Cerebral vasospasm inhibitors

Abstract

To elucidate the mechanism that the rupture of cerebral aneurysm in subarachnoid hemorrhage causes cerebral artery to twitch, leading to encephalopathy, and to provide a cerebral vasospasm inhibitor based on the mechanism and to further provide a novel cerebral vasospasm inhibitor screening method.MEANS FOR SOLVING THE PROBLEM: We have discovered the mechanism that a hematoma from the rupture of cerebral aneurysm causes the release of danger signals (DAMPs), a neutrophil and / or macrophage in bone marrow or blood binds to DAMPs through RAGE and is thus activated, and further migrates to cerebral blood vessels, causing cerebral vasospasm, and have completed the inventive cerebral vasospasm inhibitor targeting the mechanism.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an agent for suppressing cerebral vasospasm or a therapeutic agent for nerve function, an agent for suppressing cerebral vasospasm after subarachnoid hemorrhage or a therapeutic agent for nerve function, an agent for suppressing the activation of neutrophils and / or macrophages or an agent for suppressing their migration to cerebral arteries after subarachnoid hemorrhage, a therapeutic agent for subarachnoid hemorrhage, and a method for screening an agent for suppressing cerebral vasospasm or a therapeutic agent for nerve function. [Background technology]

[0002] (SAH) Subarachnoid hemorrhage (SAH) is a stroke that primarily occurs due to the rupture of a cerebral aneurysm. Many SAH patients suffer from cerebral artery spasms, known as cerebral vasospasm, which can lead to brain damage. However, the cause of cerebral vasospasm after SAH remains unknown. Rho kinase inhibitors are used clinically to treat cerebral vasospasm, but have not significantly improved the prognosis.

[0003] (RAGE) The receptor for advanced glycation endproducts (RAGE) is a single-pass transmembrane receptor belonging to the immunoglobulin superfamily. RAGE is a multiligand receptor that binds to various ligands associated with inflammation and disease (Non-Patent Document 1). It has been reported that in a rat model of subarachnoid hemorrhage, RAGE expression is increased in brain neurons and microglia (central nervous system glial cells), which are immune cells in the brain (Non-Patent Document 2). Intraperitoneal administration of the RAGE inhibitor FPS-ZM1 in a rat subarachnoid hemorrhage model improved symptoms on day 1, but the effect disappeared on day 3. It has been reported that while NF-kB-dependent intracerebral inflammation is reduced, nerve cells become more susceptible to death (Non-Patent Document 3).

[0004] In patients with subarachnoid hemorrhage whose condition worsened due to cerebral vasospasm, the level of endogenous RAGE inhibitor in the blood was low, suggesting a relationship between the severity of the patient's condition and RAGE signaling (Non-Patent Document 4), but the active ingredient contained in the cerebral vasospasm inhibitor of the present invention is unknown. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Daffu et al, Int J Mol Sci. 2013,14(10):19891-910. doi:10.3390 / ijms141019891. [Non-patent document 2] Li et al, Brain Res. 2014;1543:315-323. doi:10.1016 / j.brainres.2013.11.023. [Non-patent document 3] Li et al, Mol Neurobiol. 2017;54(1):755-767. doi:10.1007 / s12035-016-9703-y. [Non-patent document 4] Aida et al, J Neurosurg. 2019, 1.aop: 1-9. doi:10.3171 / 2019.8.JNS191269. Summary of the Invention [Problem to be solved by the invention]

[0006] The mechanism by which cerebral arteries contract after rupture of a cerebral aneurysm in subarachnoid hemorrhage, leading to brain damage, was unknown. The present invention aims to elucidate these mechanisms and provide a cerebral vasospasm inhibitor based on said mechanisms, as well as a screening method for novel cerebral vasospasm inhibitors. [Means for solving the problem]

[0007] The present inventors have discovered a mechanism by which "damage-associated molecular patterns (DAMPs), which are danger signals, are released from the hematoma formed by the rupture of a cerebral aneurysm, and neutrophils and / or macrophages in the bone marrow or blood bind to the DAMPs via RAGE, becoming activated, and then migrating to cerebral blood vessels, causing cerebral vasospasm," and have completed the cerebral vasospasm inhibitor of the present invention, which targets this mechanism.

