Prophylactic agent for secondary brain injury after onset of subarachnoid hemorrhage

Abstract

The purpose of the present disclosure is to provide a drug capable of preventing secondary brain injury after the onset of subarachnoid hemorrhage.　This prophylactic agent for secondary brain injury after the onset of subarachnoid hemorrhage comprises an inhibitor of CCR2 and CCR5, such as a FROUNT inhibitor.

Description

Drugs for preventing secondary brain damage after subarachnoid hemorrhage

[0001] The present disclosure relates to a preventive agent for secondary brain damage after the onset of subarachnoid hemorrhage. More specifically, the present disclosure relates to a preventive agent for secondary brain damage after the onset of subarachnoid hemorrhage, the preventive agent having a mechanism of suppressing infiltration of inflammatory cells into the subarachnoid space after the onset of subarachnoid hemorrhage.

[0002] Subarachnoid hemorrhage occurs when blood enters the subarachnoid space due to factors such as the rupture of a cerebral aneurysm. It has been reported that the incidence of subarachnoid hemorrhage is approximately 20 per 100,000 people per year in Japan, and approximately 10 per 100,000 people overseas. It has been reported that the mortality rate of subarachnoid hemorrhage, including sudden death, is approximately 50%, and that approximately 26% of patients are left with moderate or severe after-effects requiring assistance with daily activities after the onset of the condition. Despite recent advances in medical technology, the prognosis is known to be poor.

[0003] After the onset of subarachnoid hemorrhage, factors such as increased intracranial pressure and hematoma can cause secondary brain damage, such as ischemic brain damage and neuronal damage due to cerebral vasospasm, and this secondary brain damage is one of the main causes of poor prognosis after subarachnoid hemorrhage. The incidence of secondary brain damage after subarachnoid hemorrhage is high; for example, the incidence of cerebral vasospasm is about two-thirds in imaging diagnosis and about one-third in symptomatic (symptom-associated) diagnosis.

[0004] Infiltration of inflammatory cells into the subarachnoid space has been reported as one of the mechanisms of secondary brain injury after the onset of subarachnoid hemorrhage (Non-Patent Documents 1 to 3). Specifically, it has been reported that inflammatory cells such as macrophages and neutrophils infiltrate the hemorrhagic focus in the subarachnoid space after the onset of subarachnoid hemorrhage and produce excessive inflammatory cytokines and reactive oxygen species, resulting in secondary brain injury such as ischemic brain injury and neuronal damage caused by cerebral vasospasm.

[0005] On the other hand, inflammatory cells, such as macrophages and neutrophils, are known to migrate toward chemokines, such as CCL2, present in lesions via the chemokine receptors CCR2 and CCR5 expressed on them, resulting in inflammatory responses. Yuya Terashima, one of the present inventors, identified FROUNT as an intracellular signaling regulator that binds to the chemokine receptors CCR2 and CCR5 and mediates their signaling (Non-Patent Documents 4 and 5). Furthermore, Yuya Terashima discovered that disulfiram, a known anti-alcohol drug, binds to FROUNT and inhibits its binding to CCR2 and CCR5, making it useful as a FROUNT inhibitor (Non-Patent Document 6). It has also been reported that disulfiram can inhibit the accumulation and activation of tumor-promoting macrophages through its FROUNT inhibitory activity, thereby suppressing cancer growth and metastasis (Non-Patent Document 6). Clinical studies are currently being conducted in Japan as a potential cancer treatment. However, there have been no reports on the effect of FROUNT on the infiltration of inflammatory cells into the subarachnoid space after the onset of subarachnoid hemorrhage.

[0006] Ikram A. et al., J Stroke Cerebrovasc Dis. 30 (11): 106064 (2021)Rass V. et al., Curr Neurol Neurosci Rep. 19 (10): 78 (2019)van Lieshout JH. et al., Neurosurg Rev. 41 (4): 917-930(2018)Takemura Y. et al. al., Nat Immunol. 6 (8): 827-835 (2005)Toda E. et al., J Immunol. 183 (10): 6387-6394 (2009)Terashima U. et al., Nat Commun. 11 (1): 609 (2020)

[0007] Prevention of secondary brain injury is effective in improving the prognosis of subarachnoid hemorrhage. Conventionally, therapeutic drugs such as fasudil hydrochloride and sodium ozagrel have been administered to prevent cerebral vasospasm after the onset of subarachnoid hemorrhage. However, the effectiveness of the therapeutic drugs currently used in clinical practice is insufficient, and the development of drugs that can prevent secondary brain injury after the onset of subarachnoid hemorrhage is desired.

