Use of hyperpolarizing agents alone and in combination with other therapeutic agents for the treatment of cancer, including glioblastoma - Patent Application 20070123333

Abstract

Methods and compositions are disclosed for treating cell proliferative diseases and disorders, including cancers such as glioblastoma. The disclosed methods and compositions may utilize or include one or more modulators of the bioelectrical state of a cell, such as one or more potassium channel activators, sodium channel inhibitors in combination with mTOR inhibitors, sodium channel inhibitors in combination with proton pump inhibitors, calcium channel inhibitors in combination with mTOR inhibitors, proton pump inhibitors in combination with alkylating agents, or a combination of at least one potassium channel activator with one or more of an alkylating agent, a calcium channel inhibitor, a corticosteroid, an mTOR inhibitor, a proton pump inhibitor, and / or a sodium channel inhibitor.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Application No. 63 / 265,403, filed December 14, 2021, the entire contents of which are incorporated by reference.

[0002] The field of the invention relates to methods and compositions for treating cell proliferative diseases and disorders, such as cancer. In particular, the field of the invention relates to methods and compositions for treating brain cancer, such as glioblastoma, in a subject by administering to the subject one or more therapeutic agents that modulate the polarity of glioblastoma cells. [Background technology]

[0003] Glioblastoma is a highly lethal cancer that can recur after initial resection of the tumor due to a population of glioblastoma stem cells just outside the borders of the main mass. It is therefore important to identify ways to prevent the proliferation of these cancer cells by inducing cell cycle arrest. A depolarized resting membrane potential is associated with a proliferative state, suggesting that forced hyperpolarization could be a strategy to target the overgrowth of glioblastoma cells.

[0004] To discover potentially useful modulators of bioelectrical state, we used NG108-15 cells and U87 cells in high serum medium as models to screen ion channel modulating drugs alone, in combination with each other, or with compounds known to be somewhat effective in reducing GBM growth. In particular, we tested whether drugs or drug combinations that can efficiently hyperpolarize cells can significantly reduce the proliferation of NG108-15 cells and terminally differentiate NG108-15 cells.

[0005] As disclosed herein, the inventors have found that several agents and combinations thereof that modulate the activity of membrane channels are highly effective in reducing cell proliferation during treatment, and this effect persists even several days after treatment has ceased. Our findings have corresponding implications for treating cancers that express membrane channels. Summary of the Invention

[0006] Methods and compositions are disclosed for treating cell proliferative diseases and disorders, such as glioblastoma. The disclosed compositions include, and the methods utilize, one or more therapeutic agents that modulate the polarity of cancer cells, and may include therapeutic agents that hyperpolarize cell membranes and modulate cancer differentiation and / or growth. Cancers that may be treated by the disclosed compositions and methods include glioblastoma.

[0007] In one aspect, a method of treating a cell proliferative disease or disorder in a subject in need of such treatment is provided. The method comprises administering to the subject an effective amount of one or more potassium channel activators. The potassium channel activator can be administered in combination with one or more of an mTOR inhibitor, an alkylating agent, a corticosteroid, a proton pump inhibitor, a sodium channel inhibitor, a calcium channel inhibitor, or a peroxisome proliferator-activated receptor alpha (PPARα) activator.

[0008] In another aspect, a method for treating a cell proliferative disease or disorder in a subject in need of such treatment includes: (i) administering to the subject an effective amount of a sodium channel inhibitor (e.g., a sodium channel blocker); and (ii) administering to the subject an effective amount of mTO and administering to the subject an mTOR inhibitor. The sodium channel inhibitor is administered to the subject before, simultaneously with, or after the mTOR inhibitor is administered to the subject.

[0009] In a further aspect, a method is provided for treating a cell proliferative disease or disorder in a subject in need thereof, the method comprising: (i) administering to the subject an effective amount of a sodium channel inhibitor (e.g., a sodium channel blocker); and (ii) administering to the subject an effective amount of a proton pump inhibitor, wherein the sodium channel inhibitor is administered to the subject before, simultaneously with, or after the proton pump inhibitor is administered to the subject.

[0010] In yet a further aspect, a method is provided for treating a cell proliferative disease or disorder in a subject in need thereof, the method comprising: (i) administering to the subject an effective amount of a calcium channel inhibitor (e.g., a calcium channel blocker); and (ii) administering to the subject an effective amount of an mTOR inhibitor. The calcium channel inhibitor is administered to the subject before, simultaneously with, or after the mTOR inhibitor is administered to the subject.

[0011] In yet another aspect, a method is provided for treating a cell proliferative disease or disorder in a subject in need thereof, comprising: (i) administering to the subject an effective amount of a proton pump inhibitor, and (ii) administering to the subject an effective amount of an alkylating agent, wherein the proton pump inhibitor is administered to the subject before, simultaneously with, or after the alkylating agent is administered to the subject. [Brief description of the drawings]

[0012] [Figure 1]NG108-15 proliferation is significantly decreased with bioelectrical treatment, showing changes in cell cycle ratios. (A) Fold change (cells at day 6 / cells at day 0) starting cell counting 6 days after treatment. Lower values ​​indicate less cell proliferation. Colors indicate treatments that were followed for further analysis. Red shaded treatments correspond to positive controls that cannot be used clinically. Only treatments with significant values ​​out of 33 treatments are shown compared to DMSO control. ****: q<0.0001, ***: q<0.001, **: q<0.01, *: q<0.05 (one-way ANOVA with FDR post-hoc analysis, n>3 biological replicates). (B) FUCCI cell cycle data at day 6. Increase in red and orange fractions indicates cell cycle arrest in G1 or G1 to S transition. [Diagram 2] FIG. 1 shows that the combination of pantoprazole and bioelectric compounds significantly reduces proliferation and shows changes in cell cycle ratios compared to pantoprazole alone. (A) Percentage reduction of cells compared to control after 6 days of treatment. Treatments significantly more effective than pantoprazole alone are shown among the 32 treatments. Statistical analysis was performed on the log2 of fold change in cell number relative to control on day 6. ***: q<0.001, **: q<0.01, *: q<0.05 (One-way ANOVA with FDR post-hoc analysis, n>3 biological replicates). (B) FUCCI cell cycle data on day 6. Increase in red and orange fractions indicates cell cycle arrest at G1 or G1 to S transition. [Diagram 3] Figure 1. Recovery study of hyperpolarizing treatment in combination with pantoprazole in NG108-15 FUCCI cells. The log2 of fold change in cell counts up to day 0 was recorded for 10 days. The dotted line indicates the day drug treatment was stopped and replaced with control medium (n>3 biological replicates). The combined drug treatment slopes from day 6 to day 10 were compared to pantoprazole alone, significance is indicated by grey stars next to the corresponding line. **: p<0.01, *: p<0.05 (One-way ANOVA with Dunnett's post-hoc analysis, n>3 biological replicates). [Figure 4-A]Figure 2: Hyperpolarizing drugs, pantoprazole, and TMZ in combination with each other reduced U87 cell proliferation and altered cell cycle ratio compared to control. (A) Fold change starting cell counting 6 days after treatment (cells at day 6 / cells at day 0). Lower values ​​indicate less cell proliferation. Colors indicate treatments that were followed for further analysis. Red shaded treatments correspond to positive controls that cannot be used clinically. Only treatments with significant values ​​out of 42 treatments compared to DMSO control are shown. ****: q<0.0001, ***: q<0.001, **: q<0.01, *: q<0.05 (one-way ANOVA with FDR post-hoc analysis, n>3 biological replicates). [Figure 4-B] (B) Hyperpolarizing drugs, pantoprazole and TMZ in combination with each other, reduced proliferation of U87 cells and altered cell cycle ratios compared to controls. (B) FUCCI cell cycle data at day 6. Increases in red and orange fractions indicate cell cycle arrest at G1 or the G1 to S transition. [Diagram 5] Treatment with hyperpolarizing compounds and pantoprazole or TMZ was significantly better than TMZ alone in reducing proliferation and changing cell cycle ratios compared to control in U87 cells. (A) Percentage of cell loss compared to control after 6 days of treatment. Treatments that were significantly more effective than TMZ alone are shown among 42 treatments. Red shaded treatments correspond to positive controls that cannot be used clinically. Statistical analysis was performed on the log2 of fold change in cell number relative to control on day 6. ***: q<0.001, **: q<0.01, *: q<0.05 (one-way ANOVA with FDR post-hoc analysis, n>3 biological replicates). (B) FUCCI cell cycle data on day 6. Increase in red and orange fractions indicates cell cycle arrest in G1 or G1 to S transition. [Figure 6]Combinations of hyperpolarizing drugs with pantoprazole or TMZ or pantoprazole and TMZ showed the most significant reduction in cell proliferation and percent cell loss after 6 days of treatment compared to control (A). Treatments significantly more effective than pantoprazole alone are shown among the 42 treatments. Statistical analysis was performed on the log2 of fold change in cell number relative to control on day 6. ***: q<0.001, **: q<0.01, *: q<0.05 (one-way ANOVA with FDR post-hoc analysis, n>3 biological replicates). (B) FUCCI cell cycle data on day 6. Increase in red and orange fractions indicates cell cycle arrest in G1 or G1 to S transition. [Figure 7] Figure 1. Recovery study of hyperpolarizing treatment combined with pantoprazole or TMZ. The log2 of fold change in cell counts up to day 0 was recorded for 10 days. The dotted line indicates the day drug treatment was stopped and replaced with control medium (n>3 biological replicates). The combined drug treatment slope from day 6 to day 10 was compared to pantoprazole or TMZ alone but no significance was found (one-way ANOVA with Dunnett's post-hoc analysis, n>3 biological replicates). [Figure 8] Figure 1. Changes in resting membrane potential caused by treatment in NG108-15 FUCCI palmitoyl-mTurquoise2 cells. Changes in resting membrane potential normalized to DMSO control. More negative values ​​indicate increased hyperpolarization. (A) and (B) were performed on different days. Sample size is n=55-63 cells per condition. Significant values ​​are shown in the table. ANOVA with Dunnett post-hoc analysis was used to calculate ****: p<0.0001. [Figure 9]Differentiation analysis of NG108-15 cells reveals that treatment with pantoprazole increases neural markers after 6 days. Immunofluorescence of cells was analyzed with CellProfiler and quantified for integrated fluorescence intensity. (A) Staining for microtubule-associated protein 2 (MAP2). (B) Staining for neuron-specific class III beta-tubulin (Tuj I). (C) Staining for nerve fiber medium chain (NFM). (D) Staining for neuron-specific enolase (NSE). Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated in the color of the compared treatment. Initial fluorescence intensity was compared to their corresponding controls, and significant values ​​were indicated below the bars: ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 10] Differentiation analysis of NG108-15 cells reveals that treatment with pantoprazole increases astroglial and differentiation markers after 6 days. Cells were subjected to immunofluorescence and analyzed with CellProfiler, measuring integrated fluorescence intensity. (A) Staining for S100 calcium binding protein B (S100B). (B) Staining for glial fibrillary acidic protein (GFAP). (C) Staining for phosphorylated cAMP response element binding protein (Phospho CREB). (D) Staining for connexin 43 (Cx43). Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated in the color of the treatment compared. Initial fluorescence intensity was compared to their corresponding controls, and significant values ​​were indicated below the bars: ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 11]Senescence and proliferation analysis of NG108-15 cells reveals that treatment with pantoprazole increases senescence, decreases BrdU incorporation, and increases p27Kip1 after 6 days. Senescence-associated β-galactosidase staining was performed and scored by naked eye. Cells were subjected to immunofluorescence and analyzed with CellProfiler for integrated fluorescence intensity or presence or absence of cellular signal. (A) Staining for senescence-associated β-galactosidase staining (SA-BetaGal). (B) Staining for bromodeoxyuridine incorporation (BrdU). (C) Staining for microtubule-associated protein light chain 3 II (LC3-II). (D) Staining for cleaved caspase 3 (Casp3). (E) Staining for cyclin-dependent kinase inhibitor 1B (p27Kip). (F) Nuclear size determined by area of ​​Hoechst staining. Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated by the color of the treatment being compared. Initial fluorescence intensity was compared to the corresponding control, and significant values ​​are indicated below the bar. Logits of percent positive cells were compared between single treatments and controls, and arcsine transformation was used for zero values. Significance was expressed as ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 12] Differentiation analysis of U87 cells reveals that treatment with pantoprazole increased neuronal markers after 6 days. Cells were subjected to immunofluorescence and analyzed with CellProfiler for integrated fluorescence intensity. (A) Staining for microtubule-associated protein 2 (MAP2). (B) Staining for neuron-specific class III beta-tubulin (Tuj I). (C) Staining for nerve fiber medium chain (NFM). (D) Staining for neuron-specific enolase (NSE). Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated in the color of the compared treatment. Initial fluorescence intensity was compared to their corresponding controls, and significant values ​​were indicated below the bars: ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 13]Differentiation analysis of U87 cells reveals that treatment with pantoprazole increases astrocytic and differentiation markers after 6 days. Cells were subjected to immunofluorescence and analyzed with CellProfiler for integrated fluorescence intensity. (A) Staining for vimentin. (B) Staining for phosphorylated cAMP response element binding protein (CREB). (C) Staining for S100 calcium binding protein B (S100B). (D) Staining for glial fibrillary acidic protein (GFAP). Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated in the color of the compared treatment. Initial fluorescence intensity was compared to their corresponding controls, and significant values ​​were indicated below the bars: ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 14] Differentiation analysis of U87 cells reveals that treatment with pantoprazole increased oligodendrocyte markers after 6 days. Cells were subjected to immunofluorescence and analyzed with CellProfiler for integrated fluorescence intensity. (A) Staining for oligodendrocyte marker O4. (B) Staining for Sry-related HMg-Box gene 10 (SOX10). Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated in the color of the compared treatment. Initial fluorescence intensity was compared to their corresponding controls, and significant values ​​are indicated below the bars: ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 15]Senescence and proliferation analysis of U87 cells reveals that treatment with pantoprazole or NS164 with TMZ increases senescence, decreases BrdU incorporation and increases p27Kip1 after 6 days. Senescence-associated β-galactosidase staining was performed and scored by naked eye. Cells were subjected to immunofluorescence and analyzed with CellProfiler for integrated fluorescence intensity or presence or absence of cellular signal. (A) Staining for senescence-associated β-galactosidase staining (SA-BetaGal). (B) Staining for bromodeoxyuridine incorporation (BrdU). (C) Staining for microtubule-associated protein light chain 3 II (LC3-II). (D) Staining for cleaved caspase 3 (Casp3). (E) Staining for cyclin-dependent kinase inhibitor 1B (p27Kip). (F) Nuclear size determined by area of ​​Hoechst staining. Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated by the color of the treatment compared. Initial fluorescence intensity was compared to the corresponding control, and significant values ​​are indicated below the bar. Logits of percent positive cells were compared between single treatments and controls, and arcsine transformation was used for zero values. Significance was expressed as ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 16]Voltage dyes showed that U87 cells treated with NS1643 and a combination of NS1643 and pantoprazole for 6 days showed hyperpolarization and YAP increased its translocation to the cytoplasm in pantoprazole with NS1643 or TMZ, and pantoprazole with NS1643 treatment. Cells were subjected to immunofluorescence and analyzed in CellProfiler for integrated fluorescence intensity. Dye assays were analyzed for mean intensity, except for LysoSensor Green, which was analyzed for integrated intensity. (A) Staining of lysosomal pH using LysoSensor Green, low levels indicate alkalinization. (B) Dye indicator of membrane potential, DiBAC4(3), low levels indicate hyperpolarization. (C) Dye indicator of cytoplasmic pH, pHRodoGreen, low levels indicate alkalinization. (D) Dye indicator of cytoplasmic calcium, Fluo-4AM, high levels indicate increased calcium. (E) Nuclear to cytoplasmic Y-associated protein (YAP) ratio, with lower levels indicating translocation to the cytoplasm. Logarithms of fold changes in intensity were compared between single and combination treatments, except for the positive control, and significant values ​​were indicated in the color of the compared treatment. Initial fluorescence intensities were compared to their corresponding controls, and significant values ​​were indicated below the bars: ***: p<0.001, **: p<0.01, *: p<0.05 (one-way ANOVA with Tukey post-hoc analysis, technical replicates n>3). [Figure 17] Figure 1 shows that Live / Dead and senescence assays of human neurons after 3 days of treatment show low levels of toxicity. Lower values ​​indicate less cell death or senescence. (A) Live / Dead assay performed on human neurons cultured with drugs for 3 days. (B) Senescence assay results of senescence-associated β-galactosidase staining on human neurons cultured with drugs for 3 days. Treatments with the best reduction in proliferation in NG108-15 or U87 cells are shown from a toxicity screen of 24 samples, with significant values ​​indicated. ***: q<0.001, **: q<0.01, *: q<0.05 (one-way ANOVA with FDR post-hoc analysis, n>3 technical replicates). An increase in the percentage of dead or senescent cells indicates a toxic treatment. [Figure 18]Figure NG108-15 initial screening of compounds part A. Percentage of cell reduction compared to control after 6 days of treatment, technical replicates n>5. Color plots indicate compounds that were subsequently analyzed in NG108-15 or U87 cells. [Figure 19] Figure NG108-15 initial screening of compound part B. Percentage of cell reduction compared to control after 6 days of treatment, technical replicates n>5. Color plots indicate compounds that were subsequently analyzed in NG108-15 or U87 cells. Red shaded treatments are positive controls used in subsequent analyses. [Figure 20] Figure 21-51 shows the results of tumor tissue cytotoxicity assays for various drug treatments on both colorectal and breast cancer tissues. Figures 21-22 show an overall impact score based on the viability and morphology of cancer cells in treated tissues on a scale of 0-100, assessed by various parameters including nuclear detail, tissue cohesiveness, cytoplasmic changes, and immunohistochemical staining. On this scale, higher scores correlate with improved clinical response, while a score below 30 is considered no response. Treatments tested were: (1) 100 μM pantoprazole in combination with 10 μM retigabine, (2) 100 μM pantoprazole in combination with 100 μM lamotrigine, (3) 200 nM rapamycin in combination with 10 μM retigabine, (4) 200 nM rapamycin in combination with 30 μM minoxidil, (5) 200 nM rapamycin in combination with 10 μM zolmitriptan, (6) 200 nM rapamycin in combination with 50 μM NS1643, and (7) 50 μM NS1643 for 6 days. [Figure 21] "CRC" refers to colorectal cancer tissue and "breast" refers to breast cancer tissue. [Figure 22]"Median" indicates the median effect score of all drug treatments tested. Figures 23-51 show the histological results of tumor tissue cytotoxicity assays for various drug treatments on both colorectal and breast cancer tissues, as detailed below. [Diagram 23] FIG. 1 shows hematoxylin and eosin (H&E) stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with various treatments, including an untreated control (bottom, far right), as labeled. [Figure 24] Figure 1 shows H&E staining histological results of tumor tissue cytotoxicity assays for colorectal cancer tissues treated with various treatments, including an untreated control (bottom, far right). (NT = no tumor) [Diagram 25] FIG. 1 shows H&E staining histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with various treatments, including an untreated control (bottom, far right). [Figure 26] FIG. 1 shows H&E staining histological results of a tumor tissue cytotoxicity assay for breast cancer tissues treated with various treatments, including an untreated control (bottom, far right). [Figure 27] Figure 1 shows H&E staining histological results of tumor tissue cytotoxicity assays for breast cancer tissues treated with various treatments, including an untreated control (bottom, far right). (NT = no tumor) [Figure 28] Figure 1 shows H&E staining histological results of tumor tissue cytotoxicity assays for breast cancer tissues treated with various treatments, including an untreated control (bottom, far right). (NA = not applicable for technical reasons) [Figure 29] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with pantoprazole in combination with retigabine (top) or pantoprazole in combination with lamotrigine (bottom). [Diagram 30] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with rapamycin in combination with retigabine (top) or rapamycin in combination with minoxidil (bottom). [Diagram 31] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with rapamycin in combination with zolmitriptan (top) or rapamycin in combination with NS1643 (bottom). [Diagram 32] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with NS1643 alone (top) or untreated as a control (bottom). [Diagram 33] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with pantoprazole in combination with retigabine (top) or pantoprazole in combination with lamotrigine (bottom). [Diagram 34] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with rapamycin in combination with retigabine (top) or rapamycin in combination with minoxidil (bottom). [Diagram 35] Figure 1 shows H&E stained histological results of tumor tissue cytotoxicity assays for colorectal cancer tissues treated with rapamycin in combination with zolmitriptan (top) or rapamycin in combination with NS1643 (bottom). (NT = no tumor) [Diagram 36] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with NS1643 alone (top) or untreated as a control (bottom). [Figure 37] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with pantoprazole in combination with retigabine (top) or pantoprazole in combination with lamotrigine (bottom). [Figure 38] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with rapamycin in combination with retigabine (top) or rapamycin in combination with minoxidil (bottom). [Figure 39]FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with rapamycin in combination with zolmitriptan (top) or rapamycin in combination with NS1643 (bottom). [Diagram 40] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for colorectal cancer tissues treated with NS1643 alone (top) or untreated as a control (bottom). [Diagram 41] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for breast cancer tissues treated with pantoprazole in combination with retigabine (top) or pantoprazole in combination with lamotrigine (bottom). [Diagram 42] FIG. 1 shows H&E stained histological results of tumor tissue cytotoxicity assays for breast cancer tissues treated with rapamycin in combination with retigabine (top) or rapamycin in combination with minoxidil (bottom). [Diagram 43] FIG. 1 shows H&E stained histological results of tumor tissue cytotoxicity assays for breast cancer tissues treated with rapamycin in combination with zolmitriptan (top) or rapamycin in combination with NS1643 (bottom). [Diagram 44] FIG. 1 shows H&E staining histological results of a tumor tissue cytotoxicity assay for breast cancer tissues treated with NS1643 alone (top) or untreated as a control (bottom). [Diagram 45] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for breast cancer tissues treated with pantoprazole in combination with retigabine (top) or pantoprazole in combination with lamotrigine (bottom). [Figure 46] FIG. 1 shows H&E stained histological results of tumor tissue cytotoxicity assays for breast cancer tissues treated with rapamycin in combination with retigabine (top) or rapamycin in combination with minoxidil (bottom). [Figure 47]Figure 1 shows H&E stained histological results of tumor tissue cytotoxicity assays for breast cancer tissues treated with rapamycin in combination with zolmitriptan (top) or rapamycin in combination with NS1643 (bottom). (NT = no tumor) [Figure 48] Figure 1 shows the H&E staining histological results of a tumor tissue cytotoxicity assay for breast cancer tissues treated with NS1643 alone (top) or untreated as a control (bottom). (NT = no tumor) [Figure 49] FIG. 1 shows H&E stained histological results of a tumor tissue cytotoxicity assay for breast cancer tissues treated with pantoprazole in combination with retigabine (top) or pantoprazole in combination with lamotrigine (bottom). [Figure 50] FIG. 1 shows H&E stained histological results of tumor tissue cytotoxicity assays for breast cancer tissues treated with rapamycin in combination with retigabine (top) or rapamycin in combination with minoxidil (bottom). [Figure 51] FIG. 1 shows H&E staining histological results of tumor tissue cytotoxicity assay for breast cancer tissues treated with rapamycin in combination with NS1643. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The disclosed subject matter can be further described using the terms defined below.

