Increased sensitivity of cancer cells to tumor treatment fields (TTFELDS) by inhibiting fibrosis with a calcium channel blocker

Description

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a fibrosis inhibitor for use in a method of treating a tumor disposed in a body of a living subject, wherein the tumor is non-small cell lung cancer and the fibrosis inhibitor comprises a calcium channel blocker, in accordance with the appended claims.BACKGROUND

[0002] Tumor Treating Fields, or TTFields, are low intensity (e.g., 1-3 V / cm) alternating electric fields within the intermediate frequency range (e.g., 100-500 kHz) that inhibit cancer cell growth. This non-invasive treatment targets solid tumors and is described in US Patent 7,565,205, which is incorporated herein by reference in its entirety. TTFields are FDA approved for the treatment of glioblastoma (GBM), and may be delivered, for example, via the Optune ™< system. Optune ™< includes a 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 positioned to the left and right of the tumor, and the other pair of electrodes is positioned anterior and posterior to the tumor. In the preclinical setting, TTFields can be applied in vitro using, for example, the Inovitro ™< TTFields lab bench system.

[0003] E. Neuhaus et al., in 'Alternating Electric Fields (TTFields) Activate Cav1.2 Channels in Human Glioblastoma Cells' (2019), investigates the effects of TTFields on Ca2+-signaling, ion channel activity in the plasma membrane, cell cycle, cell death, and clonogenic survival, tested in two human glioblastoma cell lines in vitro by fura-2 Ca2+ imaging, patch-clamp cell-attached recordings, flow cytometry and pre-plated colony formation assay. E. Neuhaus et al. found that inhibition of Cav1.2 by benidipine aggravated, in one glioblastoma line, the TTFields effects and concluded that this suggests that Cav1.2-triggered signaling contributes to cellular TTFields stress response.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figures 1A-1B show the results of an exemplary study demonstrating the reduction in fold increase of tumor volume and tumor volume, respectively, resulting from the combination of TTFields at 150 kHz and sorafenib in N1S1 liver carcinoma cells; Figure 2 shows an exemplary TTFields treatment and dosing regimen for four treatment conditions (I, II, II, and IV) to evaluate fibrosis; Figure 3A shows an exemplary histopathological evaluation of the fibrosis specific mason trichrome (MT) stain (blue) of treated N1S1 liver carcinoma tumors showing (1) the original image (used for fibrosis score) (upper left), (2) Color segmentation of fibrosis in MT stained sections with the ROI- blue color representing fibrosis (for area calculation), (3) whole tissue without fibrosis (for area calculation, red- eosin, lower left); and (4) areas outside the tissue (lower right); Figure 3B shows the fibrosis score for the experiment described with respect to Figure 3A; Figure 4 shows the fibrosis area (%) for each condition calculated from the experiment described with respect to Figure 3A; Figure 5 shows the fibrosis area represented as fold change for each condition as calculated from the experiment described above with respect to Figure 3A; and Figures 6A-6D show a correlation between (1) the fibrosis score and tumor volume in TTFields treated tumors (Figure 6A), (2) the fibrosis score and tumor fold change in TTFields treated tumors (Figure 6B), (3) the fibrosis area and tumor fold change in TTFields treated tumors (Figure 6C), and (4) the fibrosis area and tumor volume in TTFields treated tumors (Figure 6D).

[0005] Results shown by the figures relate to reference examples not according to the claimed invention, but which are useful for understanding aspects of the invention.SUMMARY OF THE INVENTION

[0006] The references to methods for treatment of the human or animal body by surgery or therapy in this description are to be interpreted as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in such methods. The claimed invention is directed to a fibrosis inhibitor for use in a method of treating a tumor disposed in a body of a living subject, wherein the tumor is non-small cell lung cancer, the method comprising: applying an alternating electric field to the tumor, the alternating electric field having a frequency between 100 and 500 kHz; and administering a therapeutically effective concentration of the fibrosis inhibitor to the subject, wherein the fibrosis inhibitor comprises a calcium channel blocker.DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Ordinarily, most tumors have higher electrical conductivity than healthy surrounding tissue. And this elevated electrical conductivity may explain the specificity of TTFields towards malignant tissue. More specifically, the power loss density of an electric field, L , is defined as L = 1 2 σ E 2 where σ is the conductivity of tissue and |E| is the intensity of the electric field. (Power loss density is measured in units of milliwatts per cubic centimeter). And the elevated conductivity σ within the tumor tissue gives rise to a higher power loss density within that specific tissue.