[0008] Therefore, an object of the present disclosure is to provide a drug that can prevent secondary brain damage after the onset of subarachnoid hemorrhage.

[0009] The present inventors conducted extensive research to solve the above-mentioned problems and found that inflammatory cells infiltrate the subarachnoid space of subarachnoid hemorrhage model rats, including macrophages expressing FROUNT. Furthermore, the present inventors found that administering a FROUNT inhibitor that inhibits CCR2 and CCR5 signaling to subarachnoid hemorrhage model rats suppresses inflammatory cell infiltration into the subarachnoid space and increases survival rates. The present inventors also found that administering a FROUNT inhibitor to subarachnoid hemorrhage model rats suppresses neuronal death in the hippocampus, maintains the vascular diameter of the middle cerebral artery, and suppresses cerebral vasospasm. Based on these findings, the present inventors discovered that CCR2 and CCR5 inhibitors can be used as active ingredients for preventing secondary brain damage after subarachnoid hemorrhage.

[0010] That is, the present disclosure provides the following aspects of the invention. Item 1-1. A prophylactic agent for secondary brain damage after the onset of subarachnoid hemorrhage, comprising a CCR2 and CCR5 inhibitor. Item 1-2. The prophylactic agent according to Item 1-1, wherein the CCR2 and CCR5 inhibitor is a dual CCR2 and CCR5 inhibitor. Item 1-3. The prophylactic agent according to Item 1-2, wherein the dual CCR2 and CCR5 inhibitor is a FROUNT inhibitor. Item 1-4. The prophylactic agent according to Item 1-3, wherein the FROUNT inhibitor is at least one selected from the group consisting of disulfiram, a metal complex of diethyldithiocarbamate, a disulfide capable of generating diethyldithiocarbamate in vivo, a pharmaceutically acceptable salt thereof, and a solvate thereof. Item 1-5. The prophylactic agent according to any one of Items 1-1 to 1-4, wherein the secondary brain damage is neuronal damage or ischemic brain damage caused by cerebral vasospasm. Item 2-1. Use of a CCR2 and CCR5 inhibitor for the manufacture of a preventive drug for secondary brain damage after the onset of subarachnoid hemorrhage. Item 2-2. The use according to Item 2-1, wherein the CCR2 and CCR5 inhibitor is a dual inhibitor of CCR2 and CCR5. Item 2-3. The use according to Item 2-2, wherein the dual inhibitor of CCR2 and CCR5 is a FROUNT inhibitor. Item 2-4. The use according to Item 2-3, wherein the FROUNT inhibitor is at least one selected from the group consisting of disulfiram, a metal complex of diethyldithiocarbamate, a disulfide capable of generating diethyldithiocarbamate in vivo, a pharmaceutically acceptable salt thereof, and a solvate thereof. Item 2-5. The use according to any one of Items 2-1 to 2-4, wherein the secondary brain damage is neuronal damage or ischemic brain damage caused by cerebral vasospasm. Item 3-1. Item 3-2. A method for preventing secondary brain damage after the onset of subarachnoid hemorrhage, comprising administering an effective amount of a CCR2 and CCR5 inhibitor to a patient who has developed subarachnoid hemorrhage. Item 3-3. A method for preventing secondary brain damage after the onset of subarachnoid hemorrhage, comprising administering an effective amount of a CCR2 and CCR5 inhibitor to a patient who has developed subarachnoid hemorrhage. Item 3-4. A method for preventing secondary brain damage after the onset of subarachnoid hemorrhage, comprising administering an effective amount of a CCR2 and CCR5 inhibitor to a patient who has developed subarachnoid hemorrhage. Item 3-5. A method for preventing secondary brain damage after the onset of subarachnoid hemorrhage, comprising administering an effective amount of a CCR2 and CCR5 inhibitor to a patient who has developed subarachnoid hemorrhage. Item 3-6. A method for preventing secondary brain damage after the onset of subarachnoid hemorrhage, comprising administering an effective amount of a CCR2 and CCR5 inhibitor to a patient who has developed subarachnoid hemorrhage. Item 3-7. A method for preventing secondary brain damage after the onset of subarachnoid hemorrhage, comprising administering an effective amount of a CCR2 and CCR5 inhibitor to a patient who has developed subarachnoid hemorrhage.Item 3-4. The method for preventing according to Item 3-3, wherein the FROUNT inhibitor is at least one selected from the group consisting of disulfiram, a metal complex of diethyldithiocarbamate, a disulfide capable of generating diethyldithiocarbamate in vivo, a pharmaceutically acceptable salt thereof, and a solvate thereof. Item 3-5. The method for preventing according to any one of Items 3-1 to 3-4, wherein the secondary brain damage is neuronal damage or ischemic brain damage caused by cerebral vasospasm. Item 4-1. A CCR2 and CCR5 inhibitor used in a treatment method for preventing secondary brain damage after the onset of subarachnoid hemorrhage. Item 4-2. The CCR2 and CCR5 inhibitor according to Item 4-1, wherein the CCR2 and CCR5 inhibitor is a dual inhibitor of CCR2 and CCR5. Item 4-3. The CCR2 and CCR5 inhibitor according to Item 4-2, wherein the dual inhibitor of CCR2 and CCR5 is a FROUNT inhibitor. Item 4-4. The CCR2 and CCR5 inhibitor according to Item 4-3, wherein the FROUNT inhibitor is at least one selected from the group consisting of disulfiram, a metal complex of diethyldithiocarbamate, a disulfide capable of generating diethyldithiocarbamate in vivo, a pharmaceutically acceptable salt thereof, and a solvate thereof. Item 4-5. The CCR2 and CCR5 inhibitor according to any one of Items 4-1 to 4-4, wherein the secondary brain damage is neuronal damage or ischemic brain damage caused by cerebral vasospasm.

