Combination Treatments for Cancer

Abstract

A combination of a voltage-gated sodium channel (VGSC) blocker with at least one other substance that directly or indirectly modulates ionic mechanisms, such as a potassium channel opener, a sodium entry inhibitor, or an upstream down-regulator of VGSC expression, for treating or preventing cancer, including reducing, preventing, or inhibiting cancer metastasis and / or invasive behavior.

Description

[Technical Field]

[0001] The present invention relates to cancer therapy, particularly using a combination of a voltage-gated sodium channel (VGSC) blocker and at least one second agent, such as a potassium channel opener, another sodium entry inhibitor and / or an upstream down-regulator of VGSC expression. [Background technology]

[0002] Voltage-gated sodium channels (VGSCs) are integral membrane proteins containing pore-forming alpha subunits and smaller non-pore-forming beta subunits that transport sodium ions (Na ) across the plasma membrane of cells. + In humans, there are nine different VGSC alpha subunits or "Nav" proteins (Nav1.1 to Nav1.9), encoded by the genes SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A, and SCN11A, respectively. The VGSC alpha subunits are transmembrane proteins with four repeating transmembrane domains (designated DI, DII, DIII, and DIV), each of which contains six transmembrane spanning segments designated S1 to S6. Na + Ions flow through the pore formed by the S5 and S6 regions of all four domains. The S4 region plays a central role in voltage sensing. The smaller beta subunit contains an N-terminal extracellular immunoglobulin (Ig) loop, a transmembrane domain, and an intracellular domain.

[0003] VGSCs are expressed in metastatic cells of many cancers and enhance invasive and metastatic behavior (Brackenbury, 2012; Roger et al., 2015; Djamgoz et al., 2009; Diss et al., 2001, 2005). In some cancers, the predominant VGSC expressed is a neonatal splice variant of VGSC.

[0004] For example, more than 10 different splice isoforms of SCN5A (Nav1.5) have been described, some of which possess distinct functional properties, and different isoforms are expressed in neonatal and adulthood. The neonatal and adult forms of the Nav1.5 protein are the result of alternative splicing of exon 6, resulting in several amino acid differences in the DI:S3-S4 region of the channel protein (Figure 1). In particular, at position 211, a conserved aspartic acid (Asp or D) residue (negatively charged) in adult Nav1.5 is converted to a lysine (Lys or K) residue (positively charged) in neonatal Nav1.5.

[0005] Human Nav1.7 is encoded by the gene SCN9A. The neonatal form of Nav1.7 (nNav1.7) is a splice variant of exon 6 and is similar to nNav1.5, resulting in a conversion of the aspartic acid (Asp or D) conserved in all VGSCs (Nav1.1 to Nav1.9) to asparagine (Asn or N) at the amino acid 211 aligning with human Nav1.5.

[0006] Human Nav1.6 is encoded by the gene SCN8A. The neonatal form is spliced ​​similarly to Nav1.7 (i.e., a D to N substitution at the residue aligning with residue 211 of Nav1.5).

[0007] WO2012 / 049440 (Celex Oncology Ltd.) relates to a method for inhibiting or reducing metastatic behavior in cancer by administering ranolazine or riluzole.

[0008] WO2018 / 146313 (Celex GmbH) relates to a method for inhibiting or reducing metastatic behavior in cancer by administering eleclazine. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2012 / 049440 [Patent Document 2] WO2018 / 146313 [Non-patent literature]

[0010] [Non-Patent Document 1] Antzelevitch C et al. Circulation (2004);110:904~910 [Non-patent document 2] Abadjian et al., Adv Exp Med Biol. 2017;1036:229-257 [Non-patent document 3] Wilson and Hay, Nature Reviews Cancer 2011;11:393-410 Summary of the Invention [Problem to be solved by the invention]

[0011] However, there remains a need for improved methods of treating cancer, including methods of preventing, reducing or inhibiting the metastasis and / or invasive behavior of tumor cells. [Means for solving the problem]

[0012] The present inventors have discovered that combinations of VGSC blockers, particularly VGSC sustained current blockers, with a variety of other substances that directly or indirectly modulate ionic mechanisms, including potassium channel openers, other sodium entry inhibitors, and upstream down-regulators of VGSC expression, can be used to prevent or treat cancer. In particular, metastasis and / or invasive behavior of cancer or tumor cells may be prevented, reduced, or inhibited. Synergistic combinations are particularly preferred.

[0013] Thus, in a first aspect, the present invention relates to such a combination for use in a method of treating or preventing cancer in a subject, the combination comprising a first substance which is a sustained current blocker of a VGSC, and at least one second substance selected from, or a combination of, a potassium channel opener, a sodium entry inhibitor and an upstream regulator of VGSC expression.

[0014] In a second aspect, the present invention relates to a kit-of-parts comprising a first agent and at least one second agent for use in a method of treating or preventing cancer in a subject.

[0015] In a third aspect, the present invention relates to a method of treating or preventing cancer in a subject, comprising administering to the subject a first agent and at least one second agent.

[0016] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising as active ingredients a first substance and at least one second substance in admixture with a pharmaceutically acceptable carrier, diluent, vehicle and / or excipient.

[0017] The invention is more particularly described in the appended claims, and further details are provided below. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows alignments of nNav1.5 and aNav1.5 (SEQ ID NOs: 1 and 2, respectively), nNav1.6 and aNav1.6 (SEQ ID NOs: 3 and 4, respectively), and nNav1.7 and aNav1.7 (SEQ ID NOs: 5 and 6, respectively). Residues in the "neonatal" portion that differ from the corresponding positions in the "adult" portion are underlined. [Figure 2] Figure 1 shows the effect of ranolazine on the transient ("peak") component of VGSC currents (human highly metastatic MDA-MB-231 cells). At the concentrations used in this example (<<10 μM), there is little effect on the transient current. [Figure 3]

[0023] Figure 1 shows the effects of tetrodotoxin (TTX), ranolazine (5 μM), amiloride (100 μM), and the combination of ranolazine and amiloride on cell invasiveness under hypoxia (1% O2). A synergistic effect was observed in the combination. [Figure 4] Figure 1 shows the effect of DMSO, minoxidil (50 μM), or the combination of minoxidil (Min) and ranolazine (Ran) (5 μM) on cell invasiveness under hypoxia (1% O2). A synergistic effect was observed in the combination. [Figure 5] Figure 1 shows the effects of ranolazine, minoxidil, and drug combination treatment on invasion under hypoxia (human highly metastatic MDA-MB-231 cells). Box plots show the effect of different concentrations of treatment. (a) (i) 5 μM ranolazine, 50 μM minoxidil, and their combination, and (ii) 5 μM ranolazine, 5 μM minoxidil, and their combination (n=3 each). (b) 2.5 μM ranolazine and 2.5 μM minoxidil. Box plots show median, interquartile range; 5% and 95% confidence intervals, and outliers. ***=p<0.001. [Figure 6] Figure 1 shows the effects of a range of ranolazine (RAN), minoxidil (MIN) concentrations, and drug combination treatments on invasion under hypoxia (human highly metastatic MDA-MB-231 cells). Box plots show the effects of (a) 2.5 μM ranolazine, 2.5 μM minoxidil, and the combination treatment (n=3); (b) 1.25 μM ranolazine and 2.5 μM minoxidil, and the combination treatment (n=6); and (c) 0.625 μM ranolazine and 2.5 μM minoxidil, and the combination treatment (n=4). Box plots are shown as median, interquartile range; 5% and 95% confidence intervals, and outliers. In all cases, the effect of the combination was significantly greater than that of RAN alone (***=p<0.001). [Figure 7]Figure 1 shows the effects of ranolazine, minoxidil, and drug combination treatment on invasion under hypoxia (a) and normoxia (b) (human highly metastatic MDA-MB-231 cells). Box plots show the effects of 2.5 μM ranolazine, 2.5 μM minoxidil, and their combination on cell invasiveness (n=3 each). Box plots show median, interquartile range; 5% and 95% confidence intervals, and outliers. The results show that ranolazine is only effective under hypoxia, and the combination is significantly more effective than ranolazine alone (***=p<0.001). [Figure 8] Figure 1 shows the effect of ranolazine, minoxidil, and their combination on the invasiveness of moderately metastatic human breast cancer MDA-MB-468 cells under hypoxia. Box plots show the effect of 2.5 μM ranolazine, 2.5 μM minoxidil, and their combination on cell invasiveness under hypoxia (n=4). Box plots show median, interquartile range; 5% and 95% confidence intervals, and outliers. **=p<0.01; ***=p<0.001. [Figure 9] This figure shows the effects of ranolazine, minoxidil, and drug combination treatment on nNav1.5 protein expression under hypoxia (human highly metastatic MDA-MB-231 cells). Expression was quantified as immunofluorescence in arbitrary units (AU). Box plots show the effects of 5 μM ranolazine, 50 μM minoxidil, and drug combination treatment on nNav1.5 protein expression in MDA-MB-231 cells under (a) non-permeabilized or (b) permeabilized conditions (n ​​= 134–299 cells per condition). Box plots show median, interquartile range; 5% and 95% confidence intervals, and outliers. Ranolazine (i) reduced immunofluorescence in both conditions, and the combination (ii) was more effective than ranolazine alone, but only in non-permeabilized cells. [Figure 10] FIG. 1 shows the effects of 0.1% DMSO (control), TTX (10 μM), AG1478 (10 μM) and TTX + AG1478 on cell invasion (human highly metastatic MDA-MB-231 cells). [Figure 11]Figure 1 shows the effect of acute application of 50 μM minoxidil on VGSC activity in MDA-MB-231 cells. (a) Effect of 50 μM minoxidil on peak current. (b) Current-voltage curves for control conditions and 50 μM minoxidil (n=8 and n=6, respectively). (c) Steady-state inactivation for control cells and 50 μM minoxidil (n=7 and n=5, respectively). (d) Recovery from inactivation for control cells and 50 μM minoxidil (n=7 and n=5, respectively). Insets are shown in parts b-d. Data are presented as mean ± standard error of the mean. [Figure 12] Figure 1 shows the effect of chronic (48 h) treatment with 50 μM minoxidil under hypoxia on VGSC activity in MDA-MB-231 cells. (a) Current-voltage curves for control conditions and 50 μM minoxidil (n=7 and n=8, respectively). (b) Steady-state inactivation for control cells and 50 μM minoxidil (n=7 and n=8, respectively). (c) Recovery from inactivation for control cells and 50 μM minoxidil (n=6 and n=7, respectively). Insets apply to all figures. Data are presented as mean ± standard error of the mean. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention is based, at least in part, on the surprising discovery that a combination of a first agent that is a VGSC blocker, particularly a sustained current blocker of a VGSC, with at least one second agent that directly or indirectly modulates an ionotropic channel may be more effective than the agents individually in preventing, reducing or inhibiting metastatic and / or invasive behavior of tumor cells. The second agent may, for example, increase potassium ion efflux, decrease sodium influx and / or downregulate VGSC expression.

