Treatment of cancer / inhibition of metastasis

Abstract

To provide substances and methods for reducing or preventing metastatic behavior in voltage-gated sodium channel expressing cancer by the effect of at least reducing the persistent part of the current without eliminating the transient part of VGSC current, and to use the known drugs ranolazine and riluzole whose inhibition of metastatic cell behaviors such as detachability, lateral motility, transverse migration, and invasiveness are demonstrated.SOLUTION: There is provided ranolazine for reducing or preventing the metastasis behavior in VGSC expressing cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the treatment of cancer, particularly but not exclusively, metastatic cancer such as breast or prostate cancer. [Background technology]

[0002] The progression of metastatic cancers, such as breast and prostate cancer, is generally considered to involve five phases: 1. Genesis, i.e. the initial transformation of a normal cell into a cancer cell. 2. Proliferation, i.e., the cancer cells increase in number and form a primary tumor that gradually increases in size. 3. The switch of cancer cells from a state without the potential for metastatic behavior to a state with the potential for metastatic behavior during the developmental or proliferation phase. 4. Detachment of cancer cells from the primary tumor and their subsequent migration 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, forming secondary tumors in other organs.

[0003] The prominent change that occurs in the cells and causes the state switch in phase 3 is the expression of a functional voltage-gated sodium channel (VGSC). In breast cancer, this is the expressed Navl.5 channel, and in prostate cancer, this is the Navl.7 channel. VGSC can be expressed in neonatal and / or adult forms. In breast cancer, this is the expressed neonatal form of the Navl.5 channel (nNavl.5). In prostate cancer, it is currently not known which form is expressed. In the absence of such a channel, the cells do not have invasive potential and therefore no metastatic behavior.

[0004] In some cases, the developmental phase involves the growth of cancer cells that have metastatic potential from the beginning.

[0005] Proposal of prior art to prevent metastasis Prior to the present invention, the focus in the field was on attempting to find treatments to prevent metastasis by one or more of the following: (a) prevent the expression of functional VGSCs; (b) completely block the activity of the expressed functional VGSC; or (c) Killing cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2009 / 105230A2 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention proposes a different approach. [Means for solving the problem]

[0008] Current flows intermittently through VGSCs, i.e., the current flows in pulses. It is known that each pulse contains a transient (or peak) portion followed by a low level DC portion (known as the late or sustained current). It is also known that appropriate doses of the known drugs ranolazine or riluzole inhibit the sustained portion of the current, leaving the transient portion unaffected or only partially reduced.

[0009] Experimental work carried out in connection with the present invention, described more fully below, has demonstrated the following: (i) Inhibition of the tonic portion of Navl.5 and Navl.7 currents in breast and prostate cancer, respectively, inhibits metastatic behavior; (ii) inhibition of the transient portion of these currents is not necessary to inhibit metastatic behavior; (iii) ranolazine or riluzole at appropriate doses will inhibit metastatic behavior without preventing the growth or destroying the tumor cells; and (iv) The inhibitory effect of ranolazine and riluzole on the tonic part of the current was higher in cells previously exposed to hypoxia, a condition occurring in growing tumors and contributing crucially positively to the metastatic process.

[0010] Both ranolazine and riluzole are known for the treatment of cardiac conditions. They are further known to affect differently the magnitude of the transient and sustained parts of VGSC currents, and the effect is in a dose-dependent manner. High doses of these drugs completely block VGSC currents. Because the heart requires these currents to perform its function, the administration of these drugs or any other drug that has the effect of completely blocking VGSC currents in cardiac tissue is lethal to the patient.

[0011] However, according to one aspect of the present invention, metastatic behavior in cancer is inhibited or reduced by administering ranolazine or riluzole or another substance in a dosage appropriate to inhibit or reduce the persistent part of the VGSC current without blocking the transient part of the VGSC current, or at least without completely blocking the transient part. Thus, metastasis in cancer can be inhibited or reduced in this way without the need to administer lethal doses of drugs.

[0012] Riluzole has already been proposed in the treatment of certain cancers, particularly prostate cancer and melanoma, In both cases, it was proposed that riluzole should be administered in such a way that the cancerous cells are killed.

[0013] According to further aspects of the invention, which are described more fully below, riluzole or ranolazine are administered at dosage levels that inhibit the sustained portion of the VGSC current without blocking the transient portion of the VGSC current or without completely blocking the transient portion and without directly causing cell death.

