Glioma therapeutic agent and method for suppressing proliferation of glioma cells

Abstract

Provided is a novel pharmaceutical agent for glioma.　This glioma therapeutic agent is characterized by comprising, as an active ingredient, a synaptic activator of an inhibitory neuron. This synaptic activator is, for instance, a GABA receptor agonist, and specifically, a benzodiazepine compound such as diazepam.

Description

Glioma therapeutic agent and method for inhibiting proliferation of glioma cells

[0001] The present invention relates to a therapeutic agent for glioma and a method for inhibiting the proliferation of glioma cells.

[0002] Gliomas are malignant brain tumors that arise from neuroepithelial cells. Because the five-year survival rate for brain tumors is extremely low, the establishment of effective treatment methods is urgently needed worldwide. Specifically, in current medical practice, brain tumors are typically treated with radiation therapy or chemotherapy after surgical resection (Non-Patent Document 1). However, these post-resection treatments have been reported to have significant side effects, such as brain dysfunction and the development of secondary tumors, and many patients experience brain tumor recurrence and drug resistance. Therefore, the development of more effective and safer chemotherapy is desired.

[0003] Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. Stupp R et al., N Engl J Med. 2005 Mar 10;352(10):987-96

[0004] Therefore, an object of the present invention is to provide a new drug for glioma.

[0005] The therapeutic agent for glioma of the present invention is characterized by containing, as an active ingredient, an agent for activating synapses of inhibitory neurons.

[0006] The method of the present invention for inhibiting the proliferation of glioma cells is characterized by comprising an activation step of activating synapses of inhibitory neurons in the brain of a subject.

[0007] The method of the present invention for inhibiting the proliferation of glioma cells is characterized by comprising an activation step of activating synapses of inhibitory neurons in vitro or ex vivo where the inhibitory neurons and glioma cells coexist.

[0008] The present invention can suppress the growth of glioma by activating synapses involving inhibitory neurons.

[0009] FIG. 1A is a fluorescent micrograph showing the presence of the tumorigenesis marker EGFP protein and the cell proliferation marker Ki67 protein in Example 1. FIG. 1B is a graph showing the fluorescence intensity of Ki67 protein in Ki67-positive cells in Example 1. FIG. 2 is a graph showing the survival curve (survival rate) of a mouse model of spontaneous glioblastoma in Example 1. FIG. 3 is a graph showing glioblastoma cell proliferation in the cerebrum of a mouse model of spontaneous glioblastoma in Example 2. FIG. 4 is a graph showing glioblastoma cell proliferation in the cerebrum of a mouse model of spontaneous glioblastoma in Example 3. FIG. 5 is a graph showing the survival curve (survival rate) of a mouse model of spontaneous glioblastoma in Example 4. FIG. 6 is a graph showing the time course of calcium activity in tumor cells in the cerebrum of a mouse model of spontaneous glioblastoma after administration of a NaCh expression vector or a control vector in Example 5. Figure 7 is a graph showing the quantitative results of calcium activity in cerebral tumor cells of a mouse model of spontaneous glioblastoma after administration of a NaCh expression vector or a control vector in Example 5. Figure 8 is a graph showing the time course of calcium activity in cerebral tumor cells of a mouse model of spontaneous glioblastoma after administration of diazepam or saline in Example 6. Figure 9 is a graph showing the quantitative results of calcium activity in cerebral tumor cells of a mouse model of spontaneous glioblastoma after administration of diazepam or saline in Example 6.

[0010] The present invention includes the following embodiments. [1] A therapeutic agent for glioma, comprising as an active ingredient a synapse activator for inhibitory neurons. [2] The therapeutic agent for glioma according to [1], wherein the activator is a GABA receptor agonist. [3] The therapeutic agent for glioma according to [1] or [2], wherein the activator is a benzodiazepine compound. [4] The therapeutic agent for glioma according to [3], wherein the benzodiazepine compound is diazepam, a salt thereof, or a solvate thereof. [5] The therapeutic agent for glioma according to [1], wherein the activator is an expression vector carrying a gene encoding a voltage-gated sodium channel. [6] The therapeutic agent for glioma according to [5], wherein the gene encoding the voltage-gated sodium channel is a gene derived from a bacterium of the genus Bacillus. [7] The therapeutic agent for glioma according to [6], wherein the bacterium of the genus Bacillus is Bacillus halodurand. [8] The glioma therapeutic agent according to [1], wherein the activator is a chemogenetic molecule. [9] The glioma therapeutic agent according to [8], wherein the activator is an expression vector carrying a gene encoding a modified M3 muscarinic acetylcholine receptor.

[10] The glioma therapeutic agent according to [8] or [9], further comprising clozapine-N-oxide.

[11] The glioma therapeutic agent according to any one of [5] to

[10] , wherein the expression vector carries the encoding gene under an enhancer, and the enhancer is an enhancer of a gene specifically expressed in parvalbumin-positive inhibitory neurons.

[12] The glioma therapeutic agent according to

[11] , wherein the enhancer is an enhancer of the Scn1a gene.

[13] The glioma therapeutic agent according to any one of [5] to

[12] , wherein the expression vector is an adeno-associated virus.

[14] A method for inhibiting glioma cell proliferation, comprising an activation step of activating synapses of inhibitory neurons in the brain of a subject.

[15] The growth-inhibiting method according to

[14] , wherein the activation step is a step of administering to the brain of a subject the therapeutic agent for glioma according to any one of [1] to

[13] .

[16] The growth-inhibiting method according to

[14] or

[15] , wherein the subject is a human or a non-human animal.

[17] A method for inhibiting the proliferation of glioma cells, comprising an activation step of activating synapses of inhibitory neurons in vitro or ex vivo where the inhibitory neurons and glioma cells coexist.

[0011] Unless otherwise specified, terms used in this specification can be used in the sense commonly used in the art.

[0012] As used herein, unless otherwise specified, the term "cell" includes at least one cell, and can refer to, for example, either a single cell or a cell population.

[0013] As used herein, unless otherwise specified, the term "cell population" includes at least one type of cell, and is not limited to, for example, one type of cell, but may also refer to two or more types of cells.

[0014] As used herein, the term "treatment" refers to, for example, treatment in the broad sense, and includes prevention in addition to treatment in the narrow sense. Treatment in the narrow sense includes, for example, curing a disease, alleviating a disease, or inhibiting the progression of a disease, and prevention of a disease includes, for example, preventing contraction of a disease, preventing the onset of a disease, preventing the recurrence of a disease, etc. Treatment or prevention of a disease can also be referred to as, for example, treatment or prevention of the symptoms of a disease.