[0014] Unless otherwise specified or indicated by context, the terms "a," "an," and "the" mean "one or more." For example, "a therapeutic agent" should be interpreted as meaning "one or more therapeutic agents."

[0015] As used herein, "about," "approximately," "substantially," and "significantly" will be understood by those of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of a term that are not clear to persons of ordinary skill in the art given the context in which they are used, "about" and "approximately" will mean ±10% or less of the particular term, and "substantially" and "significantly" will mean ±10% or more of the particular term.

[0016] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising". The terms "comprise" and "comprising" should be interpreted as "open" transitional terms that allow for the inclusion of additional elements beyond those recited in the claims. The terms "consist" and "consisting of" should be interpreted as "closed" transitional terms that do not permit the inclusion of additional elements other than those recited in the claims. The term "consisting essentially of" should be interpreted as being partially closed, allowing for the inclusion only of additional elements that do not fundamentally change the nature of the claimed subject matter.

[0017] The phrase "e.g., etc." should be interpreted as "for example." Furthermore, the use of any and all exemplary language, including but not limited to "e.g., etc.", is intended merely to better clarify the invention and does not limit the scope of the invention unless otherwise claimed.

[0018] When a convention similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together). It will be further understood by one of ordinary skill in the art that substantially any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or illustration, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0019] All language such as "up to," "at least," "greater than," "less than," etc., refers to ranges that are inclusive of the recited numbers and can subsequently be divided into ranges and subranges. Ranges include individual members. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, etc.

[0020] The modal verb "may" refers to a preferred use or selection of one or more alternatives or choices among several described embodiments or features contained therein. When no alternative or choice is disclosed with respect to a particular embodiment or feature being described, the modal verb "may" refers to a positive action with respect to how to make or use the described embodiment or feature contained therein, or a definite decision to use a particular skill with respect to the described embodiment or feature contained therein. In this latter context, the modal verb "may" has the same meaning and connotation as the auxiliary verb "can."

[0021] As used herein, the phrase "effective amount" is intended to mean a drug dosage that provides a particular pharmacological response to which the drug is administered to a significant number of patients in need of such treatment. The effective amount of a drug administered to a particular patient in a particular instance is not always effective in treating the conditions / diseases described herein, even if such dosage is considered to be a therapeutically effective amount by those skilled in the art.

[0022] The terms "subject", "patient" and "individual" may be used interchangeably herein. The subject may be a human subject. The subject may refer to a human subject having or at risk of acquiring a cell proliferative disease or disorder, such as cancer, including, but not limited to, brain cancer, such as brain cancer (e.g., glioblastoma multiforme (GBM)), prostate cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, bladder cancer, kidney cancer, uterine cancer, melanoma, lymphoma (e.g., non-Hodgkin's lymphoma), leukemia, pancreatic cancer, ovarian cancer, liver and intrahepatic bile duct cancer, oral cancer, and esophageal cancer, particularly glioblastoma.

[0023] Use of Hyperpolarizing Agents Alone and in Combination with Other Therapeutic Agents to Treat Cancer The disclosed subject matter relates to compositions and methods for treating a cell proliferative disease or disorder in a subject in need thereof. The compositions include, and the methods utilize, one or more therapeutic agents that modulate the polarity of cancer cells, and may include therapeutic agents that hyperpolarize cell membranes and modulate cancer differentiation and / or growth.

[0024] In some embodiments, the disclosed subject matter relates to a method of treating a cell proliferative disease or disorder in a subject in need thereof. The method of treatment can include administering to the subject an effective amount of one or more potassium channel activators.

[0025] Suitable potassium channel activators for use in the disclosed therapeutic methods may include, but are not limited to, agents that activate KCNQ / Kv7 channels. Suitable potassium channel activators may include, but are not limited to, retigabine, which has the following chemical structure, or a suitable pharmaceutical salt thereof:

[0026] [ka]

[0027] Potassium channel activators suitable for use in the disclosed therapeutic methods can include, but are not limited to, agents that activate K(ATP) channels. Channel activators may include, but are not limited to, minoxidil, which has the following chemical structure:

[0028] [ka]

[0029] Suitable potassium channel activators for use in the disclosed therapeutic methods may include, but are not limited to, agents that activate the hERG channel. Suitable potassium channel activators may include, but are not limited to, NS1643, which has the following chemical structure, or a suitable pharmaceutical salt thereof:

[0030] [ka]

[0031] Suitable potassium channel activators for use in the disclosed therapeutic methods may include, but are not limited to, agents that activate the KCNK3 channel. Suitable potassium channel activators may include, but are not limited to, ONO-RS-082, which has the following chemical structure, or a suitable pharmaceutical salt thereof:

[0032] [ka]

[0033] Suitable potassium channel activators for use in the disclosed therapeutic methods may include combinations of potassium channel activators. Suitable combinations of potassium channel activators may include, but are not limited to, a combination comprising ONO-RS-082 and NS1643.

[0034] Potassium channel activators suitable for use in the disclosed therapeutic methods may include, but are not limited to, agents that activate BK and / or SK potassium channels. Potassium channel activators suitable for use in the disclosed methods may include, but are not limited to, chlorzoxazone, which has the following chemical structure, or a suitable pharmaceutical salt thereof:

[0035] [ka]

[0036] Suitable therapeutic agents for use in the disclosed methods of treatment may include combinations of potassium channel activators, including, but not limited to, combinations including chlorzoxazone and NS1643.

[0037] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and an mTOR inhibitor, which may be administered prior to, simultaneously with, or following administration of the one or more potassium channel activators.

[0038] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and a rapamycin having the following formula, or a suitable pharmaceutical salt thereof:

[0039] [ka]

[0040] The rapamycin may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0041] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and an alkylating agent, which may be administered prior to, simultaneously with, or following administration of the one or more potassium channel activators.

[0042] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and temozolomide, having the following formula, or a suitable pharmaceutical salt thereof:

[0043] [ka]

[0044] Temozolomide may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0045] Suitable therapeutic agents for use in the disclosed methods of treatment may include combinations of one or more potassium channel activators and corticosteroids, including but not limited to dexamethasone, benzodiazepines, and benzocaline. Corticosteroids may include, but are not limited to, xamethasone, betamethasone, triamcinolone acetonide, fluorometholone, cortisone, hydrocortisone, fludrocortisone acetate, prednisolone, prednisone, methylprednisolone, triamcinolone. The corticosteroids may be administered prior to, simultaneously with, or after administration of one or more potassium channel activators.

[0046] Suitable therapeutic agents for use in the disclosed methods of treatment may include combinations of one or more potassium channel activators and dexamethasone, having the following chemical structure, or a derivative thereof and a suitable pharmaceutical salt thereof:

[0047] [ka]

[0048] Dexamethasone or a derivative thereof may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0049] Suitable therapeutic agents for use in the disclosed therapeutic methods may include a combination of one or more potassium channel activators and a proton pump inhibitor, including, but not limited to, pantoprazole, omeprazole, lansoprazole, dexlansoprazole, esomeprazole, rabeprazole, and cilaprazole. The proton pump inhibitor may be administered before, simultaneously with, or after administration of one or more potassium channel activators.

[0050] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and pantoprazole, which has the following chemical structure, or a suitable pharmaceutical salt thereof:

[0051] [ka]

[0052] Pantoprazole may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0053] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and one or more sodium channel inhibitors (e.g., sodium channel blockers). The sodium channel inhibitors may be administered prior to, simultaneously with, or after administration of the one or more potassium channel activators.