[0014] The fact that metastatic behavior can be inhibited or reduced without causing cell death can be a significant advantage, since recent studies suggest that treating cancer by killing cells can, at least in some cases, be counterproductive in the sense that, although there is a short-term benefit, the cancer nevertheless returns and grows. Thus, the present invention offers the possibility of inhibiting or preventing metastatic behavior without the potential problems that can arise from actually killing cancer cells.

[0015] The transition process involves several steps: (a) Detachment of cells from a tumor; (b) migration of exfoliated cells into the surrounding tissue; (c) migration through peripheral tissues toward the circulatory system; and (d) migration into the circulatory system, where the cells can eventually leave and form secondary tumors.

[0016] Thus, inhibiting or reducing the activity of cells at any one or more of these steps will at least contribute to reducing metastasis. The effect of a drug on each of these substeps can be determined by a number of experiments, as described more fully below: (a) Testing the effect of drugs on cell adhesion; (b) testing the effect of drugs on the lateral motility of cells; (c) testing the effect of drugs on the transverse migration of cells; and (d) Examining the effect of drugs on cell invasiveness (i.e., the ability of cells to migrate through the medium they consume).

[0017] As described more fully below, experiments conducted in connection with the present invention demonstrate that administration of ranolazine or riluzole at various dosage levels can increase cell adhesion and / or decrease one or more of cell lateral motility, transverse migration and invasiveness.

[0018] Thus, according to another aspect of the present invention, there is provided a compound, composition or other substance that is used or intended to be used in a suitable dose to inhibit or reduce the persistent portion of VGSCs in metastatic cancer cells, preferably to inhibit or reduce metastasis without directly causing cell death, with the transient portion being unaffected or only partially reduced.

[0019] Advantages derived from the present invention, at least in certain aspects or forms, are: (a) may contain breast and prostate cancer (as well as other cancers in which VGSCs are expressed) such that patients may be able to live with such cancers without significant disadvantage; (b) as a result, the need for the patient to undergo aggressive treatment to destroy cancerous cells (such as with chemotherapy or radiation therapy) may be avoided; (c) if a patient is suspected of having breast or prostate cancer or other metastatic cancer, immediate treatment with appropriate doses of ranolazine or riluzole (or other substances with suitable properties) may be given to inhibit or prevent metastasis while awaiting the results of confirmatory tests; (d) the dosage required to achieve this only needs to be high enough to inhibit the tonic portion of the VGSC current; (e) therapeutically acceptable doses of ranolazine or riluzole achieve the required inhibition of the tonic portion of these currents, while the transient portion remains substantially unaffected; and (f) Because ranolazine and riluzole have been on the market and approved for human use for many years, the present invention can be put into clinical use without having to go through all the long-term testing for side effects, etc.