[0015] As used herein, a subject is, for example, a living organism, and may be a human or a non-human animal, and may be a living organism that has developed a disease or a living organism that has not developed a disease (also referred to as a healthy organism). As used herein, the onset of a disease refers, for example, to the onset of a brain tumor, and is not limited to the presence or absence of other diseases. When the purpose is treatment in the narrow sense, the subject may be, for example, a patient (human) or a patient animal (non-human animal), and when the purpose is prevention, the subject may be, for example, a healthy individual (human) or a healthy non-human animal.

[0016] <Therapeutic Agent for Glioma> The therapeutic agent for glioma of the present invention is characterized by containing, as an active ingredient, a synapse activator for inhibitory neurons. The therapeutic agent for glioma of the present invention is characterized by containing the activator, and other components and conditions, etc. are not particularly limited.

[0017] The therapeutic agent for glioma of the present invention is a drug for glioma, a type of brain tumor. The brain, which is part of the central nervous system, contains neurons and glial cells (glial cells) that support them, and glioma is a malignant tumor that develops from these glial cells. Gliomas are also called gliomas because they are derived from glial cells. Examples of gliomas include adult diffuse gliomas, and specific examples include astrocytoma, IDH mutation, oligodendroglioma, IHD mutation and 1p / 19q codeletion, glioblastoma, and IDH wild-type.

[0018] As a result of extensive research, the present inventors discovered that the function of inhibitory neuron synapses is involved in gliomas arising from glial cells, leading to the completion of the present invention. Although the mechanism is unknown, it is believed that by activating inhibitory neuron synapses, the microenvironment around the glioma functions to suppress glioma growth. According to the present invention, activating inhibitory neuron synapses can suppress glioma growth. Furthermore, since the present invention can suppress glioma growth, it is also possible to extend the survival time of subjects with glioma, for example.

[0019] The central nervous system contains excitatory neurons and inhibitory neurons as neural cells. Inhibitory neurons contain GABA (γ-aminobutyric acid) and / or glycine as neurotransmitters and form synapses with postsynaptic cells. Synapses involving inhibitory neurons are known to function as follows: When a neurotransmitter is released from the presynaptic membrane of an inhibitory neuron and binds to a neurotransmitter receptor in the postsynaptic membrane of a postsynaptic cell, the neurotransmitter receptor is activated. This increases the Cl ion permeability of the postsynaptic membrane, increasing the negative charge within the cell, causing the cell to become hyperpolarized, suppressing the propagation of membrane potential and ultimately suppressing neural excitation. In the present invention, activation of the synapse of an inhibitory neuron refers to activation of the above function, and may be at any stage.

[0020] In the present invention, the synapse activator may be any agent that activates the synapses of inhibitory neurons. Specific examples of the synapse activator are listed below, but the present invention is not limited to these.

[0021] (1) Binding Promoter

[0033] Examples of the synapse activator include a binding promoter that enhances the binding between a neurotransmitter in an inhibitory neuron and its corresponding neurotransmitter receptor. The therapeutic agent for glioma of the present invention contains the binding promoter as an active ingredient, thereby promoting the binding of the neurotransmitter released from an inhibitory neuron to the neurotransmitter receptor in the postsynaptic cell.

[0022] The binding promoter includes, for example, a receptor agonist for a receptor to which a neurotransmitter binds. The inhibitory neurotransmitter GABA is an inhibitory substance that functions widely in the central nervous system, and its receptors include subtypes A, B, and C. The receptor agonist includes, for example, a GABA receptor agonist, and preferably a GABA receptor agonist. Ａ It is a receptor agonist

[0023] GABA receptors (e.g., GABA Ａ The benzodiazepine receptor (receptor) is an ion channel type and forms a complex with a benzodiazepine receptor and a Cl ion channel. Because of this complex, binding of a benzodiazepine compound to the benzodiazepine receptor promotes binding between the neurotransmitter (GABA) of the inhibitory neuron and the neurotransmitter receptor (GABA receptor) of the postsynaptic cell. Therefore, among GABA receptor agonists, for example, benzodiazepine compounds are preferred as the binding promoter.

[0024] The benzodiazepine compounds have been confirmed to be safe for humans and are inexpensive, and therefore, by using the benzodiazepine compounds as the synapse activator in the therapeutic agent for glioma of the present invention, it is possible to ensure greater safety and provide the agent at a lower cost.

[0025] The benzodiazepine compound is a compound having a benzodiazepine skeleton. The benzodiazepine compound is not particularly limited, and any compound that binds to the benzodiazepine receptor can be used, preferably in a pharmaceutically acceptable form. Examples of the benzodiazepine compound include diazepam, chlordiazepoxide, midazolam, cloxazolam, clonazepam, clobazam, salts thereof, and solvates thereof. The binding promoter is preferably, for example, diazepam, a salt thereof, or a solvate thereof, and more preferably diazepam.

[0026] The type of salt is not particularly limited, and examples thereof include alkali metal salts, alkaline earth metal salts, amphoteric element salts, amine salts, inorganic acid salts, organic acid salts, etc. Examples of the alkali metal salts include sodium salts and potassium salts, examples of the alkaline earth metal salts include magnesium salts and calcium salts, and examples of the amphoteric element salts include aluminum salts.

[0027] The type of solvate is not particularly limited, and may be a hydrate or a solvate other than water (e.g., an organic solvent solvate). The organic solvent is not particularly limited, and examples thereof include alcohols such as methanol and ethanol, acetone, ethyl acetate, dimethyl sulfoxide, and methylene chloride.

[0028] The binding promoter may be, for example, GABA or a GABA analog as the GABA receptor agonist, in addition to the benzodiazepine compound. GABA is a ligand substance that binds to the GABA receptor. Therefore, by artificially administering GABA, the binding between the GABA receptor and GABA can be more efficiently achieved. GABA and a GABA analog as the GABA receptor agonist in the present invention can also be considered, for example, as a drug for replacement therapy.

[0029] When the therapeutic agent for glioma of the present invention contains the binding promoter as the synapse activator, the type of the binding promoter may be, for example, one type, or two or more types may be used in combination.

[0030] (2) Voltage-gated sodium channel expression vector Voltage-gated sodium channels (hereinafter also referred to as NaCh) can be artificially expressed in the inhibitory neurons to activate the synapses (inhibitory synapses) of the inhibitory neurons.

[0031] The NaCh expression vector has, for example, a gene encoding the NaCh. The gene encoding the NaCh is inserted into, for example, the expression vector so as to enable expression of the NaCh. The gene encoding the NaCh is, for example, a microbial gene. NaCh derived from a microbial gene is hereinafter also referred to as NaChBac.

