Increasing cancer cells' sensitivity to tumor treating fields (ttfields) by inhibiting il11 activity

Abstract

To provide a pharmaceutical composition for use in a method of treating a cancer in a subject.SOLUTION: The present invention provides a pharmaceutical composition for use in a method of treating a cancer in a subject, comprising at least one calcium channel blocker, wherein the method includes the steps of: (1) applying an alternating electric field to the target region in the subject for a predetermined time and (2) administering the pharmaceutical composition to the subject.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 882,813, filed Aug. 5, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Tumor treating electric fields, or TT fields, are low intensity (e.g., 1-3 V / cm) alternating electric fields in the mid-frequency range (e.g., 100-500 kHz) that inhibit the growth of cancer cells. This non-invasive treatment targets solid tumors and is described in U.S. Patent No. 7,565,205, which is incorporated herein by reference in its entirety. TT fields are FDA approved for the treatment of glioblastoma (GBM) and can be delivered, for example, via the Optune™ system. Optune™ includes an electric field generator and two pairs of transducer arrays (i.e., electrode arrays) that are placed on the patient's shaved head. One pair of electrodes is placed to the left and right of the tumor, and another pair of electrodes is placed in front and behind the tumor. In a preclinical setting, TT fields can be applied in vitro, for example, using the Inovitro™ TT field lab bench system. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 7,565,205 [Patent Document 2] US Patent Application Publication No. 2020 / 0016067 [Brief description of the drawings]

[0004] [Figure 1]Figure 1A shows the results of an exemplary study demonstrating the fold increase and respective decrease in tumor volume resulting from the combination of 150 kHz TT Fields with sorafenib in N1S1 hepatoma cells. Figure 1B shows the results of an exemplary study demonstrating the fold increase and respective decrease in tumor volume resulting from the combination of 150 kHz TT Fields with sorafenib in N1S1 hepatoma cells. [Diagram 2] FIG. 1 shows exemplary TTFields treatments and administration schedules for four treatment conditions (I, II, II, and IV) to assess fibrosis. [Diagram 3] Figure 3A shows an exemplary histopathological evaluation of fibrosis-specific Masson's Trichrome (MT) staining (blue) of treated N1S1 hepatoma tumors, showing (1) the original image (used for fibrosis score) (top left), (2) fibrosis color segmentation of MT stained section with ROI blue representing fibrosis (for area calculation), (3) whole tissue without fibrosis (for area calculation, red-eosin, bottom left), and (4) outer region of tissue (bottom right). Figure 3B shows fibrosis score of the experiment described with respect to Figure 3A. [Figure 4] The fibrotic area (%) for each condition is shown, calculated from the experiment described with respect to FIG. 3A. [Diagram 5] FIG. 3B shows the fibrosis area expressed as fold change for each condition, calculated from the experiment described above with respect to FIG. 3A. [Figure 6] Figure 6A shows (1) the correlation between fibrosis score and tumor volume in tumors treated with TTFields. Figure 6B shows (2) the correlation between fibrosis score and tumor fold change in tumors treated with TTFields. Figure 6C shows (3) the correlation between fibrosis area and tumor fold change in tumors treated with TTFields. Figure 6D shows (4) the correlation between fibrosis area and tumor volume (Figure 6D) in tumors treated with TTFields. Summary of the Invention [Means for solving the problem]

[0005] One aspect of the invention is directed to a first method of reducing the viability of cancer cells, the first method comprising applying an alternating electric field to the cancer cells, the alternating electric field having a frequency between 100 and 500 kHz, and inhibiting IL11 activity.

[0006] In some examples of the first method, the frequency of the alternating electric field is between 180 and 220 kHz.In some examples of the first method, the cancer cells include glioblastoma cells and hepatocarcinoma cells.

[0007] In some examples of the first method, inhibiting IL11 activity includes at least one of reducing IL11 expression, inhibiting IL11 signaling, downregulating IL11, neutralizing IL11, and blocking IL11 receptors. In some examples of the first method, inhibiting IL11 activity includes at least one of administering an IL11 antagonist, administering an IL11 neutralizing antibody, and administering an IL11 receptor alpha (IL11Ra) neutralizing antibody.

[0008] In some examples of the first method, a therapeutically effective concentration of a fibrosis inhibitor is administered to the subject.In some examples of the second method, the administration of the fibrosis inhibitor comprises administering at least one of fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, and vismodegib to the subject.In some examples, a therapeutically effective concentration of at least one of broad-spectrum tyrosine kinase inhibitors (e.g., sorafenib, sunitinib, and imatinib) is administered to the subject.