[0008] Clinical data demonstrates a significant increase in IL11 (interleukin-11) expression levels in tumors from GBM patients treated with TTFields. The data was obtained as follows. GBM tumor samples were obtained before and after a treatment according to standard chemoradiation protocol (six patients) or a protocol combining TTFields with standard chemoradiation (six patients). Gene expression analysis was performed by RNA-seq. The negative binomial generalized linear model was used to analyze expression following treatment and the differential effects of control and TTFields treatments. Raw data were analyzed using DESeq2 software. Differential gene expression analysis was calculated using the reads per kilobase million (RPKM) values of chemoradiation and TTFields treatment groups. Statistical analysis was performed using the negative binomial generalized linear model. The difference between expression before and after treatment was derived separately for each individual and the average net treatment effect was calculated for each treatment group. The difference between treatment effects was represented as fold change between the average net effects of TTFields and control. The Benjamini-Hochberg method was used to correct fold change p values for multiple comparisons. A list of 712 genes related to immune activity was compiled using the general literature, the Nanostring "nCounter ®< PanCancer immune profiling panel" and the ThermoFischer "Oncomine ™< immune response" gene lists.

[0009] A significant difference between TTFields and control treatment effects was defined as fold change >2 or <0.5 with a corrected p value <0.1. The table below presents the genes that exhibited significantly altered gene expression. GeneFold changeF.D.R P-val'IL118.50.001GPR185.90.071CD7050.079CCL264.50.031NKG2 D4.20.034T-bet3.40.019ICOS-L3.30.094ISG153.20.035Fac. XII3.10.032HGF30.041GNLY2.60.083MMP22.50.036

[0010] Notably, the differential effects of TTFields+chemoradiation treatments on expression of IL11 (as compared to the chemoradiation-only control) was an 8.5 fold increase. Without being bound by the theory of operation set forth below, this increase in IL11 can cause two problematic issues.

[0011] The first problematic issue is that IL11 is known to be a fibrotic factor. As a result, the increase in IL11 associated with the application of TTFields may produce extracellular matrix remodeling and fibrosis within the tumor tissue. Because fibrosis leads to decreased intra- and extra-cellular fluids, fibrotic tissue typically has lower conductivity than non-fibrotic tissue. Therefore, fibrosis that is promoted by the IL11 should (over time) decrease the conductivity σ in the tumor, which should in turn lower the power loss density in the tumor by operation of equation (1), thereby decreasing the effectiveness of the TTFields. This adverse chain of events can be interrupted by inhibiting IL11 activity. More specifically, inhibiting IL11 activity reduces fibrosis, which allows the conductivity σ to remain high, which allows the power loss density to remain high, which allows the effectiveness of the TTFields to remain high.

[0012] The expected effectiveness of interrupting the aforementioned adverse chain of events is supported by experiments establishing that inhibiting IL11 has been shown to prevent heart and kidney fibrosis.

[0013] The second problematic issue is that IL11 expression in tumors is associated with an aggressive phenotype and poor prognosis in a large spectrum of tumors including hepatocellular carcinoma, pancreatic cancer, gastric adenocarcinoma, non-small cell lung cancer, breast cancer, endometrial carcinoma, and chondrosarcoma.

[0014] Both of the problematic issues noted above can be ameliorated by counteracting the increase in IL11 that occurs when tumors are exposed to TTFields (e.g., by inhibiting IL11 activity).

[0015] Examples of some suitable approaches for inhibiting IL11 activity include: decreasing IL11 expression, inhibiting IL11 signaling (e.g., by administering Bazedoxifene), downregulating IL11, neutralizing IL11, blocking an IL11 receptor, administering an IL11 antagonist (e.g., W147A), administering an IL11 neutralizing antibody (e.g., ENx108A, ENx203, or ENx209), and administering an IL11 receptor α (IL11Ra) neutralizing antibody.

[0016] Inhibiting IL 11 activity may also be accomplished indirectly by inhibiting a stimulator of IL11 or by inhibiting a stimulator of IL11 receptor α (IL11Ra). For example, because IL13 is a potent stimulator of IL11 and IL11Ra, additional examples of suitable approaches for inhibiting IL11 activity include: decreasing IL13 expression, inhibiting IL13 signaling, downregulating IL13, neutralizing IL13 (e.g., by administering Dupilumab), blocking an IL13 receptor, administering an IL13 antagonist, administering an IL13 neutralizing antibody, and administering an IL13 receptor α (IL13Ra) neutralizing antibody.