[0032] The coding gene is, for example, a coding gene derived from bacteria of the genus Bacillus or Arcobacter. Examples of bacteria of the genus Bacillus include Bacillus halodurand. Sequence information for the NaCh channel of Bacillus halodurand is registered, for example, in the database GenBank under accession number BAB05220 or in the plasmid bank Addgene under plasmid number Plasmid #60658. Examples of bacteria of the genus Arbobacter include Arcobacter butzleri.

[0033] The NaCh expression vector preferably has the coding gene under an enhancer, for example. This can further promote transcription of the coding gene. As used herein, "under an enhancer" means under the control of an enhancer (the same applies hereinafter). The position of the enhancer in the NaCh expression vector is not particularly limited. The position of the enhancer may be any position in the NaCh expression vector that can control transcription of the coding gene, and may be, for example, upstream or downstream of a promoter for the coding gene.

[0034] The enhancer is preferably, for example, an enhancer of a gene specifically expressed in inhibitory neurons. By using such an enhancer, for example, even when the NaCh expression vector is introduced into various cells, transcription of the coding gene can be promoted specifically in inhibitory neurons. Furthermore, the expression of NaCh specific to the inhibitory neurons excites the inhibitory neurons and activates the inhibitory synapses of the inhibitory neurons. The origin of the enhancer sequence is not particularly limited, and is preferably the same as that of the subject to which the glioma therapeutic agent of the present invention is administered. When administered to humans, the enhancer sequence is preferably human-derived.

[0035] As a specific example, the gene specifically expressed in inhibitory neurons is the Scn1a gene, and the enhancer is enhancer E2 of the Scn1a gene. The Scn1a gene is known as a gene encoding a voltage-gated sodium channel that is specifically expressed in parvalbumin-positive (PV+) inhibitory neurons, which account for the majority of inhibitory neurons in the cerebral cortex. Enhancer E2 may be an enhancer of the Scn1a gene of a non-human animal such as a mouse, or an enhancer of the human Scn1a gene. Sequence information for enhancer E2 of the mouse Scn1a gene is registered, for example, in the plasmid bank addgene under plasmid number Plasmid #135630. Sequence information for enhancer E2 of the human Scn1a gene is registered in the genome browser Ensembl under the number #ENSG00000144285, for example.

[0036] The type of the expression vector is not particularly limited, and examples thereof include viral vectors, plasmid vectors, etc. Examples of the viral vector include adenoviral vectors, adeno-associated viral vectors (AAV), lentiviral vectors, retroviral vectors, Sendai viral vectors, etc. Among these, AAV is preferred because it has a proven track record as a vector for approved therapeutic drugs.

[0037] The constituent units of the sequences (e.g., the coding gene and enhancer) inserted into the expression vector are, for example, nucleotides, specific examples of which include ribonucleotides and deoxyribonucleotides. The coding sequence may be, for example, a ribonucleotide nucleic acid (ribonucleic acid, RNA), a deoxyribonucleotide nucleic acid (deoxyribonucleic acid, DNA), or a chimeric nucleic acid of RNA and DNA. When the coding sequence is RNA, it is, for example, pre-mRNA or mRNA, preferably mRNA. When the coding sequence is DNA, it is preferably cDNA. The coding gene is preferably cDNA. The nucleotides may be, for example, unmodified or modified. The constituent units of the coding sequence may be, for example, natural nucleic acids, artificial nucleic acids, or both. Examples of the artificial nucleic acids include LNA (locked nucleic acid).

[0038] (3) Receptor Expression Vector By artificially expressing the receptor in the inhibitory neuron, the synapse of the inhibitory neuron (inhibitory synapse) can be activated.

[0039] The receptor expression vector has, for example, a gene encoding a receptor. The encoding gene is inserted into the expression vector so as to enable expression of the receptor.

[0040] The receptor can be, for example, a chemogenetic molecule. Chemogenetics refers to, for example, a method of controlling cell function using a compound. A chemogenetic molecule is also called a chemogenetics-related molecule related to chemogenetics, and refers to, for example, an artificially designed molecule that can bind to a specific compound and control cell function through this binding.

[0041] As the chemogenetic molecule, for example, designer receptors (DREADD: Designer Receptors Exclusively Activated by Designer Drugs) that are activated only by designer drugs can be used. The designer drugs are also called, for example, "DREADD ligands" or "DREADD agonists." The DREADD is preferably, for example, G-protein, and examples thereof include modified receptors obtained by modifying an acetylcholine receptor for the neurotransmitter acetylcholine. Examples of the modified receptor include modified muscarinic acetylcholine receptors, and modified forms of human acetylcholine receptors are preferred. Specific examples include modified M3 muscarinic acetylcholine receptors (hereinafter also referred to as M3Dq) and modified M1 muscarinic acetylcholine receptors (hereinafter also referred to as M1Dq). M3Dq is a receptor that does not respond to acetylcholine but responds to, for example, clozapine-N-oxide (CNO). CNO is pharmacologically inactive and is a type of designer drug (DREADD ligand or DREADD agonist), also known as a muscarinic DREADD agonist. M3Dq is preferably human M3Dq (hM3Dq), a modified human acetylcholine receptor. The sequence information for the coding gene for human M3Dq is registered, for example, in the plasmid bank addgene under plasmid number Plasmid #50474.

[0042] Other examples of the modified muscarinic acetylcholine receptor include modified M1 muscarinic acetylcholine receptors (hereinafter also referred to as M1Dq), and a specific example that is preferred is human M1Dq (hM1Dq), which is a modified human acetylcholine receptor.

[0043] Other examples of the chemogenetic molecules include ultrapotent pharmacologically selective actuator modules (uPSEMs), pharmacologically selective actuator modules (PSAMs)-5HT3 (serotonin receptor 3), and the like.

[0044] The receptor expression vector preferably has the coding gene under an enhancer, for example. This can further promote transcription of the coding gene. The position of the enhancer in the receptor expression vector is not particularly limited. The enhancer may be located at any position in the receptor expression vector that can control transcription of the coding gene, and may be located, for example, upstream or downstream of a promoter for the coding gene.

[0045] The enhancer is preferably, for example, an enhancer of a gene that is specifically expressed in inhibitory neurons. By using such an enhancer, for example, even when the receptor expression vector is introduced into various cells, it is possible to promote the transcription of the coding gene specifically in inhibitory neurons. The origin of the enhancer sequence is not particularly limited, and for example, it is preferably the same as that of the subject to which the glioma therapeutic agent of the present invention is administered, and when administered to humans, it is preferably a human-derived enhancer sequence.

[0046] As a specific example, the specifically expressed gene may be the Scn1a gene, and the enhancer may be enhancer E2 of the Scn1a gene. The enhancer of the Scn1a gene may be the same as that described in the NaCh expression vector in (2) above.

