Application of targeted KCND2 in prevention or treatment of glioblastoma with epilepsy

Abstract

The invention provides an application of targeted KCND2 in prevention or treatment of glioblastoma with epilepsy. Specifically, the invention provides an application of a KCND2 inhibitor in preventing and / or treating epilepsy of a patient with glioblastoma. The invention also provides a pharmaceutical composition containing the KCND2 inhibitor and a method for preventing and / or treating epilepsy of a patient with glioblastoma by using the pharmaceutical composition. The invention has an application prospect in the field of treatment of diseases of glioblastoma with epilepsy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to the use of targeting KCND2 in the prevention or treatment of glioblastoma with epilepsy. Background Technology

[0002] In recent years, with the gradual discovery of the important role of tumor microenvironment components, the nervous system has become a core accomplice in tumor development and progression. The dialogue between neural innervation and tumors at the molecular and cellular level promotes the acquisition of signature capabilities, including promoting proliferative signaling, resisting cell death, and stimulating tumor invasion and metastasis. The electrical integration of central nervous system tumors with neuronal networks has prompted a re-evaluation of common clinical manifestations of gliomas, including seizures.

[0003] Glioblastoma multiforme (GBM) is the most common malignant central nervous system (CNS) tumor, characterized by its aggressive nature and a leading cause of death. The main feature of GBM is the highly invasive growth of cancer cells, which communicate directly within the local CNS microenvironment through indirect paracrine and electrochemical synapses, integrating into the tumor-invaded neural circuits and forming pathological feedback pathways. However, the nervous system is susceptible to severe neurological lesions and even damage caused by mechanical forces or tumor invasion. Neurological lesions can have devastating consequences, such as epileptic seizures. Epilepsy refers to sudden abnormal electrical activity in brain neurons, posing a significant challenge to quality of life. However, early prediction and intervention for GBM-induced epilepsy are difficult to implement easily, and the molecular mechanisms within the local neural circuits at the tumor-neuron interface and corresponding treatment methods remain to be studied.

[0004] Therefore, there is a need in this field to develop a treatment for glioblastoma with epilepsy. Summary of the Invention

[0005] The purpose of this invention is to provide a treatment method for glioblastoma accompanied by epilepsy.

[0006] In a first aspect of the invention, the use of a KCND2 inhibitor in the preparation of a pharmaceutical composition or formulation for the prevention and / or treatment of epileptic seizures in subjects with gliomas is provided.

[0007] In another preferred embodiment, the glioma is glioblastoma (GBM).

[0008] In another preferred embodiment, the KCND2 inhibitor inhibits KCND2 expression and / or inhibits KCND2 protein activity.

[0009] In another preferred embodiment, the KCND2 inhibitor is selected from the group consisting of KCND2 gene inhibitors, KCND2 protein inhibitors, or combinations thereof.

[0010] In another preferred embodiment, the KCND2 inhibitor reduces KCND2 channel-mediated efflux of cellular potassium ions.

[0011] In another preferred embodiment, the cells are glioma cells, more preferably GBM tumor cells.

[0012] In another preferred embodiment, the KCND2 inhibitor is selected from the group consisting of: small molecule drugs, anti-KCND2 specific antibodies, siRNA encoding the KCND2 gene, gene editing drugs, or combinations thereof.

[0013] In another preferred embodiment, the KCND2 inhibitor is the sole active ingredient in the pharmaceutical composition or formulation.

[0014] In another preferred embodiment, the object includes a human or a non-human mammal.

[0015] In another preferred embodiment, the non-human mammals include rodents (such as mice and rats) and primates (such as monkeys).

[0016] In a second aspect of the invention, a composition is provided for preventing and / or treating epileptic seizures in subjects with gliomas, the composition comprising:

[0017] (a) The active ingredient, wherein the active ingredient is a therapeutically effective amount of a KCND2 inhibitor; and

[0018] (b) Pharmaceutically acceptable carriers.

[0019] In another preferred embodiment, the glioma is glioblastoma (GBM).

[0020] In another preferred embodiment, the KCND2 inhibitor inhibits KCND2 expression and / or inhibits KCND2 protein activity.

[0021] In another preferred embodiment, the KCND2 inhibitor is selected from the group consisting of KCND2 gene inhibitors, KCND2 protein inhibitors, or combinations thereof.

[0022] In another preferred embodiment, the KCND2 inhibitor reduces KCND2 channel-mediated efflux of cellular potassium ions.

[0023] In another preferred embodiment, the cells are glioma cells, more preferably GBM tumor cells.

[0024] In another preferred embodiment, the KCND2 inhibitor is selected from the group consisting of: small molecule drugs, anti-KCND2 specific antibodies, siRNA encoding the KCND2 gene, or combinations thereof.