[0009] Another aspect of the invention is directed to a second method of reducing tumor volume in a living subject, the second method comprising applying an alternating electric field to the tumor, the alternating electric field having a frequency between 100 and 500 kHz, and administering to the subject a therapeutically effective concentration of an IL11 inhibitor.

[0010] In some examples of the second method, the frequency of the alternating electric field is between 180 and 220 kHz. In some examples of the second method, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step.

[0011] In some examples of the second method, administering the IL11 inhibitor comprises at least one of administering an IL11 antagonist, administering an IL11 neutralizing antibody, and administering an IL11 receptor alpha (IL11Ra) neutralizing antibody.

[0012] In some examples of the second method, administering the IL11 inhibitor comprises at least one of administering an IL13 antagonist, administering an IL13 neutralizing antibody, and administering an IL13 receptor alpha (IL13Ra) neutralizing antibody.

[0013] In some examples of the second method, a therapeutically effective concentration of a fibrosis inhibitor is administered to the subject.

[0014] In some examples of the second method, administering the fibrosis inhibitor comprises administering to the subject at least one of fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, and vismodegib.

[0015] In some examples, the second method further comprises administering to the subject a therapeutically effective concentration of at least one broad-spectrum tyrosine kinase inhibitor (e.g., sorafenib, sunitinib, and imatinib). The tumor volume of the tumor may be reduced by about 5-fold or more.

[0016] Another aspect of the invention is directed to a third method of reducing the viability of cancer cells in a living subject, the third method comprising applying an alternating electric field to the cancer cells, the alternating electric field having a frequency between 100 and 500 kHz, and administering to the subject a therapeutically effective concentration of a fibrosis inhibitor.

[0017] In some examples of the third method, the frequency of the alternating electric field is between 180 and 220 kHz. In some examples of the third method, at least a portion of the applying step is performed simultaneously with at least a portion of the administering step. In some examples of the third method, administering the fibrosis inhibitor includes administering to the subject at least one of fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, and vismodegib.

[0018] In some examples of the third method, administering the fibrosis inhibitor comprises administering a calcium channel blocker to the subject. Optionally, in some of these examples, the calcium channel blocker comprises at least one of felodipine, verapamil, diltiazem, and nifedipine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Typically, most tumors have a higher electrical conductivity than the surrounding healthy tissue, and this higher electrical conductivity may explain the specificity of TTFields for malignant tissue. More specifically, the power loss density of the electric field, L, is given by:

[0020]

number

[0021] where σ is the electrical conductivity of the tissue and |E| is the strength of the electric field (power loss density is measured in milliwatts per cubic centimeter), and a higher electrical conductivity σ in tumor tissue results in a higher power loss density in that particular tissue.

[0022] Clinical data show a significant increase in IL11 (Interleukin-11) expression levels in tumors of GBM patients treated with TTFields. Data were obtained as follows: GBM tumor samples were obtained before and after treatment according to a standard chemoradiotherapy protocol (6 patients) or a combined TTFields and standard chemoradiotherapy protocol (6 patients). Analysis of gene expression was performed by RNA-seq. A generalized linear model with negative binomial distribution was used to analyze the differences in expression after treatment and the effect of control and TTFields treatment. Raw data were analyzed using DESeq2 software. Differential gene expression analysis was calculated using the reads per kilobase million (RPKM) values ​​of the chemoradiotherapy and TTFields treatment groups. Statistical analysis was performed using a generalized linear model with negative binomial distribution. The difference in expression before and after treatment was derived separately for each individual and the mean net treatment effect was calculated for each treatment group. The difference between treatment effects was expressed as fold change between the mean net effect of TTFields and control. The Benjamini-Hochberg method was used to correct fold-change p-values ​​for multiple comparisons. A list of 712 genes associated with immune activity was compiled using the general literature, the Nanostring "nCounter® PanCancer immune profiling panel" and the ThermoFischer "Oncomine™ immune response" gene list.

[0023] Significant differences in the effect of TTFields versus control treatment were defined as a fold change >2 or <0.5 with a corrected p-value <0.1. The table below presents genes that showed significantly altered gene expression.

[0024] [Table 1]

[0025] Notably, the differential effect of TTFields + chemoradiation treatment on expression of IL11 (compared to chemoradiation only controls) was an 8.5-fold increase. Without being bound by any theory of operation below, this increase in IL11 may cause two problems.