[0008] That is, the present invention is as follows. 1. An agent for suppressing cerebral vasospasm after subarachnoid hemorrhage or for treating nerve function, which contains as an active ingredient a compound that inhibits the activation of neutrophils and / or macrophages in the bone marrow or blood or their migration to cerebral arteries in cerebral vasospasm after subarachnoid hemorrhage. 2. The agent for inhibiting cerebral vasospasm after subarachnoid hemorrhage or the agent for treating nerve function according to the preceding item 1, wherein the cerebral vasospasm is cerebral vasospasm occurring within 24 hours after subarachnoid hemorrhage. 3. A cerebral vasospasm inhibitor or neurological function therapeutic agent comprising, as an active ingredient, a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof: [ka] 4. The agent for suppressing cerebral vasospasm or therapeutic agent for nerve function according to the preceding item 3, wherein the cerebral vasospasm is cerebral vasospasm following subarachnoid hemorrhage. 5. The agent for suppressing cerebral vasospasm or therapeutic agent for nerve function according to the preceding item 4, wherein the cerebral vasospasm is caused by activation of neutrophils and / or macrophages in the bone marrow or blood after subarachnoid hemorrhage. 6. The agent for suppressing cerebral vasospasm or therapeutic agent for nerve function according to the preceding item 4, wherein the cerebral vasospasm is caused by migration of neutrophils and / or macrophages in the bone marrow or blood to cerebral arteries after subarachnoid hemorrhage. 7. An agent for inhibiting the activation of neutrophils and / or macrophages or their migration to cerebral arteries after subarachnoid hemorrhage, comprising as an active ingredient a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof: [ka] 8. A therapeutic agent for subarachnoid hemorrhage, comprising as an active ingredient a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof: [ka] 9. An agent for suppressing cerebral vasospasm or treating nerve function after subarachnoid hemorrhage, containing as an active ingredient a substance with neutrophil elastase inhibitory activity. 10. An agent for inhibiting cerebral vasospasm or treating neurological function after subarachnoid hemorrhage, containing one or more of the following substances as an active ingredient: (1) Pyrazole-5-carboxamides (2)Soluble RAGE (Soluble RAGE, sRAGE) (3) FPS-ZM1 (4)4,6-bisphenyl-2-(3-alkoxyanilino)pyrimidine (5) Azeliragon 11. A method for screening for an agent for inhibiting cerebral vasospasm or an agent for treating nerve function, which comprises determining one or more of the following substances: (1) Identify substances that inhibit the binding of RAGE and DIAPH1 (2) Identifying substances that inhibit the migration or activation of neutrophils and / or macrophages (3) Identify substances that inhibit neutrophil elastase activity (4) Identify substances that inhibit RAGE activity (5) Identify substances that inhibit the binding of DAMPs to RAGE (6) Identify substances that inhibit RAGE-Rho activation (7) Identify substances that inhibit RAGE-dependent NETosis (8) Identifying substances that inhibit Rac activation (9) Identify substances that inhibit Cdc42 activation [Effects of the Invention]

[0009] The cerebral vasospasm inhibitor of the present invention, which contains as an active ingredient a substance that has the effect of inhibiting the activation of neutrophils and / or macrophages derived from bone marrow or blood or their migration to cerebral arteries in cerebral vasospasm after subarachnoid hemorrhage, has the effect of suppressing cerebral vasospasm after subarachnoid hemorrhage and further improving neurological function. Furthermore, the activity of inhibiting the activation of neutrophils and / or macrophages or their migration to cerebral arteries has the effect of suppressing cerebral vasospasm and improving neurological function, and therefore, novel agents for suppressing cerebral vasospasm can be obtained by screening for substances that inhibit this activity. [Brief explanation of the drawings]

[0010] [Figure 1] Neurological symptoms in RAGE knockout mice. [Figure 2] Improvement in cerebral vasospasm in RAGE knockout mice (indicated by the arrowheads in the enlarged image). [Figure 3] Improvement in cerebral vasospasm and arteriolar flow in RAGE knockout mice. [Figure 4] Evaluation of RAGE mRNA expression after SAH (LCA = left cerebral artery, RCA = right cerebral artery, LCx = left cerebral cortex, RCx = right cerebral cortex, LHi = left hippocampus, RHi = right hippocampus; left bar: sham, right bar: SAH). [Figure 5] Evaluation results in vascular-specific RAGE knockout mice. [Figure 6] Results of RAGE mRNA expression analysis in leukocytes in peripheral blood and immune system organs (top left graph, bottom left graph, and center graph are for the WT SAH model. The line graph in the top right is the result for leukocytes in peripheral blood, showing that RAGE mRNA expression does not increase in RAGE- / -. The bar graph in the bottom right is for the WT model, where Spleen means spleen, LNs means lymph node, and BM means bone marrow; the left bar in each case indicates Sham, and the right bar indicates SAH). [Figure 7] Immunostaining of neutrophils accumulating in cerebral arteries after SAH. [Figure 8]Neurological symptoms in RAGE knockout mice transplanted with bone marrow cells from GFP mice. [Figure 9] Quantitative data on cerebral vasospasm in RAGE knockout mice transplanted with bone marrow cells derived from GFP mice. [Figure 10] Immunostaining observation results after SAH in RAGE knockout mice transplanted with bone marrow cells derived from GFP mice. [Figure 11] Evaluation results of cerebral vasospasm after SAH in parabiosis experiments using RAGE knockout mice and GFP mice. [Figure 12] Neurological score evaluation results and quantitative data of cerebral vasospasm after SAH in neutrophil-specific RAGE knockout mice. [Figure 13] Observation results of neutrophil nuclear staining in a transwell migration assay. "Neut" in the figure indicates neutrophil, which is synonymous with polymorphonuclear cells (PMN) in this specification. "Clot" indicates hematoma, "WBC WT" indicates wild-type neutrophils, and "WBC RAGE- / -" indicates RAGE knockout neutrophils. [Figure 14] The number of neutrophils that migrated into the hematoma in a transwell migration assay. "PMN" in the figure indicates polymorphonuclear cells, which are synonymous with neutrophils in this specification. [Figure 15] The number of neutrophils that migrated into the hematoma in a transwell migration assay and underwent NETosis, a cell death caused by neutrophil extracellular traps (NETs). [Figure 16] The effect of Compound 11 on improving neurological scores and cerebral vasospasm after SAH. [Figure 17] Western blot results of Rho pull-down assay using bone marrow-derived neutrophils (PMN) and macrophages (MN). "Rhotekin RBD" in the figure indicates Rhotekin RBD, agarose. [Figure 18] Neurological score (left panel) and cerebral vasospasm (right panel) evaluation results in mice administered a neutrophil elastase (NE) inhibitor. [Figure 19] Evaluation results of neurological score (left panel) and cerebral vasospasm (right panel) in experiments using esRAGE mice. [Figure 20] Schematic diagram of the mechanism of cerebral vasospasm after subarachnoid hemorrhage. DETAILED DESCRIPTION OF THE INVENTION

