Methods for Treating Neurodegenerative Disorders Using Alternating Electric Fields

Abstract

Provided is a method for treating neurodegenerative disorders in a subject by applying an alternating electric field to the subject's brain, and optionally also includes administering to the subject a drug for treating neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, dementia). In some examples, neurodegenerative disorders can be treated by applying an alternating electric field to the brain of a human subject whose brain does not contain a tumor.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 172,275, filed April 8, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Alternating electric fields (also known as Tumor Treatment Fields (TTFields)) are used to disrupt cell division by applying low-intensity, intermediate frequency (e.g., 50-500 kHz) alternating electric fields to cells in targeted areas of the body.

[0003] In an in vivo situation, AC electric field therapy can be delivered using a wearable and transportable device (Optune®). The delivery system includes an electric field generator, four adhesive patches (non-invasive, insulated transducer arrays), a rechargeable battery, and a carrying case. The transducer arrays are applied to the skin and connected to the device and battery. The therapy is designed to be worn around the clock for as long as possible. In a preclinical setting, TTFields can be applied in vitro, for example, using the Inovitro™ TTFields Lab Bench System. Inovitro™ includes a TTFields generator and a base plate that contains eight ceramic dishes per plate. Cells are applied on a coverslip placed inside each dish. TTFields is applied using two orthogonal pairs of transducer arrays insulated by high dielectric constant ceramics within each dish. In both in vivo and in vitro situations, the orientation of the TTFields was switched by 90° every 1 second, thus covering different directional axes of cell division.

[0004] Neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, and dementia) affect millions of people each year. These conditions are generally related to progressive damage to cells (e.g., brain cells) and connections in the nervous system.

[0005] Alzheimer's disease (AD) is associated with dementia and memory impairment and affects 25 million people worldwide. Currently, there is no effective preventative or curative treatment. Amyloid β-protein (Aβ) is implicated in the pathogenesis of AD and is associated with the formation of plaques in the brains of patients. Aβ protein can form oligomeric assemblies that are associated with memory impairment and subsequent neurodegeneration. Walsh et al., Alzheimer's disease and the amyloid βprotein, Prog Mol Biol Transl Sci., 2012;107:101-24.

[0006] Parkinson's disease (PD) is associated with loss of control over movement and is the second most common progressive neurodegenerative disorder. PD results from pathophysiological loss or degeneration of dopaminergic neurons in the substantia nigra of the midbrain and the development of neuronal Lewy bodies. Beitz, Parkinson's disease: a review, Front Biosci (Schol Ed) 2014 Jan 1;6:65-74. No effective therapy for PD is yet available. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 7,565,205 [Non-patent literature]

[0008] [Non-Patent Document 1] Walsh et al., Alzheimer's disease and the amyloid β protein, Prog Mol Biol Transl Sci., 2012;107:101 - 24 [Non - Patent Document 2] Beitz, Parkinson's disease: a review, Front Biosci(Schol Ed) January 1, 2014;6:65 - 74 [Non - Patent Document 3] Cai et al., Roles of glycogen synthase kinase 3 in Alzheimer's disease, Curr Alzheimer Res. September 2012;9(7):864 - 79 [Non - Patent Document 4] Hooper et al., The GSK3 hypothesis of Alzheimer's disease, J Neurochem. March 2008; 104(6): 1433 - 1439 [Non - Patent Document 5] Sen et al., Sulfhydration of AKT triggers Tau - phosphorylation by activating glycogen synthase kinase 3β in Alzheimer's disease, PNAS February 25, 2020, 117(8)4418 - 4427 [Non - Patent Document 6] Furlong et al., The Parkinson's disease gene PINK1 activates Akt via PINK1 kinase - dependent regulation of the phospholipid PI(3,4,5)P3, Journal of Cell Science(2019)132 [Non - Patent Document 7] Credle et al., GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein, Cell Death Differ. April 2015; 22(5): pp. 838-851 [Non-Patent Document 8] Llorens-Martin et al., GSK-3β, a pivotal kinase in Alzheimer's disease, Front Mol Neurosci. 2014; 7:46 [Non-Patent Document 9] Soutar et al., Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain, J Neurochem. 2010 November; 115(4):974–83 [Non-Patent Document 10] Phiel et al., GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides, Nature. 2003 May 22;423(6938):435-9 [Non-Patent Document 11] Uemura et al., GSK3beta activity modifies the localization and function of presenilin 1, J Biol Chem. May 25, 2007;282(21):15823~32 Summary of the Invention [Problem to be solved by the invention]