[0020] Without being bound by theory, it is possible that the second agent may result in a further reduction in intracellular sodium ion levels in VGSC-expressing tumor cells exposed to a VGSC sustained current blocker, thereby resulting in an even further reduction or inhibition of tumor cell metastatic and / or invasive behavior. Thus, opening potassium channels may hyperpolarize the membrane potential, thereby reducing the ability of the VGSC to become inactive; blocking different types of sodium channels, such as the epithelial sodium channel (ENaC), may further reduce intracellular sodium levels; and blocking EGFR-TK activity may reduce VGSC expression, thereby reducing sodium ion influx.

[0021] As described in the Examples, the present inventors have NaP Three different (independent) possible ways of generating blockade and synergy: (i) K + We tested an ATP channel opener (minoxidil); (ii) an ENaC blocker (amiloride) as a means to control sodium influx into cells independently of VGSCs; and (iii) an EGFR kinase inhibitor (AG1478) to modulate mechanisms driven by EGF signaling.

[0022] For example, the following was found in connection with the combination of ranolazine, a VGSC blocker, and minoxidil, a potassium channel opener (see Examples 3-5 for details): Matrigel® invasion in the human breast cancer MDA-MB-231 (BCa) cell line was significantly reduced in a dose-dependent manner by ranolazine under hypoxia and by minoxidil under both normoxia and hypoxia. There was evidence of a potentiating effect for the drug combination under hypoxia. Similar effects of ranolazine, minoxidil, and the drug combination on invasion were demonstrated under hypoxia for a further human BCa cell line (MDA-MB-468). Cell survival and proliferation were not affected by any of the drugs or combinations under either hypoxic or normoxic conditions. ·nNav The "total" expression of 1.5 protein was significantly reduced by both drugs under hypoxia, whereas the "plasma membrane" level was only reduced by ranolazine.

[0023] Furthermore, the combination of ranolazine with an ENaC blocker (see Example 2 for details) and the combination of the VGSC blocker TTX with an EGFR kinase inhibitor inhibited invasiveness significantly more than TTX alone (see Example 6 for details). Thus, the anti-invasive / metastatic effect of VGSC blockers such as ranolazine may be enhanced when combined in this manner with other agents that relate to one or more other aspects of the VGSC.

[0024] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the particular aspects and embodiments described, as such may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Unless otherwise specified, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] Aspects and Embodiments The following provides more details regarding certain aspects and embodiments according to the present invention.

[0026] In a first aspect, there is provided a combination for use in a method of treating or preventing cancer in a subject, the combination comprising: a) a first substance capable of at least partially blocking the sustained portion of a voltage-gated sodium channel (VGSC) current, but not completely blocking the transient portion of the VGSC current; and b) comprising at least one second agent selected from a potassium channel opener, a non-VGSC sodium entry inhibitor and an upstream down-regulator of VGSC expression, or a combination comprising any two or more thereof.

[0027] In some embodiments according to the first aspect, one or more tumors in the subject express a VGSC.

[0028] In some embodiments according to the first aspect, the VGSC is selected from Nav1.5, Nav1.7, Nav1.6, Nav1.2 and any combination of two or more thereof, and optionally one or more of Nav1.5, Nav1.7, Nav1.6 and Nav1.2 are neonatal forms.

[0029] In some embodiments according to the first aspect, one or more tumors in the subject express neonatal Nav1.5 (nNav1.5).

[0030] In some embodiments according to the first aspect, the first agent is selected from the group consisting of ranolazine (N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1-piperazineacetamide), eleclazine (4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethoxy)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one), GS-1655 (4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethyl)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one), and riluzole (6-(trifluoromethoxy)-2-benzothiazolamine), or a combination comprising any two or more thereof.

[0031] In some embodiments according to the first aspect, the first agent is ranolazine.

[0032] In some embodiments according to the first aspect, the combination comprises: a) Ranolazine and potassium channel openers; b) ranolazine and non-VGSC sodium entry blockers; c) upstream down-regulators of ranolazine and VGSC expression; d) ranolazine, a potassium channel opener and non-VGSC sodium entry inhibitor; e) ranolazine, a potassium channel opener and upstream down-regulator of VGSC expression; f) ranolazine, a non-VGSC sodium entry blocker and an upstream down-regulator of VGSC expression; or g) Ranolazine, a potassium channel opener, a non-VGSC sodium entry inhibitor, and an upstream down-regulator of VGSC expression Includes:

[0033] In some embodiments according to the first aspect, the at least one second agent comprises a potassium channel opener selected from the group consisting of minoxidil, aprikalim, bimakalim, cromakalim, diazoxide, emakalim, levcromakalim, mazokalim, naminidil, nicorandil, pinacidil, pilmakalim, salakalim, tolfenamic acid, lupirtine, retigabine, riluzole, NS1619, NS11021, benzimidazolone 1-EBIO, rottlerin, retigabine, or a combination comprising any two or more thereof.

[0034] In some embodiments according to the first aspect, the potassium channel opener is K ATP Opener, K Ca Selected from openers and Kv openers.

[0035] In some embodiments according to the first aspect, the at least one second agent comprises a sodium entry inhibitor selected from the group consisting of amiloride and digoxin, or a combination thereof.

[0036] In some embodiments according to the first aspect, the sodium entry inhibitor is an epithelial sodium channel (ENaC) blocker.

[0037] In some embodiments according to the first aspect, the at least one second agent comprises an upstream down-regulator of VGSC expression that is an EGFR kinase inhibitor selected from the group consisting of AG1478, gefitinib, erlotinib, afatinib, osimertinib, and dacomitinib, or a combination comprising any two or more thereof.

[0038] In some embodiments according to the first aspect, the combination comprises: a) ranolazine and minoxidil; b) ranolazine and amiloride; c) ranolazine and AG1478; d) eleclazine and minoxidil; e) eleclazine and amiloride; f) eleclazine and AG1478; g) Riluzole and minoxidil; h) Riluzole and amiloride; i) Riluzole and AG1478; j) GS-1655 and minoxidil; k) GS-1655 and amiloride; l) GS-1655 and AG1478; or m) Ranolazine and Riluzole Includes:

[0039] In some embodiments according to the first aspect, the cancer is breast cancer, colon cancer, prostate, non-small cell lung cancer (NSCLC), mesothelioma, cervical cancer, gastric cancer, ovarian cancer, melanoma, oral squamous cell carcinoma, astrocytoma, neuroblastoma, or any combination thereof.

[0040] In some embodiments according to the first aspect, the combination prevents, reduces or inhibits the metastatic behavior of the cancer, the invasiveness of the cancer, the subject's discomfort, the overall aggressiveness of the cancer, or a combination of any two or more thereof.

[0041] In some embodiments according to the first aspect, the first agent is administered in an amount effective to at least partially block the sustained portion of the VGSC current without completely blocking the transient portion of the VGSC current.

[0042] In some embodiments according to the first aspect, the first agent essentially blocks the sustained portion of the VGSC current.

[0043] In some embodiments according to the first aspect, the first agent and the at least one second agent are administered separately, sequentially, or simultaneously to the subject.

[0044] In a second aspect, a) a first substance capable of at least partially blocking the sustained portion of a voltage-gated sodium channel (VGSC) current, but not completely blocking the transient portion of the VGSC current; and b) at least one second substance selected from a potassium channel opener, a non-VGSC sodium entry inhibitor and an upstream down-regulator of VGSC expression, or a combination of any two or more thereof; and a kit-of-parts for separate, sequential or simultaneous use in a method of treating or preventing cancer in a subject, comprising:

[0045] In a third aspect, a) a first substance capable of at least partially blocking the sustained portion of a voltage-gated sodium channel (VGSC) current, but not completely blocking the transient portion of the VGSC current; and b) at least one second agent selected from a potassium channel opener, a non-VGSC sodium entry inhibitor and an upstream down-regulator of VGSC expression, or a combination of any two or more thereof; The present invention provides a method for treating or preventing cancer in a subject, comprising administering to the subject

[0046] In a fourth embodiment, as an active ingredient a) a first substance capable of at least partially blocking the sustained portion of the VGSC current but not completely blocking the transient portion of the VGSC current; and b) at least one second substance selected from a potassium channel opener, a non-VGSC sodium entry inhibitor and an upstream down-regulator of VGSC expression, or a combination of any two or more thereof; In accordance with the present invention, there is provided a pharmaceutical composition comprising:

[0047] In some embodiments, the kit-of-parts according to the second aspect, the method according to the third aspect or the pharmaceutical composition according to the fourth aspect further comprises any one or more features of the embodiments of the first aspect.

[0048] The following numbered embodiments are also provided:

[0049] 1. A combination for use in a method of treating or preventing cancer in a subject, comprising: a) Substances that are capable of blocking the sustained portion of voltage-gated sodium channel (VGSC) currents but do not block the transient portion of VGSC currents, and b) potassium channel openers; wherein the substance and the potassium channel opener are administered separately, sequentially or simultaneously to the subject.

[0050] 2. A combination for use according to embodiment 1, wherein the subject's primary tumor expresses a VGSC.

[0051] 3. A combination for use according to any one of embodiments 1 or 2, wherein the substance capable of blocking the sustained portion of the VGSC current but not the transient portion of the VGSC current is selected from Nav1.5, Nav1.7, Nav1.6, Nav1.2, and any combination of two or more thereof, optionally wherein one or more of Nav1.5, Nav1.7, Nav1.6 and Nav1.2 are in neonatal form.

[0052] 4. The combination for use according to embodiment 3, wherein the VGSC comprises neonatal Nav1.5 (nNav1.5).