[0011] (Subject of the present invention) The present invention relates to an agent for suppressing cerebral vasospasm or a therapeutic agent for nerve function, an agent for suppressing cerebral vasospasm or a therapeutic agent for nerve function after subarachnoid hemorrhage, an inhibitor of neutrophil and / or macrophage activation or an inhibitor of migration to cerebral arteries after subarachnoid hemorrhage, a therapeutic agent for subarachnoid hemorrhage, and a method for screening an agent for suppressing cerebral vasospasm or a therapeutic agent for nerve function.

[0012] Professor Schmidt of New York University in the United States identified the cytoskeleton-associated molecule DIAPH1 as an intracellular signaling factor for RAGE (Hudson et al., J Biol Chem. 2008, 283(49):34457-68. doi:10.1074 / jbc.M801465200.). Furthermore, a search for compounds that inhibit the binding of RAGE to DIAPH1 led to the discovery of 13 RAGE / DIAPH1 inhibitors that directly bind to the C-terminus of RAGE (Manigrasso et al., Sci Rep. 2016, 6:22450. doi:10.1038 / srep22450.). The present invention has confirmed that the compound represented by the following formula (1), which was found to be the most effective among the 13 RAGE / DIAPH1 inhibitor compounds developed by Professor Schmidt in preliminary experiments, inhibits and alleviates cerebral vasospasm and improves neurological function through the inhibition of RAGE signaling in neutrophils and / or macrophages derived from bone marrow or blood, as described in the Examples below.

[0013] [ka]

[0014] (Anti-cerebral vasospasm agent after subarachnoid hemorrhage) The cerebral vasospasm inhibitor of the present invention after subarachnoid hemorrhage (hereinafter sometimes abbreviated as "inhibitor of the present invention") targets cerebral vasospasm at any time after subarachnoid hemorrhage, for example, within 24 hours after subarachnoid hemorrhage, 0 to 24 hours, 3 to 24 hours, or 12 to 24 hours after subarachnoid hemorrhage, or 24 hours or more, or 48 hours or more. In addition, suppression includes treatment, prevention, prevention of recurrence, alleviation, complete cure, etc. of cerebral vasospasm.

[0015] (Neutrophils and / or macrophages in bone marrow or blood) As shown in the Examples below, it has been confirmed that the "neutrophils and / or macrophages" in the inhibitor of the present invention are involved in cerebral vasospasm and are derived from bone marrow or blood. In addition, inhibiting the activation of neutrophils and / or macrophages means suppressing their proliferation, migration, inflammation, and attack. Proliferation, migration, inflammation, and attack can be measured by known methods.

[0016] (Active ingredient of the inhibitor of the present invention) The active ingredient of the inhibitor of the present invention may be a high molecular weight compound or a low molecular weight compound, as long as it has the effect of inhibiting the activation of neutrophils and / or macrophages in the bone marrow or blood or their migration to cerebral arteries. Examples of polymer compounds include proteins and nucleic acid substances, and specific examples include antibodies, antibody fragments, peptides, siRNA, shRNA, etc. Substances containing low molecular weight compounds and polymer compounds may also be used. A preferred example of an active ingredient is the compound represented by formula (1). In addition, pharmacologically acceptable salts of formula (1) (e.g., addition salts, hydrochloride salts) that do not inhibit the effects of the compound represented by formula (1) are also preferred.

[0017] (subject) The subjects to which the inhibitor of the present invention is administered are humans and non-human mammals. Preferred non-human mammals include pets and livestock.