[0054] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and lamotrigine, which has the following chemical structure, or a suitable pharmaceutical salt thereof.

[0055] [ka]

[0056] Lamotrigine may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0057] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and cariporide, which has the following chemical structure, or a pharmaceutical salt thereof:

[0058] [ka]

[0059] Cariporide may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0060] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and topiramate, having the following chemical structure, or a suitable pharmaceutical salt thereof:

[0061] [ka]

[0062] Topiramate may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0063] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and one or more calcium channel inhibitors (e.g., calcium channel blockers). The calcium channel inhibitors may be administered prior to, simultaneously with, or after administration of the one or more potassium channel activators.

[0064] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and gabapentin, having the following chemical structure, or a suitable pharmaceutical salt thereof:

[0065] [ka]

[0066] Gabapentin may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0067] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and zolmitriptan, having the following chemical structure, or a suitable pharmaceutical salt thereof:

[0068] [ka]

[0069] Zolmitriptan may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0070] Suitable therapeutic agents for use in the disclosed therapeutic methods may include a combination of one or more potassium channel activators and a peroxisome proliferator-activated receptor alpha (PPARα) activator (e.g., a PPARα agonist). The PPARα activator may be administered prior to, simultaneously with, or following administration of the one or more potassium channel activators.

[0071] Suitable therapeutic agents for use in the disclosed methods of treatment may include a combination of one or more potassium channel activators and fenofibrate, having the following chemical structure, or a suitable pharmaceutical salt thereof:

[0072] [ka]

[0073] Fenofibrate may be administered prior to, simultaneously with, or following administration of one or more potassium channel activators.

[0074] In some embodiments, the disclosed subject matter relates to a method of treating a cell proliferative disease or disorder in a subject in need of such treatment. The method of treatment can include administering to the subject: (i) an effective amount of a sodium channel inhibitor (e.g., a sodium channel blocker); and (ii) an effective amount of an mTOR inhibitor, where the sodium channel inhibitor is administered to the subject before, simultaneously with, or after the mTOR inhibitor is administered to the subject.

[0075] Suitable sodium channel inhibitors for use in the disclosed methods of treatment can include, but are not limited to, agents that inhibit voltage-gated sodium channels. Suitable sodium channel inhibitors can include, but are not limited to, lamotrigine and topiramate.

[0076] Sodium channel inhibitors suitable for use in the disclosed therapeutic methods may include, but are not limited to, agents that inhibit the Na(+) / H(+) exchanger type 1 (NHE1). Suitable sodium channel inhibitors may include, but are not limited to, cariporide.

[0077] Therapeutic agents suitable for use in the disclosed methods include agents that inhibit mTOR. Suitable mTOR inhibitors may include, but are not limited to, rapamycin.

[0078] In some embodiments, the disclosed subject matter relates to a method of treating a cell proliferative disease or disorder in a subject in need of such treatment. The method of treatment can include administering to the subject: (i) an effective amount of a sodium channel inhibitor (e.g., a sodium channel blocker); and (ii) an effective amount of a proton pump inhibitor, where the sodium channel inhibitor is administered to the subject before, simultaneously with, or after the proton pump inhibitor is administered to the subject.

[0079] Suitable sodium channel inhibitors for use in the disclosed methods of treatment can include, but are not limited to, agents that inhibit voltage-gated sodium channels. Suitable sodium channel inhibitors can include, but are not limited to, lamotrigine and topiramate.

[0080] Sodium channel inhibitors suitable for use in the disclosed therapeutic methods may include, but are not limited to, agents that inhibit the Na(+) / H(+) exchanger type 1 (NHE1). Suitable sodium channel inhibitors may include, but are not limited to, cariporide.

[0081] Therapeutic agents suitable for use in the disclosed methods include agents that are proton pump inhibitors. Suitable proton pump inhibitors can include, but are not limited to, pantoprazole.

[0082] In some embodiments, the disclosed subject matter relates to a method of treating a cell proliferative disease or disorder in a subject in need of such treatment. The method of treatment can include administering to the subject: (i) an effective amount of a calcium channel inhibitor (e.g., a calcium channel blocker); and (ii) an effective amount of an mTOR inhibitor, where the calcium channel inhibitor is administered to the subject before, simultaneously with, or after the mTOR inhibitor is administered to the subject.

[0083] Suitable calcium channel inhibitors for use in the disclosed therapeutic methods may include, but are not limited to, agents that inhibit voltage-gated calcium channels. Suitable calcium channel inhibitors may include, but are not limited to, zolmitriptan.

[0084] Therapeutic agents suitable for use in the disclosed methods include agents that inhibit mTOR. Suitable mTOR inhibitors may include, but are not limited to, rapamycin.

[0085] In some embodiments, the disclosed subject matter relates to a method of treating a cell proliferative disease or disorder in a subject in need of such treatment. The method of treatment may include administering to the subject: (i) an effective amount of a proton pump inhibitor; and (ii) an effective amount of an alkylating agent, wherein the proton pump inhibitor is administered to the subject before, simultaneously with, or after the alkylating agent is administered to the subject. Suitable proton pump inhibitors may include, but are not limited to, pantoprazole. Suitable alkylating agents include temozolomide, cefolia, cefolia tetracycline ... This may include, but is not limited to,

[0086] Suitable cell proliferative diseases and disorders treated by the disclosed methods can include cancers, such as cancers expressing potassium channels (e.g., KCNQ / Kv7 channels, K(ATP) channels, hERG channels, or KCNK3 channels), sodium channels (e.g., voltage-gated sodium channels or sodium / proton exchangers such as NHE1), or calcium channels (e.g., voltage-gated calcium channels). Cancers treated by the disclosed methods can include, but are not limited to, brain cancer (e.g., glioblastoma multiforme (GBM)), prostate cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, bladder cancer, kidney cancer, uterine cancer, melanoma, lymphoma (e.g., non-Hodgkin's lymphoma), leukemia, pancreatic cancer, ovarian cancer, liver and intrahepatic bile duct cancer, oral cancer, and esophageal cancer.

[0087] The compounds utilized in the methods disclosed herein may be formulated as anti-cancer therapeutics, including therapeutics for malignancies including cancers such as brain cancer (e.g., glioblastoma multiforme (GBM)), prostate cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, bladder cancer, kidney cancer, uterine cancer, melanoma, lymphoma (e.g., non-Hodgkin's lymphoma), leukemia, pancreatic cancer, ovarian cancer, liver and intrahepatic bile duct cancer, oral cancer, and esophageal cancer.

[0088] The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions comprising (a) a therapeutically effective amount of one or more compounds disclosed herein, and (b) one or more pharma- ceutically acceptable carriers, excipients, or diluents. The pharmaceutical compositions may comprise a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, more preferably about 1 to 100 mg) of the compound. The pharmaceutical compositions may be administered to provide a daily dose of the compound of about 0.1 to about 1000 mg / kg body weight (preferably about 0.5 to about 500 mg / kg body weight, more preferably about 50 to about 100 mg / kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., about 1, 2, 3, 4, 5, or 6 hours after administration), the concentration of the compound at the site of action may be within a concentration range bounded by an endpoint selected from 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM (e.g., 0.1 μM to 1.0 μM). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., about 1, 2, 3, 4, 5, or 6 hours after administration), the concentration of the compound at the site of action may be within a concentration range bounded by an endpoint selected from 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM, 200 μM, 400 μM, 800 μM, and 1000 μM (e.g., 1.0 μM to 800 μM).

[0089] The compounds and pharmaceutical compositions comprising the compounds utilized in the methods disclosed herein can be administered in a method of treating a subject in need thereof. For example, in a method of treatment, the subject in need thereof can include a subject having a cell proliferative disease, disorder, or condition, such as cancer (e.g., brain cancer (e.g., glioblastoma multiforme (GBM)), prostate cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), colorectal cancer, bladder cancer, kidney cancer, uterine cancer, melanoma, lymphoma (e.g., cancers such as non-Hodgkin's lymphoma), leukemia, pancreatic cancer, ovarian cancer, liver and intrahepatic bile duct cancer, oral cancer, and esophageal cancer).

[0090] In some embodiments of the disclosed methods of treatment, a subject is administered 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, or more of a combination of these to treat a disease or disorder in the subject. g, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times weekly. In some embodiments, a subject may be administered a dose of the compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, , 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg may be administered once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or as high as three times weekly. Minimum and / or maximum doses of the compounds may include doses that fall within a dose range having any of these disclosed doses (e.g., 2.5 to 200 mg) as an endpoint.

[0091] In some embodiments, the minimum dosage level of the compound to achieve treatment in the disclosed therapeutic methods can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. In some embodiments, the maximum dosage level of the compound to achieve treatment in the disclosed therapeutic methods does not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. The minimum and / or maximum dose levels of the compounds for achieving treatment in the disclosed therapeutic methods can include dose levels that fall within a range having any of these disclosed dose levels as an endpoint (e.g., 500-2000 ng / kg body weight of the subject).

[0092] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions in solid dosage form, but can utilize any pharma- ceutically acceptable dosage form.Exemplary solid dosage forms include, but are not limited to, tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast-dissolving form, a controlled release form, a lyophilized form, a delayed release form, a sustained release form, a periodic release form, a mixed immediate release and controlled release form, or a combination thereof.

[0093] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions that include a carrier, for example, the carrier can be selected from the group consisting of proteins, carbohydrates, sugars, talc, magnesium stearate, cellulose, calcium carbonate, and starch gelatin paste.

[0094] The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions containing one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Fillers may include lactose monohydrate, lactose anhydrous, and various starches, and examples of binders are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Additives that affect the flowability of the powder to be compressed may be used. Suitable lubricants containing agents may include colloidal silicon dioxide such as Aerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and axulfame. Examples of flavoring agents are Magnasweet® (a trademark of MAFCO), bubble gum flavor, and fruit flavor. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0095] Suitable diluents may include pharma- ceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dibasic calcium phosphate, sugars, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate® such as Emcompress; mannitol; starch; sorbitol; sucrose; and glucose.

[0096] Suitable disintegrants include lightly cross-linked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof.

[0097] Examples of effervescent agents are effervescent couples such as organic acids and carbonates or bicarbonates. Suitable organic acids include, for example, citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid, and alginic acid, as well as anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0098] The compounds utilized in the methods disclosed herein can be formulated as pharmaceutical compositions for delivery via any suitable route. For example, pharmaceutical compositions can be administered via oral, intravenous, intramuscular, subcutaneous, topical, and pulmonary routes. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders, and granules. In some embodiments, the compounds are formulated as compositions for oral administration (e.g., in a solvent such as 5% DMSO in an oil such as vegetable oil).

[0099] The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredients with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art, which may involve mixing, granulating, and compressing or dissolving the ingredients as appropriate to the desired preparation.

[0100] Pharmaceutical compositions containing the compounds may be adapted for administration by any suitable route, for example, oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0101] Pharmaceutical compositions suitable for oral administration may be formulated as discrete units such as capsules or tablets; powders or granules; or aqueous solutions. The composition may be presented as a solution or suspension in an aqueous or non-aqueous liquid; an edible foam or whip; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.

[0102] Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time, for example, the active ingredient may be delivered from the patch by iontophoresis.

[0103] Pharmaceutical compositions suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to aid in drug penetration and emollients in the ointments and creams.

[0104] For application to the eye or other external tissues, such as mouth and skin, the pharmaceutical composition is preferably applied as a topical ointment or cream.When formulated into an ointment, the compound can be used with either a paraffinic ointment base or a water-miscible ointment base.Alternatively, the compound can be formulated into a cream containing an oil-in-water cream base or a water-in-oil base.Pharmaceutical compositions suitable for topical administration to the eye include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent.

[0105] Pharmaceutical compositions suitable for nasal administration where the carrier is a solid include coarse powders having a particle size (e.g., in the range 20 to 500 micrometers) which can be administered in a nasal manner (i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose). Where the carrier is a liquid, suitable formulations for administration as a nasal spray or nasal drops include aqueous or oil solutions of the active ingredient.

[0106] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

[0107] Tablets and capsules for oral administration may be in unit dose presentation form and may contain conventional excipients such as binders, e.g., syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, e.g., lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine; tableting lubricants, e.g., magnesium stearate, talc, polyethylene glycol, or silica; disintegrants, e.g., potato starch; or acceptable wetting agents, e.g., sodium lauryl sulfate. Tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example, sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, emulsifying agents, for example, lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, glycerin, propylene glycol, or oily esters such as ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavorings or colorings. EXAMPLES

[0108] The following examples are illustrative only and should not be construed as limiting the scope of the claimed subject matter.

[0109] Example 1 - Use of bioelectrical drugs to inhibit cancer cell behavior The present inventors have determined that a variety of hyperpolarizing and serotonergic drugs can be utilized alone or in combination with rapamycin and other small drug-like molecules to inhibit the growth and differentiation of glioblastoma cells in vitro. The present inventors have tested several known bioelectrical drugs (i.e., candidate ion sources) alone, in combination, and in combination with established drug therapies for their ability to reduce the proliferation of mammalian glioblastoma cell lines in vitro.

[0110] We tested hyperpolarizing drugs including retigabine, minoxidil, NS1643, lamotrigine, and zolmitriptan. We tested these compounds alone or in combination with other drugs including temozolomide (TMZ), pantoprazole, and rapamycin.

[0111] cAMP has been used in combination with rapamycin to differentiate and arrest the cell cycle in mouse-rat hybrid NG108-15 cells (glioblastoma and neuroblastoma). Rapamycin is known to induce an autophagic response, while cAMP hyperpolarizes the cells.

[0112] The experiments herein show that drug combinations promote differentiation and cell cycle arrest of NG108-15 glioblastoma stem cells in vitro. NG108-15 cells were transfected with a fluorescent cell cycle reporter and a fluorescent marker on the cell membrane. Cells were seeded at low confluence in 96-well plates and fresh drug was added every other day for 6 days. In some studies, drug was removed on day 6 and drug-free medium was added for an additional 4 days to test proliferation. Images of cells were taken daily and cells were counted using the Cell Profiler pipeline to determine the proportion of cells in each part of the cell cycle. Rapamycin at 100 nM, 150 nM, and 200 nM showed a highly significant decrease in proliferation and an increase in arrested cells in G1, confirming what other studies have found in human-derived glioblastoma cells. Pantoprazole at 100 μM also showed a highly significant decrease in cell proliferation and an increase in arrested cells in late S phase, confirming what other studies have found in human-derived glioblastoma cells. Temozolomide at 50 μM in combination with these two compounds was used as a comparison of the efficacy of the new combination, since temozolomide is currently the primary drug in the treatment of glioblastoma. Combinations of 10 μM zolmitriptan, 10 μM retigabine, 100 μM lamotrigine, or 30 μM minoxidil with 200 nM rapamycin showed a significant decrease in cell proliferation compared to rapamycin alone. Combinations of 100 μM lamotrigine, or 10 μM retigabine and 100 μM pantoprazole also showed a significant decrease in cell proliferation compared to pantoprazole alone. Retigabine in combination with pantoprazole also showed a highly significant increase in the number of cells arrested in late S phase with a restoration of cell proliferation.