[0020] The invention is further described with reference to the accompanying drawings and experimental data presented below. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a schematic diagram of the timeline of cancer progression from primary tumor initiation to the formation of secondary tumors (metastasis). [Diagram 2] FIG. 1 is a schematic diagram of the cellular processes that occur during cancer initiation and progression to metastasis. [Diagram 3] (3a) Sketch showing VGSC-mediated currents showing both the transient and sustained portions of the currents, and also showing currents under both normoxic and hypoxic conditions. (3b) Sketch showing the effect of different concentrations of ranolazine on the VGSC current components. (3c) Sketch showing the effect of different concentrations of riluzole on the VGSC current components. [Figure 4] FIG. 1 is a schematic diagram of a cell adhesion measuring device for measuring the adhesion of cells individually. [Diagram 5] Schematic diagram of the apparatus used to measure lateral motility of cells. (a) is a top plan view of a cell culture dish containing a semi-confluent layer of cells. (b) is a schematic cross-sectional side view of plated cells. (c) is a plan view of plated cells at time t=0, when a scar was made through the layer of cells. (d) is a plan view of plated cells at a later time (t=24 hours) when the cells have migrated and the wound has partially closed. [Figure 6] FIG. 1 is a schematic cross-sectional side view of an apparatus used to measure the transverse migration of cells. [Figure 7] FIG. 1 is a schematic cross-sectional side view of an apparatus used to measure the invasiveness of cells. [Figure 8]FIG. 1 is a graph showing the concentration-dependent effect of induced chemical hypoxia on single cell adhesion of human metastatic breast cancer MDA-MB-231 cells. [Figure 9] FIG. 1 is a graph showing the dose-dependent effect of ranolazine on single cell adhesion of MDA-MB-231 cells under normoxia and chemically induced hypoxia. [Figure 10] 1 is a histogram showing the dose-dependent effect of ranolazine on the lateral motility of MDA-MB-231 cells under normoxia and hypoxia. [Figure 11] 1 is a histogram showing the dose-dependent effect of ranolazine on the transverse migration of MDA-MB-231 cells under normoxia and hypoxia. [Figure 12] 1 is a histogram showing the dose-dependent effect of ranolazine on the invasiveness of MDA-MB-231 cells under normoxia and hypoxia. [Figure 13] FIG. 1 is a series of histograms showing the dose-dependent effect of ranolazine on the invasiveness of MDA-MB-231 cells pretreated with ranolazine for different durations under hypoxia. (a) is a histogram showing the effect of 5 μM ranolazine for cells treated for only 24 hours during the assay (i.e., no pretreatment). (b) is a histogram showing the effect of 5 μM ranolazine for cells pretreated with drug for 72 hours. (c) is a histogram showing the effect of 25 μM ranolazine for cells treated for only 24 hours during the assay (i.e., no pretreatment). (d) is a histogram showing the effect of 25 μM ranolazine for cells pretreated with drug for 48 hours. (e) is a histogram showing the effect of 25 μM ranolazine for cells pretreated with drug for 72 hours. [Figure 14] 1 is a series of histograms showing the lack of effect of ranolazine on the growth of MDA-MB-231 cells under normoxia. [Figure 15] 1 is a histogram showing the lack of effect of ranolazine on MDA-MB-231 cell viability under normoxia. [Figure 16] 1 is a histogram showing the dose-dependent effect of ranolazine on the transverse migration of highly metastatic rat prostate cancer Mat-LyLu cells under normoxia and hypoxia. [Figure 17] 1 is a histogram showing the dose-dependent effect of ranolazine on the invasiveness of Mat-LyLu cells under normoxia and hypoxia. [Figure 18] 1 is a histogram showing the lack of effect of ranolazine on proliferation of Mat-LyLu cells under normoxia and hypoxia. [Figure 19] 1 is a histogram showing the lack of effect of ranolazine on Mat-LyLu cell viability under normoxia and hypoxia. [Figure 20] 1 is a histogram showing the dose-dependent effect of riluzole on lateral motility of MDA-MB-231 cells under normoxia and hypoxia. [Figure 21] 1 is a histogram showing the dose-dependent effect of riluzole on the transverse migration of MDA-MB-231 cells under normoxia and hypoxia. [Figure 22] 1 is a histogram showing the dose-dependent effect of riluzole on the invasiveness of MDA-MB-231 cells under normoxia and hypoxia. [Figure 23] FIG. 1 is a pair of histograms showing the comparative effects on invasiveness of MDA-MB-231 cells pretreated with 5 μM riluzole for >72 hours under hypoxia. Panel (a) is a histogram showing the effect of 5 μM riluzole on cells treated for only 24 hours during the assay (i.e., no pretreatment). Panel (b) is a histogram showing the effect of 5 μM riluzole on cells pretreated with the drug for 72 hours. [Figure 24] 1 is a histogram showing the dose-dependent effect of riluzole on the growth of MDA-MB-231 cells under normoxia. [Diagram 25] 1 is a histogram showing the dose-dependent effect of riluzole on the viability of MDA-MB-231 cells under normoxia. [Figure 26]1 is a histogram showing the dose-dependent effect of riluzole on the invasiveness of Mat-LyLu cells under normoxia and hypoxia. [Figure 27] 1 is a histogram showing the dose-dependent effect of riluzole on the growth of Mat-LyLu cells under normoxia and hypoxia. [Figure 28] 1 is a histogram showing the dose-dependent effect of riluzole on the viability of Mat-LyLu cells under normoxia and hypoxia. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 1, a timeline 101 shows three successive phases in the development of a tumor: a phase 102 prior to the development of cancerous cells, a phase 103 following phase 102 during which the development of cancerous cells occurs, and a phase 104 following phase 103 during which the cancerous cells proliferate to form a growing tumor. The proliferation phase 104 can begin shortly after the development phase 103 begins.