[0018] (Administration method, dosage form) There are no particular limitations on the route of administration of the inhibitor of the present invention, but preferred routes of administration include intravenous, oral, transdermal, and transmucosal (oral, rectal, vaginal, etc.) routes. Examples of formulations for oral administration include tablets, capsules, granules, powders, syrups (dry syrups), oral jellies, etc. Examples of formulations for transdermal or transmucosal administration include patches, ointments, etc. Tablets, capsules, granules, powders, and the like can be prepared as enteric-coated preparations. For example, tablets, granules, and powders can be enteric-coated. As the enteric coating agent, a poorly soluble enteric coating agent in the stomach can be used. In addition to the active ingredient, the inhibitor of the present invention can contain a pharmacologically acceptable carrier depending on the administration form. Examples of pharmacologically acceptable carriers include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilizers, isotonicity agents, pH adjusters, stabilizers, propellants, and adhesives.

[0019] The dosage and frequency of administration of the inhibitor of the present invention may vary as appropriate depending on the subject, their age, body weight, sex, purpose (e.g., prophylactic or therapeutic), severity of symptoms, dosage form, route of administration, and other conditions. When administered to humans, the compound represented by formula (1) is administered at a dose of, for example, about 0.0001 mg / kg to 10 mg / kg body weight per day. The frequency of administration may be one or more times per day, or once every few days. For example, it may be administered 1 to 3 times, 1 to 2 times, or once per day. The inhibitor of the present invention can be made into medicines, quasi-drugs, medical devices, sanitary products, foods, beverages, and supplements.

[0020] (Treatment method) The present invention also relates to a method for preventing and / or treating cerebral vasospasm after subarachnoid hemorrhage, using a cerebral vasospasm inhibitor containing, as an active ingredient, a compound of the present invention that has the effect of inhibiting the activation of neutrophils and / or macrophages or their migration to cerebral arteries.

[0021] (Inhibitor of neutrophil and / or macrophage activation or migration to cerebral arteries after subarachnoid hemorrhage) The inhibitor of neutrophil and / or macrophage activation or migration to cerebral arteries after subarachnoid hemorrhage of the present invention can employ the same active ingredients, targets, administration methods, dosage forms, and treatment methods as the inhibitor of the present invention described above.

[0022] (Neurological function treatment after subarachnoid hemorrhage) The therapeutic agent for treating nerve function after subarachnoid hemorrhage of the present invention can employ the same active ingredients, targets, administration methods, dosage forms, and treatment methods as the inhibitor of the present invention described above. Treatment of neurological function includes, but is not limited to, improvement, prevention, prevention of recurrence, alleviation, complete cure, etc.

[0023] (Subarachnoid hemorrhage treatment) The therapeutic agent for subarachnoid hemorrhage of the present invention can employ the same active ingredients, targets, administration methods, dosage forms, and treatment methods as the inhibitor of the present invention described above. Treatment for subarachnoid hemorrhage includes, but is not limited to, improvement, prevention, prevention of recurrence, alleviation, complete recovery, and the like.

[0024] (An agent for suppressing cerebral vasospasm or treating neurological function after subarachnoid hemorrhage, containing a substance with neutrophil elastase inhibitory activity as an active ingredient) The "agent for suppressing cerebral vasospasm or therapeutic agent for neurological function after subarachnoid hemorrhage, which contains as an active ingredient a substance having neutrophil elastase inhibitory activity" of the present invention is not particularly limited as long as it contains a substance having the effect of inhibiting neutrophil elastase activity. Examples of substances having the effect of inhibiting neutrophil elastase activity include sivelestat sodium hydrate, trypsin inhibitor, soybean, 3,4-dichloroisocoumarin, elastatinal, N-(Methoxysuccinyl)-Ala-Ala-Pro-Val-chloromethyl ketone, SSR 69071, sivelestat sodium tetrahydrate, 1-(3-methylbenzoyl)-1H-indazole-3-carbonitrile, and sirtinol.

[0025] (An agent for suppressing cerebral vasospasm or treating neurological function after subarachnoid hemorrhage, containing a substance with RAGE inhibitory activity as an active ingredient) The "agent for suppressing cerebral vasospasm or therapeutic agent for neurological function after subarachnoid hemorrhage, which comprises as an active ingredient a substance having RAGE inhibitory activity" in the present invention is not particularly limited as long as it contains a substance having the effect of inhibiting the activity of RAGE. Examples of substances having the effect of inhibiting RAGE include sRAGE, FPS-ZM1, pyrazole-5-carboxamides, 4,6-bisphenyl-2-(3-alkoxyanilino)pyrimidine, and azeliragon (TTP488).