[0053] 5. A combination for use according to any one of embodiments 1 to 4, wherein the substance capable of blocking the sustained portion of the VGSC current but not the transient portion of the VGSC current is selected from ranolazine (N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2methoxyphenoxy)propyl]-1-piperazineacetamide), eleclazine (4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethoxy)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one), 4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethyl)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one, and riluzole (6-(trifluoromethoxy)-2-benzothiazolamine), or a combination of any two or more thereof.

[0054] 6. The combination for use according to any one of embodiments 1 to 5, wherein the potassium channel opener is selected from the group consisting of minoxidil, amiloride, AG1478, aprikalim, bimakalim, cromakalim, diazoxide, emakalim, levcromakalim, mazokalim, naminidil, nicorandil, pinacidil, rilmakalim, salakalim, flufenamic acid, meclofenamic acid, niflumic acid, nimesulide, rottlerin (mallotoxin), tolfenamic acid, lupirtine, retigabine and riluzole, or a combination of any two or more thereof.

[0055] 7. A combination for use according to any one of embodiments 1 to 5, wherein the potassium channel opener is selected from minoxidil, amiloride, AG1478, and combinations of any two or more thereof.

[0056] 8. The combination is Ranolazine and minoxidil; Ranolazine and amiloride; Ranolazine and AG1478; · Electrazine and minoxidil; · Elecladine and amiloride; · Elecladine and AG1478; Riluzole and minoxidil; Riluzole and amiloride; Riluzole and AG1478; 4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethyl)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one and minoxidil; 4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethyl)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one and amiloride; 4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethyl)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one and AG1478; or Ranolazine and rizuole 8. A combination for use according to any one of embodiments 1 to 7, comprising:

[0057] 9. The combination for use according to any one of embodiments 1 to 8, wherein the cancer is breast cancer, colon cancer, prostate, non-small cell lung cancer (NSCLC), mesothelioma, cervical cancer, gastric cancer, ovarian cancer, melanoma, oral squamous cell carcinoma, astrocytoma, neuroblastoma, or a combination thereof.

[0058] 10. A combination for use according to any one of embodiments 1 to 9, wherein the combination prevents, reduces or inhibits the metastatic behavior of the cancer, the invasiveness of the cancer, the subject's pain, the overall aggressiveness of the cancer, or any combination thereof.

[0059] 11. A combination for use according to any one of embodiments 1 to 10, wherein the substance is administered in an amount effective to at least partially block the sustained portion of the VGSC current without completely blocking the transient portion of the VGSC current.

[0060] 12. A combination for use according to embodiment 10, wherein the substance essentially blocks the sustained part of the VGSC current.

[0061] 13. a) Substances that can block the sustained portion of voltage-gated sodium channel (VGSC) currents but do not block the transient portion of VGSC currents; and b) potassium channel openers, 2. A kit of parts for separate, sequential or simultaneous use in a method for treating or preventing cancer in a subject, comprising:

[0062] 14. Target a) Substances that are capable of blocking the sustained portion of voltage-gated sodium channel (VGSC) currents but do not block the transient portion of VGSC currents, and b) Potassium channel opener VGSC blocker 10. A method for treating or preventing cancer in a subject, comprising administering separately, sequentially or simultaneously:

[0063] 15. As an active ingredient a) substances that can block the sustained portion of the VGSC current but do not block the transient portion of the VGSC current; and b) Potassium channel openers in admixture with a pharmaceutically acceptable carrier, diluent, vehicle and / or excipient.

[0064] 16. The kit-of-parts according to embodiment 13, the method according to embodiment 14, or the pharmaceutical composition according to embodiment 15, further comprising a feature according to any one of embodiments 1 to 12.

[0065] Thus, a combination of a first substance and at least one second substance according to any of the aspects or embodiments described herein can be used as a medicament for treating, for example, cancer. Suitable patients include mammalian patients, such as humans, monkeys, rabbits, dogs, cats, cows, horses, pigs, mice, and rats, suffering from cancer. Preferably, the patient is a human patient, such as an adult human patient. Preferably, the combination of a first substance and a second substance according to any of the aspects or embodiments described herein is synergistic.

[0066] The first agent of the combination is a voltage-gated sodium channel (VGSC) blocker, preferably a VGSC blocker, that is capable of at least partially blocking the sustained portion of VGSC current but does not completely block the transient portion of VGSC current.

[0067] As used herein, "voltage-gated sodium channels" or "VGSCs" refer to a class of integral membrane proteins that form ion channels. Integral membrane proteins allow sodium ions (Na ) into the cell through the plasma membrane of the cell. + In humans, there are nine different VGSC alpha subunits or "Nav" proteins (Nav1.1 to Nav1.9), which are encoded by nine genes (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A and SCN11A, respectively). As used herein, unless the context dictates otherwise, the term may refer to any known VGSC.

[0068] As used herein, "Nav1.5" refers to the human Nav1.5 protein (adult or neonatal form) encoded by the SCN5A gene (Genbank Gene ID: 6331). Unless otherwise indicated by context, the term Nav1.5, as used herein, is intended to encompass all forms of Nav1.5 expressed in cells (e.g., cancer cells), including neonatal Nav1.5 (also referred to herein as "nNav1.5" or the "fetal" form of Nav1.5) or other splice variants or variants known to those skilled in the art, such as splice variants and variants described in UniProtKB accession Q14524 (SCN5A_HUMAN). With reference to FIG. 1 and Q14524 (SCN5A_HUMAN), the amino acid sequence of the neonatal Nav1.5 protein differs from the adult Nav1.5 protein at least at amino acid residue 211 and may also differ at other amino acid residues, such as residues 206, 207, 209, 210, 215, and 234. Preferably, in the neonatal form, the amino acid at position 211 is K (Lys). For example, the neonatal form may contain amino acid residues V, S, N, I, K, L, and P at positions 206, 207, 209, 210, 211, 215, and 234, respectively, while the adult form contains amino acid residues T, T, F, V, D, V, and S at the same / corresponding positions. In one embodiment, in the neonatal variant, residues 206-211 are changed from TTEFVD to VSENIK, and optionally, in the neonatal variant, the amino acid residue at position 215 is changed from V to L and / or the amino acid residue at position 234 is changed from S to P. UniProtKB-H9KVD2 (H9KVD2_HUMAN) represents a specific example of the amino acid sequence of neonatal Nav1.5.

[0069] As used herein, "Nav1.7" refers to the human Nav1.7 protein (adult or neonatal form) encoded by the SCN9A gene. Unless otherwise negated by context, the term Nav1.7, as used herein, is intended to encompass all forms of Nav1.7 expressed in cells (e.g., cancer cells), including neonatal Nav1.7 (also referred to herein as "nNav1.7" or the "fetal" form of Nav1.7) or other splice variants or variants known to those skilled in the art, such as splice variants and variants described in UniProtKB accession Q15858 (SCN9A_HUMAN). Figure 1 illustrates specific amino acid residues that commonly differ between neonatal and adult forms of the Nav1.7 protein.

[0070] As used herein, "Nav1.6" refers to the human Nav1.6 protein (adult or neonatal form) encoded by the SCN8A gene. Unless otherwise indicated by context, the term Nav1.6 is intended to encompass all forms of Nav1.6 expressed in cells (e.g., cancer cells), including neonatal Nav1.6 (also referred to herein as "nNav1.6" or the "fetal" form of Nav1.6) or other splice variants or variants known to those skilled in the art, such as splice variants and variants described in UniProtKB entry Q9UQD0 (SCN8A_HUMAN). Figure 1 illustrates specific amino acid residues that commonly differ between neonatal and adult forms of the Nav1.6 protein.

[0071] As used herein, "Nav1.2" refers to the human Nav1.2 protein (adult or neonatal form) encoded by the SCN2A gene. Unless the context dictates otherwise, the term Nav1.2, as used herein, is intended to encompass all forms of Nav1.2 expressed in cells (such as cancer cells), including neonatal Nav1.2 (also referred to herein as "nNav1.2" or the "fetal" form of Nav1.2) or other splice variants or variants known to those of skill in the art, such as splice variants and variants described in UniProtKB accession Q99250 (SCN2A_HUMAN).

[0072] As used herein, the term "voltage-gated sodium channel blocker" or "VGSC blocker" refers to a substance that can at least partially block a VGSC current, preferentially the sustained (late) portion of the VGSC current. In some embodiments, a VGSC blocker is a substance that can block the sustained portion of a VGSC current but not the transient portion of the VGSC current. For example, in some embodiments, a VGSC blocker is a substance that can at least partially block the sustained portion of a VGSC current but not completely block the transient portion of the VGSC current. A VGSC blocker may, for example, be able to block the sustained current by at least about 20%, such as by at least about 30%, such as by at least about 40%, such as by at least about 50%, such as by at least about 60%, such as by at least about 70%. Preferably, particularly when provided in clinically relevant dosages, a VGSC blocker blocks the transient current by about 15% or less, such as by about 10% or less, such as by about 5% or less, such as by about 2% or less. In certain embodiments, a VGSC blocker is a substance that can block sustained currents by at least about 50%, for example, in an in vitro assay, preferably at clinically relevant concentrations, but not block transient currents by more than about 5% (see Figure 2). Suitable assays for assessing the ability of a substance to block sustained and transient VGSC currents are known to those of skill in the art; see, for example, Antzelevitch C et al., Circulation (2004); 110:904-910.

[0073] Non-limiting examples of suitable VGSC blockers include: Ranolazine (N-(2,6-dimethylphenyl)-4-[2-hydroxy-3-(2-methoxyphenoxy)propyl]-1-piperazineacetamide) · Elecladine (4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethoxy)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one) GS-1655 (4-(pyrimidin-2-ylmethyl)-7-(4-(trifluoromethyl)phenyl)-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one) Riluzole (6-(trifluoromethoxy)-2-benzothiazolamine) There is.

[0074] In certain embodiments, the (first) substance is ranolazine. For details of the use of ranolazine as a medicament for the treatment of cancer, see, for example, WO2012 / 049440 A1.

[0075] The second agent of the combination can be, for example, a potassium channel opener, a sodium entry inhibitor, or an upstream regulator of VGSC expression. Combinations comprising a VGSC blocker and two or more such second agents are also contemplated. Thus, in some embodiments, the combination comprises a VGSC blocker, a potassium channel opener, and a sodium entry inhibitor. In some embodiments, the combination comprises a VGSC blocker, a potassium channel opener, and an upstream regulator of VGSC expression. In some embodiments, the combination comprises a VGSC blocker, a sodium entry inhibitor, and an upstream regulator of VGSC expression. Combinations of potassium channel openers, sodium entry inhibitors, and upstream regulators of VGSC expression are also contemplated.

