074] [Experimental Example 4] (Examination of the inhibitory effect of CUDC-907 on the production of MCP-1 and IP-10) It was confirmed whether the inhibitory effect of CUDC-907 on the production of MCP-1 and IP-10 on the NNS disease model was sufficient compared to the production amount of the wild-type control.

[0075] Figure 12(a) is a graph showing the results of comparing the production amounts of MCP-1 in WT-iPS-MLs and MT-iPS-MLs in the presence and absence of CUDC-907 (n = 3). Figure 12(b) is a graph showing the results of comparing the production amounts of IP-10 in WT-iPS-MLs and MT-iPS-MLs in the presence and absence of CUDC-907 (n = 3). In Figures 12(a) and (b), "**", "***", "****" indicate that there are significant differences at p < 0.01, p < 0.005, p < 0.001, respectively, as a result of one-way analysis of variance (ANOVA) and Dunnett's multiple comparison, and "N.S." indicates that there is no significant difference.

[0076] As a result, when 10 nM of CUDC-907 was treated on MT-iPS-MLs, it was revealed that the production amounts of MCP-1 and IP-10 decreased to the same level as or below that of WT-iPS-MLs.

[0077] Also, Fig. 13 is a graph showing the production amounts of MCP-1 in Healthy#1 and Healthy#2, which are fibroblast cell lines derived from healthy individuals, and the production amounts of MCP-1 in NNS#1 and NNS#2, which are fibroblasts derived from NNS patients, in the presence and absence of CUDC-907 (n = 3). In Fig. 13, "**" indicates that there is a significant difference with p < 0.01 as a result of Student's t-test, and "N.S." indicates that there is no significant difference.

[0078] As a result, an increase in MCP-1 production was confirmed in NNS#1 and #2 compared to Healthy#1 and Healthy#2. Furthermore, when 30 nM of CUDC-907 was treated on NNS#1 and #2, the production amount of MCP-1 decreased to the same level as or below that of Healthy#1 and Healthy#2.

[0079] From the above results, it was revealed that the effect of CUDC-907 is sufficient to reduce the excessive production of MCP-1 and IP-10 due to NNS to normal levels.

[0080] [Experimental Example 5] (CUDC-907 inhibited the production of MCP-1 and IP-10 post-transcriptionally) To clarify the mechanism of the inhibition of MCP-1 and IP-10 production by CUDC-907, the expression levels of the CCL2 gene encoding MCP-1 and the CXCL10 gene encoding IP-10 were quantified.

[0081] After treating WT-iPS-MLs cells with 10 nM CUDC-907 for 3 hours, the cells were stimulated with 100 ng / mL TNF-α and 100 ng / mL IFN-γ for 3, 9, 15, and 21 hours, and quantitative RT-PCR was performed. The expression level of each gene was normalized by the expression level of the GAPDH gene.

[0082] Figure 14(a) is a graph showing the results of measuring the expression level of the CCL2 gene. Also, Figure 14(b) is a graph showing the results of measuring the expression level of the CXCL10 gene. In Figures 14(a) and (b), the vertical axis of the graph indicates the expression level (relative value).

[0083] As a result, different from the ELISA results, it was revealed that CUDC-907 increased the expression levels of these genes. This result indicates that CUDC-907 inhibits the production of MCP-1 and IP-10 post-transcriptionally.

[0084] Subsequently, the expression levels of MCP-1 and IP-10 were examined at the protein level. To detect the expression levels of MCP-1 and IP-10 in cells by Western blotting, the release of these cytokines to the extracellular space was inhibited by treatment with Brefeldin A. Brefeldin A is known to inhibit protein transport from the endoplasmic reticulum to the Golgi apparatus.

[0085] After treating WT-iPS-MLs cells with 10 nM CUDC-907 for 30 minutes, the cells were stimulated with 100 ng / mL TNF-α and 100 ng / mL IFN-γ for 6 hours. Brefeldin A was added 1 hour before cell collection.

[0086] Figure 15 is an image showing the results of representative Western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as a loading control. In Figure 15, "-" indicates not added, and "+" indicates added.

[0087] As a result, in the presence of brefeldin A, the intracellular levels of MCP-1 and IP-10 increased. This result indicates that brefeldin A treatment inhibited the extracellular release of MCP-1 and IP-10.

[0088] Also, in the presence of brefeldin A, CUDC-907 decreased the intracellular levels of MCP-1 and IP-10. This result indicates that CUDC-907 inhibits the production of MCP-1 and IP-10 post-transcriptionally.

[0089] [Experimental Example 6] (Inhibition of the production of MCP-1 and IP-10 by HDAC inhibitors) The inhibitory effects of HDAC inhibitors CUDC-907, JNJ-26481585, LAQ824, romidepsin, and trichostatin A on the production of MCP-1 and IP-10 by MT-iPS-MLs were examined.

[0090] Figure 16(a) is a dose-response curve for the production of MCP-1 by each compound. Figure 16(b) is a dose-response curve for the production of IP-10 by each compound. Table 4 below shows the 50% inhibitory concentration (IC 50 ) of each compound calculated based on Figures 16(a) and (b).

[0091] [Table 4] [Industrial Applicability]

[0092] According to the present invention, a therapeutic agent for Nakajo-Nishimura syndrome can be provided.

Claims

【Claim 1】 A therapeutic agent for Nakajo-Nishimura syndrome, comprising a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is at least one compound selected from the group consisting of CUDC-907, JNJ-26481585, LAQ824, romidepsin, and trichostatin A, or a pharmacologically acceptable salt thereof or a solvate thereof.

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