[0025] In another preferred embodiment, the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0026] In another preferred embodiment, the composition is an injectable formulation.

[0027] In another preferred embodiment, the composition contains 0.0001-99 wt%, preferably 0.1-90 wt%, of the KCND2 inhibitor, based on the total weight of the composition.

[0028] In another preferred embodiment, the composition further comprises the following active ingredients:

[0029] (c) Other antiepileptic drugs; and / or

[0030] (d) Anti-glioma drugs.

[0031] In another preferred embodiment, the component (a) accounts for 1-99 wt% of the total weight of the pharmaceutical composition, more preferably 10-90 wt%.

[0032] In another preferred embodiment, the anti-glioma drug is an anti-GBM drug.

[0033] In a third aspect of the invention, a medicine box is provided, the medicine box comprising:

[0034] (I) A first container containing an active ingredient, said active ingredient being a KCND2 inhibitor or a composition as described in the second aspect of the invention; and

[0035] (II) A second container containing KCND2 detection reagent.

[0036] In another preferred embodiment, the medicine box further includes an instruction manual describing the following method: using the KCND2 detection reagent to detect a sample from a glioma patient, and if the result indicates that the KCND2 expression level in the sample is higher than the reference value, then administering the active ingredient in the first container to the patient.

[0037] In another preferred embodiment, the glioma is glioblastoma (GBM).

[0038] In another preferred embodiment, the sample is a cerebrospinal fluid sample or a tissue sample.

[0039] In another preferred embodiment, the cerebrospinal fluid sample is a cellular component of cerebrospinal fluid.

[0040] In another preferred embodiment, the tissue sample is selected from the group consisting of tumor tissue, peritumoral tissue, or a combination thereof.

[0041] In another preferred embodiment, the KCND2 expression level refers to an indicator selected from the group consisting of: KCND2 protein expression level, KCND2 gene or mRNA expression level, and the number or proportion of KCND2 positive cells.

[0042] In another preferred embodiment, the expression level being higher than the reference value means that the ratio (E1 / E0) of the KCND2 expression level (E1) from the subject sample to the KCND2 expression level (E0) from the control group is ≥1.5, preferably ≥2, and more preferably ≥2.5.

[0043] In another preferred embodiment, the KCND2 detection reagent is a detection reagent for the KCND2 gene, mRNA, cDNA, or protein.

[0044] In another preferred embodiment, the detection reagent is:

[0045] i) Specific antibodies or specific binding molecules to the KCND2 protein; and / or

[0046] ii) Specific amplification primers, probes, or chips for KCND2 cDNA or mRNA.

[0047] In another preferred embodiment, the diagnostic reagent is selected from the group consisting of antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips), protein chips, or combinations thereof.

[0048] In a fourth aspect of the invention, a method for preventing and / or treating epileptic seizures in a subject with glioma is provided, characterized by comprising the step of administering to the subject a composition as described in the second aspect of the invention.

[0049] In another preferred embodiment, the object includes a human or a non-human mammal.

[0050] In another preferred embodiment, the non-human mammals include rodents (such as mice and rats) and primates (such as monkeys).

[0051] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0052] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0053] Figure 1 The neuropathology and neuronal electrophysiological manifestations in GBM patients and animal models are shown.

[0054] (A) Experimental design, including the use of magnetic resonance imaging (MRI), histological examination, and local field potential (LFP) to validate methods in GBM patients.

[0055] (B) Incidence of disease in different brain regions in patients with spontaneous epilepsy GBM (n=106 patients).

[0056] (C) Statistical chart of tumor cortical infiltration in GBM patients without epilepsy (n=700 patients) and with spontaneous epilepsy (n=106 patients).

[0057] (D and E) Representative magnetic resonance imaging (top left), hematoxylin and eosin (H&E) staining (top right), and electrocorticography (ECoG) waveforms (bottom) of GBM patients without epilepsy (D) and with spontaneous epilepsy (E). Scale bar, 50 μm.

[0058] (F) Schematic diagram of the striatal PDX model. The diagram shows the in situ transplantation of primary glioma spheroids isolated from tissues of patients with non-epileptic and spontaneously epileptic GBM to the striatum to form tumors.

[0059] Representative waveforms of cortical LFP recordings from the (G)PDX model in the non-epileptic group (n=9 mice) and the spontaneous epilepsy group (n=9 mice). The spectrograms represent the intensity of cortical oscillations at different frequencies.

[0060] Figure 2 This study showed enrichment of cellular components and progenitor-like cells in patients with spontaneous epilepsy GBM.

[0061] (A) Patient Sample Summary. Surgically resected tumor tissue from the frontal, temporal, and parietal lobes of GBM patients was collected. This included GBM patients without seizures (blue, n=4 patients) and those with spontaneous seizures (red, n=7 patients).