[0009] Glycogen synthase kinase 3 beta (GSK3β) is a proline-directed serine-threonine kinase known to phosphorylate and inactivate glycogen synthase. GSK3β is involved in the production and accumulation of Aβ in Alzheimer's disease. Cai et al., Roles of glycogen synthase kinase 3 in Alzheimer's disease, Curr Alzheimer Res. 2012 Sep;9(7):864-79; Hooper et al., The GSK3 hypothesis of Alzheimer's disease, J Neurochem. 2008 Mar;104(6):1433-1439; Sen et al., Sulfhydration of AKT triggers Tau-phosphorylation by activating glycogen synthase kinase 3β in Alzheimer's disease, PNAS 2020 Feb. 25, 117(8)4418-4427. GSK3β has been shown to be activated in neurodegenerative diseases such as Alzheimer's disease, and inhibition of GSK3β reduces Aβ pathology.

[0010] GSK-3β is regulated by the Akt pathway, which is also impaired in Parkinson's disease. Furlong et al., The Parkinson's disease gene PINK1 activates Akt via PINK1kinase-dependent regulation of the phospholipid PI(3,4,5)P3, Journal of Cell Science (2019) p. 132; Credle et al., GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein, Cell Death Differ. April 2015; 22(5): 838-851.

[0011] Taken together, it appears that inhibition of GSK3β by phosphorylation may be used to treat, reduce the symptoms of, or inhibit the development of neurodegenerative disorders such as AD and PD. [Means for solving the problem]

[0012] In one embodiment, a first method is provided, comprising administering a GSK3β inhibitor to a subject and applying an alternating electric field to the brain of the subject to treat a neurodegenerative disorder in the subject. In some embodiments, the neurodegenerative disorder may be Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), dementia, or Parkinson's disease.

[0013] In another aspect, a method is provided that includes treating a neurodegenerative disorder in a subject by administering a therapeutic agent and applying an alternating electric field to the subject's brain. In an aspect, the therapeutic agent may be an anti-amyloid monoclonal antibody to the subject and applying an alternating electric field to the subject's brain. In another aspect, the therapeutic agent may be a GSK3β inhibitor, a cholinesterase inhibitor, or an anti-amyloid monoclonal antibody. In another aspect, the GSK3β inhibitor is a cholinesterase inhibitor. In another aspect, the therapeutic agent may be a dopaminergic agent, a monoamine oxidase B (MAO-B) inhibitor, a catechol O-methyltransferase inhibitor, or an anticholinergic agent.

[0014] In another embodiment, a second method is provided, comprising administering to the subject an anti-amyloid monoclonal antibody and applying an alternating electric field to the brain of the subject, thereby treating a neurodegenerative disorder in the subject. In some embodiments, the anti-amyloid monoclonal antibody can be aducanumab.

[0015] In a further aspect, a third method is provided that includes treating Parkinson's disease in a subject by administering L-3,4-dihydroxyphenylalanine to the subject and applying an alternating electric field to the brain of the subject.

[0016] In another aspect, the method further comprises administering to the patient a therapeutic agent, such as 9-ING-41, HY-130795, TWS119, tideglusib, SAR502250, AR-A014418, TDZD8, kenpaullone, chromoib sodium, SB415286, IM-12, CP21R7, GNF4877, 1-azakempaullone, or indirubin-3'-monoxime, tacrine, galantamine, liquiritigenin, memantine, rivastigmine, donepezil, ciprofloxacin, or a combination thereof. In some embodiments, the therapeutic agent may be administered in combination with one or more of ciprofloxacin and celecoxib, or tauroursodeoxycholic acid and sodium phenylbutyrate.

[0017] A further aspect provides a method further comprising treating a neurodegenerative disorder in a human subject or diagnosing a subject with a neurodegenerative disorder by applying an alternating electric field to the brain of the human subject, wherein the brain does not comprise a tumor and the subject has not been diagnosed with having a tumor prior to applying the alternating electric field. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 provides an exemplary Western blot analysis of total GSK3β, GSK3β phosphorylated on Ser9 (pGSK3βSer9), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; housekeeping protein) in control cell lysates compared to cells treated with alternating current electric fields (TTFields) for 72 hours. [Diagram 2]FIG. 2 provides quantification of normalized relative levels of pGSK3β(Ser9) compared to total GSK3β and GAPDH in each cell line analyzed in FIG. [Diagram 3] FIG. 3 depicts a cell-free assay of amyloid beta aggregation following treatment with TTFields of various frequencies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] All references cited herein, including but not limited to patents and patent applications, are hereby incorporated by reference in their entirety.