[0076] As used herein, a "potassium channel opener" refers to a drug that acts on a potassium channel to inhibit K + These agents are substances that can facilitate ion transport, thereby generally allowing potassium flow out of the cell. These agents "antagonize" the activation of VGSCs, for example, by hyperpolarizing the membrane potential, which then induces a "sustained current" (I NaP In some embodiments, the potassium channel opener promotes ion transmission through ATP-sensitive potassium channels. ATP ) is gated by the intracellular nucleotides, ATP and ADP, and is found in a variety of tissues, including the heart, pancreatic beta cells, skeletal muscle, smooth muscle, and the central nervous system. In particular, the present invention provides a method for the detection of Ca 2+ (K Ca Potassium channels that are gated by either Kv channels or voltage (Kv channels) are also contemplated.

[0077] Non-limiting examples of potassium channel openers include: Minoxidil (6-(1-piperidinyl)pyrimidine-2,4-diamine 3-oxide) (K ATP channel openers) ·April Karim (K ATP channel openers) Bimakarim (K ATP channel openers) Cromakalim (K ATP channel openers) Diazoxide (K ATP channel openers) Emakalim (K ATP channel openers) Levcromakalim (K ATP channel openers) Masochist (K ATP channel openers) ·Naminijil(K ATP channel openers) Nicorandil (K ATP channel openers) Pinacidil (K ATP channel openers) Lirmakarim (K ATP channel openers) Sarah Karim (K ATP channel openers) Tolfenamic acid (K Ca channel activators) Lupirtin (Kv channel opener) Retigabine (Kv channel opener) Riluzole (K Ca channel activators) ·NS1619 (1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one) ( K Ca channel openers) ·NS11021 (N'-[3,5-bis(trifluoromethyl)phenyl]-N-[4-bromo-2-(2H-tetrazol-5-yl-phenyl]thiourea)( K Ca channel openers) Benzimidazolone 1-EBIO(K Ca channel openers) Rottlerin (mallotoxin) (K Ca and activators of Kv11.1 channels) Retigabine (KCNQ / Kv7 opener) There is.

[0078] Potassium channel openers of particular interest include: Minoxidil Cromakalim Levcromakalim Diazoxide NS1619 NS11021 Benzimidazolone 1-EBIO, and Retigabine There is.

[0079] In a particular embodiment, the potassium channel opener is, for example, a K channel opener selected from the group consisting of minoxidil, cromakalim, levcromakalim, and diazoxide. ATP In certain embodiments, the potassium channel opener is minoxidil.

[0080] In another particular embodiment, the potassium channel opener is a K channel opener selected from, for example, NS1619, NS11021, and benzimidazole 1-EBIO. Ca It is an open mouth medicine.

[0081] In another specific embodiment, the potassium channel opener is a KCNQ / Kv7 opener, eg, retigabine.

[0082] In some embodiments, the potassium channel opener is not riluzole, eg, in any embodiment in which the first agent is riluzole, riluzole is not the (only) second agent.

[0083] As used herein, a "sodium entry inhibitor" refers to a drug that inhibits the entry of Na into cells. + The term "sodium entry blocker" refers to a substance that has the ability to decrease the influx of sodium ions by acting on one or more non-VGSC ion channels that allow permeation of VGSC-I. Therefore, sodium entry blockers act by increasing the effect on intracellular sodium ion levels. NaP Non-limiting examples of such sodium channels include the epithelial sodium channel (ENaC), transient receptor potential channel subtype M4 (TrpM4), ATP-activated "purinergic" receptor subtype 7 (P2RX7), and Na + -K + ATPase / pump. In a specific embodiment, the sodium entry inhibitor is an ENaC blocker, which refers herein to a molecule capable of acting on ENaC to reduce sodium entry through the channel, and its action is voltage-independent. ENaC is located in the apical membrane of polarized epithelial cells, and is therefore commonly present in carcinomas. In another specific embodiment, the sodium entry inhibitor is a Na + -K + ATPase / pump blockers, as used herein, + -K + Refers to a molecule that has the ability to act on the ATPase / pump to reduce the influx of sodium ions through the channel.

[0084] Non-limiting examples of sodium entry blockers include amiloride and digoxin (Lanoxin®).

[0085] In one particular embodiment, the sodium entry inhibitor is amiloride, an ENaC blocker known as a potassium-sparing diuretic.

[0086] In certain other embodiments, the sodium entry inhibitor is Na + -K + Digoxin (Lanoxin®), an ATPase / pump inhibitor.

[0087] As used herein, an "upstream down-regulator of VGSC expression" refers to a substance that can down-regulate VGSC expression levels by acting on an upstream member of a pathway that regulates VGSC expression. In one embodiment, the substance modulates a growth factor receptor tyrosine kinase (TK). Thus, a TK inhibitor is an I NaP "EGFR kinase inhibitors" or "EGFR tyrosine kinase inhibitors" or "EGFR-TK inhibitors" are specifically intended to refer to substances capable of inhibiting the tyrosine kinase activity of the epidermal growth factor receptor (EGFR).

[0088] Non-limiting examples of EGFR-TK inhibitors include: AG1478 / Tyrphostin N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinamine, or its analogs or derivatives Gefitinib / Iressa® Erlotinib / Tarceva® Afatinib (Giotrif) Osimertinib (Tagrisso) Dacomitinib (Vidinpro) There is.

[0089] In one particular embodiment, the EGFR-TK inhibitor is AG1478. In another particular embodiment, the EGFR-TK inhibitor is gefitinib. In another particular embodiment, the EGFR-TK inhibitor is gefitinib. In another particular embodiment, the EGFR-TK inhibitor is afatinib. In another particular embodiment, the EGFR-TK inhibitor is osimertinib. In another particular embodiment, the EGFR-TK inhibitor is dacomitinib. In some embodiments, the EGFR-TK inhibitor is an analog or derivative of AG1478, gefitinib, afatinib, osimertinib, or dacomitinib, for example, an analog or derivative of AG1478 such as Ko143 {[(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester]}.

[0090] Another category of agents contemplated for combination with VGSC blockers as at least one second agent according to the present invention are inhibitors of mechanisms downstream of VGSC signaling. Non-limiting examples include: Inhibitors of the sodium-hydrogen exchanger (NHE1), such as cariporide Inhibitors of sodium-calcium exchangers (NCX), such as ORM-11372 There is.

[0091] In some embodiments, the at least one second substance does not include propranolol.

[0092] In some embodiments of the methods of treatment described herein, each of the first and at least one second agent is administered in a therapeutically effective amount or dose. By "therapeutically effective amount" and "therapeutically effective dose" is intended the amount or dosage of each agent that, when administered in combination to a patient suffering from cancer, results in a positive therapeutic response in treating the patient, such as, for example, a reduction in metastatic behavior of the cancer, prevention of metastatic behavior of the cancer, a reduction in pain, etc.

[0093] Thus, each of the first and at least one second agent is administered to the patient in an amount that is therapeutically effective for its intended purpose, and at a frequency and duration determined by a trained physician. Estimates of effective dosages and in vivo half-lives for individual agents encompassed by the present invention can be made using conventional methods or based on knowledge from in vivo testing using appropriate animal models or prior clinical use of the agent.

[0094] For example, the first agent, i.e., a VGSC blocker, is preferably administered to the patient at a dosage that at least reduces the sustained portion of the VGSC current without completely blocking the transient portion. In certain embodiments, the VGSC blocker is administered at a dosage that does not prevent the growth of cancer tumor cells or destroy the tumor. Suitable dosages of ranolazine and eleclazine can be found in WO2018 / 146313 (Celex GmbH) and WO2012 / 049440 (Celex Oncology Ltd.), both of which are incorporated herein by reference in their entireties.

[0095] The VGSC blocking agent may be administered at a dosage capable of blocking at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, such as at least about 60%, such as at least about 70%, of the sustained current. Preferably, particularly when provided in clinically relevant dosages, the VGSC blocking agent is administered at a dosage that blocks no more than about 20%, such as no more than about 15%, such as no more than about 10%, such as no more than about 5%, such as no more than about 2%, of the transient current. In certain embodiments, the VGSC blocking agent is administered at a dosage capable of blocking at least about 50% of the sustained current but not blocking the transient current by more than about 5%.

[0096] For example, in some embodiments, the VGSC blocking agent is ranolazine and is administered to the patient at a dosage that provides a ranolazine plasma concentration of, e.g., about 0.5 μM to about 20 μM, such as about 1 μM to about 10 μM, such as about 2 μM to about 5 μM, or about 0.1 μM to about 100 μM. In some embodiments, ranolazine is administered to the patient at a dosage that provides a ranolazine tumor concentration of, e.g., about 0.5 μM to about 20 μM, such as about 1 μM to about 10 μM, such as about 2 μM to about 5 μM, or about 0.1 μM to about 100 μM. In some embodiments, ranolazine is administered to a patient, e.g., an adult human, at a dosage of at least about 10 mg, such as at least about 20 mg, such as at least about 50 mg, such as at least about 100 mg, such as at least about 200 mg, such as at least about 400 mg, such as at least about 800 mg, such as at least about 1600 mg, etc. In some embodiments, ranolazine is administered to a patient, e.g., an adult human patient, at a dosage of about 100 to about 1000 mg ranolazine, such as about 375 mg to about 750 mg, e.g., 375 mg, 500 mg, or 750 mg, per administered dose, optionally in a sustained or extended release formulation, e.g., twice daily, once daily, once weekly, or once monthly.

[0097] In some embodiments, the second agent is a potassium channel blocker such as minoxidil and is administered to a patient, e.g., a human adult, in a dosage of at least about 1 mg, such as at least about 2 mg, such as at least about 5 mg, such as at least about 10 mg, such as at least about 20 mg, such as at least about 50 mg, such as at least about 100 mg. Generally, a suitable dosage will be from about 1 mg to about 100 mg of minoxidil, such as from about 2 mg to about 60 mg or from about 5 to about 40 mg per single dose administered, for example, twice daily, once daily, once weekly, or once monthly.