[0062] (B) UMAP visualization of a total of 39,420 normal cells (blue) and malignant cells (red) identified by inferCNV analysis.

[0063] (C) A UMAP visualization of a total of 39,420 cells, showing all cell populations.

[0064] (D) Cell population frequency from (C).

[0065] (E) UMAP visualization of 23,437 tumor cells from non-epileptic GBM patients (blue, 9,435 cells) and spontaneously epileptic patients (red, 14,002 cells).

[0066] (F) Two-dimensional representation of the four cellular states in GBM patients. The four quadrants correspond to the neural progenitor-like (NPC-like), oligodendrocyte precursor-like (OPC-like), astrocyte-like (AC-like), and mesenchymal-like (MES-like) states, respectively.

[0067] (G) The proportion of cell states from the non-epilepsy group and the spontaneous epilepsy group in (F).

[0068] Figure 3 This study demonstrated the association between oligodendrocyte progenitor-like cells (OPC-like) and hyperactivity of neurons around the tumor.

[0069] (A) Representative immunofluorescence (IF) staining of PDGFRA and Ki67 in GBM patients. Labeled as Ki67 (green), PDGFRA (red), and cell nuclei (DAPI, blue). Scale bar, 50 μm.

[0070] (B) Quantification of the proportion of PDGFRA+ tumor cells in tumor tissues of GBM patients without epilepsy (n=10 patients) and with spontaneous epilepsy (n=10 patients). Statistical analysis was performed using standard error (SEM), and unpaired t-tests were used to detect statistical significance (**P<0.01).

[0071] (C) Schematic diagram of an in vitro 3D model of human GBM. Individual tumor cells were isolated from tumor tissue of a patient with spontaneous epilepsy GBM, and tumor spheres were collected after approximately 4-5 weeks. Scale bar, 50 μm.

[0072] (D) Representative confocal image of the GBM sphere, labeled PDGFRA (red) and DAPI (blue). Scale bar, 50 μm.

[0073] (E) Schematic diagram of the cortical PDX model. This shows a schematic diagram of electrode placement for non-OPC-like cell and OPC-like cell-derived xenograft models used for LFP recording.

[0074] (F) Representative LFP trajectories and spectrograms recorded in the prefrontal cortex (PFC) of non-OPC (n=9 mice) and OPC-like (n=9 mice) mouse tumor models.

[0075] LFP power spectrum analysis corresponding to (G) and (F).

[0076] (H) Statistical graph of the frequency of IID events in non-OPC (n=9 mice) and OPC-like (n=9 mice) mouse models. Statistical analysis was performed using standard error (SEM), and unpaired t-tests were used to detect significant differences (**P<0.01).

[0077] Figure 4 KCND2 was found to be highly expressed in epilepsy-specific tumor cell subtypes.

[0078] (A) Enrichment score (ES) of OPC-like gene feature in tumor subsets. Clusters 7, 9, 15, and 20 did not show enrichment of this feature.

[0079] (B and C) The proportion of the C6 subset in different tumor types (B; non-epilepsy and spontaneous epilepsy) and different cell states (C; NPC-like and OPC-like).

[0080] (D) Expression map of the top 10 characteristic genes in the C6 subgroup.

[0081] (E)UMAP plot shows the expression of KCND2 in different tumor subpopulations.

[0082] (F) Expression of KCND2 in four cellular states of GBM cells.

[0083] Expression levels of (G)KCND2 in different anatomical structures in the Ivy GAP RNA-seq database GBM.

[0084] (H) Representative immunofluorescence staining of PDGFRA and KCND2 in marginal GBM tissue. Labeled as KCND2 (green), PDGFRA (red), and cell nuclei (DAPI, blue). Scale bar, 20 μm.

[0085] (I) Quantification of PDGFRA+KCND2+ cells in marginal GBM tissue. Statistical analysis was performed using standard error (SEM), and differences were detected by unpaired t-test (***P<0.001).

[0086] Figure 5 This demonstrates that KCND2 induces neuronal overexcitation through potassium homeostasis imbalance.

[0087] (A) Voltage-gated potassium channel current activation curves induced by voltage stimulation of different intensities in tumor sections of GBM patients with non-epilepsy (gray, n=6 cells) and spontaneous epilepsy (purple, n=14 cells).

[0088] (B) Representative voltage-gated potassium channel current inactivation curves of tumor cells in the spontaneous epilepsy group (n=7 cells).

[0089] (C) IV curves of K+ current corresponding to (A) and (B). The difference was detected by two-way ANOVA test. (*P<0.05, ****P<0.0001).

[0090] (D) Representative LFP records from the Ctrl group (blue, n=9 mice) and the KCND2 group (red, n=9 mice).