[0026] (Screening method) The screening method for cerebral vasospasm inhibitors or neurological function therapeutic agents of the present invention targets the following: - Identify substances that inhibit the binding of RAGE and DIAPH1 Determine substances that inhibit the migration or activation of neutrophils and / or macrophages - Identify substances that inhibit neutrophil elastase activity - Identify substances that inhibit RAGE activity - Identify substances that inhibit the binding of DAMPs, including HMGB1, to RAGE - Identify substances that inhibit Rho activation from RAGE - Identify substances that inhibit RAGE-dependent NETosis - Identify substances that inhibit Rac activation - Identify substances that inhibit Cdc42 activation Neutrophil extracellular traps (NETs) are networks of extracellular fibers that activated neutrophils release into bacteria and tissues, trapping complexes of their own DNA and proteins such as digestive enzymes. The cell death that occurs during NETs is called NETosis.

[0027] "Determining a substance that inhibits the binding of RAGE and DIAPH1" may include, for example, any one or more of the following steps. (a-1) measuring the binding ability of DIAPH1 derived from a biological sample of a subject to RAGE or the binding ability of RAGE to DIAPH1 (RAGE / DIAPH1 binding ability) in the presence of a test substance; and (a-2) A step of selecting a test substance having an effect of inhibiting the binding of RAGE and DIAPH1 (RAGE / DIAPH1 binding inhibitory effect) based on the results of (a-1) above. The binding ability in step (a-1) of the above method can be measured by a method known per se, for example, a binding assay or a method using surface plasmon resonance (e.g., Biacore (登録商標) This can be done by using Step (a-1) of the above method may further include comparing the binding ability measured in the presence of the test substance with the RAGE / DIAPH1 binding ability measured in the presence of a control substance that does not have RAGE / DIAPH1 binding inhibitory activity, and / or comparing the binding abilities measured for multiple test substances.

[0028] "Determining a substance that inhibits the migration or activation of neutrophils and / or macrophages" may include, for example, any one or more of the following steps. (b-1) measuring the migration or activation of neutrophils or macrophages derived from a biological sample of a subject to a hematoma in the presence of a test substance; and (b-2) A step of selecting a test substance having the effect of inhibiting the migration or activation of neutrophils and / or macrophages based on the results of (b-1) above. The measurement of migration or activation into the hematoma in step (b-1) of the above method can be carried out by a method known per se, for example, a cell migration assay (e.g., transwell (Corning (登録商標) ) can be performed by a migration assay using Step (b-1) of the above method may further include comparing the migration or activation of neutrophils or macrophages into the hematoma measured in the presence of the test substance with the migration or activation of neutrophils or macrophages into the hematoma measured in the presence of a control substance that does not have the effect of inhibiting the migration or activation of neutrophils and / or macrophages, and / or comparing the migration or activation of neutrophils or macrophages into the hematoma measured for multiple test substances.

[0029] (Test substance) Any substance can be used as a test substance that is a candidate therapeutic substance for use in the above screening. The type of test substance is not particularly limited, and may be an individual low molecular weight synthetic compound (e.g., siRNA), a compound present in a natural product extract, or a synthetic peptide. The test substance may be a chemical library, a phage display library, or a combinatorial library. Construction of chemical libraries, phage display libraries, and combinatorial libraries is known to those skilled in the art, and commercially available chemical libraries may also be used.

[0030] (Subjects and biological samples) In the present invention, subjects include mammals in general (including humans, cats, dogs, and horses), as well as healthy individuals, patients with subarachnoid hemorrhage, individuals suspected of having subarachnoid hemorrhage, and individuals who will experience subarachnoid hemorrhage in the future. Biological samples also include, but are not limited to, components derived from the spleen, lymph nodes, peripheral blood, blood components (serum, plasma, blood cells, white blood cells, neutrophils, etc.), mesenchymal cells, stem cells, biopsy samples, iPS cells, primary cultured cells, saliva, urine, cerebrospinal fluid, tears, sweat, hair, and tissues. [Example]

[0031] The present invention will be described in detail below using specific examples, but the present invention is not limited to these examples. The following examples were approved by the Kanazawa University Genetic Modification Experiment Safety Management Committee and Animal Experiment Committee. The materials and methods used in the examples are as follows.

[0032] (Creation of a mouse model of subarachnoid hemorrhage) Subarachnoid hemorrhage was induced in C57BL / 6J background mice by inserting a microfilament through the left external carotid artery stump, passing it through the internal carotid artery, and puncturing it at the anterior cerebral artery-middle cerebral artery bifurcation.

[0033] (Method for measuring neurological scores in mice) Neurobehavioral function was measured using the modified Garcia neurological score. The assessment consisted of six items, each scored out of 3, with higher scores indicating better function. The six items consisted of spontaneous limb activity, spontaneous movement, forelimb extension, climbing, proprioception, and whisker stimulation (see Liu et al., Mol Neurobiol. 2015, 53(7):4529-38. doi:10.1007 / s12035-015-986-9).