[0047] The type of the expression vector is not particularly limited, and for example, the description of the NaCh expression vector in (2) above can be cited, and AAV is preferred.

[0048] The constituent units of the sequences (for example, the coding gene and enhancer) to be inserted into the expression vector can be those described in the NaCh expression vector in (2) above.

[0049] The therapeutic agent for glioma of the present invention may contain, for example, only the synapse activator as an active ingredient, or may contain other active ingredients in addition to the synapse activator. The therapeutic agent for glioma of the present invention may contain, for example, only the binding promoter (1) as the synapse activator, only the NaCh expression vector (2), only the receptor expression vector (3), or a combination of any two of (1), (2), and (3), or may contain all of them.

[0050] The glioma therapeutic agent of the present invention may contain, for example, only the active ingredient, or may further contain other additives in addition to the active ingredient. The additive is preferably, for example, a pharmaceutically acceptable substance. The type of additive is not particularly limited, and examples include carriers, excipients, stabilizers, lubricants, preservatives, suspending agents, dispersants, thickeners, pH adjusters, and antifoaming agents. Therefore, some embodiments of the present invention also relate to pharmaceutical compositions for use in the treatment of glioma, comprising the glioma therapeutic agent of the present invention and pharmaceutically acceptable additives (e.g., carriers and / or excipients).

[0051] In the glioma therapeutic agent of the present invention, the content of the synapse activator is not particularly limited and can be appropriately determined depending on, for example, the purpose of administration, the presence or absence of a disease, the severity of the disease, the type, age, and sex of the subject, etc.

[0052] The administration method of the glioma therapeutic agent of the present invention is not particularly limited and can be appropriately determined depending on, for example, the type of synapse activator, the purpose of administration, the presence or absence of disease, the severity of disease, the type, age, and sex of the subject. Examples of administration methods include oral administration and parenteral administration. Examples of parenteral administration include local, intracerebral, intraventricular, intrathecal, nasal, transdermal, subcutaneous, intravenous, intraarterial, intraperitoneal, and intestinal administration. The binding promoter of (1) is preferably administered orally or intravenously, and the expression vectors of (2) and (3) are preferably administered orally, intravenously, or intracerebrally (e.g., locally in the brain).

[0053] When the synapse activator is the (1) binding promoter, the administration conditions of the therapeutic agent for glioma of the present invention can be expressed, for example, as the administration conditions of the binding promoter. The administration conditions of the therapeutic agent for glioma of the present invention are not particularly limited.

[0054] When the synapse activator is the (2) NaCh expression vector, the administration conditions of the therapeutic agent for glioma of the present invention can be expressed, for example, as the administration conditions of the NaCh expression vector. The administration conditions of the therapeutic agent for glioma of the present invention are not particularly limited.

[0055] When the synapse activator is the receptor expression vector (3), the administration conditions of the therapeutic agent for glioma of the present invention can be expressed, for example, as the administration conditions of the receptor expression vector. The administration conditions of the therapeutic agent for glioma of the present invention are not particularly limited.

[0056] When using a DREADD expression vector expressing the DREADD among the receptor expression vectors, it is preferable to further use the DREADD ligand in combination. In this case, the glioma therapeutic agent of the present invention can also be referred to as a glioma treatment kit, for example, having the DREADD expression vector and the DREADD ligand separately. When the DREADD expression vector is the modified muscarinic acetylcholine receptor expression vector, the DREADD ligand is, for example, the muscarinic DREADD agonist. As a specific example, when the modified M3 muscarinic acetylcholine receptor (M3Dq) expression vector is used, it is preferable to further use CNO, which binds to the M3Dq. Administration of CNO binds to the M3Dq expressed in the cells, increasing the intracellular calcium ion concentration and activating electrical activity. Furthermore, since M3Dq specifically binds to CNO but does not bind to, for example, acetylcholine, it is also possible to control the binding between M3Dq and CNO in cells by controlling the administration of CNO.

[0057] The M3Dq expression vector and CNO may be administered simultaneously or separately, and the administration times may be the same or different. When the M3Dq expression vector and CNO are administered separately, the glioma therapeutic agent of the present invention can also be referred to as a glioma treatment kit, as described above, containing the M3Dq expression vector and CNO separately. The same applies to other DREADD expression vectors and DREADD ligands.

[0058] The conditions for administering CNO are not particularly limited. For example, CNO is preferably administered after the administration of the M3Dq expression vector. The same applies to other DREADD ligands.

[0059] The subject to which the therapeutic agent for glioma of the present invention is administered may be, for example, a human or a non-human animal, preferably a human. The non-human animal is not particularly limited and may be, for example, a non-human mammal such as a mouse, rat, rabbit, horse, cat, dog, or monkey.

[0060] The glioma therapeutic agent of the present invention can, for example, inhibit the growth of glioma, and therefore can also be called a glioma inhibitor. Therefore, the glioma therapeutic agent of the present invention can be used in vivo as a therapeutic agent for a living body, for example, or can be used in vitro on cells or ex vivo on tissues or organs. Furthermore, the glioma therapeutic agent of the present invention can be used in vitro or ex vivo for purposes other than treatment, for example.

[0061] <Method for inhibiting proliferation of glioma cells> The method for inhibiting proliferation of glioma cells of the present invention includes a first method for use in a living body and a second method for use in a non-living body.

[0062] As described above, the first method for inhibiting glioma cell proliferation of the present invention is characterized by including an activation step of activating synapses of inhibitory neurons in the brain of a subject. The method for inhibiting glioma cell proliferation of the present invention is also referred to as a method for inhibiting glioma proliferation. The method for inhibiting glioma cell proliferation of the present invention is characterized by activating synapses of inhibitory neurons, and other configurations and conditions, etc., are not particularly limited.

[0063] In the first proliferation-inhibiting method of the present invention, the activation of synapses of inhibitory neurons is not particularly limited, and any stage of activation may be used as long as the aforementioned function of synapses of inhibitory neurons is activated. In the proliferation-inhibiting method of the present invention, the activation step of activating synapses of inhibitory neurons may be, for example, a step of administering the glioma therapeutic agent of the present invention to the brain of a subject. With regard to the administration of the glioma therapeutic agent of the present invention, the description of the administration conditions for the glioma therapeutic agent of the present invention can be used.

[0064] A specific example of the activation is, for example, enhancement of the binding between a neurotransmitter released from an inhibitory neuron and its corresponding neurotransmitter receptor. In this case, the therapeutic agent for glioma of the present invention preferably contains, for example, the binding promoter of (1) above as the synapse activator.

[0065] A specific example of the activation is the enhancement of inhibitory neurons or synaptic transmission by inhibitory neurons by expressing the voltage-gated sodium channel (NaCh). In this case, the therapeutic agent for glioma of the present invention preferably contains, for example, the NaCh expression vector of (2) above as the synapse activator.