[0091] (E) Statistical graph of the frequency of IID events in mouse models of the Ctrl group (n=9 mice) and the KCND2 group (n=9 mice). Statistical analysis was performed using standard error (SEM), and differences were detected by unpaired t-test (**P<0.01).

[0092] (F) Stained images of pyramidal neurons recorded by patch clamp on brain slices from KCND2 model mice. Recorded tumor cells are filled with avidin markers (purple; scale bar: 20 μm), tumor cells express mcherry fluorescent protein (red; scale bar: 100 μm), and neurons are labeled with NeuN (green; scale bar: 100 μm).

[0093] Membrane potential responses of pyramidal neurons to intracellular injection of various currents recorded in mice in the (G)KCND2 and Ctrl groups. Representative action potentials were generated for a 200 pA current input (duration 500 ms, left side), and the right panel shows two representative raw action potential images (arrows).

[0094] IF curves of neurons surrounding tumors in mouse models (H)Ctrl group (n=30 cells) and KCND2 group (n=28 cells). Significance of difference was determined by two-way ANOVA. (*P<0.05).

[0095] (I and J) Bar charts show the resting membrane potential Vm and action potential amplitude of patched neurons. Statistical analysis used standard error (SEM), and unpaired t-tests were used to detect significant differences. Error bars represent standard error (SEM), and unpaired t-tests showed significant differences in the means. (****P<0.0001; ns, no significant difference).

[0096] Figure 6 The results showed that KCND2 increased the firing frequency of neurons in GBM co-cultured cortical organoids.

[0097] (A) Immunofluorescence staining of GBM cell-fused human cortical organoids. Neurons (MAP2, green), GBM cells (mCherry, red), and nuclei (DAPI, blue) are labeled. Low magnification scale bar: 500 μm; high magnification scale bar: 100 μm.

[0098] (B) Representative images of organoids placed in the pores of a microelectrode array (MEA).

[0099] (C) Representative spontaneous electrical activity of organoids. The left panel shows the peak firing rates of organoids in the control and KCND2 groups. The right panel shows extracellular neuronal peak activity recorded at two single electrodes in the control and KCND2 groups within 15 ms.

[0100] The MEA record analysis of organoids in the (D and E)Ctrl group (n=6 organoids) and the KCND2 group (n=6 organoids) quantified the changes in the number of peak discharges (D) and the average discharge frequency (E). Standard error (SEM) was used for statistical analysis. The two-tailed Mann-Whitney test was used to detect significant differences (*P<0.05).

[0101] (F) Displays representative MEA raster plots of single discharge (point), burst discharge activity (blue spike cluster), and synchrotron network burst discharge activity (pink).

[0102] The MEA record analysis of organoids in the (G and H)Ctrl group (n=6 organoids) and the KCND2 group (n=6 organoids) quantified the changes in burst discharge activity frequency (G) and synchronous discharge index (H), and the standard error (SEM) was used to statistically analyze the changes. The two-tailed Mann-Whitney test was used to detect the significance of the differences (*P<0.05).

[0103] Figure 7 The study showed that inhibition of KCND2 expression reduced the excitability of peritumoral neurons.

[0104] (A) Relative levels of KCND2 mRNA in OPC-shCtrl and OPC-shKCND2 cells. n=6 per group. Error bars represent standard error (SEM). Unpaired t-test showed significant differences in means. (****P<0.0001; ns, no significant difference).

[0105] (B) Representative confocal images of KCND2 (red) and cell nuclei (DAPI, blue) staining in OPC-shCtrl and OPC-shKCND2 tumor spheroids. Scale bar, 20 μm.

[0106] (C) Power spectral density analysis of local field potentials (LFP) at different frequencies. n = 9 mice.

[0107] Quantitative results of (DI) delta wave (0.5-3Hz, D), theta wave (3-8Hz, E), alpha wave (8-13Hz, F), beta wave (13-30Hz, G), low gamma wave (30-70Hz, H), and high gamma wave (70-100Hz, I) power. n = 9 mice. Error bars represent standard error (SEM). Unpaired t-tests showed significant differences in means. (ns, no significant difference; *P<0.05; **P<0.01).

[0108] Quantitative statistical results of the frequency of (M)IID. n = 9 mice. Error bars represent standard error (SEM). Unpaired t-test showed that the difference in means was significant (*P<0.05). Detailed Implementation