[0113] [Example 2 - Ion channel drugs suppress cancer phenotype in NG108-15 cells: Towards a novel electroneutical for glioblastoma] Glioblastoma is a highly lethal brain cancer that commonly recurs after tumor resection and chemotherapy treatment. Depolarized resting membrane potential and acidic intertumor extracellular pH are associated with proliferative status and drug resistance, suggesting that forced hyperpolarization and disruption of proton pumps in the plasma membrane may be a successful strategy to target glioblastoma hyperproliferation. A study was performed to determine the efficacy and safety of 139 compounds, their concentration and ion modulation in the NG108-15 rodent neuroblastoma / glioma cell line. A screen of drug combinations was performed. A subset of these were then tested in the U87 human glioblastoma cell line. The FUCCI cell cycle reporter was stably integrated into both cell lines and the effects on proliferation and cell cycle responses were monitored by live imaging. Immunocytochemistry, electrophysiology, and a panel of physiological dyes reporting voltage and pH were used to characterize the responses. The most effective treatments for proliferation in U87 cells were NS1643 in combination with pantoprazole; retigabine in combination with pantoprazole; and pantoprazole or NS1643 in combination with temozolomide. Marker analysis and physiological dye signatures suggest that exposure to bioelectropharmaceuticals significantly reduces proliferation, senesces cells, and promotes differentiation. These results, along with the low toxicity observed in human neurons, demonstrate the high efficacy of electropharmacology utilizing FDA-approved repurposed drug combinations.

[0114] In addition to standard biochemical factors and biomechanical forces, cellular and tissue-level order is now known to be regulated by bioelectrical signaling between many cell types [11, 20]. The cellular transmembrane potential (V mem ) are regulated by ion channels, which are not only important targets in embryonic channelopathies [21-23] but are increasingly being viewed as cancer targets as well [21, 24-35]. Overall, adult non-proliferating cells tend to have hyperpolarized membrane potentials, whereas stem cells, embryonic cells, and other highly proliferative cells are much more depolarized [21, 36]. Cancer cells V memTransformed cells tend to resemble those of embryonic cells and have a much more depolarized membrane potential than their noncancerous counterparts [36, 37]. Indeed, transformed cells can be detected in vivo in animal models using voltage-sensitive fluorescent dyes based on their abnormal bioelectrical signature

[38] . Importantly, the resting membrane potential is not only a marker but is also functionally informative on cell behavior. The classical test by Cone showed that V mem are important regulators of proliferation in terminally differentiated cells or cancer [39-41], and this is confirmed by recent studies linking depolarization to a plastic, undifferentiated, highly proliferative state. For example, treatment of mesenchymal stem cells with depolarizing drugs inhibits their differentiation into adipocytes or osteoblasts, and depolarized V mem It has been suggested that hyperpolarization is not only correlated with stemness but is required for differentiation [42–46]. Importantly, in vivo experiments have shown that co-expression of hyperpolarizable ion channels in the context of human oncogenes expressed in Xenopus tadpoles or optogenetic activation of the channels

[47] can prevent tumors from forming when the oncogenes are expressed alone [33, 38, 48]. In mammalian models, several recent studies have shown that ion channelopathies are present in many cancers and play important roles in cell proliferation, progression through the cell cycle, and metastasis [24, 28, 30, 35, 49–55].

[0115] The location of many ion channels within the outer cell membrane also makes them attractive targets. memAgonists or antagonists used to modulate IL-1 can be selected for binding sites on the outside of the membrane, avoiding the challenges posed by increased drug efflux transporters in glioblastoma stem cells (GSCs). In addition, ion channel transcripts have been shown to be upregulated in GSCs, including SCN8A, encoding a sodium channel, KCNB1, encoding a voltage-gated potassium channel, and GRIA3, encoding an ionotropic glutamate receptor nonselective for monovalent cations

[53] . Furthermore, intracellular alkalinization induced by dysfunction of proton transport has been shown to increase drug resistance in glioblastoma tumors (GBM), providing an optimized niche for GSCs, and contributing to extracellular acidification that promotes the release of large oncosomes, a type of extracellular vesicle that can transform neighboring cells [56-58]. Thus, due to the location and importance of these channels in cancer cell proliferation, migration, and metastasis, ion channels provide optimal targets for the development of new GBM therapies.

[0116] To discover effective interventions for glioblastoma among known small molecule modulators of bioelectrical state, the NG108-15 cell line was first used to study differentiation in glioblastoma cells. V that can potentially promote mem We screened for modulating compounds

[59] . NG108-15 cells are a hybrid of mouse N18TG2 neuroblastoma cells and rat C6-BU-1 glioma cells and are a common model system in neuronal differentiation studies. This line was selected due to its extensive use in determining factors required for neuronal differentiation, its close similarity to NSCs and GSCs, and its well-characterized membrane properties and electrophysiology [60-62]. The goal was to find compounds that could safely abrogate the proliferative potential in NG108-15 cells under high serum conditions that are normally prohibitive for differentiation, revealing robust candidates for subsequent evaluation in the U87 (ATCC) human glioblastoma line and for future testing in patient-derived GSCs under serum-free conditions [63-66].

[0117] To screen candidate drugs targeting ion channels and pumps, as well as their combinations, we created a new tool: NG108-15 and U87 stable lines expressing the fluorescent FUCCI cell cycle indicator

[67] and a plasma membrane mTurquoise tag to reveal whole cell morphology. We used automated electrophysiology on NG108-15 cells to determine what the combinations did to the overall transmembrane potential. For U87 cells, we used dyes to determine cytoplasmic calcium levels, internal pH (pHi), transmembrane potential, lysosomal pH, and performed Live / Dead assays to obtain a comprehensive profiling of the effects of drugs on cell physiology. Finally, we used β-galactosidase staining and immunocytochemistry to analyze the senescence, cell cycle inhibitor levels, and differentiation status of treated NG108-15 and U87 cells. Several combinations of compounds that appear to promote reduced proliferation and induced differentiation were identified, demonstrating that this class of electropharmacology, most of which are already FDA approved for other conditions, is a good candidate for cancer repair in GBM.

[0118] [method] Reagents: Rapamycin (R8781, Sigma), retigabine (SML0325, Sigma), minoxidil (M4145, Sigma), NS1643 (sc-2041353, Santa Cruz Biotechnology), lamotrigine (L3791, Sigma), zolmitriptan (SML0248, Sigma), cariporide (SML1360, Sigma), topiramate (T0575, Sigma), pantoprazole sodium hydrate (P0021, Sigma), fenofibrate (F6020, Sigma), acetazolamide (A6011, Sigma), quercetin (Q4951, Sigma), temozolomide (2706, Tocris) Stocks of tamoxifen (D4902, Sigma), ONO-RS-082 (O0766, Sigma), topotecan hydrochloride (4562, Tocris), CKD602 (5125, Tocris), (Z)-4-hydroxytamoxifen (3412, Tocris), lansoprazole (2582, Tocris), chlorzoxazone (C4397, Sigma), and sodium butyrate (B5887, Sigma) were made at 1000x in DMSO. Stocks of gabapentin (G154, Sigma) and cisplatin (232120, EMD Millipore) were made at 1000x in water. Dibutyryl cAMP sodium salt (D0627, Sigma) was the only compound that was directly dissolved in cell culture medium at 1 mM concentration.

[0119] Cell culture: NG108-15 cells (ATCC), passages 7–10, were cultured in growth medium containing DMEM medium with high glucose and without phenol red or sodium pyruvate (31053-028, ThermoFisher) supplemented with 2 mM Glutamax, 10% FBS, HAT supplement, and 10 U / mL penicillin / streptomycin. Cells were dissociated with Accutase, passaged when 70% confluent, and maintained at 37°C with 5% CO2. Medium was changed every 2 days. For live cell assays, phenol red cells were cultured in imaging medium containing Fluorobrite DMEM (A1896701, ThermoFisher) without supplements. Plates were coated with a 1 / 50 dilution of Nole Red growth factor reduced (GFR) Matrigel (356231, Corning). For all live cell imaging experiments, except for cAMP with rapamycin treatment, which was performed in exactly the same medium except for 1% FBS, cell culture medium was replaced with Fluorobrite 1000 with 2 mM Glutamax, 10% FBS, HAT supplement, and 10 U / mL penicillin / streptomycin. All drug screens, including controls, were performed using 0.02% DMSO in high serum Fluorobrite DMEM.

[0120] U87 (ATCC) cells were cultured under the same conditions as NG108-15 cells, excluding HAT supplement, and were used for experiments at passages 7–10. All cells seeded for antibody staining and senescence assays were cultured on polyethylenimine (181978, Sigma)-coated plates at 25 μg / mL in 150 mM NaCl solution for 1 h at room temperature, followed by rinsing with PBS as above and an additional coating of 1 / 50 Matrigel.

[0121] Human neural cells were differentiated from human induced neural stem cells (hiNSCs) (passages 7–10) as previously described

[68] and kindly provided by David Kaplan. Briefly, hiNSCs were grown on mouse embryonic fibroblast (MEF) feeder cells until they were ready to be differentiated into neurons. Cells were then seeded at a density of 128,000 cells / mL in 96-well plates coated with poly-D-lysine (A3890401, ThermoFisher) and laminin (L2020, Sigma). Cells were differentiated in Neurobasal (12348017, ThermoFisher) medium supplemented with 2% B27 (17504044, Gibco), 1% Glutamax, and 1% antibiotic-antimycotic for 7 days, with medium changes every 2 days. At the end of the 7 days, treatments were added to the differentiation medium for 3 days.

[0122] Molecular biology: The ES-FUCCI construct containing the hygromycin resistance cassette was subcloned using XmnI and SalI from the plasmid ES-FUCCI, a gift from Pierre Neveu (Addgene plasmid #62451; http: / / n2t.net / addgene:62451; RRID:Addgene_62451)

[67] . The CAG pPalmitoyl-mTurquoise2 construct was subcloned using BamHI and NotI from the plasmid pPalmitoyl-mTurquoise2, a gift from Dorus Gadella (Addgene plasmid #36209; http: / / n2t.net / addgene:36209; RRID:Addgene_36209)

[69] . All subcloned fragments were cloned into the pENTR1A plasmid, which contains the CAG promoter and multiple cloning site (MCS), and subsequently cloned into SV40 poly(A) using the same sites used to excise the fragments from the parental plasmid. For ES-FUCCI, the CAG promoter, MCS, and poly(A) were removed from the pENTR1A plasmid with SpeI and SalI blunted prior to ligation with the fragment. The resulting pENTR1A ES-FUCCI was then Gateway LR clonase-transformed into pmhyGENIE-3, a highly active piggyBac transposase-based helper-independent self-inactivating delivery system, a gift from Stefan Moisyad [70,71] (11791020, ThermoFisher). pENTR1A CAG pPalmitoyl-mTurquoise2 was cloned into pmhyGENIE-3, which contains a neomycin resistance gene in the backbone. The resulting plasmids, HypG3 Hygro ES-FUCCI and HypG3 NeoBB CAG pPalmitoyl-mTurquoise2, were used for subsequent transfections.

[0123] Generation of stable lines: All transgenic cell lines were cultured in 24 wells containing 500 μL of culture medium. HypG3 plasmids were generated by transfecting cells at 30% confluence with 500 ng of the appropriate HypG3 plasmid via 1 μL Lipofectamine 3000 (L3000008, ThermoFisher) per well of a 96-well plate. After 24 hours, the reagent was removed and fresh culture medium was added. Cells were allowed to recover for 24 hours before selection with 1000 μg / mL G418 or 200 μg / mL hygromycin. After selection, cells were serially diluted into 96-well plates and single colony clones were expanded. Clones showing strong growth and strong expression were selected for subsequent experiments.

[0124] Proliferation and FUCCI assays: Both cell lines were seeded at 5,000 cells / mL in black-walled flat-bottom 96-well plates coated with 1 / 50 dilution of Matrigel. Images of cells were taken on day 0 and daily thereafter for 10 days using a Zeiss Axio1 with an on-stage incubator kept at 37°C with 5% CO2. A 5x objective was used with filters for YFP, RFP, and CFP, and careful calibration of each plate was performed to ensure that the same field of view was imaged each day. Drugs were added after the first image was taken and then changed every 2 days until day 6. For recovery experiments, after imaging on day 6, all drugs were removed and cells were placed in imaging medium without any drugs added. This medium was changed every 2 days until day 10. Analysis of proliferation and snapshots of FUCCI reporters over 6 or 10 days was performed using the CellProfiler

[72] pipeline designed by Broad. Decimal differences in cell counts were calculated by dividing the total number of cells on each day by the total number of cells from day 0. The proportion of cells in each cell cycle stage per day was determined by counting all nuclei of one color and dividing it by the total number of nuclei. Blind quality controls were performed on all images to ensure that fibers or image artifacts were not falsely counted by the program.

[0125] Antibody staining, BrdU and senescence assays: NG108-15 cells were seeded at 15,000 or 30,000 cells / mL and U87 cells at 10,000 cells / mL on PEI and Matrigel coated plates and grown in imaging medium for 6 days with drug treatment. Fresh medium was added every 2 days. On day 6, for all antibody staining, cells were fixed with 4% formaldehyde in PBS for 30 min, except for BrdU, which was fixed for 15 min, and senescence assay, which was fixed for 20 min using Senescence Beta-Galactosidase Staining Kit (9860, CellSignaling), and then stained according to the manufacturer's protocol. After fixation, cells were washed twice with PBS, permeabilized with 3% Triton-X in PBS for 15 min, and then washed twice again with PBS. Cells for BrdU assay were further treated with 1N HCl on ice for 10 min, followed by 2N HCl at room temperature for 50 min. Cells were then blocked with standard blocking buffer consisting of TBS containing 10% goat serum, 1% BSA, and 0.05% Tween 20, or without detergent for antibodies against phosphorylated proteins or monoclonal antibodies, except for anti-YAP and anti-BrdU staining, which were performed according to the manufacturer's protocol. Primary antibodies were added at the following concentrations: 1:250 anti-S100 beta (GTX129573, Genetex), 1:500 anti-GFAP (AB5804, EMD-Millipore), 1:300 anti-SOX10 (ab155279, Abcam), 1:100 MAP2 (4542, CellSignaling), 1:400 anti-cleaved caspase 3 (9661, CellSignaling), 1:300 anti-Cx43 (STJ2411, St. John's Laboratory), 1:800 anti-p27 Kip1(3698, CellSignaling), 1:150 anti-BrdU (5292, CellSignaling), 1:125 anti-LC3-II (2775, CellSignaling), 8μg / mL anti-O4 (MAB1326, R&D Systems), 1:1000 anti-TH (RPCA-TH, Encor), 1:4000 anti-NSE (RPCA-NSE, Encor), 1:4000 anti-NFM (RPCA-NF-M, Encor), 0.5 μg / mL anti-TujI (801202, Biolegend), 1:40 Antibodies were diluted in the respective blocking buffer and incubated overnight at 4°C. The next day, cells were washed three times for 5 min each with TBS-T or TBS, and secondary antibodies were added as follows: 1:1000 donkey anti-mouse 647 (A-31571, Thermo-Fisher) or 1:1000 donkey anti-rabbit 647 (A-31573, Thermo-Fisher). Each was diluted in blocking buffer with 2.5 μg / mL Hoechst33342 and incubated for 1 h at room temperature. Cells were then washed again three times for 5 min each with TBS-T or TBS and covered with gelvatol. Cells were imaged on an EVOS M7000 system, with at least 50% of each well in a 96-well plate imaged and analyzed using the CellProfiler pipeline to measure integrated intensity, mean intensity, or nuclear to cytoplasmic ratio of mean intensity

[73] .