[0066] Furthermore, a specific example of the activation is, for example, the enhancement of inhibitory neurons or synaptic transmission by inhibitory neurons by expressing the receptor. In this case, the therapeutic agent for glioma of the present invention preferably uses, for example, a therapeutic agent containing the receptor expression vector of (3) as the synapse activator. Furthermore, when the DREADD expression vector (e.g., the M3Dq expression vector) is used as the receptor expression vector, it is preferable to use the DREADD ligand (e.g., CNO) in combination, as described above.

[0067] In the first growth-inhibiting method of the present invention, the subject is, for example, a human or a non-human animal, preferably a human. The above-mentioned descriptions of the non-human animal may also be used. The growth-inhibiting method of the present invention is also referred to as a method for treating glioma. Accordingly, some embodiments of the present invention relate to, for example, a method for treating glioma in a subject in need of treatment. The method includes administering to the subject a compound that activates inhibitory neuronal synapses in the brain of the subject. Here, the compound may be a glioma therapeutic agent described herein. Furthermore, some embodiments of the present invention relate to, for example, a method for treating glioma in a subject in need of treatment. The method includes administering to the subject an expression vector encoding DREADD and administering to the subject a designer drug (DREADD ligand) that activates DREADD, wherein the DREADD activated by the DREADD ligand activates inhibitory neuronal synapses in the brain of the subject. Furthermore, some embodiments of the present invention relate to a method for treating glioma, for example, in a subject in need thereof, wherein inhibitory neurons of the subject have been previously modified to express DREADD, and the method comprises administering a DREADD ligand to the subject.

[0068] Next, the second method of the present invention for inhibiting the proliferation of glioma cells is characterized by including, as described above, an activation step of activating the synapses of inhibitory neurons in vitro or ex vivo where inhibitory neurons and glioma cells coexist.

[0069] Specific examples of the activation are not particularly limited, and include the same means as those in the first proliferation-inhibiting method, and the description of the first proliferation-inhibiting method can be cited.

[0070] In the second proliferation-inhibiting method of the present invention, "in vitro where inhibitory neurons and glioma cells coexist" refers to, for example, a state in which inhibitory neurons and glioma cells are cultured in the same medium. The inhibitory neurons may be, for example, cells isolated from a living body or a passaged cell line. The glioma cells may be, for example, cells isolated from a living body or a passaged cell line.

[0071] In this case, the activation step may include, for example, culturing a mixture of inhibitory neurons and glioma cells in a medium containing the glioma therapeutic agent of the present invention (the glioma inhibitor). The seeding amount of each cell type in the medium and the amount of the glioma therapeutic agent added to the medium are not particularly limited. The medium and culture conditions are not particularly limited, and general medium and culture conditions for neurons can be used.

[0072] In the second growth-inhibiting method of the present invention, the "ex vivo location where inhibitory neurons and glioma cells coexist" refers to, for example, a tissue or organ containing glioma isolated from the brain of a living organism. The tissue is brain tissue containing glioma (also called a glioma mass), and the organ is a brain containing glioma.

[0073] In this case, the activation step may include, for example, culturing the tissue or organ in a medium containing the therapeutic agent for glioma.

[0074] <Uses> The present invention relates to a benzodiazepine compound for use in the treatment of glioma. The present invention relates to the use of a benzodiazepine compound in the manufacture of a therapeutic agent for glioma. The same description as for the therapeutic agent for glioma of the present invention can be used for the benzodiazepine compound.

[0075] The present invention relates to a voltage-gated sodium channel expression vector for use in the treatment of glioma. The present invention relates to use of a voltage-gated sodium channel expression vector in the manufacture of a therapeutic agent for glioma. The same description as for the therapeutic agent for glioma of the present invention can be applied to the benzodiazepine compound.

[0076] The present invention relates to a modified M3 muscarinic acetylcholine receptor expression vector for use in the treatment of glioma. The present invention also relates to the use of a modified M3 muscarinic acetylcholine receptor in the manufacture of a therapeutic agent for glioma. The modified M3 muscarinic acetylcholine receptor can be used in conjunction with the description of the therapeutic agent for glioma of the present invention.

[0077] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0078] Example 1 The effect of artificially expressing NaCh in inhibitory neurons in the cerebrum of a mouse in which glioblastoma was induced on glioblastoma was confirmed.

[0079] (1) Glioblastoma Cell Proliferation Rosa26-LSL-EGFP mice (5-10 weeks old, JAX) were used (n = 3 / group). These mice contained a transcriptional repressor sequence flanked by loxP sequences and an EGFP gene encoding green fluorescent protein knocked into the Rosa26 locus. Glioblastoma-inducing sgRNA, SpCas9, and Cre genes were injected into the cerebral ventricles of the mice by electroporation, and the genes were introduced into cells in the ventricular zone. The sgRNA used knocked out the Trp53, Nf1, and Pten genes (Trp53 / Nf1 / Pten) (Zuckermann et al. 2015 Nat Commun). The administration conditions were as follows: A mixture of sgRNA and SpCas9 gene for the Trp53 gene, a mixture of sgRNA and SpCas9 gene for the Nf1 gene, and a mixture of sgRNA and SpCas9 gene for the Pten gene were prepared (0.25 μl each). The final concentrations of the sgRNA and gene in the three mixtures were 1 μg / μl. Then, a total of 0.75 μl of each of the 0.25 μl was injected as described above.

[0080] The mice were grown for 35 to 70 days, and glioblastoma was induced in the cerebrum to obtain a spontaneous glioblastoma mouse model (hereinafter also referred to as a mouse model). The mouse model is also referred to as a model mouse.

[0081] A NaCh expression vector carrying the mCherry gene as a fluorescent marker was administered to the same site of the cerebrum of the mouse model using a syringe. AAV was used as the expression vector, and the NaCh gene was inserted under enhancer E2. The enhancer E2 was the enhancer E2 of the mouse Scn1a gene, and the NaCh gene was the NaChBac gene derived from Bacillus halodurans. The administration conditions were as follows: 1 μl of the NaCh expression vector (concentration 1×10 １０ A single dose of 100 mg / ml was administered.

[0082] The mouse model (E2-NaChBac mouse) administered with the NaCh expression vector was used as an example mouse model (n=3). The example mouse model was further grown, and 21 days after administration of the NaCh expression vector, glioblastoma cell proliferation was confirmed.

[0083] As a control, a control vector without the NaCh gene inserted downstream of the enhancer E2 was used instead of the NaCh expression vector. The mouse model was treated in the same manner as above, except for the use of the control vector, to prepare a control mouse model (n=3). Cell proliferation was then confirmed for the control mouse model in the same manner as for the mouse model of the example.