[0109] Through extensive and in-depth research, the inventors have, for the first time, developed the use of KCND2 inhibitors in the prevention and / or treatment of epilepsy in patients with glioblastoma. This invention focuses on glioblastoma patients with clinically significant seizures, exploring the complex interactions between heterogeneous glioblastoma subtypes and neuronal networks. Using single-cell sequencing, electrophysiological studies in patient-derived xenograft mouse models, and the construction of glioma-cortical organoid models, the profound impact of gliomas on surrounding neural networks and their interrelationships was comprehensively evaluated. The results showed that progenitor-like tumor cells predominate in glioblastoma patients with seizures, promoting the excitability of peritumoral neurons. Specifically, epilepsy-specific progenitor-like glioma subpopulations exhibit KCND2 upregulation. High KCND2 expression significantly affects epilepsy by inducing potassium ion homeostasis imbalance in the tumor microenvironment, leading to enhanced neuronal activity. Furthermore, this invention utilizes KCND2 knockdown to verify the potential of KCND2 as a target for the prevention or treatment of seizures in GBM patients. Therefore, this invention provides the use of KCND2 inhibitors in the prevention and / or treatment of epilepsy in patients with glioblastoma. This invention also provides pharmaceutical compositions containing KCND2 inhibitors and methods of using them to treat, prevent, and / or treat epilepsy in patients with glioblastoma. Based on this, the invention was completed.

[0110] the term

[0111] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0112] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0113] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0114] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0115] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0116] KCND2

[0117] KCND2, also known as Kv4.2, is a member of the potassium voltage-gated channel family D. It is a protein primarily expressed on neurons under physiological conditions, mediating transmembrane potassium transport across excitable membranes. Kv4.2 is a major component of neuronal A-type currents, rapidly activated at membrane potentials below the action potential threshold, inactivated, and reactivated. It is a key regulator of dendritic excitation in hippocampal pyramidal neurons and is crucial for dendritic signal integration.

[0118] Detection and Diagnostic Methods

[0119] This invention provides a composition for the prevention and / or treatment of epileptic seizures in patients with gliomas. The pharmaceutical composition provided by this invention preferably contains 0.1-99 wt% of a first active ingredient, a KCND2 inhibitor, with the remainder being a second active ingredient, a pharmaceutically acceptable carrier, a diluent or solution, or a saline solution.

[0120] The first active ingredient of this invention can be directly applied to prevent and / or treat epileptic seizures in patients with gliomas. Furthermore, it can be used in combination with other therapeutic agents, i.e., the second active ingredient.

[0121] The second active ingredient may be any pharmaceutical ingredient that can prevent and / or treat glioma or epilepsy, including but not limited to chemotherapeutic agents, targeted therapies, sedatives, etc.

[0122] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of this invention. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., which are conventional in the pharmaceutical field.

[0123] The compounds and pharmaceutical compositions provided by this invention can be in various forms, such as tablets, injections, capsules, powders, syrups, solutions, suspensions, and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterilization devices for injection or infusion.

[0124] The pharmaceutical compositions of this invention are suitable for clinical use in mammals, including humans and animals. The optimal dosage for an individual should be determined based on the specific treatment. Generally, a low dose is started, and the dose is gradually increased until the most suitable dosage is found.

[0125] The inhibitors of the present invention can be administered in various ways, such as by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, into the body such as muscle, intradermal, subcutaneous, vein, mucosal tissue; or by being mixed with or encapsulated by other substances and then introduced into the body.

[0126] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0127] To prepare the active ingredient of this invention into an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder injection, mannitol, glucose, etc., can also be added as a support agent.

[0128] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0129] The active ingredients or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs.

[0130] When the active ingredient of this invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0131] The main advantages of this invention include:

[0132] 1) This invention is the first to discover the difference in KCND2 protein expression between GBM patients with and without epilepsy, and to discover the potential of KCND2 as a therapeutic target.

[0133] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0134] Example 1: The inherent ability of tumor cells to cause glioblastoma-related epilepsy

[0135] To understand the common mechanisms underlying tumor-associated neurological discharges in the brain, we conducted a systematic clinical screening focusing on abnormal neural activity in brain regions prone to seizures in GBM patients. Of a total of 806 GBM patients, 106 (13.2%) experienced spontaneous epileptic seizures (Epi), with a higher incidence in the following brain regions: frontal lobe (57.5%), temporal lobe (28.3%), and parietal lobe (6.6%). Figure 1(A and 1B). Notably, GBM patients with spontaneous seizures (spontaneous seizure group) generally exhibited more extensive cortical involvement with a wider tumor location (spontaneous seizures: 42.5% vs. no seizures: 19.3%), while GBM patients without seizures (no seizure group) had tumors primarily confined to the subcortical region ( Figure 1 (A and 1C). These findings suggest a potential interaction between neurons in the cortical gray matter and the tumor, which may influence the epileptogenic mechanisms in GBM patients. These tumor samples were evaluated using magnetic resonance imaging (MRI) and histopathological examination, with standard features including heterogeneous enhancement, nuclear atypia, cell division, angiogenesis, and necrosis. Figure 1 D and 1E). Notably, the spontaneous epilepsy group showed tumor cell invasion of the cerebral cortex compared to the non-epilepsy group, which is consistent with our clinical screening analysis results. Figure 1 D and 1E). Intraoperative electrocorticography (ECoG) recorded spontaneous epileptic waves in the tumor area and surrounding region of epileptic GBM patients. In contrast, tumor growth and invasiveness in non-epileptic GBM patients were confined to the subcortical region and showed consistent, orderly patterns of minute voltage fluctuations (D and 1E). Figure 1 (D and 1E).