[0011] According to the preventive drug of the present disclosure, by using a CCR2 and CCR5 inhibitor such as a FROUNT inhibitor, it is possible to suppress the infiltration of inflammatory cells into the subarachnoid space after the onset of subarachnoid hemorrhage. Furthermore, the preventive drug of the present disclosure can prevent secondary brain damage by suppressing the infiltration of inflammatory cells into the subarachnoid space, thereby improving the prognosis of subarachnoid hemorrhage.

[0012] These are hematoxylin-eosin stained images of brain sections from a rat model of subarachnoid hemorrhage 24 hours after autologous blood infusion and from a rat that did not receive autologous blood infusion. In A, the lower image shows an enlargement of the area circled by the dotted line in the upper image. The asterisk in the upper image of A indicates the optic nerve region. In B, a further enlargement of the image of a rat model of subarachnoid hemorrhage shown in the lower image of A. In image B, cells with crescent-shaped nuclei (lobulated nuclei) are neutrophils, and round ones are erythrocytes (subarachnoid hemorrhage hematomas). These are images of brain sections from a rat model of subarachnoid hemorrhage 24 hours after autologous blood infusion, immunostained for CD68 and myeloperoxidase, and stained with DAPI. (A) Immunostained images for FROUNT and CD68 of brain sections from a rat model of subarachnoid hemorrhage 24 hours after autologous blood infusion, as well as a merged image of the immunostained and DAPI-stained images. (B) An enlarged image of the area enclosed by the dotted line in the merged image of (A). Coexpression of FROUNT and CD68 is observed in the cells marked with arrows in (B). (C) Immunostained images for CD68 and myeloperoxidase of brain sections from a rat model of subarachnoid hemorrhage treated with disulfiram (disulfiram group) or no drug (vehicle group). (D) Survival rates were measured up to 1 day after drug administration in rat models of subarachnoid hemorrhage treated with no drug (vehicle group), cyanamide (cyanamide group), disulfiram (disulfiram group), or DSF-41 (DSF-41 group). The figures show the results of measuring the number of apoptotic neurons in the hippocampus in subarachnoid hemorrhage model rats that were not administered a drug (vehicle group) or that were administered disulfiram (disulfiram group). The bars in the figures represent the mean ± standard error. The figures show the results of measuring the diameter of the middle cerebral artery in subarachnoid hemorrhage model rats that were not administered a drug (vehicle group) or that were administered disulfiram (disulfiram group). The bars in the figures represent the mean ± standard error.

[0013] 1. Terms Unless otherwise specified, terms used herein have the meanings that are commonly understood by those skilled in the art of medicine, pharmacology, molecular biology, microbiology, organic chemistry, etc. If a term defined herein does not have the same meaning as commonly understood, the description in this specification shall take precedence.