[0084] Glioblastoma cell proliferation was assessed by analyzing Ki67 protein, a cell proliferation marker (also known as a cell proliferation potential marker). Specifically, the procedure was as follows. Twenty-one days after administration of the vector (the NaCh expression vector or the control vector), the mouse model was fixed with paraformaldehyde, and the cerebral cortex containing tumor tissue was excised. Brain slices were prepared from the cerebral cortex, and Ki67 protein was visualized by immunohistochemical staining using an anti-Ki67 antibody to detect Ki67-positive cells. Furthermore, the fluorescence intensity of Ki67 protein was measured for each Ki67-positive cell in the brain slice.

[0085] Figure 1 shows the results of glioblastoma cell proliferation in the cerebrum of a mouse model of spontaneous glioblastoma. First, Figure 1A is a fluorescent micrograph showing the presence of the tumorigenesis marker EGFP protein and the cell proliferation marker Ki67 protein in a brain slice prepared from the cerebral cortex of the mouse model 21 days after vector administration. In Figure 1A, the left column is a photograph of EGFP protein, the right column is a photograph of Ki67 protein, the top row is the result of the control mouse model, and the bottom row is the result of the mouse model of the example (E2-NaCh).

[0086] As shown in the left column of Figure 1A, when the control and the example (E2-NaCh) were compared for EGFP fluorescence intensity in EGFP-positive cells (i.e., tumor cells), the results showed that they showed almost the same fluorescence intensity. Also, as shown in the right column of Figure 1A, when the control and the example (E2-NaCh) were compared for fluorescence intensity of Ki67 protein, a cell proliferation marker in tumor cells, the results showed that the fluorescence intensity of the example was lower than that of the control.

[0087] 1B is a graph showing the fluorescence intensity of Ki67 protein in Ki67-positive cells in brain slices prepared from the cerebral cortex of the mouse model 21 days after vector administration. As shown in FIG. 1B, the fluorescence intensity of the cell proliferation marker Ki67 protein was significantly reduced in the Ki67-positive cells of the example mouse model administered with the NaCh expression vector, compared to the Ki67-positive cells of the control mouse model not administered with the NaCh expression vector.

[0088] (2) Glioblastoma Tumor Progression Rate In the same manner as in (1) above, an example mouse model (E2-NaChBac, n=20) administered with the NaCh expression vector and a control mouse model (n=18) administered with the control vector were grown, and the tumor progression rate of glioblastoma was confirmed. The tumor progression rate of glioblastoma was analyzed as a mouse survival curve. Specifically, this was done as follows. That is, after the treatment (after the administration of the vector), the mouse model was maintained in a mouse breeding room until neurological symptoms (gait abnormalities, head enlargement, or weight loss) were observed. The mouse models in which symptoms were observed were then treated with CO ２ After euthanasia, the whole brain was collected and tumor formation was confirmed under a microscope using EGFP as an indicator. For mouse models in which tumors were confirmed, the postnatal days were calculated and survival curves were created.

[0089] The survival curve (survival rate) of the spontaneous glioblastoma mouse model is shown in Figure 2. As shown in Figure 2, the survival rate of the example mouse model administered with the NaCh expression vector was significantly higher than that of the control mouse model.

[0090] The E2 enhancer of the NaCh expression vector functions specifically in GABAergic inhibitory neurons, allowing NaCh to be expressed specifically in the inhibitory neurons, thereby specifically activating the inhibitory neurons. This example demonstrates that activation of the inhibitory neurons suppresses glioblastoma cell proliferation and improves the survival rate of glioblastoma mice.

[0091] Example 2 The effect of artificially expressing the DREADD in inhibitory neurons in the cerebrum of a mouse transplanted with human glioblastoma on glioblastoma was confirmed.

[0092] NSG mice (JAX) aged 2.5 to 3 months were used (n=3-5). A DREADD expression vector (M3Dq) carrying the mCherry gene encoding a red fluorescent protein was administered to the somatosensory cortex (3 mm deep from the surface) of the mouse cerebrum using a Hamilton syringe. Specifically, AAV was used as the expression vector (M3Dq), and the DREADD gene was inserted downstream of enhancer E2. Enhancer E2 was that of the mouse Scn1a gene, and the DREADD gene was the human M3Dq gene. The administration conditions were as follows: 2 μl of the expression vector (concentration 1×10 １０ The dose was 100 mg / ml.

[0093] The mice (DREADD mice) administered with the DREADD expression vector (M3Dq) were grown for one week, and then transplanted with human glioblastoma to create a glioblastoma-transplanted mouse model (hereinafter also referred to as a mouse model). Specifically, Greenfire-expressing PDXs were administered to the same site of the mouse's cerebrum using a syringe. The Greenfire-expressing PDXs are human grade 4 glioblastoma cells that express the EGFP gene and the Luciferase gene. This allows visualization of the transplanted human cells using EGFP and non-invasive intraviral imaging by administering Luciferin. The administration conditions were as follows: 4 μl (5 × 10 ５ Therefore, this study was used to confirm whether the results obtained in the mouse tumors of Example 1 would have the same effect in human subjects.

[0094] The mouse model was grown for an additional 5-6 weeks to allow glioblastoma to develop in the cerebrum, after which the DREADD ligand CNO was administered intraperitoneally. The administration conditions were as follows: Administration period: 13 hours and 3 hours before tumor collection; Number of administrations: 2; Amount of CNO per administration: 2 μg per gram of mouse body weight. 13 hours after the first CNO administration, glioblastoma cell proliferation in the cerebrum of the mouse model was confirmed (Example 2-1: E2_M3Dq_CNO).

[0095] In Example 2-2 (E2_NaChBac), the NaChBac expression vector in Example 1 was administered to the NSG mice instead of the DREADD expression vector (M3Dq). A glioblastoma-implanted mouse model (hereinafter also referred to as a mouse model) was prepared in the same manner as in Example 2-1. Glioblastoma cell proliferation in the cerebrum was confirmed in the same manner as in Example 2-1, except that the mouse model was not administered CNO.

[0096] Additionally, the following two mouse strains were prepared as controls, and cell proliferation was similarly confirmed. That is, for Control 2-1 (E2_RFP), the control vector in Example 1 was administered to the NSG mice instead of the DREADD expression vector (M3Dq), and CNO was not administered. Otherwise, the mouse model was prepared and cell proliferation was confirmed in the same manner as in Example 2-1. For Control 2-2 (E2_M3Dq_noCNO), the NSG mice were administered the DREADD expression vector (M3Dq), but CNO was not administered. Otherwise, the mouse model was prepared and cell proliferation was confirmed in the same manner as in Example 2-1.