[0136] Example 2: Mouse model of GBM patient tumor cells transplanted and its neuronal excitability detection

[0137] To determine whether the inherent ability of tumor cells might induce seizures, we performed primary in vitro cultures of tumor tissue obtained from GBM patients in either the non-epileptic or spontaneously epileptic groups. Subsequently, we orally transplanted glioma cells from either the non-epileptic or spontaneously epileptic groups into the striatum of mice, and performed 12-hour local field potential (LFP) recordings in the mouse cortex on day 45 post-transplantation. Figure 1 F). Surprisingly, we found strong, active neuronal firing activity in the tumor-infiltrating cortex of spontaneously epileptic mice, accompanied by a significant enhancement of low-frequency activity in the 0-10 Hz range, indicating the presence of significant synchronous firing activity. Figure 1 G). However, LFP recordings showed no significant changes in cortical network activity in tumor-transplanted mice without epilepsy (G). Figure 1 G). These results demonstrate the inherent ability of tumor cell populations in GBM to trigger rhythmic activity in the brain during seizure intervals.

[0138] Example 3: Tumor heterogeneity in GBM epilepsy patients metastasizing to progenitor-like populations

[0139] To gain a deeper understanding of the molecular and cellular changes in glioma-associated epilepsy, we performed single-cell nuclear sequencing (snRNA-seq) on tumor samples from the frontal, temporal, and parietal lobes of four patients without epilepsy and seven patients with spontaneously occurring GBM. Figure 2 A). A total of 39,420 cells passed quality control and were used for further analysis. First, we used the inferCNV algorithm to infer large-scale copy number variations (CNVs) to distinguish malignant cells from normal cells. Figure 2 B). Subsequent unsupervised clustering analysis of all individual cells enabled us to identify major cell categories, including stromal cells, neurons, oligodendrocytes, lymphocytes, myeloid cells, and tumor cells. Figure 2 (C and 2D). Although the immune microenvironment plays an important role in the occurrence, progression, and evolution of GBM, its potential role in the pathogenesis of glioma-associated epilepsy has not been fully explored. We analyzed the differences in immune cell composition and function between the non-epileptic and spontaneously epileptic groups. We observed a significant decrease in the proportion of immune components in the spontaneously epileptic group (14.75%) compared to the non-epileptic group (35.05%), particularly myeloid cells (spontaneous epileptic group: 13.05% vs. non-epileptic group: 30.53%) and lymphocytes (spontaneous epileptic group: 1.70% vs. non-epileptic group: 1.70%).

[0140] 4.52% Figure 2 D). These results indicate that an immune remodeling process mediated by abnormal neural networks exists during tumor-induced epilepsy.

[0141] To investigate changes in intratumoral heterogeneity in tumors of the spontaneously epileptic and non-epileptic groups, we scored the plasticity of individual tumor cell states and compared their distribution in the spontaneously epileptic and non-epileptic groups. Figure 2 E and 2F). Interestingly, the tumor cells in the spontaneous epilepsy group contained a higher proportion of progenitor-like cells, including neural progenitor-like (NPC-like) subsets with high levels of expression of TCF12, MAP2, SOX11, and DCX (spontaneous epilepsy group: 39.37% vs. non-epilepsy group: 11.46%) and oligodendrocyte progenitor-like (OPC-like) subsets with high levels of expression of OLIG1, PDGFRA, VCAN, and PLP1 (spontaneous epilepsy group: 15.63% vs. non-epilepsy group: 6.30%). Figure 2G). However, in the non-epileptic group, the proportions of tumor cells in the mesenchymal-like (MES-like) subset (non-epileptic group: 37.48% vs. spontaneously epileptic group: 28.98%) and astrocyte-like (AC-like) subsets were increased (non-epileptic group: 44.77% vs. spontaneously epileptic group: 16.02%). These results suggest that OPC-like cell subsets may be associated with the development of epilepsy in GBM patients.