[0126] Dye staining protocol: For the Live / Dead assay, 1 μM calcein green AM and 0.5 μM ethidium homodimer-1 (L3224, ThermoFisher) were added to PBS with 10 μg / mL Hoechst to make the staining solution. Half of the medium was removed from the human neurons incubated with treatment for 3 days and replaced with the staining solution. This was done four times to ensure that the cells did not detach. The cells were then incubated at 37°C for 15 minutes and imaged. For resting membrane potential staining, DiBAC4(3) was used. U87 cells were seeded at 10,000 cells / mL on Matrigel-coated plates as above. The cells were treated with drugs for 6 days, with the medium changed every other day. On day 6, the medium was removed and washed once with dye buffer (pH 7.4) consisting of Hanks' balanced salt solution (HBSS) containing 20 mM HEPES, then staining solution consisting of dye buffer containing 2 μM DiBAC4(3) was added to the cells and incubated at 37° C. for 30 minutes. The staining solution was then removed and fresh staining solution containing treatment was added to the cells. For cytoplasmic calcium staining, Fluo-4AM was used. The staining solution consisted of dye buffer containing 4 μM Fluo-4AM and a 1:1 ratio of Pluronic F-127 (20% in DMSO) and incubated at room temperature for 30 minutes. The staining solution was then removed and fresh dye buffer was added and incubated at 37° C. for 20 minutes. Dye buffer containing treatment was then added to the cells. For cytoplasmic pH staining, pHrodo Green was used. The staining solution consisted of dye buffer containing a 1:1000 dilution of pHrodo Green stock and a 1:100 dilution of PowerLoad concentrate (P35373, ThermoFisher). Cells were incubated for 30 min at 37°C. The staining solution was removed and washed once with dye buffer, then fresh dye buffer containing treatments was added and incubated for 5 min at 37°C. A calibration curve was performed by adding instead the components of the Intracellular pH Calibration Kit (P35379 ThermoFisher). For lysosomal pH staining, LysoSensor Green was used.The staining solution consisted of dye buffer containing 1 μM dilution of LysoSensor Green DND-187 (L7535 ThermoFisher). Cells were incubated as above, and the staining solution was removed and replaced with dye buffer containing 2.5 μg / mL Hoechst33342 for 10 min. Hoechst was removed and cells were washed once with dye buffer before adding dye buffer containing treatments. Cells were imaged using an EVOSM7000 system equipped with a GFP filter cube for staining and a DAPI filter for Hoechst. Images were then analyzed using the CellProfiler pipeline.

[0127] Electrophysiology: Automated patch clamp experiments were performed using the SyncroPatch 384PE platform (Nanion Technologies®). This system provides gigaohm resistance seals during simultaneous recording in a 384-well plate format. Provide 1-2x10 cells in a T175 culture flask (Falcon). 6Cells were harvested 48–72 h after seeding, then rinsed with PBS (5 mL), treated with 3 mL of Accutase (STEMCELL technologies) for 5 min at 37 °C, resuspended in 10 mL of serum-free medium, and pelleted at 1000 rpm for 3 min at room temperature. The supernatant was discarded and the cells were resuspended in serum-free DMEM medium (31053-028, ThermoFisher) containing high glucose and without phenol red or sodium pyruvate, and physiological extracellular solution (pECS) 50% (v:v). Cells were kept in a temperature-controlled dedicated reservoir at 10 °C until the moment of the experiment, shaking at 200 rpm as described

[74] . Experiments were performed within 1 h after the harvesting process. Assays were performed in single-hole chips with resistances of 4–5 MΩ after priming the chip with the following solutions (in mM): physiological extracellular solution (pECS) 10 HEPES, 140 NaCl, 5 glucose, 4 KCl, 2 CaCl2, 1 MgCl2, 295–305 mOsm pH 7.4 (NaOH); internal recording solution (in mM) 20 EGTA, 50 KCl, 10 NaCl, 60 KF, 10 HEPES at pH 7.2, and 285 mOsm. 15 µL of cell suspension (50% v / v pECS / medium, no serum) was added to each well to a final density of 50–80 K cells / mL. Cell capture was facilitated by holding a negative pressure of -100 mbar for 20 s. After successive hyperpolarizing steps from -30 mV to -100 mV, the capture seal was enhanced, followed by the transient addition of a high Ca2+ extracellular solution (80 mM NaCl, 3 mM KCl, 35 mM CaCl2, 10 mM MgCl2, 10 mM HEPES) at pH 7.4 and 298 mOsm. The high Ca2+ solution is washed out by successive external exchanges replacing half the well's volume with the external recording solution each time. All recording solutions were prepared using ultrapure MilliQ water (18 MΩ-cm). After the formation of a Giga-seal, a pressure of 250 mbar was applied to rupture the membrane patch. Once in the whole-cell configuration, different voltage protocols were applied to analyze the different conductances of NG108-15 cells. To monitor the membrane potential, signals were recorded in current clamp mode "I=0" (passive conditions) without current injection. The membrane potential was recorded for seven sweeps of 1 sec, with an inter-sweep time of 10 sec.The average membrane potential of seven sweeps was reported.

[0128] [result] Combination of bioelectric compounds and the proton pump inhibitor, pantoprazole, arrests proliferation of NG108-15 cells in high serum and shifts the proportion of cells in late S, G2, and M stages NG108-15 cells containing the FUCCI cell cycle reporter and palmitoyl-mTurquoise2 fluorescent membrane tag were incubated with compounds alone and in combination in medium containing high serum (known to be differentiation inhibitory in this line) and tested for their ability to inhibit proliferation (Figures 18-19). Compounds selected for testing included compounds known to alter the membrane potential of cells by decreasing proton efflux, increasing potassium efflux, or decreasing sodium influx, as well as other compounds selected for their potential combinatorial effects with ionotropic agents, including cell cycle specific disruptors and autophagy inducing compounds (Table 1). Additionally, temozolomide, the most clinically relevant compound currently used in standard treatment of glioblastoma, was tested. Table 1 shows a list of the most effective compounds that were tested in experiments using mouse / rat neuroblastoma / glioma NG108-15 cells containing the FUCCI cell cycle reporter and palmitoyl-mTurquise2 fluorescent membrane tag, human glioblastoma U87 cells containing the FUCCI cell cycle reporter and palmitoyl-mTurquoise2 fluorescent membrane tag, and / or induced neural human stem cells.

[0129] [Table 1]

[0130] The subset of compounds showing the highest efficacy in the initial screen is shown in Table 2.

[0131] [Table 2]

[0132] Many of the compounds tested showed significant differences in proliferation from controls after 6 days of treatment, but only a few of these were examined in more detail due to their novelty (Table 2). Figure 1 shows the best of the selected individual and combination treatments when compared to controls at day 6. Note that chlorzoxazone was not included in the combination treatments in the NG108-15 analysis shown due to its poor performance in the U87 cell line. The stacked bar graphs show the proportion of cells in different parts of the cell cycle as shown by the FUCCI cell cycle reporter.

[0133] The proliferation of NG108-15 cells treated alone with NS1643 human Ether-a-go-go (hERG) potassium channel activator at 20 μM and 50 μM was able to significantly reduce proliferation by approximately 1.6-fold and 2.4-fold, respectively, compared to the control (Figure 1-A). In fact, NS1643 at 20 μM was highly effective in reducing cell proliferation when combined with pantoprazole, a proton pump inhibitor known to inhibit the expression of vacuolar ATPase (V-ATPase) in human gastric adenocarcinoma

[79] , and this combination worked significantly better than pantoprazole or NS1643 alone, showing a 7.2-fold reduction. NS1643 at 50 μM also significantly reduced proliferation when combined with rapamycin (an autophagy inducer), reducing proliferation by 4.6-fold, working better than rapamycin or NS1643 alone. Retigabine [80, 81], which opens the voltage-activated potassium channel Kv7, significantly reduced cell proliferation with a reduction of about 1.6-fold compared to the control, while its combination with rapamycin or pantoprazole performed better than either of these compounds alone with a reduction of 2.9-fold and 6-fold compared to the control, respectively. However, pantoprazole at 100 μM, alone (5.1-fold reduction) or in combination with lamotrigine

[82] , which blocks voltage-gated sodium channels, or NS1643, or rapamycin, was the most effective compound with a reduction of 6.8, 7.2, and 9.3-fold compared to the control, respectively. These three combinations performed better than one of the positive controls consisting of a treatment of 1 mM cAMP with 200 nM rapamycin in full serum medium (5.1-fold reduction). This same positive control treatment is known to terminally differentiate these cells when in low serum

[83] , and this was confirmed (Figures 9-10). However, the use of cAMP is clinically problematic due to its many off-target effects

[84] . The cell cycle data in Figure 11-B reveal that pantoprazole increases the percentage of cells in early S, and the combination can also increase the percentage of cells in G1. Rapamycin treatment alone increased the percentage of cells in G1, whereas NS1643 treatment did not seem to affect the cell cycle percentage (Figure 1B).It is worth noting that these compounds were effective in NG108-15 cells, whereas the standard glioblastoma treatment, temozolomide (TMZ), was not, a situation seen in many GBM cases

[85] . .

[0134] The combination of 100 μM lamotrigine, 20 μM NS1643, and 100 nM rapamycin was the only combination that showed significantly greater efficacy than pantoprazole alone in reducing cell proliferation after 6 days of treatment, with the combination with rapamycin showing the most significant difference (Figure 2-A). The reduction in cell number for these combinations compared to the control was 85%, 86%, and 90%, respectively. The percentage of cells in G1 and early S was only slightly increased with pantoprazole treatment in combination with lamotrigine and NS1643, whereas the combination with rapamycin increased the percentage of G1 (Figure 2-B). Taken together, these data reveal that several FDA-approved drugs for human use can be combined and repurposed to significantly reduce cancer cell proliferation.

[0135] Hyperpolarizing drugs in combination with pantoprazole reduce NG108-15 proliferation after treatment is stopped: To understand whether these treatments have a sustained effect on the cells (stopping cell proliferation even after the drugs are discontinued), recovery studies were performed (Figure 3). Cells were treated with drugs for 6 days, then the drugs were removed (bounded by dashed lines), after which the cells were cultured in drug-free medium for an additional 4 days.

[0136] The three combinations with pantoprazole showed significantly less cell proliferation after 6 days of treatment, but the cells showed some recovery after treatment was stopped (Figure 3). The slope of each combination treatment from day 6 to day 10 was compared to pantoprazole alone. Pantoprazole combined with retigabine was the only treatment combination that was significantly different, in addition to the positive control, which showed no recovery, and showed terminally differentiated cells. One thing to note was that while not significantly different from pantoprazole on day 6, pantoprazole treatment with retigabine showed fewer cells than pantoprazole treatment with lamotrigine on day 10. Therefore, it was decided to perform further analysis on this combination.

[0137] Many of the significant treatments in NG108-15 cells were also significantly more effective in human glioblastoma U87 cells compared to the control after 6 days (Figure 4). In particular, NS1643 at 50 μM significantly reduced cell proliferation by 1.7-fold compared to the control, but was much more effective when combined with pantoprazole, rapamycin, or temozolomide (TMZ), resulting in 3.3-fold, 2.7-fold, and 2.5-fold reductions relative to the control, respectively. Pantoprazole also worked very well in this cell line, with a significant percent reduction in cells relative to the control of 54%, and when combined with rapamycin, retigabine, NS1643, lamotrigine, and TMZ, showed highly significant differences in cell proliferation compared to the control, with percent reductions of cells of 60%, 72%, 72%, 61%, and 61%, respectively (Figure 5-A). TMZ was highly effective in reducing cell number in U87 cells compared to control (43% reduction), but combinations with rapamycin, pantoprazole, or NS1643 significantly increased cell efficacy by 55%, 61%, and 61%, respectively, compared to control (Figure 5-A). The cell cycle data in Figure 4-B show that some, but not all, of the most effective combinations increased the G1 and early S fraction of cells. Pantoprazole showed its characteristic increase in early S fraction of cells, and rapamycin showed the increase in G1 fraction seen in NG108-15 cells. TMZ and NS1643 treatment did not show a significant change in the fraction of cells in each phase of the cell cycle compared to control. The combination of TMZ and rapamycin increased the fraction of cells in G1 compared to TMZ alone, and the combination with NS1643 increased the fraction of cells in early S (Figure 5-B). The pantoprazole combination consistently showed a greater percentage of cells in early S, with a complementary reduction in cells in late S, G2, and M, compared to TMZ alone (Figure 5-B).

[0138] Unfortunately, we found no evidence that FUCCI reporter signal was expressed in the cytoplasm, which obscured the FUCCI reporter signal. Due to autofluorescence aggregation in the cells, it was not possible to obtain an exact percentage of cells in each cell cycle stage for the best treatment of pantoprazole with retigabine in U87 cells.

[0139] The same combinations that were significantly better than pantoprazole in NG108-15 cells were also significant in U87 cells with the added combination of TMZ or retigabine (Figure 6-A). Pantoprazole in combination with rapamycin, lamotrigine, NS1643, TMZ or retigabine were all significantly better than pantoprazole alone. The most significant combinations were with NS1643 or retigabine, which were 1.6-fold less effective than pantoprazole alone. The combination of pantoprazole with NS1643 was so effective that reducing the pantoprazole concentration in half and combining it with 50 μM NS1643 was significantly more effective than 100 μM pantoprazole alone (1.2-fold less effective). The characteristic increase in G1 when pantoprazole was combined with rapamycin and with NS1643 increased the proportion of cells in early S, with complementary decreases in late S, G2, and M (Figure 6B). Thus, NS1643, retigabine, rapamycin, lamotrigine, and pantoprazole are also effective in human glioblastoma cell lines, and NS1643 or pantoprazole potentiate the effects of standard TMZ treatment.

[0140] Bioelectrical drug combinations with pantoprazole reduce U87 proliferation after treatment is stopped. The combination of pantoprazole with rapamycin shows a similar recovery slope after 6 days as pantoprazole alone (Figure 7). However, the combination of pantoprazole with 50 μM retigabine or NS1643 shows a reduced recovery rate compared to pantoprazole alone, although not significantly. TMZ treatment alone showed only a slight increase in proliferation after treatment was stopped, while NS1643 alone showed a high increase in proliferation. However, the combination of NS1643 with TMZ did not show an increase in proliferation after treatment was stopped, although the recovery slope was not significantly different from TMZ alone. Surprisingly, the positive control using cAMP in combination with rapamycin started to proliferate after 8 days, revealing that bioelectrical drugs have a more stable effect on cells than a strong cAMP signal.

[0141] Electrophysiology of NG108-15 cells shows changes in resting membrane potential induced by treatment Electrophysiological measurements were used to determine the changes in resting membrane potential caused by treating NG108-15 cells with compounds that significantly reduced cell proliferation compared to controls immediately after application. Untreated cells were patched and a baseline measurement was taken, then drugs were added and cell V mem changes were recorded.