[0014] In the present disclosure, FROUNT is a CCR2 and CCR5 signal-promoting molecule, a cytoplasmic protein that binds to the intracellular C-terminal regions of the chemokine receptors CCR2 and CCR5. The amino acid sequence of FROUNT and the nucleotide sequence of the FROUNT gene are registered in the NCBI database GenBank under the accession numbers AF498261 and NM#024844.

[0015] In the present disclosure, CCR2 and CCR5 inhibitors are substances that can inhibit signal transduction mediated by the chemokine receptor CCR2 and signal transduction mediated by the chemokine receptor CCR5. CCR2 and CCR5 inhibitors include dual inhibitors that can inhibit both CCR2 and CCR5 with a single molecule, and combinations of CCR2 and CCR5 inhibitors. CCR2 and CCR5 inhibitors include not only CCR2 and CCR5 antagonists, but also FROUNT inhibitors that bind to FROUNT and co-inhibit the binding between FROUNT and CCR2 and between FROUNT and CCR5.

[0016] In the present disclosure, subarachnoid hemorrhage refers to a state in which blood flows into the subarachnoid space due to causes such as the rupture of a cerebral aneurysm.

[0017] In the present disclosure, secondary brain damage after the onset of subarachnoid hemorrhage refers to brain damage caused by factors such as increased intracranial pressure and hematoma after the onset of subarachnoid hemorrhage, and specifically includes nerve cell damage (damage to brain nerve cells, axonal damage, nerve cell death, etc.), ischemic brain damage caused by cerebral vasospasm (damage to brain nerve cells, axonal damage, nerve cell death, etc.), etc.

[0018] In the present disclosure, a preventive agent for secondary brain damage after the onset of subarachnoid hemorrhage is a drug that is administered after the onset of subarachnoid hemorrhage in order to prevent or delay the onset of secondary brain damage.

[0019] 2. Preventive Agent for Secondary Brain Injury After Subarachnoid Hemorrhage The preventive agent of the present disclosure is a preventive agent for secondary brain injury after subarachnoid hemorrhage, and is characterized by comprising an inhibitor of CCR2 and CCR5 as an active ingredient. The preventive agent of the present disclosure is described in detail below.

[0020] [CCR2 and CCR5 inhibitors] The prophylactic agent of the present disclosure contains CCR2 and CCR5 inhibitors as active ingredients.

[0021] In a preferred embodiment of the prophylactic agent of the present disclosure, a dual inhibitor of CCR2 and CCR5 is used as the CCR2 and CCR5 inhibitor. A preferred example of a dual inhibitor of CCR2 and CCR5 is a FROUNT inhibitor.

[0022] Specific examples of FROUNT inhibitors include disulfiram, metal complexes of diethyldithiocarbamate, disulfides capable of generating diethyldithiocarbamate in vivo, pharmaceutically acceptable salts thereof, and solvates thereof.

[0023] The structure of disulfiram (chemical name: tetraethylthiuram disulfide) is shown below in general formula (1).

[0024] The structure of diethyldithiocarbamate (DDC) is as shown in the following general formula (2). The diethyldithiocarbamate metal complex may be a complex of any metal. The metal may be monovalent or divalent or higher. One embodiment of the DDC metal complex is a complex of a divalent or higher metal. Specific examples of the metal complex include complexes of monovalent metals such as sodium complexes and lithium complexes; and complexes of divalent or higher metals such as copper complexes, iron(II) complexes, iron(III) complexes, zinc complexes, platinum complexes, gold complexes, aluminum complexes, magnesium complexes, vanadium complexes, selenium complexes, cobalt(II) complexes, and cobalt(III) complexes. These DDC metal complexes may be used alone or in combination of two or more.

[0025] Disulfides capable of generating DDC in vivo may be any disulfide capable of serving as a prodrug for DDC, including, for example, compounds that generate at least one molecule of DDC upon cleavage of the S—S bond of one molecule of the disulfide compound. An example of a disulfide capable of generating DDC in vivo is the compound shown in general formula (3) below. In general formula (3), the structural portion beyond the wavy line is omitted. The structural portion beyond the wavy line may be any structure as long as it does not prevent cleavage of the S—S bond. Specific examples of disulfides capable of generating DDC in vivo include DDC-adducted disulfide compounds formed by an S-S exchange reaction between a disulfide compound such as oxidized glutathione (Glutathione-SS-Glutathione) and disulfiram (compounds in which the structure at the end of the wavy line in general formula (3) is glutathione); and DDC-adducted proteins formed by an S-S exchange reaction between a protein having at least one pair of functional cysteine ​​residues, such as thioredoxin, and disulfiram (for example, compounds in which the structure at the end of the wavy line in general formula (3) is thioredoxin). These disulfides may be used singly or in combination of two or more.