[0142] Example 4: OPC-like tumor cells affect the excitability of peritumoral neurons

[0143] Recent studies have highlighted the importance of OPC-like cells in gliomas in integrating tumor-neural circuit activity and cortical excitability; OPC-like tumor cells are characterized by the expression of genes such as PDGFRA. We found PDGFRA in tumor tissue from patients with spontaneously epileptic GBM. + The proportion of OPC-like tumor cells increased ( Figure 3 A and 3B), consistent with snRNA-seq data. To investigate whether OPC-like tumor cells can alter neuronal activity and promote seizures, we sorted primary OPC-like tumor cells (PDGFRA) from spontaneously epileptic GBM tumors. + (cells) and non-OPC-like tumor cells (PDGFRA) - Cells were injected into the prefrontal cortex (PFC) of mice to establish a cortical GBM transplantation model. Figure 3 C and 3D). LFP recordings of neural network activity in the prefrontal cortex of model mice were performed and sampled for 12 hours 45 days after tumor transplantation. Figure 3 E). Compared to non-OPC-like tumor cells, OPC-like tumor cells result in a higher incidence of interictal epileptic-like discharges (IIDs) and enhanced low-frequency activity in adjacent cortical regions. Figure 3 (F-3H). Overall, our results indicate that OPC-like tumor cells can enhance the excitability of surrounding cortical PCs.

[0144] Example 5 KCND2 + OPC-like tumor cells are located at the tumor-neural interface.

[0145] 5.1 Differential Expression Analysis of KCND2 Gene

[0146] To further explore specific tumor subpopulations of glioma-associated epilepsy, we extracted all tumor cells from a snRNA-seq dataset containing cells from both the non-epilepsy and spontaneous epilepsy groups for a more comprehensive analysis. Through unsupervised clustering analysis, we identified 21 cell subpopulations with different gene expression patterns and scored the cell state of these subpopulations. Since OPC-like tumor cells have the potential to increase neuronal excitability and induce epilepsy-like activity, we focused on subpopulation 6 (C6), which exhibited significant progenitor cell characteristics. Notably, tumor cells in subpopulation C6 were predominantly derived from the spontaneous epilepsy group (non-epilepsy group: 4% vs. spontaneous epilepsy group: 96%) and exhibited the strongest OPC characteristics. Figure 4 A-4C).

[0147] To further elucidate the molecular mechanisms by which OPC-like tumor cells may induce epilepsy, we focused on analyzing the top ten differentially expressed genes in the C6 subset, finding significant upregulation of DSCAM, FGF12, PCDH15, KCND2, and TNR. Figure 4 D and 4E). Furthermore, we found that KCND2 is primarily expressed in progenitor-like cells, particularly in the OPC-like subset (D and 4E). Figure 4 F). This suggests that KCND2 may play a specific role in the development of brain tumors and may be involved in the pathogenesis of glioma-associated epilepsy.

[0148] 5.2KCND2 Spatial Expression Analysis

[0149] We analyzed the spatial expression patterns of KCND2 in different anatomical structures of GBM patient tumor tissues from the IVY-GAP database and found that KCND2 expression was significantly higher in the tumor frontal and invasive tumor regions than in the tumor core regions, such as microvascular proliferation and necrosis areas. Figure 4 G). To verify this finding, we obtained marginal tumor tissue and confirmed KCND2. + OPC-like cells were more abundant in tumor tissues of spontaneously epileptic GBM patients, but less abundant in tumor tissues of non-epileptic GBM patients. Figure 4 (H and 4I). In summary, these results indicate that KCND2-expressing OPC-like tumor cells are specifically located at the tumor-nerve interface, which may be a potential cause of epilepsy in GBM patients.

[0150] Example 6: Identification of the functional voltage-gated potassium channel Kv4.2 in tumor cells.

[0151] To determine KCND2-associated K in tumor cells +Functional assembly of the channel: We performed whole-cell recording of tumor cells on fresh tumor tissue sections from GBM patients with and without epilepsy and spontaneous epilepsy to detect the presence of K+. + Current. When we set the holding potential to -70mV, and selectively applied sodium channel blocker tetrodotoxin (TTX, 1μM) and calcium channel blocker cadmium ions (Cd2+), the current was measured. + Voltage-gated K+ was detected in tumor cells of spontaneously epileptic GBM patients in the presence of 100 μM. + Current ( Figure 5 A-5C), but this current was not detected in non-epilepsy tumor slices. Figure 5 A and 5C). Recorded K + The current exhibits rapid activation and deactivation mechanical characteristics, which is characteristic of type A K. + Characteristics of the current. Furthermore, when the cell is maintained at a more positive potential (-20mV), K... + The current is completely deactivated, with K V 4.2 The mediated current exhibits consistent biophysical properties (i.e., complete inactivation at depolarization membrane potential). Figure 5 B and 5C). In summary, these results strongly support the functional KCND2 (Kv4.2) voltage-gated K in tumor cells of patients with epileptic GBM. + The existence of the passage.