[0026] The first problem is that IL11 is known to be a fibrotic factor. As a result, the increase in IL11 associated with the application of TT-fields can result in extracellular matrix remodeling and fibrosis in tumor tissue. Because fibrosis causes a decrease in intracellular and extracellular fluid, fibrotic tissues usually have lower electrical conductivity than non-fibrotic tissues. Thus, IL11-promoted fibrosis will (gradually) decrease the tumor's electrical conductivity σ, which in turn will decrease the tumor's power loss density by operation of equation (1), thereby decreasing the efficacy of TT-fields. This deleterious sequence of events can be interrupted by inhibiting IL11 activity. More specifically, inhibiting IL11 activity reduces fibrosis, allowing the electrical conductivity σ to remain high, allowing the power loss density to remain high, and allowing the efficacy of TT-fields to remain high.

[0027] The expected benefits of interrupting the aforementioned deleterious sequence of events are supported by experiments demonstrating that inhibition of IL11 has been shown to prevent cardiac and renal fibrosis.

[0028] The second issue is that tumor expression of IL11 is associated with an aggressive phenotype and poor prognosis in a wide range of tumors, including hepatocellular carcinoma, pancreatic cancer, gastric adenocarcinoma, non-small cell lung cancer, breast cancer, endometrial carcinoma, and chondrosarcoma.

[0029] Any of the above problems can be ameliorated by counteracting the increase in IL11 that occurs when tumors are exposed to TTFields (e.g., by inhibiting IL11 activity).

[0030] Examples of some suitable approaches for inhibiting IL11 activity include reducing IL11 expression, inhibiting IL11 signaling (e.g., by administering bazedoxifene), downregulating IL11, neutralizing IL11, blocking the IL11 receptor, administering an IL11 antagonist (e.g., W147A), administering an IL11 neutralizing antibody (e.g., ENxl08A, ENx203, or ENx209), and administering an IL11 receptor alpha (IL11Ra) neutralizing antibody.

[0031] Inhibition of IL11 activity can also be achieved indirectly by inhibiting stimulators of IL11 or by inhibiting stimulators of IL11 receptor alpha (IL11Ra). For example, since IL13 is a potent stimulator of IL11 and IL11Ra, further examples of suitable approaches for inhibiting IL11 activity include reducing IL13 expression, inhibiting IL13 signaling, downregulating IL13, neutralizing IL13 (e.g., by administering dupilumab), blocking IL13 receptors, administering IL13 antagonists, administering IL13 neutralizing antibodies, and administering IL13 receptor alpha (IL13Ra) neutralizing antibodies.

[0032] Considering the above, one approach to treat tumors and reduce the viability of cancer cells is to apply TT Fields to cancer cells and inhibit IL11 activity. This approach is expected to improve the efficacy of TT Fields treatment by improving both of the above problems. In an in vivo situation, inhibition of IL11 activity can be achieved by administering a therapeutically effective concentration of an IL11 inhibitor to the subject.

[0033] An alternative approach to treat tumors and reduce cancer cell viability is to apply TT Fields to cancer cells and generally inhibit fibrosis. This approach is expected to increase the efficacy of TT Field treatment by improving the first problem above (regardless of whether the second problem above is improved). More specifically, since fibrotic tissue is usually less conductive than non-fibrotic tissue, IL11-induced fibrosis will (gradually) decrease the tumor conductivity σ, which in turn will decrease the tumor power loss density by the operation of equation (1), thereby decreasing the efficacy of TT Fields. This deleterious sequence of events can be countered by using a different approach to inhibit IL11-induced fibrosis.

[0034] In some embodiments, this is accomplished using at least one of the following drugs: fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, and vismodegib (each of which includes a compound known to inhibit fibrosis). In other embodiments, this is accomplished using a calcium channel blocker known to inhibit fibrosis (e.g., felodipine, verapamil, diltiazem, and / or nifedipine). In other embodiments, this is accomplished using any of a variety of other compounds known to inhibit fibrosis.

[0035] In some embodiments, this can be accomplished using at least one compound that is both anti-tumor and anti-fibrotic (e.g., sorafenib or any suitable broad-spectrum receptor tyrosine kinase inhibitor (RTKI), such as sunitinib and imatinib). In this aspect, sorafenib can reduce fibrosis, reduce tumor size, and improve tumor response to TTFields treatment.

[0036] As with the embodiments described above based on inhibiting IL11 activity to inhibit fibrosis, the reduced level of fibrosis in these embodiments can keep the electrical conductivity σ high, can keep the power loss density high, and can keep the efficacy of the TTFields high.