[0020] As described herein, an alternating electric field can be applied to the brain or brain regions of a subject's body prior to or during treatment for a neurodegenerative condition, such as AD, PD, or dementia, to inhibit activity of GSK3β, for example, by phosphorylation on serine 9. Without being bound by theory, it is believed that the combination of treating the brain with an alternating electric field in combination with standard therapeutic treatments for neurodegenerative diseases can synergistically enhance the treatment or prevention of neurodegenerative diseases.

[0021] As described herein, TTFields reduce the activity of GSK3β. GSK3β activation has been shown to be upregulated in neurodegenerative diseases such as Alzheimer's disease. Inhibition of GSK3β has been shown to improve neurological conditions in mice. Llorens-Martin et al., GSK-3β, a pivotal kinase in Alzheimer's disease, Front Mol Neurosci. 2014; 7:46. GSK3β has also been implicated in Parkinson's disease. Credle et al., GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein, Cell Death Differ. 2015 Apr; 22(5): 838-851.

[0022] For example, as described herein, GSK3β is involved in the production and accumulation of Aβ protein in Alzheimer's disease. Additionally, GSK3β is regulated by the Akt pathway, which is also involved in Parkinson's disease.

[0023] In AD patients, high amounts of phosphorylated Akt along with active GSK3β means that phosphorylated Akt could not inactivate GSK3β. Sen et al., Sulfhydration of AKT triggers Tau-phosphorylation by activating glycogen synthase kinase 3β in Alzheimer's disease, PNAS 2020 Feb 25, 117(8) 4418-4427. Intracellular hydrogen sulfide (H 2 For induction of intracellular H, total and phosphorylated Akt were sulfhydrylated with C77. 2Increased S levels result from the induction of the proinflammatory cytokine IL-1β, a pathological hallmark of AD. Sulfhydrylated Akt does not interact with, and therefore does not phosphorylate, GSK3β.

[0024] Overexpression of tau disrupts axonal transport and leads to vesicle aggregation, a phenomenon that is reversed by GSK-3β inhibitors. Soutar et al., Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain, J Neurochem. 2010 Nov;115(4):974-83. Consistent with this, GSK-3β inhibition has been shown to reduce Aβ production in AD mouse models. Phiel et al., GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides, Nature. 2003 May 22;423(6938):435-9.

[0025] In vitro studies suggest that GSK-3β affects PS1 function that is required for the generation of toxic Aβ. Uemura et al., GSK3beta activity modifies the localization and function of presenilin 1, J Biol Chem. 2007 May 25;282(21):15823-32.

[0026] Application of an alternating electric field to the brain or regions of the brain can be used to inhibit activation of GSK3β, in conjunction with treatment of the subject with GSK3β inhibitors and other drugs for the treatment of neurodegenerative disorders.

[0027] The AC electric fields discussed herein are similar to Tumor Treatment Electric Fields (TTFields) and can be applied using hardware similar to Novocure's Optune® device at the same 200 kHz frequency used by Optune, or at different frequencies (e.g., 50 kHz to 1 MHz). The size and shape of the transducer array used to apply the AC electric fields to the subject's body will vary depending on the anatomical location to which the AC electric fields are applied. As used herein and in the drawings, the term "TTFields" is synonymous with the term "AC electric fields."

[0028] In one aspect, a first method is provided that includes treating a neurodegenerative disorder in a subject by applying an alternating electric field to the brain of the subject.

[0029] In some instances, the neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, dementia, or amyotrophic lateral sclerosis (ALS).

[0030] In some cases, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step. In some cases, the applying step has a duration of at least 24 hours, 48 ​​hours, 72 hours, 7 days, 14 days, 30 days, or longer. The applying step may be continuous or discontinuous with breaks (e.g., breaks at 1, 2, 3, 6, 12, or 24 hours).

[0031] In some examples, the frequency of the alternating electric field is 50 to 1 MHz, 75 to 500 kHz, 80 kHz to 300 kHz, 100 kHz to 200 kHz, or 150 kHz.