[0152] Example 7: Establishment and Analysis of a GBM Xenograft Model Overexpressing KCND2

[0153] We constructed a tumor cell xenograft model overexpressing KCND2 (KCND2 group) and recorded LFP in the mouse cortex for 12 hours on day 25 post-transplantation. The results showed that compared with the KCND2-deficient control group, the KCND2-overexpressing tumor cell xenograft model mice exhibited more severe epileptiform discharges and seizures. Figure 5 D). Furthermore, the frequency of IIDs events was also increased in the KCND2 group mice. Figure 5 E). Subsequently, we examined the electrophysiological properties of peritumoral pyramidal neurons (PCs). Figure 5 F), we found that PCs adjacent to KCND2-overexpressing tumor cells generated more action potentials during stepwise current injection. Figure 5 G and 5H). This increase in excitability is mainly due to the depolarized resting membrane potential (Vm) (G and 5H). Figure 5 I). Further analysis showed a decrease in the action potential amplitude and its maximum depolarization slope. Figure 5 Our results indicate that KCND2 expression in tumor cells increases the excitability of neurons in the peritumoral region, accompanied by epileptiform activity and seizures.

[0154] Example 8: Establishment and Analysis of Cortical Organoids from Tumor Cells Overexpressing KCND2

[0155] To further verify the effect of KCND2 on the excitability of neurons in the human cerebral cortex, we constructed cortical organoids derived from human induced pluripotent stem cells, and then co-cultured KCND2-expressing tumor cells with human cortical organoids. Figure 6 A). Five days after co-culture, the co-cultured organoids were analyzed using a microelectrode array (MEA) to detect peak neuronal firing activity. Figure 6 B). Notably, we observed that tumor cells overexpressing KCND2 significantly increased the frequency of neuronal peak firing and burst firing (B). Figure 6 C-6G), and networked discharge activity has emerged ( Figure 6 F and 6H). This indicates that KCND2 significantly increases neuronal excitability in human cortical organoids. Overall, our results reveal that KCND2-expressing OPC-like tumor cells are distributed at the tumor periphery, disrupting the KCND2-mediated tumor-neuronal interface. + Homeostasis leads to neuronal overexcitation, which in turn causes epilepsy in GBM patients.

[0156] Example 9: Effect of KCND2 Inhibition on Epilepsy Seizures

[0157] To investigate the effect of KCND2 expression in OPC-like cells on neural activity, we generated OPC-like cells with stable KCND2 knockdown (OPC-shKCND2). Figure 7 (A and 7B). We then constructed orthotopic xenograft models of OPC-shCtrl and OPC-shKCND2 cells and monitored cortical local field potential (LFP) activity on day 45 post-inoculation. Subsequent analysis of LFP at different frequencies showed that KCND2 knockdown significantly reduced the power of low-frequency brain oscillations (δ, θ, α waves) and high-frequency gamma oscillations (A and 7B). Figure 7 C-7I). Furthermore, after KCND2 knockdown, the frequency of IID events significantly decreased (C-7I). Figure 7 Our results indicate that KCND2 expression in tumor cells increases the excitability of peritumoral neurons, accompanied by epileptiform activity and seizures.

[0158] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of KCND2 inhibitors in the preparation of pharmaceutical compositions or formulations for the prevention and / or treatment of epileptic seizures in patients with gliomas.

2. The use as described in claim 1, characterized in that, The glioma mentioned is glioblastoma (GBM).

3. The use as described in claim 1, characterized in that, The KCND2 inhibitor is selected from the group consisting of KCND2 gene inhibitors, KCND2 protein inhibitors, or combinations thereof.

4. The use as described in claim 1, characterized in that, The KCND2 inhibitors mentioned are selected from the group consisting of: small molecule drugs, anti-KCND2 specific antibodies, siRNA encoding the KCND2 gene, gene editing drugs, or combinations thereof.

5. The use as described in claim 1, characterized in that, The objects mentioned include humans or non-human mammals.

6. A composition for preventing and / or treating epileptic seizures in patients with gliomas, characterized in that, The composition comprises: (a) The active ingredient, wherein the active ingredient is a therapeutically effective amount of a KCND2 inhibitor; and (b) Pharmaceutically acceptable carriers.

7. The composition according to claim 6, characterized in that, The KCND2 inhibitor is selected from the group consisting of KCND2 gene inhibitors, KCND2 protein inhibitors, or combinations thereof.

8. The composition according to claim 6, characterized in that, The composition also contains the following active ingredients: (c) Other antiepileptic drugs; and / or (d) Anti-glioma drugs.

9. A medicine box, characterized in that, The medicine box includes: (I) A first container containing an active ingredient, said active ingredient being a KCND2 inhibitor or the composition as described in claim 6; and (II) A second container containing KCND2 detection reagent.

10. The medicine box as described in claim 9, characterized in that, The medicine box further includes an instruction manual, which describes the following method: using the KCND2 detection reagent to detect samples from glioma patients, and if the results indicate that the KCND2 expression level in the sample is higher than the reference value, then the active ingredient in the first container is administered to the patient.

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