[0142] Rapamycin, retigabine, NS1643, TMZ, and lamotrigine all significantly hyperpolarized the cells compared to the control (Figure 8). Although retigabine, lamotrigine, and NS1643 are known hyperpolarizing agents, TMZ has been published to depolarize glioma cells

[86] , an effect opposite to that observed in NG108-15 cells. Rapamycin also hyperpolarized NG108-15 cells, a novel effect suggesting that existing cancer drugs may have an as yet unrecognized bioelectrical mechanism of action. Surprisingly, pantoprazole had no immediate effect on the membrane potential of the cells, but its combination with retigabine and rapamycin, both of which individually hyperpolarize cells, depolarized the cells instead. Combinations of pantoprazole with lamotrigine and NS1643 did not observably change the membrane potential compared to the control.

[0143] NG108-15 cells express neuronal markers upon drug treatment after 6 days The next question was whether, in addition to effects on proliferation, such treatments also had differentiation effects, which could be informative regarding the future behavior of treated cells in vivo. Differentiation markers of neuronal lineages were used to stain NG108-15 cells incubated for 6 days with the most effective treatments observed in the proliferation data (Figure 9). 50 μM NS1643 or 100 μM NS1643 were used to stain NG108-15 cells incubated for 6 days with the most effective treatments observed in the proliferation data (Figure 9). Treatment with 100 μM pantoprazole in combination with nM rapamycin consistently showed a significant increase in neuronal differentiation markers, including microtubule-associated protein 2 (MAP2)

[87] , neuron-specific class III beta-tubulin (TujI)

[88] , neuron-specific enolase (NSE)

[89] , and neurofilament medium chain (NFM)

[90] . The combination of pantoprazole with 10 μM retigabine showed an increase in all but MAP2. The combination of pantoprazole with either NS1643 or retigabine showed a significant increase in neural markers compared to either drug alone. The combination of NS1643 and pantoprazole showed higher levels of both MAP2 and TujI than pantoprazole alone. Retigabine in combination with pantoprazole showed higher immunoreactivity of NFM compared to pantoprazole alone. These findings suggest that these treatments are pushing the treated cells toward a more differentiated state.

[0144] Staining cells treated for 6 days with S100 calcium binding protein B (S100B)

[91] and glial fibrillary acidic protein (GFAP)

[92] markers revealed differentiation of NG108-15 cells into astrocyte / oligodendrocyte or astrocytic lineages, respectively (Figure 10-A and -B). Both markers were significantly upregulated in all combination treatment groups as well as pantoprazole alone. The combination of NS1643 with pantoprazole showed significantly higher S100B and GFAP immunoreactivity than pantoprazole. All treatments except NS1643 alone showed a significant increase in CREB, which is known to play an important role in promoting differentiation [15, 93-96] (Figure 10-C). Pantoprazole alone, along with the combination, also showed an increase in connexin 43 (Cx43) expression, a known marker of glioblastoma differentiation

[97] .

[0145] The next question was whether any of the treatments would induce senescence, as this would be an important outcome for the course of malignant tumors in vivo. Treatment of NG108-15 cells with pantoprazole alone or in combination with NS1643, retigabine and rapamycin all showed highly significant increases in senescence-associated β-galactosidase activity staining

[98] (Figure S1A). To explain this increase, we used p27 Kip1 Since high levels of p27 are known to inhibit the cell cycle and induce senescence [99-106], Kip1 The levels of p27 in all combination treatments except for pantoprazole with rapamycin were examined (Figure 11-E). Kip1 Immunoreactivity levels of α-Caspase-3 were increased. To confirm the senescent phenotype, the size of the nuclei was examined and found to be significantly larger in pantoprazole, NS1643, and pantoprazole combined with NS1643, retigabine, or rapamycin treated groups (Figure 11-F). Cells were examined for cleaved caspase-3, a marker of apoptosis (Figure 11-D), and LC3-II, a marker of autophagy (Figure 11-C). Although the levels of both these markers were found to be very low after 6 days of treatment, there was a significant increase in cleaved caspase-3 staining for cells treated with pantoprazole, or pantoprazole with NS1643, retigabine, or rapamycin. Surprisingly, it was observed that treatment with retigabine or NS1643 significantly reduced cleaved caspase-3 positive cells compared to the control. Furthermore, LC-II staining only showed a significant increase in cells treated with rapamycin, as expected (Figure 11-C). Proliferation of NG108-15 cells, measured by BrdU incorporation, was significantly decreased after 6 days with treatment of pantoprazole alone, retigabine alone, and combinations of pantoprazole with NS1643, retigabine, and rapamycin (Figure 11-B), consistent with the viable cell counts obtained in Figure 1-A. Thus, the combination treatment that resulted in the lowest cell proliferation also showed an increase in markers of senescence and did not show a large proportion of apoptotic or autophagic cells.

[0146] U87 cells showed a significant increase in neuronal markers upon combination treatment with TMZ compared to the control (Figure 12). NFM and NSE were similar to those seen in NG108-15 cells. As well as increased in cells treated with pantoprazole or combination treatment of pantoprazole with NS1643 or retigabine (Figure 12-C). TujI was highly elevated only in U87 cells treated with NS1643 and TMZ (Figure 12-B). MAP2 showed high levels of immunoreactivity when cells were treated with combination of TMZ and NS1643 or pantoprazole (Figure 12-A). It should be noted that cell staining was very heterogeneous, with some cells staining brighter than others with the same treatment.

[0147] Astrocyte differentiation markers were increased in the treatment with pantoprazole and NS1643, retigabine, rapamycin, and pantoprazole in combination with TMZ, as well as NS1643 in combination with TMZ (Figure 13). Vimentin, a known marker for astrocytes [107,108], was significantly increased when cells were treated with NS1643, TMZ, and pantoprazole in combination with rapamycin, and NS1643 in combination with TMZ (Figure 13-A). CREB, a known marker for differentiation (Figure 13-B), and S100B (Figure 13-C) and GFAP (Figure 13-D), markers for astrocyte differentiation, were also increased by these same treatments (Figure 13-B). These results were in good agreement with those found in NG108-16 cells.

[0148] U87 cells showed an increase in O4 for all treatments except those containing retigabine (Figure 14-A). Sox10 levels (Figure 14-B) were also increased for all treatments except retigabine alone. Taken together, the above data suggest that the differentiation activity of these treatments is not specific to one species or one type of cell line.

[0149] U87 cells were tested for the same proliferation, senescence, autophagy and apoptosis markers tested in NG108-15 cells and similar results were found (Figure 15). All treatments showed an increase in senescence markers, the most significant being the combination of pantoprazole with NS1643, TMZ and NS1643 with TMZ (Figure 15-A). The nuclear size of the combination treatment with the highest level of senescent cells was also significantly larger than the control, confirming this phenotype (Figure 15-F). p27 Kip1 Similar changes were observed in NG108-15 cells when the levels of p27 were examined (Figure 15-E). Kip1 NS1643 in combination with TMZ showed the highest increase in levels. Cleaved caspase 3 (Fig. 15-D) and LC3-II (Fig. 15-C) levels showed very low, non-significant levels of expression, indicating that all cells were not undergoing high levels of apoptosis or autophagy, respectively. BrdU incorporation was reduced in all treatments that showed lower proliferation in Fig. 6-A. These data indicate that the lower proliferation of U87 cells treated with the most successful drug combinations was driven by increased levels of senescence.

[0150] The voltage dye DiBAC4(3) [109, 110] was used to measure the resting membrane potential of U87 cells treated for 6 days (Figure 16-B). NS1643 alone and in combination with pantoprazole were found to exhibit significant hyperpolarization compared to the control. To investigate further, pHRodo Green was used to examine whether the internal pH of the cells was altered in response to treatment (Figure 16-C). A dramatic increase in pH was observed with all treatments incorporating NS1643, and a slight increase in pH was observed with pantoprazole with retigabine. In addition to cytoplasmic pH, lysosomal pH was tested because of reports that pantoprazole deacidifies the lumen of lysosomes [111, 112]. The dye, LysoSensor Green (Figure 16-A) showed a dramatic alkalinization of lysosomes when treated with NS1643 alone or in combination. Pantoprazole did not show alkalinization of lysosomes in U87 cells, which has been previously reported when pantoprazole was delivered in neutral cell culture medium at pH 7.4

[0113] . Indeed, pantoprazole and its combination with TMZ showed a significant increase in lysosomes, which was consistent with another study performed in neutral pH medium. This is consistent with the study of

[0114] . In addition to the dye, the ratio of cytoplasmic to nuclear YAP was also tested (Figure 16-E). Pantoprazole has been found to reduce YAP activity in ovarian cancer and liver [115, 116]. As YAP has been found to be a master regulator of the cell cycle, especially in cancer, the effect of such treatment on this protein has been investigated [116-119]. Pantoprazole treatment alone was found to have no significant effect on the cytoplasmic to nuclear ratio of YAP, but when combined with NS1643 or TMZ, it showed a significant decrease indicating less YAP in the nucleus compared to the cytoplasm. This significant decrease in the YAP nuclear to cytoplasmic ratio was also evident with NS1643 combined with TMZ treatment. Taken together, these data indicate that NS1643 in combination or alone can increase cytoplasmic calcium levels, increase cytoplasmic pH, and increase lysosomal pH, but a significant decrease in the YAP nuclear-to-cytoplasmic ratio is only seen when combined with pantoprazole or TMZ.

[0151] Neuronal cytotoxicity was minimal after 3 days of treatment with the best-performing drugs and drug combinations. To determine whether the observed effects were specific to cancer cells and could be expected to be usable in vivo without damaging native neurons, these compounds were tested on fibroblast-derived human induced pluripotent stem cells committed to the neural stem cell lineage. These hiNSCs were differentiated in neural medium for 7 days and then treated with electropharmacology for 3 days. The short treatment time was necessary to allow for the Live / Dead assay to be performed without too much cell detachment. Figure 9 shows the percentage of cells killed after treatment compared to the control.

[0152] Toxicity analysis by Live / Dead staining in human neurons shows that only 3 out of 24 treatments showed significant toxicity (Figure 17-A). Pantoprazole showed a slight increase in significant toxicity compared to the control, and pantoprazole with lamotrigine also showed a slight increase, but it was more significant than pantoprazole alone when compared to the control. NS1643 at 50 μM in combination with TMZ showed the highest toxicity when compared to the control. However, the difference between the control toxicity and the most toxic combination of NS1643 at 50 μM and TMZ was still only 5.7% higher than the control. Senescence assay was also performed on neurons under all treatments tested by Live / Dead staining, but no significant differences were found (Figure 17B), and all senescence levels were all less than 1.75%, in stark contrast to the senescence levels of more than 50% in NG108-15 and U87 cells after 6 days of treatment (Figures 11-A and 15-A).

[0153] [Consideration] <Bioelectric Drug> Drug is V memThese drugs were selected based on their predicted effects on ion channel targeting, which have been shown in amphibian models in vivo to prevent and reverse tumorigenesis and metastatic behavior [120, 121]. Indeed, a number of drugs with bioelectrical targets, such as ivermectin (a chloride channel drug) [122–125], salinomycin and monensin (ionophores) [122, 126], various potassium channel drugs [55, 127–131], and drugs targeting proton pumps

[0132] , have been found to have anticancer activity in various screens [133–137]. Thus, the combination of these compounds represents a novel entry into the field of electropharmacology, repurposing known ion channel targeting drugs to manipulate complex cellular outcomes [138, 139]. This approach has already been used to design interventions to repair brain birth defects [140, 141]. A better understanding of the control of cell behavior individually and in tissues is likely to enable much more precisely targeted electropharmaceutical intervention in cancer, as part of the goal of normalizing cells as an alternative to traditional chemotherapy

[0142] .

[0154] Proliferation: The NG108-15 hybrid cell line used in this study has characteristics characteristic of cancer stem cells. It shows a potent anti-inflammatory effect, can be easily transfected and selected, and has been used to test neuronal differentiation for many years. To find treatments that would be robust, all selected compounds were screened in high serum medium, which is normally inhibitory for NG108-15 differentiation [83, 143, 144]. Furthermore, high serum medium provided abundant growth factors that have been shown to be secreted in the peripheral area of ​​resected GBM tumors and are believed to drive migration and proliferation of GBM stem cells in the area [9]. The best performing novel combinations were pantoprazole with retigabine, lamotrigine, NS1643, or rapamycin, which reduced proliferation by 80%, 85%, 86%, and 90%, respectively, when compared to controls. FUCCI analysis showed that the cell cycle percentage was dominated by cells in either G1 or early S. All of these compounds are already FDA approved for other conditions, except for NS1643.

[0155] The best novel combinations were then used to inform compound screening against the human glioblastoma cell line U87 (ATCC). Proliferation assays showed 13 drugs or drug combinations that significantly reduced proliferation compared to controls compared to the preceding glioblastoma treatment, TMZ. Of these 13, the top four treatments were pantoprazole or NS1643 in combination with TMZ, and pantoprazole in combination with retigabine or NS1643, which reduced proliferation by 62%, 63%, 71%, and 74%, respectively, when compared to controls. FUCCI analysis showed that cell cycle percentages were again dominated by cells in either G1 or early S.

[0156] Differentiation: Differentiation therapy for GBM is an alternative treatment strategy that could possibly overcome the problem of recurrence after tumor resection

[14] . For differentiation therapy to be successful, the treatment needs to be effective at clinically relevant concentrations, and differentiation needs to be durable without re-entry into the cell cycle after treatment cessation. This study showed that using a hyperpolarizing compound in combination with pantoprazole, it is possible to arrest the cell cycle of proliferating cells and direct them towards partial differentiation and senescence.

[0157] NG108-15 cells treated with the most successful drug combinations displayed neuronal, astrocytic and oligodendrocyte differentiation markers as well as senescence markers. The mixed nature of differentiation markers in NG108-15 cells has been reported previously

[0145] and may be due to their hybrid neuroblastoma / glioma status. Many of the drug combinations tested in NG108-15 cells were also successful in U87 cells, which showed dramatically less proliferation, less cell cycle progression, and a significant increase in differentiation markers that required cell cycle arrest long enough to accumulate

[0146] . The mixed nature of differentiation markers elevated in U87 cells indicates that this line can differentiate into neuronal, astrocytic and oligodendrocyte lineages, consistent with previous studies [147, 148]. It is important to note that many of the differentiation markers are expressed heterogeneously and that there may be multiple differentiated phenotypes in each sample rather than just one type. Connexin 43 was also upregulated in treated NG108-15 cells, indicating that this marker of cell-cell communication increased as cells became more differentiated, as supported by previous studies

[97] .