[0097] Glioblastoma cell proliferation was measured by analyzing Ki67 protein, a cell proliferation marker, in the same manner as in Example 1 above.

[0098] Figure 3 shows the results of glioblastoma cell proliferation in the cerebrum of a glioblastoma-implanted mouse model. Specifically, Figure 3 is a graph showing the percentage of cells positive for the cell proliferation marker Ki67 protein in the tumor of collected cerebral cortical tissue, in the area projected by neural cells infected with the viral vector (mCherry positive). Compared to the two controls on the left (E2_RFP, E2_M3Dq_noCNO), the two examples on the right (E2_M3Dq_CNO, E2_NaChBac) significantly reduced the percentage of cells positive for the cell proliferation marker Ki67 protein in the tumor. In other words, it was found that glioblastoma proliferation could be suppressed by expressing M3Dq in inhibitory neurons and then activating them by administering CNO, or by constitutively activating them by expressing NaChBac in inhibitory neurons.

[0099] The E2 enhancer of the DREADD expression vector functions specifically in GABAergic inhibitory neurons. Therefore, by administering the vector to the cerebrum of a glioblastoma-bearing subject, DREADD can be expressed specifically in the inhibitory neurons. Furthermore, by administering the DREADD ligand CNO, the inhibitory neurons can be specifically activated. It was found that activation of the inhibitory neurons in this way suppresses glioblastoma cell proliferation.

[0100] Example 3 Diazepam was administered to mice in which glioblastoma was induced, and the effect on glioblastoma was confirmed.

[0101] A spontaneous glioblastoma mouse model was prepared in the same manner as in Example 1. Diazepam was then administered intraperitoneally to the mouse model using a syringe. The administration conditions were as follows: Administration period: 7 weeks to 14 days after birth Number of times: twice daily Amount of diazepam per administration: 10 μg per gram of mouse body weight

[0102] After 14 days of administration, glioblastoma cell proliferation in the cerebral cortex of the mouse model was confirmed by analyzing Ki67 protein, a cell proliferation marker, in the same manner as in Example 1.

[0103] As a control (n=3), physiological saline was administered to the mouse model instead of diazepam, and cell proliferation was confirmed in the same manner as above.

[0104] Figure 4 shows the results of glioblastoma cell proliferation in the cerebrum of a mouse model of spontaneous glioblastoma. Figure 4 is a graph showing the percentage of cells positive for the cell proliferation marker Ki67 protein among tumor cells in the collected cerebral cortex tissue. As shown in Figure 4, administration of diazepam significantly reduced the percentage of cells positive for the cell proliferation marker Ki67 protein compared to the control.

[0105] Diazepam can specifically activate these inhibitory neurons, and it was found that intraperitoneal administration of diazepam activates these inhibitory neurons in the cerebral cortex, thereby suppressing glioblastoma cell proliferation.

[0106] Example 4 Diazepam was administered to mice in which glioblastoma was induced, and the effect on glioblastoma was confirmed.

[0107] A spontaneous glioblastoma mouse model was prepared in the same manner as in Example 1. Diazepam was then administered intraperitoneally to the mouse model using a syringe. The administration conditions were as follows: Administration period: from 7 weeks after birth until weight loss was confirmed Number of times: once daily Amount of diazepam per administration: 10 μg per gram of mouse body weight

[0108] The tumor progression rate of glioblastoma in the cerebrum was confirmed for the mouse model. The tumor progression rate of glioblastoma was analyzed as a mouse survival curve, as in Example 1 (2). Specifically, the procedure was carried out as follows. That is, after the treatment (administration of diazepam), the mouse models (n=30) were maintained in a mouse breeding room until neurological symptoms (gait abnormalities, head enlargement, or weight loss) were observed. The mouse models in which symptoms were observed were then treated with CO ２After euthanasia, the mice were euthanized by IVF, and the whole brain was collected and examined under a microscope using EGFP as an indicator for tumor formation. For mouse models in which tumors were confirmed, the postnatal days were calculated and a survival curve was created. Note that for the control mice (n=29), only saline was administered instead of diazepam, and survival was similarly confirmed, and a survival curve was created.

[0109] Figure 5 shows the survival curve (survival rate) of the spontaneous glioblastoma mouse model. In Figure 5, * indicates P<0.05. As shown in Figure 5, the survival rate of the example mice administered diazepam was significantly higher than that of the control mice.

[0110] [Example 5] The NaCh expression vector was administered to mice in which glioblastoma had been induced, and the effect on calcium activity in glioblastoma cells was confirmed.

[0111] A spontaneous glioblastoma mouse model was prepared in the same manner as in Example 1. Then, the NaCh expression vector of Example 1(1) was administered to the cerebral ventricles of the mouse model using a syringe to prepare example mice (E2-NaChBac, n=14). Furthermore, the control vector of Example 1(1) was administered to the mice instead of the NaCh expression vector to prepare control mice (n=6). Then, the example mice and the control mice were each grown for 7 days after administration of the vector.

[0112] Calcium activity in glioblastoma cells was analyzed using in vivo calcium imaging in mice. Specifically, the procedure was as follows. Specifically, during the preparation of the mouse model, a fluorescent protein expression vector (PB-CAG-jGCaMP8m) was administered simultaneously with the NaCh expression vector or the control vector to express jGCaMP8m. jGCaMP8m is a fluorescent protein whose fluorescence intensity fluctuates in correlation with intracellular calcium concentration, allowing changes in calcium ion concentration to be detected by changes in fluorescence intensity. After 7 days of growth, the fluorescence intensity of jGCaMP8m-expressing tumor cells in the cerebrum of the mouse model was monitored over time using a two-photon microscope in vivo. These results are shown in Figures 6 and 7.

[0113] Figure 6 is a graph showing the time course of calcium activity in the cerebrum of a mouse model on day 7 after administration of the vector (the NaCh expression vector or the control vector). In Figure 6, the upper graph shows the time course of the fluorescence intensity of the calcium sensor for the control mouse model (n=1), and the lower graph shows the time course of the fluorescence intensity of the calcium sensor for the mouse model of the example (n=1). Figure 6 shows the results for 10 locations in the cerebrum for each of the mouse model of the example and the control mouse model, and the bars on the vertical axis represent the normalized amount of change in fluorescence intensity (10 df / F ０ ), and the horizontal bar represents time (30 s).

[0114] As shown in Figure 6, the example mouse model administered with the NaCh expression vector (lower panel) had fewer regions showing fluctuations in fluorescence intensity and smaller amplitudes of fluorescence intensity fluctuations than the control mouse model not administered with the NaCh expression vector (upper panel). This indicates that expression of the NaCh expression vector can reduce calcium activity in glioblastoma cells in the mouse cerebrum. Note that Figure 6 shows the results for one individual from each of the control mouse (n = 6) and example mouse (n = 14), but similar results were obtained for the remaining individuals.