[0098] In some embodiments, the second agent is an ENaC blocker such as amiloride and is administered to a patient, e.g., a human adult, in a dosage of at least about 1 mg, such as at least about 2 mg, such as at least about 3 mg, such as at least about 4 mg, such as at least about 5 mg, such as at least about 8 mg, such as at least about 10 mg. Generally, a suitable dosage will be about 1 to about 10 mg of amiloride per kg of body weight, such as about 2 to about 8 mg or about 3 to about 5 mg, per kg of body weight, per single dose administered, for example, twice daily, once daily, once weekly, or once monthly.

[0099] In some embodiments, the second agent is an EGFR-TK inhibitor, such as erlotinib, and is administered to a patient, e.g., a human adult, at a dosage of at least about 5 mg, such as at least about 10 mg, such as at least about 20 mg, such as at least about 50 mg, such as at least about 100 mg, such as at least about 150 mg, etc. Generally, a suitable dosage will be about 5 to about 150 mg of AG1478 per kg of body weight, such as about 10 to about 100 mg or about 20 to about 50 mg per single dose administered, for example, twice daily, once daily, once weekly, or once monthly.

[0100] An appropriate treatment regimen may generally include repeating at least one administration at daily, weekly, or monthly intervals, etc., until a therapeutic benefit or response is observed or anticipated. Delivery vehicles for the first and / or second substances, including sustained release formulations, may be utilized as needed. Appropriate dosing regimens may be envisioned and implemented by those skilled in the art, e.g., physicians.

[0101] Maintenance therapy is also contemplated. The phrase "maintenance therapy" or "maintenance period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used to maintain a subject during disease treatment for an extended period of time (months or years), e.g., to reduce the risk of metastasis. Maintenance regimens may utilize continuous therapy (e.g., administering a drug at regular intervals (e.g., weekly, monthly, yearly, etc.)) or intermittent therapy (e.g., discontinuation treatment, intermittent treatment, treatment upon recurrence, or treatment upon achievement of certain predetermined criteria (e.g., pain, disease symptoms, etc.)).

[0102] The VGSC blocking agent and at least one second agent may be administered to the subject simultaneously, separately or sequentially.

[0103] In some embodiments, the VGSC blocking agent and at least one second substance are co-administered. As used herein, the term "co-administration" refers to administering a first and second substance simultaneously or substantially simultaneously, for example, within 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or less. Optionally, co-administration can include administering the two substances via the same route of administration. In some embodiments, co-administration is achieved by administering a pharmaceutical composition containing both the first and second substances.

[0104] In some embodiments, the VGSC blocking agent and at least one second substance are administered separately. As used herein, the term "separate administration" refers to administering the first and second substances on separate occasions and / or according to separate but overlapping dosing regimens. Thus, the first and second substances may be administered in any order, multiple hours apart, multiple days apart, or more than one day apart. In some embodiments, the separate administration of the first and second substances occurs within at most one month or at most one week apart. Separate or similar routes of administration may be used.

[0105] In some embodiments, the VGSC blocking agent and at least one second substance are administered sequentially. As used herein, the term "sequential administration" refers to administering a first and second substance on separate occasions in a predetermined order. Thus, the first and second substances may be administered in a predetermined order at least 5 minutes apart, at least 10 minutes apart, at least 15 minutes apart, at least 30 minutes apart, at least 1 hour apart, at least 1 day apart, or at least 2 days apart. In some embodiments, the sequential administration of the first and second substances occurs within at most one month or at most one week apart. Separate or similar administration routes may be used.

[0106] In some embodiments, the first and at least one second agent are both administered at intervals such that both agents are at or above pharmacologically effective levels in the tumor and / or at or above predetermined levels in the blood, serum, or reference tissue. Generally, the pharmacologically effective level in the tumor is predetermined or correlated with a particular level in the blood or serum.

[0107] Particularly contemplated for combination treatment are cancer types that express, or are at least known to be associated with the expression of, one or more VGSCs, and therefore metastatic behavior. Table 1 below shows the associations that have been found between some specific cancer types and their VGSC subtype expression.

[0108] [Table 1]

[0109] In some embodiments, the cancer includes one or more tumors, e.g., primary tumors, comprising cancer cells known to express or be associated with the expression of Nav1.5, Nav1.6, Nav1.7, Nav1.2, or combinations thereof in adult and / or neonatal form.

[0110] In some embodiments, the cancer comprises one or more tumors, e.g., primary tumors, comprising cancer cells known to express or be associated with the expression of Nav1.5 in adult or neonatal form.

[0111] In certain embodiments, the cancer comprises one or more tumors, e.g., primary tumors, comprising cancer cells that express, or are known to be associated with, neonatal Nav1.5 expression.

[0112] As used herein, "treating" or "treatment" of cancer includes, but is not limited to, reducing the metastatic behavior of cancer, preventing the metastatic behavior of cancer, reducing pain, reducing the invasiveness of cancer, reducing the overall aggressiveness of cancer, or any combination thereof. Thus, in individual and specific embodiments, a method of treatment according to the present invention can be (i) reducing the metastatic behavior of cancer, (ii) preventing the metastatic behavior of cancer, (iii) reducing pain in a patient afflicted with cancer, (iv) reducing the invasiveness of cancer, or (v) a combination of two or more of (i)-(iv).

[0113] The progression of metastatic cancers such as breast, colon and prostate cancer goes through five main stages: 1. Development, i.e., the initial transformation of normal cells into cancer cells; 2. Proliferation, i.e., the cancer cells increase in number to form a primary tumor that generally has a smooth, well-defined surface and increases in size; 3. During the transformation, development, or proliferation stage, it is generally characterized by the dissolution and spread of cancerous borders by cancer cells from a state without potential for invasive or metastatic behavior to a state with potential; 4. The detachment of cancer cells from the primary tumor, followed by the migration of these cells into the circulatory system and into surrounding areas of tissue within the same organ; 5. Metastasis, i.e., the migration of detached cells via the circulation (blood or lymph) to other organs and the creation of secondary tumors in those organs. It is generally believed to include at least some of the following:

[0114] It should be noted, however, that metastasis can occur without an initial growth stage, in which case metastases can be found in patients without an identifiable primary tumor.

[0115] By "reducing metastatic behavior" of cancer is intended any reduction in behavior associated with detached cancer cells migrating through the circulation (blood or lymph) and accumulating in other organs and / or creating secondary tumors, or locally invading surrounding tissues. Typically, the patient is in stage 3, 4, or 5, such as stage 4 or 5. Reducing metastatic behavior can include, for example, one or more of: (i) reducing the transcription, translation, and / or expression of neonatal and / or adult VGSCs (e.g., Nav1.5) in cancer cells compared to controls, generally reducing neonatal Nav1.5 (nNav1.5); (ii) reducing the invasiveness of cancer cells; (iii) reducing the peak density of VGSC currents in cancer cells; (iv) reducing the proportion of cancer cells exhibiting VGSC currents; (v) reducing the motility of cancer cells (e.g., reducing lateral motility), (vi) reducing the migration of cancer cells (e.g., transverse migration), and (vii) reducing the sustained portion of VGSC currents without eliminating the transient portion. The VGSC may be Nav1.5 (in adult and / or neonatal forms), such as nNav1.5. "Motility" reflects the ability of tumor cells to initially migrate to and through the basement membrane into surrounding tissue; cellular "invasiveness" reflects the ability of tumor cells to invade surrounding tissue and migrate through that tissue towards the circulatory system; and "migratory ability" reflects the ability of tumor cells to migrate from surrounding tissue through its wall into the circulatory system.

[0116] By "preventing metastatic behavior" of cancer, it is intended to refer to prophylactically treating cancer patients who are at risk for, but have not yet been diagnosed with, metastatic disease to prevent or reduce the risk of metastatic behavior of the cancers described above. Generally, the patient is at stage 1, 2, or 3. Preventing metastatic behavior can include, for example, preventing or reducing expression of a VGSC, e.g., one or more Navl.5, e.g., adult and / or neonatal forms, such as nNavl.5.

[0117] As used herein, the term "benign" refers to a first- or second-stage tumor or cancer. As used herein, a tumor may also or alternatively be characterized as benign if it (a) does not invade nearby tissues (invasive); (b) does not metastasize to other parts of the body (spreading); (c) tends to have well-defined borders; and / or (d) grows slowly.

[0118] The term "malignant condition" as used herein refers to a third, fourth or fifth stage tumor or cancer.

[0119] In quantitative or qualitative terms, by "reducing the overall grade of a cancer" is intended a reduction in any behavior associated with cancer progression. In some embodiments, reducing the grade of a cancer refers to the reversal of a cancer from any one of stage 3, 4, or 5 to a lower-numbered stage, including, but not limited to, from stage 3 to stage 2 or lower, from stage 4 to stage 3 or lower, and from stage 5 to stage 4 or lower. In some embodiments, reducing the grade of a cancer refers to the reversal of a malignant cancer or tumor to a benign cancer or tumor. In some embodiments, by "reducing the overall grade of a cancer" is intended a reduction of a cancer to a non-metastatic, but not necessarily non-invasive, state.

[0120] By "reducing cancer invasiveness" is intended a significant decrease in the invasiveness of cancer cells under predetermined conditions, e.g., normoxic or hypoxic conditions. Examples of suitable assays for determining invasiveness are provided elsewhere herein (see, e.g., the section entitled "Functional Characteristics"). A significant decrease in invasiveness includes, for example, a decrease of at least about 10%, 20%, or more, such as at least 30%, 40%, 50%, 60%, 70%, or 80%, compared to a control.

[0121] Cancers suitable for treatment by the present invention include, but are not limited to, breast cancer, colon cancer, prostate cancer, non-small cell lung cancer (NSCLC), mesothelioma, cervical cancer, gastric cancer, ovarian cancer, melanoma, oral squamous cell carcinoma, astrocytoma, neuroblastoma, and any combination thereof. In some embodiments, the cancer is breast cancer, colorectal cancer, lung cancer, ovarian cancer, neuroblastoma, or any combination thereof. In some embodiments, colorectal cancer may be characterized herein as colon cancer or rectal cancer, generally depending on the origin of the cancer tissue. However, unless otherwise indicated by context, the terms colorectal cancer and colon cancer may be used interchangeably. In one embodiment, the cancer is colorectal cancer, colon cancer, or both.

[0122] In certain embodiments, the cancer is breast cancer.