[0158] Electrophysiology, pH and Cell Cycle Regulation NS1643, a hERG channel opener and potassium modulator, was shown to hyperpolarize both cell lines tested and was one of the most successful single treatments. In combination with pantoprazole, it was able to arrest the NG108-15 cell cycle long enough to allow differentiation in high serum medium and to work well in U87 cells. Treatment with NS1643 in combination with pantoprazole showed significant effects not seen with either treatment alone. Assays in U87 cells showed that this novel combination treatment dramatically alkalized the pH of the cytoplasm and lysosomes. Cytoplasmic calcium inhibits the cell cycle inhibitor p27 Kip1We showed that YAP translocation and p27 significantly increased with an increase in p27 expression and an increase in senescence-associated β-galactosidase-positive cells. Furthermore, combination treatment with NS1643 also led to a decrease in the nuclear-cytoplasmic ratio of YAP, the elevated levels of which are known to accelerate cancer cell cycle progression, proliferation, treatment resistance, and metastasis [118, 119, 150]. Thus, YAP translocation and p27 Kip1 Increased levels of both NS1643 and TMZ were found in cells that showed the most senescence, supporting what other studies have reported as the involvement of both proteins in senescence [104, 151]. Proliferation under these senescence-inducing treatments was dramatically reduced, and U87 cells did not re-enter the cell cycle until 4 days after treatment was stopped. The combination of NS1643 and TMZ was also highly effective in reducing U87 cell proliferation, but did not hyperpolarize U87 cells by voltage dye assay. However, cells significantly expressed various differentiation markers after 6 days of treatment. Cells treated with NS1643 and TMZ, like other NS1643-containing treatments, also showed increased lysosomal and cytoplasmic pH, p27 kip1 The combination of these dyes showed an increase in membrane potential, and a significant decrease in the YAP nuclear-to-cytoplasmic ratio. As has been reported for other compounds, the voltage dyes may not have accurately reported membrane potential in this combination due to interactions with the dye molecule itself [152, 153].

[0159] Consistent with the results herein, NS1643 has also been shown to induce senescence in melanoma and breast cancer [55, 127, 154]. This senescent phenotype is thought to occur via an increase in internal calcium levels (response to hyperpolarization) that triggers activation of calcineurin, which dephosphorylates NFAT, leading to its translocation to the nucleus

[0131] . In the nucleus, the translocated NFAT and activated calcineurin increase the p21 promoter activity, also found by NS1643, by a mechanism similar to that seen in differentiating keratinocytes, where calcineurin, in combination with NFAT, increases Sp1 / Sp3-dependent transcription and p21 promoter activity

[0155] . WAF1 / CIP1 The same study in keratinocytes showed that calcineurin inactivation increases the expression of p27kip1 It has also been shown that mitochondrial ROS production is reduced by calcineurin activation in neurons

[0155] , and ROS levels were found to be elevated in breast cancer cells treated with NS1643

[0127] . Increased ROS levels have been found to decrease proteasome function

[0157] , downregulating p27 expression through decreased ubiquitination and degradation. kip1

[0158] Currently, there are no publications showing the efficacy of NS1643 against glioblastoma, or its efficacy in combination with rapamycin, pantoprazole, or TMZ.

[0160] Another successful combination included a KCNQ channel opener and the FDA-approved epilepsy treatment, retigabine

[80] . Application of retigabine alone slightly hyperpolarized NG108-15 cells and only slightly reduced proliferation by itself, but its ability to arrest NG108-15 proliferation was most significantly increased by pantoprazole. Pantoprazole, a proton pump inhibitor, worked well alone in reducing proliferation in these two cell lines. However, when treatment was stopped, the cells immediately re-entered the cell cycle, indicating that treatment with pantoprazole alone does not arrest the cell cycle long enough to allow terminal differentiation. When pantoprazole was combined with retigabine, a synergistic effect was achieved in which fewer cells re-entered the cell cycle after treatment was stopped. Interestingly, when NG108-15 cells were treated with the combination, depolarization of NG108-15 cells was observed, but no change was seen in the membrane potential of U87 cells.

[0161] Pantoprazole alone has been reported to increase lysosomal alkalinity by inhibiting the V-ATPase channel

[79] . However, no lysosomal alkalinization was observed, and lysosomal signals were increased. These results are consistent with previous studies of pantoprazole at neutral pH. This is consistent with a recent study showing that pantoprazole in situ does not inhibit the V-ATPase channel, but instead increases lysosomal biogenesis

[0113] . Interestingly, this same study showed that pantoprazole interferes with proteosome function, which may explain why it worked so well as a combination treatment. The recent study also showed that pantoprazole inhibits p27 Kip1 These results indicate that proteosome dysfunction with increased PTEN leads to senescence and endoreplication, resulting in cells with large nuclei

[0159] . Furthermore, loss of PTEN in U87 (ATCC) cells leads to cells that preferentially senesce in response to stress

[0160] . The presence of endoreplication in strongly senescent cells was evidenced by the size of the nuclei in both cell lines and may explain why the BrdU incorporation results were not as low as the cell count data suggested. Treatment with pantoprazole alone showed less differentiation and senescence in U87 cells when compared to pantoprazole in combination with retigabine, NS1643, TMZ, or rapamycin. Pantoprazole has been shown to be effective against glioblastoma in vitro

[77] , but to our knowledge, this is the first study to test its efficacy in combination with NS1643, lamotrigine, retigabine, rapamycin, or temozolomide.

[0162] TMZ, the primary GBM treatment, was not significantly effective in reducing proliferation of NG108-15 cells. This has also been observed in some GBM cases. However, as evidenced by the recovery assay, the combination of TMZ and pantoprazole in NG108-15 cells significantly reduced their proliferation compared to pantoprazole alone, but did not terminally differentiate them. However, TMZ itself was not effective in NG108-15 cells, but was significantly effective in U87 cells. This effectiveness in U87 cells may explain why the combination of pantoprazole or NS1643 with TMZ was so effective in reducing proliferation and increasing differentiation markers in these cells but not in the NG108-15 line. It should be noted that these combinations also dramatically increased senescence markers in these cells and caused a significant decrease in the YAP nuclear to cytoplasmic ratio.

[0163] Drug Concentrations and FDA Status: The drug concentrations used in these studies were within the C-value reported for the highest dose that did not produce unacceptable toxicity. max Retigabine, approved by the FDA for epilepsy, was used at 10 μM, which is the upper reported C limit obtained at the 1200 mg / day dose of approximately 2250 ng / mL or approximately 7.4 μM. max The values ​​are close to those of the 0.1% to 0.2% values ​​

[0161] . The blood-brain barrier permeability is suitable for retigabine, whose free plasma concentration is nearly the same as its free brain concentration

[0162] . Retigabine also has a high plasma protein binding affinity of about 80%, so the choice to use high serum to test drugs and drug combinations also helps to strengthen the clinical relevance of such treatment data

[0162] . However, the binding of compounds in fetal bovine serum may differ from that in human serum

[0163] .

[0164] For immunological studies, rapamycin, an FDA-approved immunosuppressant, was used at a dose of 100 nM. This concentration is less than the maximum dose of 40 mg / day of the nanoamorphous oral formulation in the fasted state and is maxThe mean plasma concentration of rapamycin was 219 ng / mL or approximately 239 nM, and toxicity at this level was considered manageable.

[0164] Rapamycin has also been used in Phase I clinical trials for glioblastoma and has been found to effectively cross the blood-brain barrier.

[0165] .

[0165] Pantoprazole, an FDA-approved proton pump inhibitor, was used at a dose of 100 μM. The maximum dose of pantoprazole administered for Zollinger-Ellison syndrome is 240 mg / day, and if the Cmax is proportional to that administered at a dose of 30 mg / day (as is the case for doses up to 80 mg / day), then the Cmax would be 42 mg / L or approximately 110 μM.

[0166] However, pantoprazole has a blood-brain barrier permeability of only 2%.

[0167] Therefore, its use in glioblastoma therapy must rely on novel methods for delivery across the blood-brain barrier, of which many new strategies are being developed. R

[0168] However, it should be noted that all tests performed on pantoprazole in this study were performed at neutral pH, so the efficacy of pantoprazole may be increased in an in vivo environment where the tumor microenvironment is more acidic.

[0169] .

[0166] The hERG activator NS1643 is not currently approved by the FDA; however, it has recently been used in a breast cancer xenograft model in immunocompromised mice and showed no apparent toxicity to cardiac or normal breast epithelial cells.

[0167] Limitations of this study: Electrophysiological analysis in NG108 cells showed how the compounds had immediate changes in membrane potential, but did not show changes in potential over time. Pantoprazole had no significant effect on the membrane potential of NG108-15 cells alone, but the effect of pantoprazole on the abrogation of hyperpolarization seen with retigabine, lamotrigine, NS1643, and rapamycin is quite interesting considering how quickly it occurred. If pantoprazole was simply blocking the function of these other compounds, it is intriguing why the combination of compounds would be more effective than pantoprazole alone. Furthermore, the lack of hyperpolarization in NG108-15 cells with combination treatment points to the fact that ionomodulators do not necessarily need to hyperpolarize the membrane potential to have an effect. The depolarization of NG108-15 cells seen with the combination of pantoprazole with retigabine or rapamycin may affect the ability to progress through the cell cycle if the hyperpolarization level required to progress through S phase is not reached, an effect seen especially in OPCs [170-172]. However, this is not the case with the combination of pantoprazole with NS1643 or lamotrigine, which did not have any significant changes in the resting membrane potential compared to controls in NG108-15 cells.

[0168] In this example, only the DiBAC4(3)-based analysis of resting membrane potential was obtained and may have had some interference with the dye caused by the compound itself.

[0169] Conclusions: The use of the NG108-15 cell line in high serum conditions for initial screening of many compounds and combinations provides a subset of drugs that showed significant reduction in proliferation in the human U87 glioblastoma cell line treated for 6 days, and showed reduced growth up to 4 days after treatment was stopped. These results suggest that treatment can be administered intermittently. The top combinations provided robust candidates that were able to induce terminal differentiation and senescence in NG108-15 cells under normally prohibitive conditions, and showed terminal differentiation and senescence in U87 cells.

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[0359] Example 3 - Ion channel drugs arrest the cell cycle of NG108-15 and U87 cells The most promising compounds (e.g., showing the greatest reduction in cancer cell proliferation) from the results of experiments using mouse / rat neuroblastoma / glioma NG108-15 cells containing the FUCCI cell cycle reporter and palmitoyl-mTurquoise2 fluorescent membrane tag were retested in at least three biological replicates. Those compound combinations that showed significant activity against NG108-15 cells were then screened against the human glioblastoma U87 cell line containing the FUCCI cell cycle reporter and palmitoyl-mTurquoise2 fluorescent membrane tag. Follow-up biological replicates were performed for the positive hits from the initial U87 cell screen (showing a reduction in glioblastoma cell proliferation). Differentiation, proliferation, and apoptosis were also investigated. Antibodies against various cell markers for cellular markers were used to stain both NG108-15 and U87 cells that had been treated with the most promising drug combinations for 6 days and then fixed. Senescence and BrdU incorporation assays were performed on cells treated in the same manner. To confirm the mechanism of action of these treatments (control of the bioelectrical state within the cells), automated patch clamp data were obtained on NG108-15 cells treated with some of the most promising drug combinations immediately after application to examine the effect on the resting membrane potential. A determination of functional maturation was also made by examining calcium signaling for some of the best performing compounds and combinations. Finally, live / dead assays were performed on human induced neural stem cells that had been differentiated for 7 days prior to 3 days of treatment with the most promising compound combinations in both NG108-15 and U87 cells.

[0360] Strategy: Compounds selected for testing included those known to alter the membrane potential of cells by either creating an acidic environment inside the cell and an alkaline environment outside the cell, increasing potassium efflux, or decreasing sodium influx. Rapamycin was selected for its ability to induce autophagy and for its combinatorial effect with ionotropic drugs. The most effective combinations were able to arrest the cell cycle long enough to allow terminal differentiation, senescence, or apoptosis. All compounds and combinations tested were compared to temozolomide treatment, the standard clinical therapy for glioblastoma. A list of the most effective compounds tested and their functions are listed in Table 2 above.

[0361] For NG108-15 cells, Figure 1 shows the best individual and combination treatments compared to the control on day 6. In these bar graphs, higher (e.g., control bar) represents more cells and lower represents reduced cancer cell proliferation (the effect we are looking for). The stacked bar graphs show the proportion of cells in different parts of the cell cycle. An increase in the red fraction (G1) and orange fraction (early S) indicates cell cycle arrest. A decrease in the yellow fraction (actively dividing) and an increase in either the red or orange fraction (in transition to start dividing) is desirable.

[0362] FIG. 1-A shows that NG108-15 cells treated with 20 μM and 50 μM NS1643 (hERG activator) alone were able to significantly reduce proliferation. These two concentrations of NS1643 were highly effective in reducing cell proliferation when combined with pantoprazole (a proton pump inhibitor), working better than pantoprazole or NS1643 alone. 50 μM NS1643 also significantly reduced proliferation when combined with rapamycin (an autophagy inducer), working better than rapamycin or NS1643 alone. Retigabine (a voltage-activated potassium channel opener) significantly reduced cell proliferation by itself, but the combination of retigabine with rapamycin or pantoprazole worked better than either compound alone. Pantoprazole at 100 μM was the most effective compound, either alone or in combination with lamotrigine (which blocks voltage-gated sodium channels), NS1643, and rapamycin. These three combinations performed better than one of the positive controls, cAMP with 200 nM rapamycin in full serum medium, which is known to cause terminal differentiation of these cells in low serum (the other positive control). However, cAMP cannot be used clinically due to its many off-target effects.

[0363] The cell cycle data in Figure 1-B reveal that pantoprazole increases the percentage of cells in early S and the combination can also increase the percentage of cells in G1. Rapamycin treatment increased the percentage of cells in G1, whereas NS1643 treatment did not seem to affect the cell cycle percentage. Currently, there are no publications showing the efficacy of NS1643 against glioblastoma, or its efficacy in combination with rapamycin or pantoprazole. However, this drug has been published as effective against breast cancer and melanoma. Pantoprazole Pantoprazole has been shown to reduce glioblastoma growth in vitro, but there are no studies showing the efficacy of treatment with NS1643, lamotrigine, retigabine, or rapamycin. It is worth noting that temozolomide (a standard glioblastoma treatment) was not effective against NG108-15 cells.

[0364] Figure 2 shows that the combination of pantoprazole performed significantly better than pantoprazole alone in NG108-15 cells. Figure 2-A shows that the combination of pantoprazole with 20 μM NS1643, 100 μM lamotrigine and 100 nM rapamycin was the only combination that showed significantly higher efficacy than pantoprazole alone in reducing cell proliferation after 6 days of treatment, with the combination with rapamycin showing the most significant difference. Figure 2-B shows that the percentage of cells in G1 and early S was only slightly increased by pantoprazole treatment in combination with NS1643 and lamotrigine, while the combination with rapamycin significantly increased the percentage of G1.

[0365] To understand whether these drug treatments terminally differentiate and therefore arrest cell proliferation after the drugs are withdrawn, recovery studies were performed (Figure 3). Cells were treated with drugs for 6 days, then the drugs were removed (bounded by the dashed line in Figure 3) and the cells were placed in drug-free medium for an additional 10 days.

[0366] Figure 3 shows that the three combinations with pantoprazole showed significantly less cell proliferation after 6 days of treatment, but showed some recovery after treatment was stopped, lower than pantoprazole alone, but not significantly. However, the positive control showed no recovery and more cells that were terminally differentiated or dying after treatment.