[0034] (RAGE inhibitor Compound 11) The RAGE / DIAPH1 inhibitor compound was discovered by Professor Schmidt of New York University in the United States, who identified the cytoskeleton-associated molecule DIAPH1 as an intracellular signaling factor for RAGE (see Hudson et al., J Biol Chem. 2008, 283(49):34457-68. doi:10.1074 / jbc.M801465200.), and further explored compounds that inhibit the binding of RAGE to DIAPH1 (see Manigrasso et al., Sci Rep. 2016, 6:22450. doi:10.1038 / srep22450.). The present inventors were provided with Compound 11 (C11: a compound represented by formula (1)), one of the RAGE / DIAPH1 inhibitor compounds synthesized by Professor Yasuhiko Yamamoto of Kanazawa University with permission from the developer, Professor Schmidt.

[0035] (co-culture experiment) Transwell (Corning), a septum-walled culture dish with 3 μm pores at the bottom of the upper layer, was used. (登録商標) ) was used, and hematoma was placed in the lower dish and LPS-stimulated neutrophils were placed in the upper dish, followed by incubation at 37°C for 45 minutes. [Example]

[0036] The present inventors carried out the following confirmation. (Neurological symptoms in RAGE knockout mice) RAGE knockout mice (RAGE - / - A mouse model of subarachnoid hemorrhage (SAH) was established in wild-type (WT) and control mice, and the modified Garcia neurological score was calculated. The calculation results confirmed that neurological symptoms were significantly improved in RAGE knockout mice 12 and 24 hours after SAH (FIG. 1).

[0037] (Improvement of cerebral vasospasm) RAGE knockout mice (RAGE - / -Subarachnoid hemorrhage (SAH) mouse models of wild-type (WT) and control mice, as well as sham-operated wild-type mice, were evaluated for spasm of the main basal cerebral arteries 24 hours after SAH by cardiac perfusion fixation with 4% PFA followed by perfusion with India ink-containing gelatin. This evaluation confirmed that cerebral vasospasm and the total arteriolar length were significantly improved in RAGE knockout mice (FIG. 2). Furthermore, quantification of the diameter of the left internal carotid artery (ICA) and the total vascular length of the left cerebral arterioles revealed improvements in vasospasm and arteriolar flow in RAGE knockout mice (Figure 3).

[0038] (Evaluation of RAGE mRNA expression after SAH) The RAGE mRNA expression level was assessed 12 hours after SAH in wild-type mice with a subarachnoid hemorrhage (SAH) mouse model and in sham-operated (sham) wild-type mice. The results of this evaluation showed that RAGE mRNA expression increased in cerebral arteries, the cerebral cortex, and the hippocampus 12 hours after SAH (Figure 4).

[0039] (Evaluation in vascular-specific RAGE knockout mice) We hypothesized that cerebral vasospasm and the resulting brain damage are caused by RAGE expressed in cerebral blood vessels. To test this hypothesis, we evaluated vascular-specific RAGE knockout mice. Specifically, control mice (RAGE equivalent to wild-type mice) fl / fl ) and vascular endothelial cell-specific RAGE knockout mice (Tie2 Cre RAGE fl / fl ) Neurological scores were calculated and the diameter of the left internal carotid artery (ICA) was quantified. Surprisingly, the evaluation results showed no difference between the neurological score and the degree of cerebral vasospasm, confirming that RAGE expressed in cerebral blood vessels is not involved in the pathology of subarachnoid hemorrhage (Figure 5). [Example]

[0040] (Analysis of inflammatory cells and the immune system) RAGE knockout mice (RAGE - / - RAGE mRNA expression levels were assessed in peripheral blood leukocytes and immune system organs in wild-type (WT) and control subarachnoid hemorrhage (SAH) mouse models, as well as in sham-operated (sham) wild-type mice. RT-qPCR confirmed that RAGE mRNA expression increased in leukocytes, including neutrophils and macrophages in peripheral blood after SAH, as well as in immune system organs such as the spleen and lymph nodes, and that mRNA expression of the cytoskeleton-regulating proteins RhoA and Rock1 also increased in leukocytes (Figure 6). We observed inflammation and immune cells after SAH using immunostaining. At 3 hours into the hyperacute phase, neutrophils accumulated inside and outside the lumen of the internal carotid artery, but no accumulation was observed in RAGE knockout mice (Figure 7). Furthermore, at 24 hours, a large number of neutrophils accumulated in the outer wall of the internal carotid artery, whereas almost no accumulation was observed in RAGE knockout mice (Figure 7).

[0041] (Immune system RAGE evaluation) As donors, bone marrow cells from GFP mice, which have wild-type RAGE genes, were used, and as recipients, wild-type (WT) or RAGE knockout mice (RAGE - / - ) to create a mouse model of subarachnoid hemorrhage (SAH). In RAGE knockout mice, the neurological scores and the degree of cerebral vasospasm were deteriorated to the same extent as in wild-type mice, indicating that RAGE in inflammatory and immune cells controls the pathology after subarachnoid hemorrhage (Figures 8 and 9). Furthermore, immunohistochemistry confirmed that RAGE knockout mice transplanted with bone marrow cells derived from GFP mice exhibited neutrophil accumulation after SAH (Figure 10). The results of this example confirmed that cerebral vasospasm and the resulting brain damage are caused by RAGE in bone marrow-derived immune cells.