[0017] In view of the above, one approach for treating tumors and reducing viability of cancer cells, which approach is not defined by the appended claims, is to apply TTFields to the cancer cells, and to inhibit IL11 activity. This approach is expected to increase the efficacy of the TTFields treatment by ameliorating both of the problematic issues noted above. In the in vivo context, the inhibition of IL11 activity may be accomplished by administering a therapeutically effective concentration of an IL11 inhibitor to the subject.

[0018] An alternative approach for treating tumors and reducing viability of cancer cells, in accordance with the present invention, is to apply TTFields to the cancer cells, and generically inhibit fibrosis. This approach is expected to increase the efficacy of the TTFields treatment by ameliorating the first problematic issue noted above (without regard to whether the second problematic issue noted above is ameliorated). More specifically, because fibrotic tissue typically has lower conductivity than non-fibrotic tissue, the fibrosis that is triggered by the IL11 should (over time) decrease the conductivity σ in the tumor, which should in turn lower the power loss density in the tumor by operation of equation (1), thereby decreasing the effectiveness of the TTFields. This adverse chain of events can be counteracted by using a different approach for inhibiting the fibrosis that is triggered by the IL11.

[0019] In some embodiments not defined by the appended claims, this is accomplished using at least one of the following drugs: fasudil, pirfenidone, nintedanib, losartan, hyaluronidase, tranilast, and vismodegib, (each of which comprises a compound known to inhibit fibrosis). In accordance with the present invention, this is accomplished using a calcium channel blocker that is known to inhibit fibrosis (e.g., Felodipine, Verapamil, Diltiazem, and / or Nifedipine). In other embodiments not defined by the appended claims, this is accomplished using any of a variety of other compounds that are known to inhibit fibrosis.

[0020] In some embodiments, this can be accomplished using at least one compound that is both anti-tumorigenic and anti-fibrotic (e.g., in embodiments not defined by the appended claims, sorafenib or any suitable broad spectrum receptor tyrosine kinase inhibitors (RTKI) such as sunitinib and imatinib). In this aspect, sorafenib can decrease fibrosis and decrease tumor size, improving the tumor response to TTFields treatment.

[0021] As in the embodiments described above that rely on inhibiting IL11 activity to inhibit fibrosis, the reduction in the level of fibrosis in these embodiments will allow the conductivity σ to remain high, which allows the power loss density to remain high, which allows the effectiveness of the TTFields to remain high.

[0022] In the in vivo context, the inhibition of fibrosis may be accomplished by administering a therapeutically effective concentration of a fibrosis inhibitor to the subject.

[0023] In the in vivo context, the administering of the relevant molecule or molecules (e.g., an IL11 inhibitor or a fibrosis inhibitor) to the cancer cells can occur continuously from a first time (t 1 ) when the molecule(s) are circulating in the patient's body (e.g., after administering it systemically) or introduced into the vicinity of the cancer cells until such time (t 2 ) as the molecule(s) are eliminated from the patient's body or exhausted. As a result, if TTFields are applied to the cancer cells between t 1 and t 2 , the applying step will be simultaneous with at least a portion of the administering step. The administration of the molecule(s) to the subject may be performed using any of a variety of approaches including but not limited to intravenously, orally, subcutaneously, intrathecal, intramuscularly, intraventricularly, and intraperitonealy. And the application of the alternating electric field to the cancer cells may be performed using the Novocure Optune ®< system or a variant thereof that operates at a different frequency.

[0024] In some instances, fibrosis can be reduced using factors that are both anti-fibrotic and anti-tumorigenic. For example, it is known that the combination of TTFields and sorafenib synergistically reduces tumor volume in hepatocellular carcinoma N1S1 cells. See US Patent Application Publication 2020 / 0016067; Figure 1. As shown in Figure 1, the combination of TTFields and sorafenib, which is not defined by the appended claims, results in at least a 10 fold decrease in tumor volume.

[0025] The effects of TTFields alone and in combination with sorafenib were evaluated with respect to their impact on fibrosis in N1S1 tumors. TTFields in combination with sorafenib significantly reduced the fibrosis score relative to TTFields alone as evaluated by a pathologist. Figures 3A-3B. The fibrosis area of the tumors was measured by percent area and fold change and showed a similar reduction in Fibrosis. Figures 4-5.