[0023] It has been demonstrated that human breast and prostate cancer cells may not contain any functional VGSC at first, and that tumor cells will not become invasive unless such channels are expressed in the tumor. However, in many such tumors, even if no VGSC is present at first, functional VGSC is expressed at some point. This induces a change to the conditions under which tumors can spread. Figure 1 shows a situation in which cells do not contain any functional VGSC at first, but at some point during time 105, functional VGSC begins to be expressed. This can occur at any time after the start of the development phase 103.

[0024] Timeline 106 in FIG. 1 shows the phase occurring after time 105, at which point the cancer becomes metastatic.

[0025] In the first phase 107 after time 105, metastatic cells detach from the tumor. Then, in phase 108, they invade and migrate through surrounding areas within the same organ toward the circulatory system (particularly the vascular and / or lymphatic systems). In phase 109, metastatic cells enter the circulatory system and may then migrate to other organs throughout the body where they may cause the formation of secondary tumors.

[0026] The above phases are illustrated pictorially in Figure 2, where reference numeral 200 denotes a portion of an organ such as the breast or prostate. Healthy cells 201 of the breast or prostate are shown supported on a basement membrane 202 and surrounding a cancerous tumor 203. The cancerous tumor 203 is presumed to have passed through the development phase 103 and entered the proliferation phase 104.

[0027] Certain cells 204 of a cancerous tumor 203 are shown detaching from the tumor 203 and passing through a degraded region 202a of the basement membrane 202 into an adjacent region 205 of the organ containing the tumor 203, which may contain primarily collagen fibers. Cancer cells 206, having detached from the tumor and passing through the basement membrane 202, are shown passing through region 205 toward a blood vessel 207. Cancerous cells 208 are shown migrating through the wall of blood vessel 207 into the bloodstream 209.

[0028] Cells 210 already in the bloodstream are shown being carried within the bloodstream to region 211 where cells 212 are shown to migrate outward through the wall of blood vessel 207 toward another organ 213, such as a lymph node or the liver, where they may form a secondary tumor (not shown).

[0029] Reference numeral 214 denotes dormant cancerous cells that are simply settled on or adjacent to the wall of blood vessel 207 .

[0030] As will be explained more fully below, the present invention provides treatments or means for preventing or reducing one or more of the metastatic behavior of cancer cells occurring in the various phases described. In particular, the present invention provides treatments or means for: (a) increasing the adhesiveness of cells in the tumor, making them less likely to detach; and / or (b) reducing the motility of detached cells, making them less likely to migrate into and through the basement membrane into the surrounding tissue; and / or (c) decreasing the invasiveness of cells that have entered the surrounding tissue by decreasing the ability of the cells to migrate through the tissue toward the circulatory system; and / or (d) reducing the ability of cells to migrate from the tissue through the wall into the circulatory system.

[0031] It has been explained above that cancerous cells that do not express functional VGSCs do not behave invasively. Furthermore, it is known that current passes through the VGSC in pulses, each of which includes a transient or peak portion followed by a sustained or late portion at a much lower level. According to one aspect of the present invention, one or more of the above metastatic behaviors are inhibited or reduced by inhibiting or reducing the sustained portion of the current without eliminating the peak portion, making it possible to use drugs that preferentially reduce the sustained portion of the current.

[0032] Some such drugs are known for treating cardiac conditions such as arrhythmia or angina pectoris. When treating the heart, it is important to ensure that the peak portion of the current is not eliminated, as it is essential for maintaining the functionality of the heart and its rhythm. Thus, according to one aspect of the present invention, known drugs such as ranolazine or riluzole, which have been used previously to inhibit or reduce the tonic portion of VGSC current without eliminating the peak portion, are used to inhibit or reduce metastatic behavior in cancer, particularly breast or prostate cancer.

[0033] The nature of the VGSC current, and the effect of treatment with ranolazine or riluzole thereon, is further illustrated with reference to Figures 3(a), 3(b) and 3(c).

[0034] Referring to Figure 3(a), a continuous line curve 301 shows a current pulse passing through a functional VGSC under normoxic conditions, with the horizontal axis being time and the vertical axis being the amplitude or magnitude of the current. As can be seen, this current pulse includes a peak or transient portion 302 and a sustained or late portion 303. In fact, the duration of the sustained portion 303 is much greater than that of the transient portion 302, but this is not shown in the figure since Figure 3(a) is a graphical sketch rather than a curve actually derived from experimental data.