[0042] (Evaluation in parabiosis) Wild-type (WT) and RAGE knockout mice (RAGE - / - We performed parabiosis between wild-type GFP mice and RAGE-knockout mice to share peripheral circulation and generate a mouse model of subarachnoid hemorrhage (SAH). The analysis results showed that RAGE-knockout mice did not show improvement in cerebral vasospasm (Figure 11). This is consistent with the results of bone marrow transplantation experiments (Figure 9). The results of this example confirmed that RAGE in the immune system is involved in cerebral vasospasm after SAH.

[0043] (Evaluation in neutrophil-specific RAGE knockout mice) We established a mouse model of subarachnoid hemorrhage (SAH) using neutrophil-specific RAGE knockout mice and evaluated neurological symptoms. Control mice (RAGE corresponding to wild-type mice) fl / fl ) and neutrophil-specific RAGE knockout mice (LysM Cre RAGE fl / fl ) Neurological scores were calculated and the diameter of the left internal carotid artery (ICA) was quantified. The evaluation results showed that the neutrophil-specific RAGE knockout mice had improved neurological symptoms and cerebral vasospasm compared to the control group (FIG. 12). These findings confirm that cerebral vasospasm and the resulting brain damage after SAH are caused by RAGE in immune cells, particularly neutrophils. Although most of the cells that accumulate in spasmed blood vessels are neutrophils (Figure 7), because LysM is also expressed in macrophages, it is possible that macrophage RAGE is also involved in the pathogenesis. [Example]

[0044] (Transwell migration assay of neutrophils into blood clots) The upper dish contains LPS-stimulated wild-type (WT) mice and RAGE knockout mice (RAGE - / - ) derived from 1 × 10 5neutrophils, or 1 × 10 IgG, anti-HMGB1 neutralizing antibody (αHMGB1, antibody with inhibitory effect on HMGB1), solvent (Vehicle), or Compound 11 (RAGEi C11) 5 Wild-type neutrophils were plated and placed in a hematoma in the bottom dish, and a transwell migration assay was performed. Migration of neutrophils into hematomas from RAGE knockout mice was suppressed, unlike that of neutrophils from wild-type mice (FIG. 13). Transwell assays showed that migration into the hematoma (clot) was inhibited in neutrophils (polymorphonuclear cells (PMN)) from RAGE knockout mice, wild-type mice treated with anti-HMGB1 neutralizing antibody (αHMGB1), and wild-type mice treated with Compound 11 (Figure 14). Furthermore, cell death (netosis) caused by neutrophil extracellular traps in migrated neutrophils (polymorphonuclear cells) was inhibited in neutrophils from RAGE knockout mice, wild-type mice treated with anti-HMGB1 neutralizing antibody (αHMGB1), and wild-type mice treated with Compound 11 (Figure 15). These results confirmed that neutrophils migrate into hematomas in a RAGE-dependent manner and cause netosis, and that this neutrophil migration and netosis are suppressed by inhibition of HMGB1, a ligand for RAGE, and by the RAGE inhibitor Compound 11. [Example]

[0045] In this example, the improvement of neurological function after subarachnoid hemorrhage (SAH) by Compound 11 (C11) was confirmed, as was the inhibitory effect on cerebral vasospasm, and the mechanism of action was also confirmed. C11 was intraperitoneally administered twice at a dose of 5 mg / kg to wild-type mice: simultaneously with SAH induction and 12 hours after SAH induction. Control wild-type mice received an intraperitoneal injection of a solvent mixture of ethanol and peanut oil. Evaluation of these mice confirmed that C11 administration improved neurological scores and cerebral vasospasm after SAH (Figure 16). From the results shown in FIGS. 13 to 16, it was confirmed that C11 has the effect of reducing subarachnoid hemorrhage, the effect of reducing cerebral vasospasm, and the effect of improving neurological function through the inhibition of RAGE signaling. [Example]

[0046] (Rho pull-down assay using bone marrow-derived neutrophils and macrophages) Neutrophils and macrophages were isolated from the bone marrow of wild-type and RAGE knockout mice on a C57BL / 6J background by density gradient centrifugation using Histopaque (Merck). The isolated neutrophils and macrophages were stimulated with LPS for 1 hour, and activated Rho signaling was detected using a Rho pull-down assay kit (Merck). Activated Rho in LPS-stimulated neutrophils and macrophages was detected by Western blotting using Rhotekin RBD Agarose Beads (agarose beads with the Rho-binding domain RBD of Rhotekin, a protein that binds to Rho). As shown in Figure 17, activated Rho was elevated in wild-type (WT) neutrophils (PMN), whereas the RAGE knockout (RAGE - / - ) Expression was significantly reduced in neutrophils. A similar trend was observed in macrophages (MN), but the expression of wild-type RAGE was lower than that in neutrophils. These findings suggest that in activated neutrophils and macrophages, intracellular Rho is activated as a downstream signaling molecule of RAGE. Fasudil hydrochloride is currently used clinically as a drug to prevent cerebral vasospasm after subarachnoid hemorrhage (SAH). This drug is thought to target Rho kinase, an upstream molecule of Rho in vascular smooth muscle. In practice, fasudil hydrochloride is administered several days after the onset of SAH, but no conclusion has been reached as to whether it significantly improves patient prognosis. In this example, the RAGE / Rho signaling of neutrophils can be targeted early on the day of onset (particularly, within 5 hours, 15 hours, 24 hours, 36 hours, 48 ​​hours, and 62 hours), which differs from the therapeutic mechanism of conventional drugs for preventing cerebral vasospasm, and therefore this may be a superior treatment for cerebral vasospasm compared to conventional preventive drugs. In addition, Rac and Cdc42, which are Rho family small G proteins like Rho, are thought to exhibit the same behavior as Rho, so Rac and Cdc42 signals in neutrophils and / or macrophages may also be therapeutic targets for cerebral vasospasm. [Example]