[0032] In one embodiment, a second method is provided, comprising applying an alternating electric field to the brain of the subject and further administering a therapeutic agent to treat the neurodegenerative disorder in the subject.For example, the therapeutic agent may comprise one or more of a GSK3β inhibitor, a cholinesterase inhibitor, and an anti-amyloid monoclonal antibody.In another example, the GSK3β inhibitor is a cholinesterase inhibitor.

[0033] For example, the therapeutic agent may be one or more of tacrine, galantamine, liquiritigenin, memantine, rivastigmine, and donepezil. In some examples, the GSK3β inhibitor or cholinesterase inhibitor is one or more of tacrine, galantamine, liquiritigenin, memantine, rivastigmine, and donepezil.

[0034] In some examples, the GSK3β inhibitor consists of, or consists essentially of, at least one component selected from the group consisting of 9-ING-41, HY-130795, TWS119, tideglusib, SAR502250, AR-A014418, TDZD8, kenpaullone, chromoib sodium, SB415286, IM-12, CP21R7, GNF4877, 1-azakempaullone, and indirubin-3'-monoxime.

[0035] In some cases, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step. In some cases, the applying step has a duration of at least 24 hours, 48 ​​hours, 72 hours, 7 days, 14 days, 30 days, or longer. The applying step may be continuous or discontinuous with breaks (e.g., breaks at 1, 2, 3, 6, 12, or 24 hours).

[0036] In some examples, the frequency of the alternating electric field is 50 to 1 MHz, 75 to 500 kHz, 80 kHz to 300 kHz, 100 kHz to 200 kHz, or 150 kHz.

[0037] In some instances, the neurodegenerative disorder is Alzheimer's disease, Parkinson's disease, dementia, or amyotrophic lateral sclerosis (ALS).

[0038] In another embodiment, a third method is provided that includes treating a neurodegenerative disorder in a subject by administering to the subject an anti-amyloid monoclonal antibody and applying an alternating electric field to the brain of the subject.

[0039] In some examples, the anti-amyloid monoclonal antibody is aducanumab.

[0040] In some cases, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step. In some cases, the applying step has a duration of at least 24 hours, 48 ​​hours, 72 hours, 7 days, 14 days, 30 days, or longer. The applying step may be continuous or discontinuous with breaks (e.g., breaks at 1, 2, 3, 6, 12, or 24 hours).

[0041] In some examples, the frequency of the alternating electric field is 50 to 1 MHz, 75 to 500 kHz, 80 kHz to 300 kHz, 100 kHz to 200 kHz, or 150 kHz.

[0042] In some instances, the neurodegenerative disorder is dementia, ALS, Alzheimer's disease, or Parkinson's disease.

[0043] In a further aspect, a fourth method is provided that includes treating a neurodegenerative disorder (e.g., Parkinson's disease) in a subject by administering to the subject one or more of levodopa (L-3,4-dihydroxyphenylalanine) and carbidopa (N-amino-α-methyl-3-hydroxy-L-tyrosine monohydrate) and applying an alternating electric field to the brain of the subject.

[0044] In some cases, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step. In some cases, the applying step has a duration of at least 24 hours, 48 ​​hours, 72 hours, 7 days, 14 days, 30 days, or longer. The applying step may be continuous or discontinuous with breaks (e.g., breaks at 1, 2, 3, 6, 12, or 24 hours).

[0045] In some examples, the frequency of the alternating electric field is 50 to 1 MHz, 75 to 500 kHz, 80 kHz to 300 kHz, 100 kHz to 200 kHz, or 150 kHz.

[0046] In a further aspect, a fifth method is provided that includes treating a neurodegenerative disorder in a subject (e.g., ALS, Parkinson's disease, Alzheimer's disease, dementia) by administering a therapeutic agent to the subject and applying an alternating electric field to the brain of the subject.

[0047] In some examples, the therapeutic agent is one or more of ciprofloxacin, celecoxib, tauroursodeoxycholic acid, and sodium phenylbutyrate. For example, the therapeutic agent may include a combination of ciprofloxacin and celecoxib. For example, the therapeutic agent may include a combination of tauroursodeoxycholic acid and sodium phenylbutyrate.

[0048] In some examples, the therapeutic agent is a dopamine agonist. Suitable dopamine agonists include, but are not limited to, one or more of pramipexole, ropinirole, rotigotine, and apomorphine.

[0049] In some examples, the therapeutic agent is a monoamine oxidase type B (MAO-B) inhibitor. Suitable MAO-B inhibitors include, but are not limited to, selegiline, rasagiline, and safinamide.