[0123] In some embodiments, one or more tumors of a patient are hypoxic or at risk of becoming hypoxic. In some embodiments, one or more tumors of a patient are predicted or determined to be hypoxic by a trained physician. The presence of hypoxia can be determined by a variety of techniques known to those skilled in the art, including, but not limited to, magnetic resonance imaging (MRI) (see, e.g., Abadjian et al., Adv Exp Med Biol. 2017;1036:229-257) or staining a sample of tumor tissue with pimonidazole (see, e.g., Wilson and Hay, Nature Reviews Cancer 2011;11:393-410).

[0124] Cancer-expressing VGSCs (eg, nNav1.5) are generally at stage 3, 4 or 5 as described above.

[0125] In one embodiment, the patient is at stage 3, 4 or 5, such as stage 4 or 5. In one embodiment, the cancer is at stage 1, 2 or 3, such as stage 1 or 2.

[0126] In one embodiment, the cancer is stage 3. Patients with stage 3 cancer have not generally been diagnosed with metastatic disease, but are at risk for metastatic behavior of the cancer, i.e., progression to stage 4 or 5. Patients with stage 3 cancer may therefore be treated according to the present invention to prevent metastatic behavior of the cancer.

[0127] In one embodiment, the cancer is at stage 4. Patients with stage 4 cancer may not be diagnosed with metastatic disease, but the cancer has progressed to metastatic behavior. Patients with stage 4 cancer may therefore be treated with the present invention to reduce the metastatic behavior of the cancer.

[0128] In one embodiment, the cancer is at stage 5. Patients with stage 5 cancer may be diagnosed with metastatic disease, and the cancer is characterized by metastatic behavior. Patients with stage 5 cancer may therefore be treated with the present invention to reduce the metastatic behavior of the cancer.

[0129] In some embodiments, the patient may have a cancer associated with VGSC expression and / or risk of metastatic behavior, but the VGSC expression (e.g., nNav1.5 expression) and / or metastatic behavior has not yet been determined. Cancers prone to metastatic behavior include, for example, colon cancer, breast cancer, lung cancer, and ovarian cancer. For example, immunohistochemical analysis of a cancer cell-containing sample, such as a tumor biopsy or blood sample obtained from the patient, may show that the tumor cells in the sample do not express nNav1.5 or any other VGSC tested. Thus, the cancer may be stage 1 or (more likely) stage 2.

[0130] In one embodiment, the cancer is stage 2. Patients with stage 2 cancer have not generally been diagnosed with metastatic disease, but are at risk for VGSC (e.g., nNav1.5) expression and metastatic behavior of the cancer, i.e., progression to stage 3, 4, or higher. Patients with stage 2 cancer may therefore be treated according to the present invention to prevent nNav1.5 expression or metastatic behavior of the cancer.

[0131] A patient suffering from any one of stages 1 to 5 of cancer, such as any one of stages 2 to 5, may also suffer from pain resulting from the cancer, e.g., the primary tumor, and therefore may be treated by the present invention to reduce pain.

[0132] In one embodiment, when used in a method according to the invention, the combination reduces or prevents metastatic behavior in nNav1.5-expressing cancers without killing the cancer cells.

[0133] In one embodiment, when used in a method according to the invention, the combination reduces or prevents metastatic behavior in nNav1.5-expressing cancers without substantially affecting cancer cell proliferation.

[0134] In one embodiment, treatment of cancer cells with the combination causes neonatal morphology of Navl.5 expression in the cancer cells (e.g., determined as mRNA, protein, or plasma membrane levels) to be significantly lower than a predetermined control value, expression in cancer cells not exposed to the combination, or a control, such as cancer cells exposed to a reference compound, e.g., the first or second agent. In one embodiment, treatment of cancer cells with the combination causes the invasiveness, motility, and / or migration ability of the cancer cells treated with the combination to be significantly lower than a predetermined control value, expression in cancer cells not exposed to the combination, or a control, such as cancer cells treated with the first agent alone or the second agent alone.

[0135] Generally, the first agent and at least one second agent are administered in the form of joint or separate compositions. Each may be formulated in a single composition, generally a pharmaceutical composition, for administration to a subject by any suitable route, including, but not limited to, oral, buccal, sublabial, sublingual, rectal, intravenous, subcutaneous, intradermal, intramuscular, transdermal, and intranasal administration, and / or direct administration to a tumor, primary tumor, etc. In some embodiments, the VGSC blocker and second agent, e.g., a potassium channel opener, are formulated in the same composition, generally a pharmaceutical composition, for administration to a patient by any suitable route.

[0136] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and the tumor or cancer to be treated. Sustained release systems may be used to release the agent over a particularly long period of time. Delivery can also be carried out by local (e.g., intratumoral) or systemic administration of a composition, such as a pharmaceutical composition comprising a VGSC blocker and / or a second agent, e.g., a potassium channel opener, to a subject.

[0137] The subject is generally a patient, preferably a human patient such as a human cancer patient, or a human subject at risk of cancer, particularly a cancer associated with VGSC expression. In some embodiments, the cancer patient has one or more tumors, e.g., primary tumors, that are at risk of metastasis and / or invasive behavior. In some embodiments, the cancer patient has one or more tumors, e.g., primary tumors, that are at risk of metastasis and / or invasive behavior. Human cancer patients are particularly intended for cancer treatment according to the present invention.

[0138] In some embodiments, the treatment methods described herein include testing whether the cancer or one or more tumors, e.g., primary tumor, contains cells that express VGSC, optionally in neonatal form, such as neonatal Navl.5, Navl.6, and / or Navl.7. This step may be performed prior to administering a VGCS blocker, and optionally prior to administering a potassium channel blocker. A sample may be taken, for example, by tumor biopsy, from the tumor of the subject intended for treatment, and the tumor sample or tumor cells analyzed for expression of the relevant VGSC mRNA, VGSC protein, or both.

[0139] In some embodiments, the patient is suffering from a cancer comprising Nav1.5-expressing cancer cells. Such cancers may be identified, for example, by immunohistochemistry or analysis of a cancer cell-containing sample (such as a tumor biopsy or blood sample) obtained from the patient using detectable monoclonal or polyclonal antibodies specific for nNav1.5, aNav1.5, or both to detect expression of Nav1.5 by the cancer cells, or by analyzing the sample for the presence of Nav1.5 mRNA. In one embodiment, the cancer cells express both aNav1.5 and nNav1.5. In certain embodiments, the cancer expresses Nav1.5 in adult and / or neonatal forms, e.g., neonatal Nav1.5. Preferably, the cancer cells express Nav1.5 at least predominantly in the neonatal form. In a preferred embodiment, the treatment method includes determining that the cancer comprises cells expressing nNav1.5 prior to administering the first agent, the second agent, or both.

[0140] This invention is further illustrated by the following examples, which should not be construed as limiting. [Example]

[0141] Example 1 Materials and Methods This example describes the materials and methods used in the experimental work reported in Examples 2-7.

[0142] Cell culture: Cells were cultured in 60 x 15 mm tissue culture dishes (Falcon®, Becton Dickinson, Oxford, UK) in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Paisley, UK) supplemented with 4 mmol / L L-glutamine (Invitrogen) and 5% fetal bovine serum (FBS) (Invitrogen). For seeding, cells were treated with trypsin-EDTA (Sigma-Aldrich®, Dorset, UK) for 5-10 minutes at 37°C in an incubator; FBS-supplemented DMEM was added after trypsinization to stop further reaction. Cells were pelleted by centrifugation at 1700 rpm for 1 minute and resuspended in medium. The concentration of the suspension was determined using a hemocytometer. For subsequent assays, cells were left overnight in an incubator (Heraeus, Hanau, Germany) at 37°C, 5% CO2 and 100% humidity 24 hours before pretreatment with a given drug for a given time.

[0143] Pharmacology: 1. Tetrodotoxin (TTX) was obtained from Alomone Labs (Jerusalem, Israel) and used at concentrations of 10-20 μM to partially block the voltage-gated sodium channels (VGSCs) present. TTX serves as a positive control because it is a known blocker of VGSCs.

[0144] 2. AG1478 was obtained from Sigma-Aldrich (Dorset, UK) and used at a concentration of 10 μM. It was dissolved in 0.1% DMSO, which was also used as a control solution.

[0145] 3. Ranolazine was obtained from Sigma-Aldrich (Dorset, UK). Four concentrations of ranolazine were used: 0.625, 1.25, 2.5, and 5 μM.

[0146] 4. Minoxidil was obtained from Alfa Aesar™ (Thermo Fisher Scientific, UK). Three concentrations of minoxidil were used: 2.5, 5, and 5 μM, except for one early immunocytology experiment where 50 μM was used. Minoxidil was dissolved in 2.5% DMSO, which was also used as the corresponding control solution.

[0147] Experiments were designed in pairs (i.e., cells were treated in parallel with pharmacological agents and their combinations) to allow for direct comparison and statistical analysis. In all cases, cells were pretreated with a given agent for 24 hours before the functional assay began (with a given treatment lasting the entire duration of the assay).

[0148] Ranolazine (2 mM) and minoxidil (31 mM) stock solutions were prepared by dissolving the drugs in DMEM and 100% dimethyl sulfoxide (Sigma-Aldrich), respectively, and frozen at -20°C until use. Control solutions for minoxidil and combination treatments were made by substituting DMSO for the final concentration of minoxidil. Fresh solutions were made at the desired concentrations by dilution into DMEM and warmed to 37°C before each experiment.

[0149] Treatments were either short-term / acute or long-term / 48 hours (functional assays). Normoxic incubations were performed in a humidified chamber, and hypoxic incubations (37°C, 5% CO2, 100% humidity and 1% O2) were performed in a hypoxic chamber (Micro Galaxy, RS Biotech Laboratory Equipment Ltd, Irvine, UK).