[0115] Figure 7 is a quantitative graph of calcium activity in the cerebrum of mice 7 days after administration of each vector. In Figure 7, the left panel shows the number of areas (spots) that showed a transient increase in calcium ion concentration per 100 cells, the middle panel shows the frequency of calcium activity (Hz), and the right panel shows the amplitude of calcium activity (dF / Fb). In Figure 7, ** means P<0.01.

[0116] The example mouse model administered with the NaCh expression vector showed a reduced number of areas showing calcium activity (left panel in Figure 7) and amplitude (right panel in Figure 7) compared to the control mouse model not administered with the NaCh expression vector.

[0117] The E2 enhancer of the NaCh expression vector can specifically express NaCh in the inhibitory neurons and specifically activate the inhibitory neurons. This example demonstrates that activation of the inhibitory neurons can suppress calcium activity in glioblastoma, thereby suppressing glioblastoma cell proliferation and improving the survival rate of glioblastoma mice.

[0118] Example 6 Diazepam was administered to mice in which glioblastoma was induced, and the effect on calcium activity in glioblastoma cells was confirmed.

[0119] A spontaneous glioblastoma mouse model was prepared in the same manner as in Example 1. Then, in the same manner as in Example 4, diazepam was administered intraperitoneally to the mouse model using a syringe to prepare example mice (n=8). Furthermore, physiological saline was administered to the mice instead of diazepam to prepare control mice (n=8). The diazepam administration conditions were as follows: Administration period: 7-8 weeks after birth Number of times: once Amount of diazepam per administration: 10 μg per gram of mouse body weight

[0120] Calcium activity in glioblastoma cells was analyzed using in vivo calcium imaging in mice. Specifically, the procedure was as follows, similar to that described in Example 5. Specifically, jGCaMP8m, whose fluorescence intensity correlates with intracellular calcium concentration, was expressed during the preparation of the mouse model. Then, 30 minutes after diazepam administration, the fluorescence intensity of jGCaMP8m-expressing tumor cells was monitored over time using a two-photon microscope. These results are shown in Figures 8 and 9.

[0121] Figure 8 is a graph showing the time course of calcium activity in the cerebrum of mice administered with PBS or diazepam. In Figure 8, the upper graph shows the time course of fluorescence intensity of the calcium sensor for the control mouse model (administered with physiological saline, n=1), and the lower graph shows the time course of fluorescence intensity of the calcium sensor for the mouse model of the example (administered with diazepam). Figure 8 shows the results for 10 locations in the cerebrum for each of the mouse model of the example and the control mouse model, with the bars on the vertical axis representing the normalized amount of change in fluorescence intensity (10 df / F ０ ), and the horizontal bar represents time (30 s).

[0122] As shown in Figure 8, the mouse model of the present invention administered with diazepam (bottom panel) showed fewer fluorescence intensity peaks than the control mouse model administered with saline (top panel). This indicates that administration of diazepam reduces calcium activity in glioblastoma cells in the mouse cerebral cortex. Note that Figure 8 shows the results for one mouse from each of the control mouse model (n = 8) and the mouse model of the present invention (n = 8), but similar results were obtained for the remaining mice.

[0123] Figure 9 is a quantitative graph of calcium activity in the cerebrum for a control mouse model administered PBS and an example mouse model administered diazepam. In Figure 9, the left panel shows the number of areas (spots) showing transient increases in calcium ion concentration per 100 cells, the middle panel shows the frequency of calcium activity (Hz), and the right panel shows the amplitude of calcium activity (dF / Fb). In Figure 7, * indicates P<0.05.

[0124] The frequency of calcium activity in each region (center panel in FIG. 9) was significantly reduced in the example mouse model administered with diazepam compared to the control mouse model administered with saline.

[0125] Diazepam can specifically activate these inhibitory neurons. The results suggest that intraperitoneal administration of diazepam activated these neurons in the cerebrum, resulting in the suppression of calcium activity in glioblastoma, which may have led to the suppression of cell proliferation and improved survival rates in mice.

[0126] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0127] This application claims priority based on Japanese Patent Application No. 2024-70495, filed April 24, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0128] The present invention can suppress the growth of glioma by activating synapses involving inhibitory neurons.

Claims

1. A therapeutic agent for glioma, containing as an active ingredient an inhibitory neuron synapse activator.

2. The glioma therapeutic agent according to claim 1, wherein the activating agent is a GABA receptor agonist.

3. The glioma therapeutic agent according to claim 1 or 2, wherein the activator is a benzodiazepine compound.

4. The therapeutic agent for glioma according to claim 3, wherein the benzodiazepine compound is diazepam, a salt thereof, or a solvate thereof.

5. The therapeutic agent for glioma according to claim 1, wherein the activator is an expression vector carrying a gene encoding a voltage-gated sodium channel.

6. The therapeutic agent for glioma according to claim 5, wherein the coding gene is derived from a bacterium of the genus Bacillus.

7. The therapeutic agent for glioma according to claim 6, wherein the bacterium of the genus Bacillus is Bacillus halodurand.

8. The glioma therapeutic agent according to claim 1, wherein the activating agent is a chemogenetic molecule.

9. The therapeutic agent for glioma according to claim 8, wherein the activating agent is an expression vector carrying a gene encoding a modified M3 muscarinic acetylcholine receptor.

10. The therapeutic agent for glioma according to claim 8 or 9, further comprising clozapine-N-oxide.

11. A glioma therapeutic agent described in any one of claims 5 to 10, wherein the expression vector has the coding gene under an enhancer, and the enhancer is an enhancer of a gene that is specifically expressed in parvalbumin-positive inhibitory neurons.

12. The glioma therapeutic agent according to claim 11, wherein the enhancer is an enhancer of the Scn1a gene.

13. The glioma therapeutic agent according to any one of claims 5 to 12, wherein the expression vector is an adeno-associated virus.

14. A method for inhibiting the proliferation of glioma cells, comprising an activation step of activating synapses of inhibitory neurons in the brain of a subject.

15. The method for inhibiting proliferation described in claim 14, wherein the activation step is a step of administering a glioma therapeutic agent described in any one of claims 1 to 13 to the brain of a subject.

16. The method for inhibiting proliferation according to claim 14 or 15, wherein the subject is a human or non-human animal.

17. A method for inhibiting the proliferation of glioma cells, comprising an activation step of activating synapses of inhibitory neurons in vitro or ex vivo where the inhibitory neurons and glioma cells coexist.

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