[0050] In some examples, the therapeutic agent is a catechol O-methyltransferase inhibitor. Suitable catechol O-methyltransferase inhibitors include, but are not limited to, one or more of entacapone, opicapone, and tolcapone.

[0051] In some instances, the therapeutic agent is an anticholinergic. Suitable anticholinergic agents include, but are not limited to, one or more of benztropine and trihexyphenidyl.

[0052] In some examples, the therapeutic agent is amantadine. In other embodiments, the therapeutic agent is one or more of riluzole and edaravone.

[0053] In some instances, the neurodegenerative disorder is dementia, ALS, Alzheimer's disease, or Parkinson's disease.

[0054] In some cases, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step. In some cases, the applying step has a duration of at least 24 hours, 48 ​​hours, 72 hours, 7 days, 14 days, 30 days, or longer. The applying step may be continuous or discontinuous with breaks (e.g., breaks at 1, 2, 3, 6, 12, or 24 hours).

[0055] In some examples, the frequency of the alternating electric field is 50 to 1 MHz, 75 to 500 kHz, 80 kHz to 300 kHz, 100 kHz to 200 kHz, or 150 kHz.

[0056] In some examples, a therapeutic agent (e.g., a GSK3β inhibitor, a cholinesterase inhibitor, an anti-amyloid monoclonal antibody, L-3,4-dihydroxyphenylalanine, etc.) is provided to a subject in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount of drug or a dose of drug sufficient to achieve its intended purpose of improving, preventing, treating, or curing the indicated disease or condition. One skilled in the art can determine a therapeutically effective amount of a drug, for example, from the label of the drug or product, or from the results of experiments or clinical trials designed to determine the therapeutically effective dose, pharmacokinetics, or other properties of the drug.

[0057] For example, one of ordinary skill in the art can determine the therapeutically effective dose of a drug by consulting the drug label approved by the U.S. Food and Drug Administration (FDA). One of ordinary skill in the art can consult the product label approved by the FDA or other regulatory agency for any approved drug or treatment referenced herein. As used herein, the term "treating" refers to prescribing, administering, or instructing another to administer a drug or treatment to a subject.

[0058] In some embodiments, the application of the electric field can be interrupted by rest. For example, six sessions each having a duration of 12 hours include a 2 hour rest between sessions. In another embodiment, the step of applying the electric field has a duration of at least 3 hours. In some examples, the AC electric field can be applied at an intensity of 175V / m (RMS), 150kHz, for 24, 48, 72 hours or longer.

[0059] In some embodiments, the strength of the alternating electric field is 0.1 V / cm to 20 V / cm (RMS) or 1.0 V / cm to 2.5 V / cm (RMS).

[0060] In yet another embodiment, the frequency of the alternating electric field is 50-1 MHz, 100-500 kHz, 125-175 kHz, or 150 kHz. In another embodiment, the drug is delivered to a tissue, location, or cell at a therapeutically effective concentration, and the alternating electric field has a field strength of at least 1 V / cm in at least a portion of the tissue, location, or cell.

[0061] In yet another aspect, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step.

[0062] A further embodiment provides a method of treating a neurodegenerative disorder (e.g., Alzheimer's disease, Parkinson's disease, dementia, ALS) in a human subject, comprising applying an alternating electric field to the brain of a human subject, the brain of which does not contain a tumor.

[0063] In some examples, the applying step has a duration of at least 24 hours, 48 ​​hours, 72 hours, 7 days, 14 days, or 30 days. As described herein, the alternating electric field can inactivate or reduce the activity of GSK3β and be used to treat neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, dementia, ALS). Neurodegenerative disorders may be slowly progressing diseases, and therefore, in some examples, the alternating electric field can be applied for a longer duration (e.g., at least 30 days or longer).

[0064] In some examples, the frequency of the alternating electric field is 50 kHz to 1 MHz, for example, 50 to 500 kHz.

[0065] Figure 1 is an exemplary Western immunoblot showing the levels of GSK3β and phosphorylated GSK3β (anti-pGSK3βSer9) after treatment with AC electric field for a duration of 72 hours. Analysis was performed in the following cell lines: A2780 (ovarian cancer), A549 (lung epithelial carcinoma), Hela (cervical epithelial adenocarcinoma) and MSTO (metastatic lung cancer). As shown in Figure 1, application of AC electric field (+) for 72 hours increased the level of pGSK3βSer9 compared to control cells that did not receive AC electric field (-), indicating inactivation of GSK3β or reduction of its activity.