[0026] Salts of the compounds (disulfiram, DDC metal complexes, and disulfides capable of generating DDC in vivo) are not particularly limited, as long as they are pharmaceutically acceptable, and may be either acid addition salts or base addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate; and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and paratoluenesulfonate. Examples of base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, and ammonium salt; and organic base salts such as triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt. These salts may be used alone or in combination of two or more.

[0027] Solvates of the compounds (disulfiram, metal complexes of DDC, and disulfides capable of generating DDC in vivo) and their salts are not particularly limited as long as they are pharmaceutically acceptable, and examples include hydrates, ethanolates, etc. These solvates may be used singly or in combination of two or more.

[0028] Another example of a FROUNT inhibitor is DSF-41 (CAS No. 20231-01-0). The structure of DSF-41 is shown in the following general formula (4).

[0029] Further, another example of the FROUNT inhibitor is a compound represented by the following general formula (5): In general formula (5), X 1 and X 2 are the same or different and each represents a halogen atom, preferably a chlorine atom or a bromine atom. In general formula (5), R represents an alkyl group having 1 to 5 carbon atoms, preferably a methyl group or an ethyl group.

[0030] Specific examples of dual inhibitors of CCR2 and CCR5 other than FROUNT inhibitors include cenicriviroc (CAS No. 497223-25-3), BMS-813160 (CAS No. 1286279-29-5), and TAK-779 (CAS No. 229005-80-5).

[0031] In another embodiment of the prophylactic agent of the present disclosure, a CCR2 inhibitor and a CCR5 inhibitor are used in combination as CCR2 and CCR5 inhibitors. Specific examples of CCR2 inhibitors include CCR2 Antagonist (CAS No. 445479-97-0), CCR2 Antagonist 1 (CAS No. 1683534-96-4), CCR2 Antagonist 4 (CAS No. 226226-39-7), INCB3344 (CAS No. 1262238-11-8), RS504393 (CAS No. 300816-15-3), and CCX-140B (CAS No. 1100318-47-5). Specific examples of CCR5 inhibitors include Aplaviroc (CAS No. 461443-59-4), Vicriviroc (CAS No. 541503-81-5), Maraviroc (CAS No. 376348-65-1), CCR5 Antagonist 1 (CAS No. 716354-86-8), and DAPTA (CAS No. 106362-34-9). When a CCR2 inhibitor and a CCR5 inhibitor are used in combination, the ratio of these inhibitors is determined based on the IC values ​​of the CCR2 inhibitor and the CCR5 inhibitor used. 50 It should be set appropriately taking into consideration the above.

[0032] In the prophylactic agent of the present disclosure, one type of CCR2 and CCR5 inhibitor may be used alone, or two or more types may be used in combination.

[0033] Among CCR2 and CCR5 inhibitors, preferred are dual CCR2 and CCR5 inhibitors, more preferred are FROUNT inhibitors, and even more preferred are disulfiram, metal complexes of DDC, disulfides capable of generating DDC in vivo, pharmaceutically acceptable salts thereof, and solvates thereof.

[0034] [Formulation] The prophylactic agent of the present disclosure is provided by preparing a pharmaceutical composition in a desired dosage form containing a CCR2 and CCR5 inhibitor and pharmaceutically acceptable carriers and / or additives. Examples of pharmaceutically acceptable carriers and / or additives include excipients, antioxidants, buffers, stabilizers, surfactants, chelating agents, binders, sterile water, physiological saline, etc. The pharmaceutical composition may be formulated by conventional methods depending on the dosage form, and the content of the CCR2 and CCR5 inhibitor in the pharmaceutical composition may be appropriately determined depending on the amount of the CCR2 and CCR5 inhibitor per dose.

[0035] [Target Animal] The target animal to which the prophylactic agent of the present disclosure is administered is not particularly limited and may be, for example, a mammal such as a human, mouse, rat, hamster, guinea pig, rabbit, cat, dog, goat, sheep, pig, cow, or monkey, but is preferably a human.