[0026] The reduction in tumor fold change (Figure 1A) and tumor volume (Figure 1B) correlated with the increase in fibrosis score (Figures 6A-6B) in TTFields treated tumors. The reduction in tumor fold change and tumor volume correlated with fibrosis area increase (Figures 6C-6D).

[0027] Taken together, TTFields can be combined with anti-IL11 and / or additional anti-fibrotic agents and agents that can reduce tumor volume synergistically. In some instances not defined by the appended claims, sorafenib can be used to reduce tumor volume and also as an anti-fibrotic agent as described herein.

[0028] The term "reducing viability of cancer cells" as used herein, refers to reducing the growth, proliferation, or survival of the cancer cells. In some aspects, the reduction in viability of the cancer cells comprises reducing clonogenic survival of the cancer cells, increasing cytotoxicity of the cancer cells, inducing apoptosis in the cancer cells, and decreasing tumor volume in a tumor formed from at least a portion of the cancer cells.

[0029] The term "clonogenic survival" refers to the ability of a single cancer cell to grow into a colony of cancer cells. In one aspect, a "colony" is at least fifty cells.

[0030] The term "cytotoxicity" refers to a measure of the ability of drug or treatment to kill a cell.

[0031] The term "apoptosis" refers to the phenomena termed "programmed cell death" referring to the death of cell as part of the controlled cell cycle of cell growth and development.

[0032] The term "therapeutically effective concentration," as used herein, refers to the concentration of the relevant compound sufficient to achieve its intended purpose (e.g., inhibiting IL 11 activity, inhibiting fibrosis, etc.).EXAMPLES

[0033] The following examples relate to reference examples not according to the claimed invention, but which are useful for understanding aspects of the invention.Example 1 - TTFields (150 kHz) / sorafenib combination led to a significant reduction in tumor volume in vivo

[0034] As shown in Figures 1A and 1B and in US Patent Application Publication 2020 / 0016067 (hereby incorporated by reference in its entirety), the combination of TTFields at 150 kHz and sorafenib significantly reduced the fold increase in tumor volume in N1S1 liver carcinoma cells. Tumor volume was determined by MRI. Figures 1A and 1B shows the results of an in vivo study described below with respect to N1S1 tumor volume in Sprague Dawley rats treated with heat, TTFields alone, sorafenib alone, and the combination of TTFields and sorafenib. In this experiment, the volume of the tumor was measured using MRI on day 6 (one day before treatment start) and day 14 (one day after the end of treatment); and each data point in Figure 1A represents the volume of the tumor on day 14 divided by the volume of the tumor on day 6. Thus, numbers greater than 1 represent growth in the tumor volume, and numbers smaller than 1 represent a tumor that has shrunk between day 6 and day 14. In this figure,"*" represents p < 0.1, "**" represents p < 0.01, and "****" represents p < 0.0001. As shown in Figure 1, there is a synergistic decrease in tumor volume following treatment with a combination of TTFields (2.86 V / cm) and sorafenib (10mg / kg / day). Figure 1B represents the volume of the tumor.Example 2 - Fibrosis and TTFields Treatment

[0035] Male SD rats, 8-weeks of age (Envigo Ltd, Israel), were anesthetized by I.P. injection of Ketamine (75 mg / kg) and Xylazine (10 mg / kg)(Figure 2). After loss of postural reflexes, the fur was removed from the upper abdominal area using a trimmer. The left lobe of the liver was surgically exposed and 10µl of cell suspension containing 50,000 N1-S1 hepatocellular carcinoma cells in equal volumes of serum free medium and Matrigel (CORNING, Bedford, MA) were injected directly into the left lobe using a syringe with a 31-gauge needle. The peritoneum and abdominal muscles incisions were closed using surgical sutures and the skin were close by clips. The rats were allowed to recover for 6 days.

[0036] On day 6 all animals underwent a first MRI scan and were randomly divided into the following treatment groups: Group I - rats were treated with sham heating electrodes and vehicle injection. Group II - rats were treated with 150 kHz TTFields and vehicle injection. Group III - rats were treated with 10 mg / kg / day Sorafenib. Group IV - rats were treated with 150 kHz TTFields and 10 mg / kg / day Sorafenib.