[0066] Figure 2 provides a graphical representation of the quantification of the band intensity of the Western blot (Figure 1) and its relative value to the housekeeping gene (GAPDH) in the control sample. Treatment with AC electric field increased the level of phosphorylated GSK3β in A2780 (ovarian cancer), A549 (lung epithelial cancer), Hela (epithelial cervical adenocarcinoma) and MSTO (lung metastasis cancer) cell lines compared to GAPDH control, indicating that AC electric field inactivates GSK3β or reduces its activity. Fiji ImageJ software was applied to measure the band intensity of the Western blot. In one aspect, an increase in phosphorylation of serine 9 indicates inhibition of GSK3β activity. As discussed above, inactivation of GSK3β or reduction of its activity can be used as a treatment for neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and dementia.

[0067] Figure 3 depicts a cell-free assay of amyloid beta aggregation after treatment with TTFields at various frequencies (100, 200, 300, 400, 500 kHz) for 24, 48, and 72 hours. The Thioflavin T (ThT) intensity of amyloid beta samples treated with TTFields at the above frequencies was compared to the ThT intensity of samples not exposed to TTFields and incubated at 37°C (positive control) or 4°C (negative control). PBS alone served as a negative control (background). As shown in Figure 1, treatment with TTFields for 72 hours reduced the aggregation signal of amyloid beta compared to the positive control (results are the average of two independent experiments. Statistical analysis * P < 0.05; ** P < 0.01, **** P < 0.0001; two-way ANOVA, Sidak test).

[0068] Amyloid beta peptides were prepared by dissolving HFIP-treated human amyloid beta (Sigma-Aldrich, AG968) in DMSO (180 μl) and further diluting in PBS to a final concentration of 10 μM. Samples were treated with TTFields (1.62 V / cm, 37°C) at various frequencies (100, 200, 300, 400, 500 kHz) for a total duration of 72 h. Incubation of amyloid beta peptides at 4°C served as a negative control for amyloid beta fibrillization, and at 37°C served as a positive control for amyloid beta fibrillization.

[0069] Thioflavin T (ThT) is a cationic benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid. ThT (Sigma-Aldrich, T3516) was added to the samples and measured at each time point (24, 48, 72 h) at room temperature (approximately 24° C.) using a SYNERGY / H1 plate reader (BioTek) through the top of a black 96-well plate with an excitation filter of 450 nm and an emission filter of 482 nm.

[0070] Cell lines and cultures All cell lines were obtained from ATCC (A2780 (human ovarian cancer cell line), Hela (cervical cancer), MSTO and A549 (human lung cancer)). Cells were cultured in Dulbecco's modified Eagle's medium (Biological Industries), RPMI (GIBCO), F12K (ATCC) medium supplemented with 10% fetal bovine serum and antibiotics.

[0071] Application of TTFields TTFields were applied to cell cultures using the Inovitro™ system (Novocure Ltd) as previously described. Cells were seeded on coverslips at a density of 5000–20,000 cells in 500 μL and treated with the same nominal intensity (e.g., 1.75 V / cm RMS) at a given exemplary optimal frequency (e.g., A2780 (200 kHz), MSTO (150 kHz), Hela (200 kHz), and A549 (150 kHz)). TTFields were applied from two directions, changed by 90° every 1 s, as previously described. Culture medium (2 ml per dish) was added 24 h after seeding and covered in Parafilm (P7793, Sigma Aldrich) to avoid medium evaporation.

[0072] Cell lysates and immunoblotting Cell extracts were prepared using RIPA lysis buffer (R0278, Sigma Aldrich) supplemented with a protease cocktail (Complete Mini, Roche) and phosphatase inhibitors (Halt #78420, Thermo Scientific). After protein concentration was determined (BCA Protein Assay Kit, ab102536, Abcam), 30 μg of protein was resolved by SDS-polyacrylamide gel electrophoresis (Bolt 12% Bis-Tris base gel NW00080BOX, Thermo-Fischer) under reducing conditions (Bolt Sample reducing agent, #2060435 and Sample buffer #2045289, Novex). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and probed with appropriate primary antibodies (GAPDH (SC-32233, Santa Cruz), GSK3β (Cell Signaling, 9832S) and pGSK3βSer9 (Cell Signaling, 5558S)) followed by horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit 7074, Cell Signaling and goat anti-mouse 7076, Cell Signaling) and chemiluminescent substrate (WBLUF0100, Sigma-Aldrich). Quantification of bands was performed by Image J software.