[0036] [Dosage and Administration] The prophylactic agent of the present disclosure is administered to patients who have developed subarachnoid hemorrhage. The prophylactic agent of the present disclosure inhibits the infiltration of inflammatory cells into the subarachnoid space after the onset of subarachnoid hemorrhage, thereby inhibiting the onset of secondary brain damage, and is therefore used to prevent secondary brain damage after the onset of subarachnoid hemorrhage. In addition, the prophylactic agent of the present disclosure can improve the survival rate and reduce sequelae after the onset of subarachnoid hemorrhage, and can therefore also be used as an agent for improving the prognosis of subarachnoid hemorrhage.

[0037] Among secondary brain injuries associated with subarachnoid hemorrhage, neuronal injury and cerebral vasospasm are known to be induced by infiltration of inflammatory cells into the subarachnoid space. Therefore, suitable examples of secondary brain injuries to be prevented by the prophylactic drug of the present disclosure include neuronal injury and ischemic brain injury caused by cerebral vasospasm.

[0038] The method of administration of the prophylactic agent of the present disclosure is not particularly limited, and examples include intrathecal administration, intravascular (intraarterial or intravenous) injection, continuous infusion, subcutaneous administration, intramuscular administration, enteral administration, intraperitoneal administration, topical administration, pulmonary administration (inhalation), oral administration, etc. Among these administration methods, a preferred example is intrathecal administration.

[0039] The prophylactic agent of the present disclosure is preferably administered promptly after the onset of subarachnoid hemorrhage, but if surgery is performed after the onset of subarachnoid hemorrhage, it may be administered before, during, or after surgery.

[0040] The dosage of the prophylactic agent of the present disclosure may be appropriately determined to an amount effective for preventing secondary brain injury depending on the age, body weight, severity of subarachnoid hemorrhage, type of active ingredient used, and other factors of the subject. For example, the dosage of a CCR2 and CCR5 inhibitor per adult may be appropriately set within a range of approximately 1 to 50 mg / kg, preferably approximately 5 to 30 mg / kg. The prophylactic agent of the present disclosure may be administered once, or may be administered continuously for a total of approximately four weeks, spaced one or several days apart. The above dosage range refers to the dosage when a dual CCR2 and CCR5 inhibitor is used, and refers to the dosage of each of the CCR2 inhibitor and CCR5 inhibitor when a CCR2 inhibitor and a CCR5 inhibitor are used in combination.

[0041] The present invention will be described in more detail below with reference to examples, but the present invention should not be construed as being limited to the following examples.

[0042] Test Example 1: Analysis of cells accumulating in the subarachnoid space early after subarachnoid hemorrhage 1. Test method Using an animal model of subarachnoid hemorrhage prepared by injecting autologous blood into the anterior chiasmatic cistern, cells accumulating in the subarachnoid space early after subarachnoid hemorrhage were analyzed. The specific test method is as follows.

[0043] Blood was collected from the femoral vein of male SD rats (9 weeks old). Then, with the rat's head fixed in a stereotaxic apparatus, a hole was drilled in the midline of the skull, 6.5 mm anterior to the bregma. A syringe needle was inserted stereotaxically into the hole, reaching the base of the skull. The needle was inserted with the tip pointing caudally and tilted at a 30-degree angle relative to the vertical. Next, 200 μl of the collected autologous blood was injected to induce subarachnoid hemorrhage. Twenty-four hours after autologous blood injection, the brain was harvested, and frozen sections of the brain subarachnoid space (the bleeding site) were prepared. Frozen sections were also prepared using the same procedure as above, except that autologous blood injection was omitted.

[0044] The frozen sections were stained with hematoxylin and eosin. Furthermore, the sections were immunostained for CD68, myeloperoxidase, and FROUNT, as well as stained with DAPI (4',6-diamidino-2-phenylindole). For immunostaining, primary antibodies were mouse anti-CD68 monoclonal antibody (#ab31630, Abcam, Cambridge, UK), rabbit anti-myeloperoxidase polyclonal antibody (#ab9535, Abcam), and mouse anti-FROUNT monoclonal antibody (prepared by Yuya Terashima, Institute of Life and Medical Sciences, Tokyo University of Science). Secondary antibodies were fluorescently labeled with each primary antibody. Detection was performed using a confocal microscope system (Olympus FV-3000).