[0037] In an in vivo context, inhibition of fibrosis can be achieved by administering to a subject a therapeutically effective concentration of a fibrosis inhibitor.

[0038] In an in vivo situation, administration of the relevant molecule or molecules (e.g., an IL11 inhibitor or a fibrosis inhibitor) to cancer cells may be performed continuously from a first time (t1) when the molecules are circulating in the patient's body (e.g., after systemic administration) or when introduced near the cancer cells, until a time (t2) when the molecules are cleared or exhausted from the patient's body. As a result, if a TT Field is applied to the cancer cells between t1 and t2, the applying step is performed simultaneously with at least a portion of the administering step. Administration of the molecules to the subject can be performed using any of a variety of approaches, including, but not limited to, intravenous, oral, subcutaneous, intrathecal, intramuscular, intracerebroventricular, and intraperitoneal. Additionally, application of an alternating electric field to the cancer cells can be performed using the Novocure Optune® system or variants thereof operating at different frequencies.

[0039] In some cases, fibrosis can be reduced using agents that are both anti-fibrotic and anti-tumor. For example, it is known that the combination of TTFields and sorafenib synergistically reduces tumor volume in hepatocellular carcinoma N1S1 cells. See Figure 1 in US Patent Application Publication No. 2020 / 0016067. As shown in Figure 1, the combination of TTFields and sorafenib results in a 10-fold or greater reduction in tumor volume.

[0040] The effect of TTFields alone and in combination with sorafenib was evaluated for its effect on fibrosis in N1S1 tumors. TTFields in combination with sorafenib significantly reduced fibrosis scores compared to TTFields alone, as assessed by a pathologist (Figures 3A-B). Tumor fibrotic area, as measured by percent area and fold change, showed a similar reduction in fibrosis (Figures 4-5).

[0041] The reduction in tumor fold change (Figure 1A) and tumor volume (Figure 1B) correlated with an increase in fibrosis score (Figures 6A-6B) in TTFields-treated tumors. The reduction in tumor fold change and tumor volume correlated with an increase in fibrotic area (Figures 6C-6D).

[0042] In summary, TTFields can be combined with anti-IL11 and / or additional anti-fibrotic agents and agents that can synergistically reduce tumor volume. In some cases, sorafenib can be used to reduce tumor volume and as an anti-fibrotic agent as described herein.

[0043] As used herein, the term "reducing the viability of cancer cells" refers to reducing the growth, proliferation, or survival of cancer cells. In some aspects, reducing the viability of cancer cells includes reducing the clonogenic survival of cancer cells, increasing cytotoxicity of cancer cells, inducing apoptosis in cancer cells, and reducing tumor volume in tumors formed from at least a portion of the cancer cells.

[0044] The term "clonogenic survival" refers to the ability of a single cancer cell to grow into a colony of cancer cells. In one embodiment, a "colony" is at least 50 cells.

[0045] The term "cytotoxicity" refers to the measure of the ability of a drug or treatment to kill cells.

[0046] The term "apoptosis" refers to a phenomenon also known as "programmed cell death," which refers to the death of a cell as part of the regulated cell cycle of cell growth and development.

[0047] As used herein, the term "therapeutically effective concentration" refers to the concentration of the relevant compound sufficient to achieve its intended purpose (eg, inhibiting IL11 activity, inhibiting fibrosis, etc.). EXAMPLES

[0048] Example 1 The TTFields (150 kHz) / sorafenib combination caused a significant reduction in tumor volume in vivo. As shown in Figures 1A and 1B and in US Patent Publication No. 2020 / 0016067 (incorporated herein by reference in its entirety), the combination of 150 kHz TT fields with sorafenib significantly reduced the fold increase in tumor volume in N1S1 hepatoma cells. Tumor volume was determined by MRI. Figures 1A and 1B show the results of the in vivo study described below for N1S1 tumor volume in Sprague Dawley rats treated with heat, TT fields alone, sorafenib alone, and the combination of TT fields and sorafenib. In this experiment, tumor volume was measured using MRI on day 6 (one day before treatment began) and day 14 (one day after treatment ended), with each data point in Figure 1A representing the tumor volume on day 14 divided by the tumor volume on day 6. Thus, a number greater than 1 represents an increase in tumor volume, and a number less than 1 represents a tumor that shrank between days 6 and 14. In this figure, "*" represents p<0.1, "**" represents p<0.01, and "****" represents p<0.0001. As shown in Figure 1, tumor volume was synergistically reduced after treatment with a combination of TTFields (2.86 V / cm) and sorafenib (10 mg / kg / day). Figure 1B shows the tumor volume.