[0073] The in vitro experiments described herein were carried out using the Novocure Inovitro™ system. In these experiments, the direction of the AC electric field was switched between two perpendicular directions at 1 second intervals. However, in alternative embodiments, the direction of the AC electric field can be switched at a faster rate (e.g., at intervals of 1-1000 ms) or at a slower rate (e.g., at intervals of 1-100 seconds).

[0074] In the in vitro experiments described herein, the direction of the AC electric field was switched between two perpendicular directions by applying AC voltage to two pairs of electrodes arranged in an alternating sequence at 90° to each other in 2D space. However, in alternative embodiments, the direction of the AC electric field can be switched between two non-perpendicular directions, or between three or more directions (assuming additional electrode pairs are provided), by rearranging the electrode pairs. For example, the direction of the AC electric field can be switched between three directions, each determined by the arrangement of its own electrode pair. If necessary, these three pairs of electrodes can be arranged so that the resulting electric field is arranged at 90° to each other in 3D space. In other alternative embodiments, the electrodes do not need to be arranged in pairs. See, for example, the electrode arrangement described in U.S. Pat. No. 7,565,205, which is incorporated herein by reference. In other alternative embodiments, the direction of the electric field remains constant.

[0075] In the in vitro experiments using the Inovitro™ system described herein, the Inovitro™ system used conductive electrodes placed on the outer surface of the sidewall of the dish, and the ceramic material of the sidewall acts as a dielectric, so that the electric field was capacitively coupled in the culture. However, in alternative embodiments, the electric field can be applied directly to the cells without capacitive coupling (e.g., by modifying the Inovitro™ system configuration so that the conductive electrodes are placed on the inner surface of the sidewall rather than the outer surface of the sidewall).

[0076] The methods described herein can also be applied in an in vivo context by applying an AC electric field to a target area of ​​a living subject's body (e.g., using the Novocure Optune® system). For example, this can be accomplished by placing electrodes on or under the subject's skin such that application of an AC voltage between a selected subset of these electrodes imposes an AC electric field in the target area of ​​the subject's body.

[0077] For example, in situations where the relevant cells are located in the subject's brain, one pair of electrodes can be placed on the front and back of the subject's head, and a second pair of electrodes can be placed on the right and left sides of the subject's head. In some embodiments, the electrodes are capacitively coupled to the subject's body (e.g., by using an electrode that includes a conductive plate and also has a dielectric layer placed between the conductive plate and the subject's body). However, in alternative embodiments, the dielectric layer may be omitted, in which case the conductive plate would be in direct contact with the subject's body. In another embodiment, the electrodes can be inserted subcutaneously under the patient's skin. An AC voltage generator applies an AC voltage of a selected frequency (e.g., 200 kHz) between the right and left electrodes for a first time period (e.g., 1 second) to induce an alternating electric field in which many significant components of the field lines are parallel to the transverse axis of the subject's body.

[0078] The AC voltage generator then applies an AC voltage of the same frequency (or a different frequency) between the anterior and posterior electrodes for a second time (e.g., 1 second), inducing an alternating electric field in which many significant components of the field lines are parallel to the sagittal axis of the subject's body. This two-step sequence is then repeated for the duration of the treatment. Optionally, thermal sensors may be included in the electrodes, and the AC voltage generator may be configured to reduce the amplitude of the AC voltage applied to the electrodes if the sensed temperature at the electrodes becomes too high. In some embodiments, one or more additional electrode pairs may be added and included in the sequence. In alternative embodiments, only a single electrode pair is used, in which case the field lines do not switch direction. It should be noted that any of the parameters for this in vivo embodiment (e.g., frequency, field strength, duration, speed of switching direction, and electrode placement) may be varied as described above in relation to the in vitro embodiment. However, care must be taken in the in vivo situation to ensure that the electric field remains safe for the subject at all times.

[0079] It should be noted that in the experiments described herein, the AC electric field was applied for an uninterrupted time interval (e.g., 72 hours). However, in alternative embodiments, the application of the AC electric field may be interrupted, preferably by short breaks. For example, a 24-hour time interval can be filled by applying the AC electric field for six 4-hour blocks, with a 1- or 2-hour break between each of these blocks.