[0045] 2. Test Results The results of hematoxylin and eosin staining of brain sections obtained from subarachnoid hemorrhage model rats and rats that did not receive autologous blood injection are shown in Figure 1. As a result, it was confirmed that in the subarachnoid hemorrhage model rats, inflammatory cells (neutrophils, etc.) infiltrated around the hematoma of subarachnoid hemorrhage early after subarachnoid hemorrhage.

[0046] Furthermore, the results of immunostaining for CD68 and myeloperoxidase, as well as DAPI staining, for brain sections obtained from a rat model of subarachnoid hemorrhage are shown in Figure 2. As a result, the presence of cells positive for CD68, a macrophage marker, and myeloperoxidase, a neutrophil marker, was observed in the subarachnoid space (site of hemorrhage), confirming the infiltration of macrophages and neutrophils into the subarachnoid space.

[0047] Furthermore, the results of immunostaining for FROUNT and CD68 in brain sections obtained from a rat model of subarachnoid hemorrhage are shown in Figure 3. As a result, cells co-expressing CD68 and FROUNT were observed in the subarachnoid space, demonstrating that macrophages expressing FROUNT had infiltrated the subarachnoid space.

[0048] Test Example 2: Verification of the effect of FROUNT inhibitor on secondary brain damage after subarachnoid hemorrhage 1. Test method Using an animal model of subarachnoid hemorrhage, the inhibitory effect of FROUNT inhibitor on secondary brain damage after subarachnoid hemorrhage was verified. The specific test method is as follows.

[0049] Blood was collected from the femoral vein of male SD rats (9 weeks old). Then, with the rat's head fixed in a stereotaxic apparatus, a hole was drilled in the midline of the skull 6.5 mm anterior to the bregma. A syringe needle was inserted stereotaxically into the drilled hole to the base of the skull. The needle was inserted with the tip pointing caudally and tilted at a 30-degree angle relative to the vertical. Next, 200 μl of the collected autologous blood was injected to induce subarachnoid hemorrhage. Immediately after the injection of the autologous blood, the test solutions listed in Table 1 were administered intrathecally. After administration of the test solutions, the rats were kept in a normal breeding environment.

[0050]

[0051] Brains were collected from one rat each in the vehicle and disulfiram groups 24 hours after administration of the test solution, and frozen sections of the subarachnoid space (hemorrhage site) were prepared. Immunostaining of CD68 and myeloperoxidase was performed on the frozen sections using the method described in Test Example 1 above.

[0052] The rats in each group were kept for one day after administration of the test solution, and the number of surviving and dead rats was counted to determine the survival rate.

[0053] 2. Test Results The results of immunostaining for CD68 and myeloperoxidase on frozen brain sections obtained from rats in the vehicle and disulfiram groups are shown in Figure 4. It was confirmed that in the subarachnoid hemorrhage model rats administered disulfiram, the infiltration of macrophages and neutrophils around the hematoma of subarachnoid hemorrhage was suppressed.

[0054] Table 2 shows the number of surviving and dead animals for each group up to one day after administration of the test solution, and Figure 5 shows the survival rate for each group up to one day after administration of the test solution. While the improvement in survival rate was limited in the subarachnoid hemorrhage model rats administered cyanamide, a significant improvement in survival rate was observed in the subarachnoid hemorrhage model rats administered the FROUNT inhibitors disulfiram or DSF-41. Like disulfiram, cyanamide is a compound that has aldehyde dehydrogenase inhibitory activity but does not have FROUNT inhibitory activity. These results demonstrate that the improvement in survival rate in the subarachnoid hemorrhage model rats is due to the inhibition of FROUNT.

[0055]

[0056] These results demonstrate that inhibiting FROUNT suppresses the infiltration of inflammatory cells (macrophages and neutrophils) into the subarachnoid space, which induces secondary brain injury after subarachnoid hemorrhage, and prevents secondary brain injury. Furthermore, because FROUNT inhibition results in dual inhibition of CCR2 and CCR5, these results also demonstrate that inhibiting CCR2 and CCR5 suppresses the infiltration of inflammatory cells into the subarachnoid space after subarachnoid hemorrhage and prevents secondary brain injury.

[0057] Test Example 3: Verification of the effect of FROUNT inhibitor on neuronal cell death in the hippocampus and middle cerebral artery after the onset of subarachnoid hemorrhage 1. Test method Using an animal model of subarachnoid hemorrhage, the inhibitory effect of FROUNT inhibitor on neuronal cell death in the hippocampus after the onset of subarachnoid hemorrhage was verified. The specific test method is as follows.