[0049] Example 2 Fibrosis and TTFields treatment Eight-week-old male SD rats (Envigo Ltd, Israel) were anesthetized by IP injection of ketamine (75 mg / kg) and xylazine (10 mg / kg) (Figure 2). After the postural reflex was lost, the hair was removed from the epigastric region using a trimmer. The left lobe of the liver was surgically exposed, and 10 μl of a cell suspension containing 50,000 N1-S1 hepatocellular carcinoma cells in equal volumes of serum-free medium and Matrigel (CORNING, Bedford, MA) was injected directly into the left lobe using a syringe with a 31-gauge needle. The peritoneum and abdominal muscle incisions were closed using surgical sutures, and the skin was closed by clips. The rats were given 6 days to recover.

[0050] On day 6, all animals underwent a first MRI scan and were randomized into the following treatment groups: Group I-rats were treated with sham heating electrodes and vehicle injections. Group II-rats were treated with a 150 kHz TT field and vehicle injections. Group III-rats were treated with sorafenib 10 mg / kg / day. Group IV - rats were treated with 150 kHz TT fields and sorafenib 10 mg / kg / day.

[0051] On day 7, electrodes (TT-fields or sham heating) were placed on the animals. Animals were treated with TT-fields or sham heating for 6 days. Sorafenib or control vehicle was administered by IP injection on 5 of the 6 treatment days. All electrodes were removed on day 13, and animals underwent a second MRI on day 14 and were euthanized. Tumor volumes were determined based on MRI scans on days 7 and 14 after tumor implantation. The MRI system was a Bruker Icon system operating at 1 Tesla installed in the company's animal facility. Animals were anesthetized with isoflurane and placed prone in a rat body coil. After localizer images, T2-weighted coronal anatomical images were scanned using a RARE sequence and the following parameters: TR / TE 1900 / 51ms, number of slices 10, slice thickness 1mm, FOV 55-65mm, acquisition matrix 140, average 8, acquisition time 4min 18s. Tumor volumes were measured by manual segmentation of the tumors using ITK-SNAP version 3.6.0-rcl free software (t-test; *P<0.05, **P<0.01, and ***P<0.001) (Figure 1).

[0052] Tumors were harvested for histological assessment of fibrosis grade using a semiquantitative grading scale. Paraffin blocks were cut to a thickness of approximately 4 microns. Sections were placed on glass slides and stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT). Masson's trichrome stained sections were analyzed for fibrosis using a scoring grade scale and digital morphometry.

[0053] H&E stained sections were examined by a single pathologist and scored by a semiquantitative scoring system for the presence of pathological changes as follows (Figure 3 and Figure 3A): Grade 0: No signs of fibrosis Grade 1: Very mild signs of fibrosis Grade 2: Mild signs of fibrosis Grade 3: Moderate signs of fibrosis Grade 4: Severe fibrosis Grade 5: Very severe fibrosis

[0054] Color segmentation of the blue stained areas (MT: fibrosis) was performed using MATLAB color-based segmentation using the L*a*b* color space, as shown in Figure 3. The L*a*b* color space is derived from the CIE XYZ tristimulus values. The L*a*b* space consists of a lightness "L*" or luminance layer, a chromaticity layer "a*" that indicates where the color falls along the red-green axis, and a chromaticity layer "b*" that indicates where the color falls along the blue-yellow axis.

[0055] Example 3 Morphometric measurements (fold change in fibrotic area, percent fibrotic area) As shown in Figure 4, the percentage of the stained area (Figure 3: top right) out of the total tissue (Figure 3: top right + bottom left) was calculated.

[0056] Morphometry of percent fibrosis shows the highest fibrotic area in tumors from rats receiving TTFields alone (34% increase compared to control) and the lowest fibrotic area in tumors from rats receiving sorafenib (anti-fibrotic) alone (30.4% decrease compared to control).

[0057] Combined TTFields and sorafenib treatment resulted in a 20.8% reduction in fibrotic area compared to the control group. These results are summarized in "Fibrosis area (fold change)" (Figure 5). The percentage change in fibrosis area is summarized in Figure 4.