[0037] On day 7, electrodes (TTFields or sham heating) were placed on the animals .The animals were treated for 6 days with TTFields or sham heat. Sorafenib or the control vehicle were administered through IP injection on 5 out of 6 days of treatment. On day 13 all electrodes were removed, and the animals underwent a second MRI on day 14 and euthanized. Tumor volume were determined based on MRI scans on days 7and 14 after tumor implantation. The MRI system is a Bruker Icon system working at 1 Tesla placed inside the animal facility of the company. Animals were anesthetized with isoflurane and placed prone in a rat body coil. After a localizer image, a T2 weighted coronal anatomical image were scanned with a RARE sequence and the following parameters: TR / TE 1900 / 51 ms, number of slices 10, slice thickness 1mm, FOV 55-65mm, acquisition matrix 140, 8 averages, acquisition time 4 m 18 s. Tumor volume was measured by manual segmentation of the tumor using ITK-SNAP version 3.6.0-rc1 free software. (t-test; * P<0.05, ** P<0.01, and *** P<0.001)(Figure 1).

[0038] Tumors were harvested for histological evaluation of the grade of fibrosis using a semi-quantitative evaluation scale. Paraffin blocks were sectioned at approximately 4 microns thickness. The sections were put on glass slides and stained with Hematoxylin & Eosin (H&E) and Masson Trichrome (MT). Masson Trichrome stained sections were analyzed for fibrosis, using a scoring grade scale and digital morphometry.

[0039] H&E stained sections were examined by one pathologist and scored by a semi-quantitative scoring system for the presence of pathological changes (Figures 3 and 3A), as follows: 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: High grade of fibrosis Grade 5: Very high grade of fibrosis

[0040] Color segmentation of blue stained areas (MT: fibrosis) was performed, as demonstrated in Figure 3, with MATLAB Color-Based Segmentation, using the L*a*b* Color Space. The L*a*b* color space is derived from the CIE XYZ tri-stimulus values. The L*a*b* space consists of a luminosity 'L*' or brightness layer, chromaticity layer 'a*' indicating where color falls along the red-green axis, and chromaticity layer 'b*' indicating where the color falls along the blue-yellow axis.Example 3 - Morphometry (Fibrosis area fold change, Fibrosis area percent)

[0041] The percent of the stained areas (Figure 3: top-right) out of the whole tissue (Figure3: top-right + bottom-left) was calculated as shown in Figure 4.

[0042] Morphometry of fibrosis percent shows highest fibrotic area in tumors from rats that received TTFields alone (34% increase relative to control), and lowest fibrotic area in tumors from rats that received sorafenib (anti-fibrotic) alone (30.4% decrease relative to control).

[0043] The combined treatment of TTFields and sorafenib resulted in 20.8% reduction in fibrotic area compared to control group. These results are summarized in "Fibrosis area (fold change)" (Figure 5). The percent change in fibrosis area is summarized in Figure 4.Example 4 - Correlation of Response to TTFields Measured as Tumor Volume Reduction and Increase in Fibrosis

[0044] Figures 6A-6D show a correlation between (1) the fibrosis score and tumor volume in TTFields treated tumors (Figure 6A), (2) the fibrosis score and tumor fold change in TTFields treated tumors (Figure 6B), (3) the fibrosis area and tumor fold change in TTFields treated tumors (Figure 6C), and (4) the fibrosis area and tumor volume in TTFields treated tumors (Figure 6D). Without being bound by theory, it is believed that reducing fibrosis associated with TTFields treatment makes the tumor more susceptible to treatment with TTFields alone, and in combination with an IL11 inhibitor, fibrosis inhibitor, or an anti-tumorigenic factor / fibrosis inhibitor (e.g., sorafenib).

[0045] While the present invention has been disclosed with reference to certain embodiments, numerous modifications, alterations, and changes to the described embodiments are possible without departing from the scope of the present invention, as defined in the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but that it has the full scope of the appended claims.

Claims

1. A fibrosis inhibitor for use in a method of treating a tumor disposed in a body of a living subject, wherein the tumor is non-small cell lung cancer, the method comprising: applying an alternating electric field to the tumor, the alternating electric field having a frequency between 100 and 500 kHz; and administering a therapeutically effective concentration of the fibrosis inhibitor to the subject, wherein the fibrosis inhibitor comprises a calcium channel blocker.

2. The fibrosis inhibitor for use of claim 1, wherein the frequency of the alternating electric field is between 180 and 220 kHz.

3. The fibrosis inhibitor for use of claim 1 or claim 2, wherein at least a portion of the applying step is performed simultaneously with at least a portion of the administering step.

4. The fibrosis inhibitor for use of any preceding claim, wherein the calcium channel blocker comprises at least one of felodipine, verapamil, diltiazem, and nifedipine.