[0150] Cytotoxicity and proliferation: Cell viability was determined using a trypan blue dye exclusion assay. The assay was performed under both normoxic and hypoxic conditions. After the end of the treatment period, the medium was aspirated and replaced with 0.2 mL of 0.4% trypan blue (Sigma-Aldrich) and 0.8 mL of DMEM medium. The dishes were mixed and incubated in a humidified chamber for 10 minutes. The trypan blue solution was replaced with 1 mL of DMEM, and the dishes were observed under an inverted microscope (ID 03, Zeiss) at 100x magnification with phase contrast. Cell metabolic activity was assessed by determining the levels of NAD(P)H-dependent cellular oxidoreductase using the MTT assay. Cells were cultured at 2 × 10 4 Cells were seeded at 100 μL / well into 24-well plates (Becton Dickinson, Oxford, UK) and allowed to stand overnight before treatment. During treatment, 500 μL of solution was added to each well, and assays were performed under normoxic and hypoxic conditions. After treatment, 400 μL of DMEM and 100 μL of MTT were added to each well. The plates were incubated at 37°C for 3 hours under normoxic conditions. The MTT solution was aspirated and replaced with 500 μL of DMSO and 75 μL of Sörenson's glycine buffer. The plates were shaken at 150 cycles / min for 5 minutes to mix the DMSO and formazan. The absorbance of the resulting formazan was measured at 570 nm using a multiplate reader (ELX800 Universal Microplate Reader, Bio-Tek Instruments, UK). Duplicate measurements were recorded for each result, 5 minutes apart, and the average was calculated. A standard curve showing a linear relationship between cell number and absorbance values ​​was plotted, and raw absorbance values ​​were used for further analysis.

[0151] Matrigel Invasion: Cells were initially seeded at 5 x 10 cells / dish in 35 x 10 mm dishes (Becton Dickinson) and allowed to settle overnight. The total treatment time was 48 h, and cells were pretreated with drugs before the start of the assay. For the MDA-MB-231 cell line, the pretreatment time was 36 h, and for the MDA-MB-468 cell line, the pretreatment time was 28 h. Eight-micrometer pore transwell filters (Becton Dickinson) were placed in 24-well plates and coated with 50 mL of Matrigel® (Becton Dickinson) diluted to a 1.25 mg / mL to 10 mg / mL stock in FBS-free DMEM. The Matrigel was allowed to solidify overnight in an incubator. After the pretreatment period, inserts were hydrated with 500 μL of FBS-free DMEM before seeding and incubated at 37°C on the day of seeding. Cells were trypsinized, resuspended in treatment solution supplemented with 1% FBS, and placed in the upper chamber of the insert at 2 × 10 4 Cells were seeded at 100 μL per insert. A chemotactic gradient was created by adding 300 μL of a solution supplemented with 1% FBS to the upper chamber and 300 μL of a solution supplemented with 5% FBS to the lower chamber. The plates were incubated under hypoxic / normoxic conditions. The FBS-supplemented solution in both chambers was aspirated, and after the assay, the top of the insert was wiped with a cotton swab to remove non-invaded cells and Matrigel. Invaded cells were fixed with 300 μL of ice-cold 100% methanol for 15 minutes and stained with 300 μL of 0.5 g / mL crystal violet diluted in 25% methanol for 15 minutes, both of which were added to the lower chamber. The inserts were then washed with distilled water and allowed to dry before counting the invaded cells. Stained cells in 20 independent fields were counted at 400x magnification using an inverted microscope (Carl Zeiss, Hertfordshire, UK).

[0152] Immunocytology: Cells were plated at 5 × 10 in a 35 × 10 mm dish (Becton Dickinson). 5Cells were initially seeded at 1 cell / dish and allowed to settle overnight. Cells were pretreated with treatment solutions for 32 hours. Assays were performed only under hypoxic conditions. Prior to seeding, 13 mm cover glasses were placed in 24-well plates, and 500 μL of poly-L-lysine (Sigma) was added to each well for 20 minutes. After the pretreatment period, cells were trypsinized and 2 × 10 cells were added to 500 μL of the respective treatment solution. 4Cells were seeded at 1 cell per coverslip. Plates were incubated under hypoxic conditions, and the assay was allowed to proceed for 16 hours. At least two coverslips were prepared for each experiment. After the assay, coverslips were washed with phosphate-buffered saline (PBS) before fixation with 500 μL of 4% paraformaldehyde (Sigma) diluted in PBS. Fixation was for 10 minutes at room temperature on an orbital shaker. Coverlips were then washed three times for 5 minutes in PBS. One coverslip per condition was permeabilized with 500 μL of 0.1% saponin / PBS solution (Sigma) for 4 minutes, followed by three 5-minute PBS washes. All permeabilized and non-permeabilized coverslips were treated with 500 μL of 5% bovine serum albumin (BSA) / PBS (pH 7.4, volume / volume; Dako, Cambridge, UK) blocking agent for 1 hour. The coverslips were transferred from the 24-well plate to a humidity chamber and incubated overnight at 3–5°C with 100 μL of a 1:200 dilution of a 0.7 mg / mL stock primary antibody (NESO pAb) on an orbital shaker. The primary antibody used was NESO pAb, which specifically recognizes and binds to an external epitope sequence of the neonatal isoform of Nav1.5 VGSC (nNav1.5) (Chioni et al., 2005). The coverslips were then rinsed 3 times with PBS for 5 minutes and incubated with 100 μL of a 1:100 concentration of the secondary antibody, goat anti-rabbit IgG Alexa Fluor® 568 (Dako), in the dark for 1 hour at room temperature. A negative control coverslip (incubated with the secondary antibody alone) was also prepared to confirm the specificity of the primary antibody. Coverslips were then rinsed 3 x 5 min in PBS + 0.1% BSA and immersed in distilled water before mounting onto glass slides using mounting medium (Dako) and storing in the dark at 3-5°C. Images were taken with a Canon® digital camera inserted into an Axioimager immunofluorescence and phase-contrast inverted microscope (Zeiss) and processed with Remote Capture Software® (Canon). Corrected total cellular fluorescence was analyzed and quantified using ImageJ.

[0153] Electrophysiology: Whole-cell patch-clamp recordings are performed with cells superfused with mammalian physiological saline (MPS). Details of whole-cell recordings have been described previously (Fraser et al., 2003, 2005; Grimes et al., 1995; Laniado et al., 1997). Briefly, MPS contains (in mM): 144 NaCl, 5.4 KCl, 1 MgCl, 2.5 CaCl, 5 HEPES, and 5.6 D-glucose (adjusted to pH 7.3 with NaOH). Patch pipettes (tip resistance, approximately 5 MΩ) are inserted into the outward direction of the K + The cells were filled with a solution adjusted to pH 7.4 with 1 M CsOH designed to block currents (in mM): NaCl 5, CsCl 145, MgCl 2 2, CaCl 2 1, HEPES 10, and EGTA 11. Estimated intracellular free Ca 2+ The concentration is approximately 15 nM (Laniado et al., 2001). A holding potential of -100 mV is applied. A standard voltage clamp protocol is used to study the electrophysiological properties of VGSC currents. The following characteristics are primarily studied: peak current (and its density), current-voltage curve (current normalized to peak), steady-state inactivation / "efficacy" = test current (I) / maximum current (I max ); Recovery from inactivation = test current (I t ) / contrast current (I c ). To accurately determine the acute effects of minoxidil on peak current blockade, only currents greater than 200 pA are used. Further details of the voltage clamp protocol, data analysis, and curve fitting have been published previously (Onkal et al., 2008).

[0154] Data Analysis: A minimum of three biological replicates, each consisting of at least three technical replicates, were performed. For invasion assays, a minimum of three biological replicates, each consisting of two inserts, were performed. Normality of the data was confirmed by the Shapiro-Wilk W test. Parametric data were analyzed by Student's t-test and displayed by bar graphs (mean ± standard error of the mean). Nonparametric data were analyzed using the Mann-Whitney U test and displayed by box plots (median, interquartile range; 5% and 95% confidence intervals, and outliers shown). Significant results are indicated as * (P < 0.05), ** (P < 0.01), or *** (P < 0.001).

[0155] Example 2 Ranolazine and amiloride combination This example compares the effects of tetrodotoxin (TTX), ranolazine (5 μM), amiloride (100 μM), and the combination of ranolazine and amiloride (5 μM and 100 μM, respectively) on cell invasiveness under hypoxia (1% O2) using the materials and methods described in Example 1. TTX (20 μM) was used as a positive control. The results are shown in Figure 3.

[0156] Example 3 Ranolazine and minoxidil combination This example compares the effects of ranolazine, minoxidil, and their combination on cell invasiveness under hypoxia or normoxia using the materials and methods described in Example 1, particularly the Matrigel invasion assay.

[0157] Figure 4 shows the initial results.

[0158] Under hypoxia, minoxidil (50 μM and 5 μM) significantly reduced MDA-MB-231 cell invasion by 89% and 56%, respectively. Ranolazine (5 μM) reduced invasion by 42%, and its combination with 50 μM and 5 μM minoxidil resulted in a 100% and 54% reduction (P<0.001 for all) (Fig. 5a). In addition, the 11% increase in the degree of reduction between 50 μM minoxidil and the combined treatment was significant (P<0.05), but not with 5 μM minoxidil treatment (Fig. 4a). Reducing minoxidil to 2.5 μM resulted in a significant 48% reduction in invasion, while 5 μM ranolazine significantly reduced invasion by 43%, and the combined treatment caused a significant 58% reduction (Fig. 4b; P<0.001 for all). In addition, a significant enhancement was observed with the combination treatment compared with minoxidil alone (Fig. 5b; P<0.001).

[0159] The effect of decreasing ranolazine concentrations was also examined. 2.5 μM ranolazine significantly reduced infiltration by 36%, 2.5 μM minoxidil significantly reduced infiltration by 24%, and the combination treatment caused a significant 55% reduction (Figure 6a; P<0.001 for all). The combination treatment produced a significant enhancement compared to ranolazine treatment alone (Figure 6a; P<0.001). 1.25 μM ranolazine significantly reduced infiltration by 32%, and 2.5 μM minoxidil significantly reduced infiltration by 41%, and the combination treatment caused a significant 71% reduction (Figure 6b; P<0.001 for all). The combination treatment produced a significant enhancement compared to minoxidil treatment alone (Figure 6b; P<0.001). Finally, 0.625 μM ranolazine significantly reduced infiltration by 24%, 2.5 μM minoxidil significantly reduced infiltration by 42%, and the combination treatment caused a significant 53% reduction (Fig. 6c; P < 0.001 for all). The combination treatment was not significantly different from minoxidil treatment alone (Fig. 6c).