[0058] Example 4 Correlation of response to TTFields measured as reduction in tumor volume and increase in fibrosis 6A-6D show correlations between (1) fibrosis score and tumor volume in tumors treated with TTFields (FIG. 6A), (2) fibrosis score and tumor fold change in tumors treated with TTFields (FIG. 6B), (3) fibrosis area and tumor fold change in tumors treated with TTFields (FIG. 6C), and (4) fibrosis area and tumor volume in tumors treated with TTFields (FIG. 6D). Without being bound to a particular theory, it is believed that the reduction in fibrosis associated with TTFields treatment renders tumors more susceptible to treatment with TTFields alone and in combination with IL11 inhibitors, fibrosis inhibitors, or anti-tumorigenic factors / fibrosis inhibitors (e.g., sorafenib).

[0059] Although the present invention has been disclosed with reference to specific embodiments, numerous modifications, changes, and variations to the described embodiments are possible without departing from the field and scope of the invention, as defined in the appended claims. Accordingly, the present invention is not limited to the described embodiments, but rather has full scope defined by the language of the following claims and their equivalents.

Claims

1. 1. A pharmaceutical composition comprising at least one calcium channel blocker for use in a method of treating cancer in a subject, said method comprising: (1) applying an alternating electric field to a target area of ​​a subject for a period of time; and (2) administering the pharmaceutical composition to a subject 13. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition of claim 1, wherein the frequency of the alternating electric field is between 100 and 500 kHz.

3. The pharmaceutical composition according to claim 1, wherein at least a portion of step (1) is carried out simultaneously with at least a portion of step (2).

4. The pharmaceutical composition of claim 1, wherein the subject has at least one of glioblastoma and hepatocellular carcinoma.

5. 2. The pharmaceutical composition of claim 1, wherein the calcium channel blocker is selected from felodipine, verapamil, diltiazem, nifedipine, and combinations thereof.

6. The method further comprising: (3) administering to the subject a therapeutically effective concentration of at least one additional composition; 2. The pharmaceutical composition of claim 1, wherein the at least one additional composition comprises at least one component selected from the group consisting of sorafenib, sunitinib, imatinib, fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, vismodegib, and combinations thereof.

7. 1. A pharmaceutical composition for use in a method of reducing tumor volume in a living subject, comprising at least one calcium channel blocker, said method comprising: (1) applying an alternating electric field to the tumor for a period of time; and (2) administering the pharmaceutical composition to a subject 13. A pharmaceutical composition comprising:

8. 8. The pharmaceutical composition of claim 7, wherein the frequency of the alternating electric field is between 100 and 500 kHz.

9. The pharmaceutical composition according to claim 7, wherein at least a portion of step (1) is carried out simultaneously with at least a portion of step (2).

10. The pharmaceutical composition of claim 7, wherein the subject has at least one of glioblastoma and hepatocarcinoma.

11. 8. The pharmaceutical composition of claim 7, wherein the calcium channel blocker is selected from felodipine, verapamil, diltiazem, nifedipine, and combinations thereof.

12. The method further comprising: (3) administering to the subject a therapeutically effective concentration of at least one additional composition; 8. The pharmaceutical composition of claim 7, wherein the at least one additional composition comprises at least one component selected from the group consisting of sorafenib, sunitinib, imatinib, fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, vismodegib, and combinations thereof.

13. The pharmaceutical composition of claim 7, wherein the tumor volume of the tumor is reduced by about 5-fold or more.

14. 1. A pharmaceutical composition for use in a method of reducing the viability of cancer cells in a living subject, comprising at least one calcium channel blocker, said method comprising: (1) applying an alternating electric field to the cancer cells for a certain period of time; and (2) administering the pharmaceutical composition to a subject 13. A pharmaceutical composition comprising:

15. 15. The pharmaceutical composition of claim 14, wherein the frequency of the alternating electric field is between 100 and 500 kHz.

16. The pharmaceutical composition according to claim 14, wherein at least a portion of step (1) is carried out simultaneously with at least a portion of step (2).

17. The pharmaceutical composition of claim 14, wherein the subject has at least one of glioblastoma and hepatocellular carcinoma.

18. 15. The pharmaceutical composition of claim 14, wherein the calcium channel blocker is selected from felodipine, verapamil, diltiazem, nifedipine, and combinations thereof.

19. The method further comprising: (3) administering to the subject a therapeutically effective concentration of at least one additional composition; 15. The pharmaceutical composition of claim 14, wherein the at least one additional composition comprises at least one component selected from the group consisting of sorafenib, sunitinib, imatinib, fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, vismodegib, and combinations thereof.

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