[0047] (Neutrophil elastase inhibitor administration experiment in mice) Sivelestat (sodium salt hydrate), a neutrophil elastase inhibitor (NE) (already clinically approved for the treatment of acute lung injury associated with systemic inflammatory response syndrome) (purchased from Cayman Chemical Company), was intraperitoneally administered at 25 mg / kg to wild-type mice twice: simultaneously with subarachnoid hemorrhage (SAH) induction and 6 hours after SAH induction. Control wild-type mice received intraperitoneal administration of the vehicle PBS alone. Neurological scores were measured 12 and 24 hours after SAH, and 24 hours after SAH, mice were cardiac perfused with 4% PFA and subsequently perfused with India ink gelatin to observe and evaluate spasm of the basilar major cerebral arteries. The NE inhibitor-treated mice showed improved neurological scores (Fig. 18, left panel) and reduced cerebral vasospasm (Fig. 18, right panel) compared with the vehicle-treated group, suggesting that NE from neutrophils induces cerebral vasospasm and brain damage. As a result, substances with NE inhibitory activity (NE inhibitors) can be used as therapeutic agents (reducing agents) for subarachnoid hemorrhage, suppressants of cerebral vasospasm, and agents for improving neurological function. [Example]

[0048] (Experiment using esRAGE mice) We received esRAGE mice, a transgenic mouse model expressing endogenous secretory RAGE (esRAGE), an endogenous RAGE inhibitor, from Professor Yasuhiko Yamamoto of Kanazawa University. Subarachnoid hemorrhage (SAH) was induced in wild-type and esRAGE mice. Neurological scores were measured 12 and 24 hours after SAH, and 24 hours later, the mice were fixed by cardiac perfusion with 4% PFA. Subsequently, India ink gelatin was perfused to observe and evaluate spasm of the main basal cerebral arteries. The esRAGE mice group had improved neurological scores (Fig. 19, left panel) and reduced cerebral vasospasm (Fig. 19, right panel) compared to the wild-type mice group, demonstrating that RAGE inhibition by secreted RAGE has a therapeutic effect in mice. As a result, substances with RAGE inhibitory activity (RAGE inhibitors) can be used as therapeutic agents (reducing agents) for subarachnoid hemorrhage, suppressants of cerebral vasospasm, and agents for improving neurological functions.

[0049] (General remarks) Based on the results of Examples 1 to 7, the causes of cerebral vasospasm after subarachnoid hemorrhage are considered to be as follows (see FIG. 20). 1) DAMPs are released from hematomas formed by rupture of cerebral aneurysms. 2) RAGE in neutrophils and / or macrophages in the bone marrow or blood binds to DAMPs. 3) Neutrophils and / or macrophages bound to DAMPs become activated and migrate into the cerebral blood vessels. 4) Neutrophils and / or macrophages that migrate into cerebral blood vessels cause cerebral vasospasm. As a result, a substance that inhibits any of the above steps 1) to 4) can be used as a suppressant or therapeutic agent for subarachnoid hemorrhage, a suppressant or therapeutic agent for cerebral vasospasm after subarachnoid hemorrhage, and an agent for improving or treating neurological function after subarachnoid hemorrhage.

Claims

1. 1. An agent for inhibiting cerebral vasospasm or treating nerve function after subarachnoid hemorrhage, comprising, as an active ingredient, a compound represented by the following formula (1) or a pharmacologically acceptable salt thereof: 【Chemical 1】

2. The agent for inhibiting cerebral vasospasm or therapeutic agent for nerve function after subarachnoid hemorrhage according to claim 1, characterized in that the cerebral vasospasm is caused by activation of neutrophils and / or macrophages in the bone marrow or blood after subarachnoid hemorrhage.

3. 2. The agent for inhibiting cerebral vasospasm or therapeutic agent for nerve function after subarachnoid hemorrhage according to claim 1, characterized in that the cerebral vasospasm is caused by migration of neutrophils and / or macrophages in the bone marrow or blood to cerebral arteries after subarachnoid hemorrhage.

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