[0367] Figure 4 shows the results of U87 human glioblastoma cells analyzed in the same way as NG108-15 cells above. Figure 4-A shows that in human glioblastoma U87 cells, the same drug treatments that were significantly effective in NG108-15 cells were also significantly effective after 6 days compared to the control. In particular, NS1643 at 50 μM significantly reduced cell proliferation compared to the control, but was much more effective when combined with pantoprazole, TMZ, and rapamycin. Pantoprazole treatment also worked very well in this cell line, showing highly significant differences in cell proliferation compared to the control when combined with rapamycin, retigabine, NS1643, lamotrigine, or TMZ. The data showed that in U87 cells, TMZ was highly effective in reducing cell number compared to the control, but combinations with minoxidil, lamotrigine, pantoprazole, rapamycin, retigabine, and NS1643 increased the effectiveness of TMZ. The cell cycle data in Figure 4-B show that some, but not all, of the most effective combinations increased the G1 and early S fraction of cells. Pantoprazole showed its characteristic increase in early S fraction of cells, and rapamycin showed the increase in G1 fraction seen in NG108-15 cells. TMZ and NS1643 treatments did not show significant changes in the percentage of cells in each stage of the cell cycle compared to the control. Unfortunately, the percentage of cells in each cell cycle stage for the best treatment in U87 cells, i.e., pantoprazole with retigabine, was not observed due to autofluorescence aggregation in the cytoplasm of cells that caused false results in the FUCCI reporter data.

[0368] Figure 5-A shows that pantoprazole alone was significantly more effective at reducing cell proliferation after 6 days of treatment than TMZ alone. All combinations that were more effective than TMZ alone were with TMZ or pantoprazole. Rapamycin, NS1643, and pantoprazole all significantly increased the efficacy of TMZ, with NS1643 and pantoprazole being the most significant of these combinations with TMZ. Figure 5-B shows that TMZ in combination with rapamycin increased the percentage of cells in G1, and NS1643 increased the percentage of cells in G2, and NS1643 increased the percentage of cells in G3, and NS1643 increased the percentage of cells in G4, and NS1643 increased the percentage of cells in G5, and NS1643 increased the percentage of cells in G6, and NS1643 increased the percentage of cells in G1, and NS1643 increased the percentage of cells in G2 ... The results showed that combinations with TMZ and 3 increased the percentage of cells in early S compared to TMZ alone. The pantoprazole combination consistently showed a greater percentage of cells in early S, with a complementary decrease in cells in late S, G2, and M compared to TMZ alone.

[0369] Figure 6 examines in more detail the significance between different pantoprazole concentrations and pantoprazole alone in U87 cells. Figure 6-A shows that in U87 cells, the same combinations that were significantly better than pantoprazole in NG108-15 cells were also significant with the added combination of TMZ or retigabine. Pantoprazole combined with rapamycin, lamotrigine, NS1643, TMZ or retigabine were all significantly better than pantoprazole alone. The most significant combination was the combination with NS1643 and retigabine. The combination of pantoprazole and NS1643 was so effective that reducing the pantoprazole concentration by half and combining it with NS1643 at 50 μM was significantly more effective than pantoprazole alone at 100 μM. FIG. 6-B shows the characteristic increase in G1 when pantoprazole is combined with rapamycin, and that combination with NS1643 increases the proportion of cells in early S, with a complementary decrease in late S, G2, and M.

[0370] Recovery tests were also performed on U87 cells, and Figure 7 shows the only treatments that were significantly better than pantoprazole in reducing cell proliferation at day 6. Figure 7 shows that the combination of pantoprazole and rapamycin shows similar recovery after 6 days as pantoprazole alone. However, the combination of pantoprazole with 50 μM retigabine or NS1643 shows a much lower recovery rate than pantoprazole alone. This suggests that the treatment is a permanent change to the cells so that proliferation capacity cannot be restored. Surprisingly, the positive control using cAMP in combination with rapamycin started to proliferate after 8 days.

[0371] To investigate the mechanistic characteristics of these treatments, automated patch clamp data were collected examining the electrophysiology of NG108-15 cells treated with some of the drug combinations that performed better in the cell proliferation assay (Figure 8). Untreated cells were patched and baseline measurements were taken, then drugs were added and changes in their resting membrane potential were recorded.

[0372] Figure 8 shows that rapamycin, retigabine, NS1643, TMZ, and lamotrigine all significantly hyperpolarized the cells compared to the control. Surprisingly, pantoprazole had no immediate effect on the membrane potential of the cells, but its combination with retigabine and rapamycin depolarized the cells instead of hyperpolarizing them. The combination of pantoprazole with lamotrigine and NS1643 did not significantly change the membrane potential compared to the control. However, this electrophysiological analysis shows what immediate changes the compound has in the membrane potential, not what the change in potential is over time. Pantoprazole is known to have an inhibitory effect on the transcription of V-ATPase in cancer cells, which may result in changes in membrane potential over a longer period of time. The sustained depolarization of the cells seen with the combination of pantoprazole with retigabine and rapamycin may affect the ability of the cell cycle to progress if the hyperpolarization level required to progress through S phase is not reached. The FUCCI data showed that pantoprazole and the combination increased the proportion of cells in early S stage, and the proliferation data showed that the cells significantly slowed their proliferation.

[0373] In addition to this electrophysiology data, calcium event analysis was performed on some of the most promising combinations of drugs on NG108-15 cells, but no significant findings were observed after multiple tests. This may indicate that rather than terminally differentiating the cells, the treatment is slowing the cell cycle long enough that the cells can no longer re-enter. This may result in senescent cells or may indicate some other form of cell cycle exit. To test this, antibody staining of NG108-15 and U87 cells was performed on some of the best The procedure was carried out using

[0374] In addition to these assays, we investigated the neurotoxicity of the best performing compounds. Human induced pluripotent stem cells derived from fibroblasts and committed to a neuronal stem cell lineage, called hiNSC, were differentiated in neuronal medium for 7 days and then treated with drugs for 3 days. The cells used in this assay did not adhere well to the plate and showed some cell death even in the control, so we limited the treatment to 3 days so that we could perform the Live / Dead assay without too much cell detachment. Figure 25 shows the percentage of cells that died after treatment compared to the control.

[0375] Toxicity analysis in hiNSCs differentiated into neurons for 7 days and then treated with drugs for 3 days shows that only 3 treatments showed some toxicity. Pantoprazole showed a slight increase in significant toxicity compared to the control, and pantoprazole with lamotrigine also showed a slight increase, but it was more significant than pantoprazole alone when compared to the control. NS1643 at 50 μM in combination with TMZ showed the highest toxicity when compared to the control. However, the difference between the control toxicity and the most toxic combination of NS1643 at 50 μM and TMZ was still only 5.7% higher than the control. Considering the high mortality rate of the control cells and the delicate nature of these cells, toxicity seems to be negligible. The remaining treatments showed no significance compared to the control.

[0376] In summary, initial screening of multiple drug treatments on NG108-15 cells with cell cycle reporters successfully provided many positive hits that were also effective in inhibiting proliferation of human glioblastoma U87 cells. At least 13 drugs and / or drug combinations were found to be significantly more effective than the current gold standard in glioblastoma therapy. These drug combination treatments showed growth retardation even after drug removal after 6 days of treatment. Electrophysiology and calcium event data suggest that some of these drug combinations may not be acting by hyperpolarization followed by terminal differentiation, but rather by cell cycle disruption that causes cells to lose their ability to proliferate. Neuronal toxicity data revealed that the most promising drug treatments at some of their highest concentrations did not show very high levels of undesirable toxicity compared to the control. Only three treatments showed undesirable toxicity compared to the control, with the most toxic one showing only a 5.7% increase in toxicity.

[0377] Example 4 - Tumor tissue effects of ion channel drugs and drug combinations A tumor tissue cytotoxicity assay was used to evaluate the sensitivity of different tumor tissues to different drugs and drug combinations. This tumor tissue cytotoxicity assay uses a tissue platform that preserves cancer tissue architecture, including vasculature and immune tissue, thereby more accurately reflecting cancer growth in vivo (see, e.g., Ben-Hamo, R. et al., Predicting and affecting response to cancer therapy based on pathway-level biomarkers. Nature Communications 11(1), 3296(2020)). This tumor tissue cytotoxicity assay can provide a quantitative readout of the functional effect of different drugs on a particular tumor in the context of the unique microenvironment of the tumor tissue. The tumor microenvironment can affect the efficacy of drugs, for example, via hypoxic response, changes in the extracellular matrix, and immune response. For example, the tumor microenvironment can confer drug resistance in vivo to tumor cells that are otherwise drug-sensitive in vitro. Furthermore, this tumor tissue cytotoxicity assay allows for the evaluation of antitumor drugs whose mechanism of action occurs via the microenvironment and / or tissue features such as the vasculature or immune tissue. Furthermore, this assay is able to maintain the structural morphology of tumor tissue even after 10 days in culture, allowing the evaluation of drugs that require many days to achieve cytotoxicity.

[0378] Different drugs and drug combinations (7) were evaluated for their cytotoxic effects on breast and colorectal cancer tissues using the tumor tissue cytotoxicity assay described below. The experiment included analysis of the cytotoxicity of seven drug treatments on colorectal cancer tumors (N=3) and breast cancer tumors (N=3). Prior to the initiation of each drug combination treatment, calibration experiments (N=2) were performed for each type of tissue using three different concentrations of the individual drugs to determine the most appropriate concentration. Tumor slices were cultured for at least 5 days in the presence of the drug or drug combination. At selected time points (e.g., day 5 or day 10), tumor slices were fixed and processed to produce stained sections with morphological and histological markers. The drugs tested were pantoprazole, retigabine, lamotrigine, rapamycin, minoxidil, zolmitriptan, and NS1643, either alone or in the combinations shown in Table 3.

[0379] [Table 3]

[0380] The viability and morphology of cancer cells in treated tissues were analyzed and scored on a scale of 0 to 100, assessed by a variety of parameters including nuclear detail, tissue cohesiveness, cytoplasmic changes, and immunohistochemical staining. On this scale, higher scores correlate with improved clinical response, with scores above 50 being more likely to result in clinical benefit. A score below 30 is considered no response.

[0381] Results of tumor tissue cytotoxicity assays for drug treatments on either colorectal or breast cancer tissues are shown in Figures 21-51. Figures 23-51 show examples of histological results of tumor tissue cytotoxicity assays for various drug treatments on both colorectal and breast cancer tissues. Each tissue tested responded best (higher scores) to a different drug or drug combination (Figures 21-22). No single combination showed a strong response in all samples (Figures 21-22). The combination of rapamycin and minoxidil showed scores of 50 or higher in four of six tissues and above the median in five of six samples (Figures 21-22). One colorectal and one breast cancer tumor sample were highly resistant, with no combinations demonstrating a significant effect on these tissues (see Figures 21-22). These two samples were resistant to the combined treatment with rapamycin and minoxidil (see Figure 22).

[0382] Since each patient's unique tumor may respond differently to treatment (creating the need for personalized cancer therapy), it is not surprising that each tissue tested responded best to a different drug treatment. However, in terms of robustness and population trends, the rapamycin + minoxidil combination was found to be effective in 66% of the tissues tested, and in 5 of 6 cases (83%) this treatment scored higher than the median. Only tissues that were generally resistant to the various drug treatments did not respond to this combination.

[0383] It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein may be combined with any one or more of the above-mentioned components not specifically disclosed herein. The present invention may be suitably implemented without any number of elements, one or more limitations. The terms and expressions used are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude the features shown and described or equivalents of some thereof, but it is recognized that various modifications are possible within the scope of the present invention. Thus, although the present invention has been illustrated by specific embodiments and optional features, it is to be understood that modifications and / or variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

[0384] A number of patent and non-patent literature are cited herein. The cited references are incorporated herein by reference in their entirety. If there is a discrepancy in the definition of a term in this specification compared to the definition of the term in the cited reference, the term shall be interpreted according to the definition in this specification.

Claims

1. 1. A therapeutic agent that modulates the polarity of cancer cells for use in a subject in need of treatment for a cell proliferative disease or disorder, wherein the therapeutic agent modulates the polarity of cancer cells is selected from the group consisting of one or more potassium channel activators, one or more sodium channel inhibitors, one or more calcium channel inhibitors, one or more proton pump inhibitors, and combinations thereof.

2. The therapeutic agent for use according to claim 1, wherein the potassium channel is one or more of a KCNQ / Kv7 channel, a K(ATP) channel, a human Ether-a-go-go (hERG) channel, a KCNK3 channel, a BK channel, and an SK channel.

3. A therapeutic agent for use as described in claim 1 or claim 2, wherein the one or more potassium channel activators include one or more of retigabine, minoxidil, NS1643, ONO-RS-082, and chlorzoxazone.

4. A therapeutic agent for use according to claim 1 or claim 2, wherein the cell proliferative disease or disorder is a cancer selected from brain cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, bladder cancer, kidney cancer, uterine cancer, melanoma, lymphoma, leukemia, pancreatic cancer, ovarian cancer, liver and intrahepatic bile duct cancer, oral cancer, and esophageal cancer.

5. A therapeutic agent for use as described in claim 1 or claim 2, further comprising an mTOR inhibitor for administration to the subject before, simultaneously with, or after administering an effective amount of one or more of the potassium channel activators, one or more sodium channel inhibitors, one or more calcium channel inhibitors, or a combination thereof to the subject.

6. A therapeutic agent for use as described in claim 1 or claim 2, further comprising an alkylating agent for administration to the subject before, simultaneously with, or after administration of one or more of the potassium channel activators and / or one or more proton pump inhibitors to the subject.

7. A therapeutic agent for use as described in claim 1 or claim 2, further comprising temozolomide, dexamethasone, or pantoprazole for administration to the subject before, simultaneously with, or after administering an effective amount of one or more of the potassium channel activators to the subject.

8. A therapeutic agent for use as described in claim 1 or claim 2, wherein one or more proton pump inhibitors are administered to the subject before, simultaneously with, or after one or more potassium channel activators and / or one or more sodium channel inhibitors are administered to the subject.

9. A therapeutic agent for use as described in claim 1 or claim 2, wherein one or more sodium channel inhibitors are administered to the subject before, simultaneously with, or after administering one or more potassium channel activators to the subject.

10. The therapeutic agent for use according to claim 9, wherein one or more of the sodium channel inhibitors are selected from lamotrigine, cariporide and topiramate.

11. A therapeutic agent for use as described in claim 1 or claim 2, wherein one or more calcium channel inhibitors are administered to the subject before, simultaneously with, or after administering one or more potassium channel activators to the subject.

12. The therapeutic agent for use according to claim 11, wherein one or more of the calcium channel inhibitors are selected from gabapentin and zolmitriptan.

13. A therapeutic agent for use as described in claim 1 or claim 2, further comprising a peroxisome proliferator-activated receptor alpha (PPARα) activator for administration to the subject before, simultaneously with, or after administration of one or more of the potassium channel activators to the subject.

14. A therapeutic agent for use as described in claim 13, wherein the PPARα activator is fenofibrate.

15. A therapeutic agent for use according to claim 1 or claim 2, wherein one or more of the sodium channels are selected from voltage-gated sodium channels and Na(+) / H(+) exchanger type 1 (NHE1) channels.

16. A therapeutic agent for use as described in claim 1 or claim 2, wherein one or more of the sodium channel inhibitors are selected from lamotrigine, topiramate and cariporide.

17. The therapeutic agent for use according to claim 5, wherein the mTOR inhibitor is rapamycin.

18. A therapeutic agent for use as described in claim 1 or claim 2, wherein one or more of the proton pump inhibitors are selected from pantoprazole, omeprazole, lansoprazole, dexlansoprazole, esomeprazole, rabeprazole, and ilaprazole.

19. A therapeutic agent for use as described in claim 1 or claim 2, wherein one or more of the calcium channels are voltage-activated calcium channels.

20. A therapeutic agent for use as described in claim 1 or claim 2, wherein one or more of the calcium channel inhibitors is zolmitriptan.

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