[0160] Next, the effects of the drugs were investigated under normoxia versus hypoxia. Under hypoxia, 2.5 μM ranolazine significantly reduced infiltration by 30% (P<0.001), but had no effect under normoxia (Figure 7). 2.5 μM minoxidil significantly reduced infiltration by 49% under hypoxia (Figure 7a) and 53% under normoxia (Figure 7b; P<0.001 for both), but these were not different from each other (P=0.49). In addition, combined treatment resulted in a significant reduction of 56% under hypoxia and 57% under normoxia (P<0.001 for both), a significant enhancement observed compared with minoxidil treatment under hypoxia (P<0.001), but not normoxia (P=0.89).

[0161] In a final set of experiments, the effects of ranolazine, minoxidil, and combination treatment under hypoxia were also tested on an additional BCa cell line (MDA-MB-468) also known to express functional VGSC activity (Aydar et al., 2016). 2.5 μM ranolazine significantly reduced invasion by 33% (Figure 8; P<0.01), and 2.5 μM minoxidil significantly reduced it by 44% (Figure 8; P<0.001). The combination treatment caused a significant 55% reduction in invaded cells (Figure 8; P<0.001), and this reduction was significantly greater when compared with minoxidil treatment (Figure 8; P<0.001).

[0162] To summarize: The results in Figures 5 and 6 show that the effects of ranolazine (RAN) were dose-dependent up to subclinical levels (625 nM), and that minoxidil (MIN) was also anti-invasive, potentiating the effects of RAN at all concentrations tested (p<0.001). The results in Figure 7 show that the anti-invasive effect of ranolazine (RAN) was only observed under hypoxia and was dose-dependent up to subclinical levels (625 nM), that MIN was also anti-invasive under both normoxia and hypoxia, and that the anti-invasive effect of RAN + MIN was greater than that of RAN alone under both normoxia and hypoxia. The results in Figure 8 show that RAN also inhibits the invasiveness of MDA-MB-468 cells, MIN is also anti-invasive, and the anti-invasive effect of RAN + MIN is greater than that of RAN alone.

[0163] Example 4 Control experiment The method used is described in Example 1.

[0164] PCR for three cell lines (MBA-MD-231, MBA-MD-468, and mouse 4T1) revealed that K ATP It was confirmed that the channel subunits K6.1, 6.2 and SUR1 / 2A / 2B mRNAs were expressed.

[0165] The highest concentrations of ranolazine (5 μM), minoxidil (50 μM), and their combination used in the experiment had no effect on cell viability of either MDA-MB-231 or MDA-MB-468 cell lines over 48 hours under normoxic or hypoxic treatment.

[0166] Similarly, the drug concentrations used in the motility and invasion studies had no effect on the growth of both MDA-MB-231 and MDA-MB-468 cell lines over a 48-hour period.

[0167] Example 5 Effect on nNav1.5 expression This example investigates the effects of ranolazine, minoxidil, and their combination on nNav1.5 expression levels. See Example 1 for materials and methods.

[0168] Immunocytochemical staining for nNav1.5 protein was performed under non-permeabilized and permeabilized conditions (Figures 9a and 9b, respectively). As expected, compared with the non-permeabilized condition, fluorescence levels were significantly higher in both DMEM and DMSO controls by 12% and 48%, respectively (both P<0.001). This is consistent with the non-permeabilized condition detecting primarily "plasma membrane" expression, whereas the permeabilized condition detected "total" (i.e., both plasma membrane and intracellular nNav1.5 protein). Ranolazine (5 μM) significantly reduced nNav1.5 protein expression by 30% and 43%, respectively, under both non-permeabilized and permeabilized conditions (both P<0.001). Minoxidil (50 μM) and the drug combination significantly reduced nNav1.5 expression in permeabilized cells by 49% and 37%, respectively (Figure 9b; P<0.001 and P<0.01, respectively). In contrast, in non-permeabilized cells, neither minoxidil nor the drug combination had a significant effect (Fig. 9a; P = 0.58 and P = 0.18, respectively). In addition, the reducing effect of the combination treatment on nNav1.5 expression was significant in non-permeabilized cells compared with ranolazine (Fig. 9a; P < 0.05). However, there was no significant difference in the effect of the drug combination in permeabilized cells.

[0169] Example 6 Combination of AG1478 and TTX This example compares the effects of AG1478 (10 μM), TTX (10 μM), and their combination on the invasiveness of the MDA-MB-231 cell line in normoxia. The control is 0.1% DMSO.

[0170] The results are shown in Figure 10. Briefly, Figure 10a shows typical microscopic images of invaded cells under the four treatment conditions. Quantitative data, plotting invasiveness normalized to the control, are shown in Figure 10b. TTX has a small effect, but it is not significant (the concentration was kept low so as not to obscure any combined effect of the combination). AG1478 has a significant inhibitory effect on invasiveness. The combination of TTX and AG1478 is significantly greater than the individual effects of TTX or AG1478 (p = 0.02 and 0.006, respectively).

[0171] Example 7 Electrophysiological measurements The results are shown in Figures 11 and 12. Briefly, short-term (acute) application of minoxidil (50 μM) had no effect on peak currents (consistent with the lack of effect of minoxidil on plasma membrane nNav1.5 protein expression), but delayed recovery times and shifted steady-state inactivation to more hyperpolarized potentials, meaning that VGSCs were less likely to activate (Figure 11). The lack of any change in VGSC characteristics after chronic (48 h) treatment with 50 μM minoxidil (followed by washout) indicates that minoxidil must be present to produce any effect (Figure 12).

[0172] Each reference cited in this application, including each reference listed below, is specifically incorporated herein by reference in its entirety. (References) TIFF0007783630000002.tif134170

Claims

1. 1. A composition for use in treating or preventing cancer in a subject, the composition comprising ranolazine in combination with minoxidil.

2. a) ranolazine, and b) Minoxidil 2. A kit of parts for separate, sequential or simultaneous use in a method for treating or preventing cancer in a subject, comprising:

3. A pharmaceutical composition comprising ranolazine, used in combination with minoxidil, for treating or preventing cancer in a subject.

4. A pharmaceutical composition comprising minoxidil, used in combination with ranolazine, for treating or preventing cancer in a subject.

5. A pharmaceutical composition for treating or preventing cancer in a subject, comprising as active ingredients ranolazine and minoxidil in admixture with a pharmaceutically acceptable carrier, diluent, vehicle and / or excipient.

6. Use of ranolazine in combination with minoxidil in the preparation of a medicament for treating or preventing cancer in a subject.

7. Use of minoxidil in combination with ranolazine in the preparation of a medicament for treating or preventing cancer in a subject.

8. The composition of claim 1, the kit of parts of claim 2, or the pharmaceutical composition of any one of claims 3 to 5, wherein one or more tumors in a subject express a VGSC.

9. 8. The use according to claim 6 or 7, wherein one or more tumors in the subject express a VGSC.

10. A composition according to claim 1 or 8, a kit of parts according to claim 2 or 8, or a pharmaceutical composition according to any one of claims 3 to 5 and 8, wherein the VGSC is selected from Nav1.5, Nav1.7, Nav1.6, Nav1.2 and any combination of two or more thereof, and optionally one or more of Nav1.5, Nav1.7, Nav1.6 and Nav1.2 is in neonatal form.

11. 10. The use of any one of claims 6, 7 and 9, wherein the VGSC is selected from Nav1.5, Nav1.7, Nav1.6, Nav1.2 and any combination of two or more thereof, and optionally one or more of Nav1.5, Nav1.7, Nav1.6 and Nav1.2 is in neonatal form.

12. 11. The composition, kit-of-parts, or pharmaceutical composition of claim 10, wherein one or more tumors in the subject express neonatal Nav1.5 (nNav1.5).

13. 12. The use of any one of claims 6, 7, 9, and 11, wherein one or more tumors in the subject express neonatal Nav1.5 (nNav1.5).

14. The composition of any one of claims 1, 8, 10 and 12, the kit of parts of any one of claims 2, 8, 10 and 12, or the pharmaceutical composition of any one of claims 3 to 5, 8, 10 and 12, wherein the cancer is breast cancer, colorectal cancer, prostate, non-small cell lung cancer (NSCLC), mesothelioma, cervical cancer, gastric cancer, ovarian cancer, melanoma, oral squamous cell carcinoma, astrocytoma, neuroblastoma, or any combination thereof.

15. 12. The use of any one of claims 6, 7, 9 and 11, wherein the cancer is breast cancer, colon cancer, prostate, non-small cell lung cancer (NSCLC), mesothelioma, cervical cancer, gastric cancer, ovarian cancer, melanoma, oral squamous cell carcinoma, astrocytoma, neuroblastoma, or any combination thereof.

16. A composition according to any one of claims 1, 8, 10 and 12, a kit of parts according to any one of claims 2, 8, 10 and 12, or a pharmaceutical composition according to any one of claims 3 to 5, 8, 10 and 12, which prevents, reduces or inhibits the metastatic behaviour of cancer, the invasiveness of cancer, the subject's pain, the overall aggressiveness of cancer, or a combination of any two or more thereof.

17. 14. The use of any one of claims 6, 7, 9, 11, and 13, wherein the metastatic behavior of the cancer, the invasiveness of the cancer, the subject's pain, the overall aggressiveness of the cancer, or a combination of any two or more thereof, is prevented, reduced, or inhibited.

18. A composition described in any one of claims 1, 8, 10 and 12, a kit of parts described in any one of claims 2, 8, 10 and 12, or a pharmaceutical composition described in any one of claims 3 to 5, 8, 10 and 12, wherein ranolazine is administered in an amount effective to at least partially block the sustained portion of the VGSC current without completely blocking the transient portion of the VGSC current.

19. The use described in any one of claims 6, 7, 9, 11, and 13, wherein ranolazine is administered in an amount effective to at least partially block the sustained portion of the VGSC current without completely blocking the transient portion of the VGSC current.

20. The composition, kit of parts, or pharmaceutical composition of claim 18, wherein ranolazine essentially blocks the sustained portion of the VGSC current.

21. The use of claim 19, wherein ranolazine essentially blocks the sustained portion of the VGSC current.

22. A composition described in any one of claims 1, 8, 10, and 12, a kit of parts described in any one of claims 2, 8, 10, and 12, or a pharmaceutical composition described in any one of claims 3 to 5, 8, 10, and 12, wherein ranolazine and minoxidil are administered to a subject individually, sequentially, or simultaneously.

23. The use described in any one of claims 6, 7, 9, 11, and 13, wherein ranolazine and minoxidil are administered to a subject separately, sequentially, or simultaneously.

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