[0058] Blood was collected from the femoral vein of male SD rats (9 weeks old). Then, with the rat's head fixed in a stereotaxic apparatus, a hole was drilled in the midline of the skull 6.5 mm anterior to the bregma. A syringe needle was inserted stereotaxically into the drilled hole to the base of the skull. The needle was inserted with the tip pointing caudally and tilted at a 30-degree angle relative to the vertical. Next, 200 μl of the collected autologous blood was injected to induce subarachnoid hemorrhage. Immediately after the injection of the autologous blood, the test solutions listed in Table 3 were administered intrathecally. After administration of the test solutions, the rats were kept in a normal breeding environment.

[0059]

[0060] Twenty-four hours after administration of the test solution, the brains were harvested and 5-μm-thick coronal sections were prepared 4 mm posterior to the bregma. The number of apoptotic neurons in the hippocampus and the diameter of the middle cerebral artery were measured using the sections.

[0061] The number of apoptotic neurons in the hippocampus was determined by immunostaining for cleaved caspase-3, an apoptotic marker (n = 3 per group). Specifically, sections were blocked with 3% goat serum (#AB_2337258, Jackson ImmunoResearch, Baltimore, MD) in phosphate buffer, then incubated with a primary antibody against cleaved caspase-3 (rabbit monoclonal anti-cleaved caspase-3 antibody (#9661S, Cell Signaling Technology, Danvers, MA)) and a secondary antibody conjugated with Alexa Fluor 647-conjugated donkey anti-rabbit IgG H&L antibody (#A31573, Thermo Fisher Scientific, Waltham, MA). Images were then captured using a confocal microscope (FV3000, Olympus, Tokyo, Japan, or LSM880, Carl Zeiss, Göttingen, Germany). The number of cleaved caspase-3-positive cells in the hippocampus was then counted.

[0062] The diameter of the middle cerebral artery was measured using Elastica van Gieson staining (n = 4 per group). Specifically, the sections were stained with Elastica van Gieson staining, and images were captured using a microscope system. The diameter of the middle cerebral artery was then measured.

[0063] 2. Test Results Figure 6 shows the results of measuring the number of apoptotic neurons in the hippocampus, and Figure 7 shows the results of measuring the diameter of the middle cerebral artery. The number of apoptotic neurons in the hippocampus was lower in the disulfiram group than in the vehicle group, confirming that disulfiram administration inhibits hippocampal neuronal death after subarachnoid hemorrhage. Furthermore, the diameter of the middle cerebral artery was larger in the disulfiram group than in the vehicle group, confirming that disulfiram administration inhibits cerebral vasospasm after subarachnoid hemorrhage and maintains a large vascular diameter. These results demonstrate that inhibition of CCR2 and CCR5 can prevent neuronal damage after subarachnoid hemorrhage and ischemic brain damage caused by cerebral vasospasm.

[0064] The present disclosure is not limited in any way to the description of the embodiments and examples of the invention. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of the documents and the like shown in this specification are hereby incorporated by reference in their entirety.

Claims

1. A prophylactic agent for secondary brain injury after the onset of subarachnoid hemorrhage, comprising an inhibitor of CCR2 and CCR5.

2. The prophylactic agent according to claim 1, wherein the inhibitor of CCR2 and CCR5 is a dual inhibitor of CCR2 and CCR5.

3. The prophylactic agent according to claim 2, wherein the dual inhibitor of CCR2 and CCR5 is a FROUNT inhibitor.

4. The prophylactic agent according to claim 3, wherein the FROUNT inhibitor is at least one selected from the group consisting of disulfiram, a metal complex of diethyldithiocarbamate, a disulfide capable of generating diethyldithiocarbamate in vivo, pharmaceutically acceptable salts thereof, and solvates thereof.

5. The prophylactic agent according to claim 1 or 2, wherein the secondary brain injury is neuronal injury or ischemic brain injury caused by cerebral vasospasm.

6. Use of an inhibitor of CCR2 and CCR5 for the manufacture of a prophylactic agent for secondary brain injury after the onset of subarachnoid hemorrhage.

7. A method for preventing secondary brain injury after the onset of subarachnoid hemorrhage, comprising administering an effective amount of an inhibitor of CCR2 and CCR5 to a patient who has developed subarachnoid hemorrhage.

8. An inhibitor of CCR2 and CCR5, which is used in a treatment method for preventing secondary brain injury after the onset of subarachnoid hemorrhage.

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