[0080] Although the present invention has been disclosed with reference to certain specific embodiments, several modifications, changes, and variations to the described embodiments are possible without departing from the sphere and scope of the invention as defined in the appended claims. Accordingly, the present invention is not limited to the described embodiments, but it is intended that it have the full scope defined by the language of the claims recited below, and equivalents thereof.

Claims

1. A system for treating a neurodegenerative disorder in a subject, comprising an AC voltage generator and one or more electrode pairs, wherein the AC voltage generator applies an alternating electric field to the subject's brain.

2. 13. The system of claim 1 for use in combination with administration to a subject of a therapeutic agent for treating a neurodegenerative disorder.

3. The system of claim 1 or 2, wherein the neurodegenerative disorder is Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), dementia, or Parkinson's disease.

4. 3. The system of claim 2, wherein the therapeutic agent comprises one or more of a GSK3β inhibitor, a cholinesterase inhibitor, or an anti-amyloid monoclonal antibody.

5. The system of claim 4 , wherein the therapeutic agent is a GSK3β inhibitor.

6. The system of claim 5 , wherein the GSK3β inhibitor is a cholinesterase inhibitor.

7. 3. The system of claim 2, wherein the therapeutic agent is 9-ING-41, HY-130795, TWS119, tideglusib, SAR502250, AR-A014418, TDZD8, kenpaullone, chromoib sodium, SB415286, IM-12, CP21R7, GNF4877, 1-azakempaullone, or indirubin-3'-monoxime.

8. 3. The system of claim 2, wherein the therapeutic agent is tacrine, galantamine, liquiritigenin, memantine, rivastigmine, or donepezil.

9. The system of claim 4, wherein the therapeutic agent is an anti-amyloid monoclonal antibody.

10. The system of claim 9, wherein the anti-amyloid monoclonal antibody is aducanumab.

11. 3. The system of claim 2, wherein the therapeutic agent is one or more of ciprofloxacin, celecoxib, tauroursodeoxycholic acid, and sodium phenylbutyrate.

12. 3. The system of claim 2, wherein the therapeutic agent is one or more of levodopa and carbidopa.

13. The system of claim 2, wherein the therapeutic agent is L-3,4-dihydroxyphenylalanine.

14. The system of claim 2 , wherein the therapeutic agent is a dopamine agonist.

15. 15. The system of claim 14, wherein the dopamine agonist is one or more of pramipexole, ropinirole, rotigotine, and apomorphine.

16. The system of claim 2 , wherein the therapeutic agent is an MAO-B inhibitor.

17. 17. The system of claim 16, wherein the MAO-B inhibitor is one or more of selegiline, rasagiline, and safinamide.

18. The system of claim 2, wherein the therapeutic agent is a catechol O-methyltransferase inhibitor.

19. 19. The system of claim 18, wherein the catechol O-methyltransferase inhibitor is one or more of entacapone, opicapone, and tolcapone.

20. The system of claim 2 , wherein the therapeutic agent is an anticholinergic agent.

21. 21. The system of claim 20, wherein the anticholinergic agent is one or more of benztropine and trihexyphenidyl.

22. The system of claim 2, wherein the therapeutic agent is amantadine.

23. 3. The system of claim 2, wherein the therapeutic agent is one or more of riluzole and edaravone.

24. The system of claim 2 , wherein at least a portion of the applying step is performed simultaneously with at least a portion of the administering step.

25. 3. The system of claim 1 or 2, wherein the applying step has a duration of at least 24 hours.

26. 3. The system of claim 1 or 2, wherein the applying step has a duration of at least 72 hours.

27. 3. The system of claim 1 or 2, wherein the applying step has a duration of at least 30 days.

28. 3. The system according to claim 1 or 2, wherein the frequency of the alternating electric field is between 50 kHz and 1 MHz.

29. 3. The system according to claim 1 or 2, wherein the strength of the alternating electric field is from 0.1 V / cm to 20 V / cm (RMS).

30. 30. The system of claim 29, wherein the intensity is between 1.0 V / cm and 2.5 V / cm.

31. The system of claim 1 or 2, wherein the brain does not contain a tumor.

32. The system described in claim 1 or 2, wherein the subject has been diagnosed with a neurodegenerative disorder.

33. 3. The system of claim 1 or 2, wherein the subject has not been diagnosed as having a tumor prior to application of the alternating electric field.

34. A pharmaceutical agent for use in a method of treating a neurodegenerative disorder in a subject, comprising: applying an alternating electric field to the brain of a subject; and Administering a therapeutic agent to the subject A pharmaceutical comprising:

Images