[0035] The dashed-dotted curve 304 shows the pulse of VGSC current under hypoxic conditions. As can be seen, the peak portion 305 of the current under hypoxic conditions is smaller than the peak portion 302 under normoxic conditions, while the sustained portion 306 under hypoxic conditions is larger than the sustained portion 303 under normoxic conditions. As will become apparent from a review of the experimental results described below, the difference between these curves under hypoxic and normoxic conditions is significant because many of the cells in a cancerous tumor are hypoxic due to partial sequestration from the blood circulation by other cancerous cells.

[0036] 3(b) is a sketch showing the effect of increasing doses of ranolazine on the transient and tonic portions of the VGSC current, respectively. As can be seen, the horizontal axis shows the ranolazine dosage level and the vertical axis shows the normalized VGSC current. The solid curve 305 shows the magnitude of the transient portion of the current plotted against increasing dosages, and the dashed curve 306 shows the magnitude of the tonic portion of the current against increasing ranolazine dosages, with dosage levels shown on the horizontal axis.

[0037] As can be further seen from Figure 3(b), at dosage levels in the range 1-10 μM (therapeutically acceptable to humans), the sustained portion of the current is reduced more significantly than the transient portion. The difference between the two reductions is indicated in Figure 3(b) by the double arrow 307.

[0038] A similar effect with increasing doses of riluzole can be seen in Figure 3(c), where solid curve 308 shows the magnitude of the transient portion of the current plotted against increasing doses of riluzole, and dashed curve 310 shows the magnitude of the tonic portion of the current. Therapeutically acceptable doses of riluzole for humans include the range of 1 μM to 10 μM. As can be seen in Figure 3(c) and as indicated by double arrow 311, the decrease in the magnitude of the tonic portion of the current is much greater than the decrease in the magnitude of the transient portion.

[0039] As with FIG. 3(a), the curves in FIGS. 3(b) and 3(c) are not based on specific experimental results but are sketches intended to illustrate current flow.

[0040] The experiments described below more fully were carried out to measure the effect of various dosage levels of ranolazine and riluzole on one or more of the metastatic behaviors of certain cancer cell lines.Specifically, in these experiments, one or more of the following measurements were carried out for each of these drugs at various dosage levels: cell adhesion, cell lateral motility, cell invasion, and cell transverse migration.Furthermore, experiments were carried out to determine the effect of some of these doses on cell proliferation activity and cell viability (i.e., whether the drug kills cells).

[0041] Before describing the experiments and the quantitative results obtained therefrom, the following table qualitatively summarizes the results obtained. From these tables, and the subsequent detailed consideration of the experiments and the results obtained, it can be seen that the reduction of various metastatic behaviors can be achieved at a therapeutically acceptable level without affecting cell proliferation or killing cells. The latter may be particularly important, since it has recently been suggested that treating cancer by killing cells may be counterproductive, since cancer may reappear in a more aggressive form after the killing treatment is discontinued. Thus, preventing or reducing the invasiveness of cancerous cells without killing them may be a treatment that has considerable advantages over conventional cell-killing treatments.

[0042] Tables 1 and 2 summarize the results of experiments with various dosage levels of ranolazine on human breast cancer cells and rat prostate cancer cells (rat prostate cells are similar to human prostate cells). Tables 3 and 4 summarize the results obtained by treating cells of the same cell lines with various dosage levels of riluzole.

[0043] [Table 1]

[0044] [Table 2]

[0045] [Table 3]

[0046] [Table 4]

[0047] Single cell adhesion assay FIG. 4 is a schematic diagram of the single-cell adhesion measurement apparatus (SCAMA) first described in the paper by Palmer et al. (2008).

[0048] Human breast cancer cells derived from the MDA-MB-231 cell line were cultured at 2.5 × 10 4 Cells were plated at a density of 1000 cells / ml and left to rest in cell culture dishes 401 for 48 hours before measurements. The medium was removed and 2 ml of study drug was applied for 10 min. Adhesion was measured using a glass micropipette 402 connected to a vacuum pump 403 via plastic tubing 404. The tip of the micropipette was withdrawn to a tip diameter of approximately 20 μm (range 17-24 μm). A vacuum pump was used to generate negative pressure inside the reservoir 405 and this negative pressure was applied to the tip of the micropipette by pressing a thumb against the open end of a sealable T-piece 406. Cells were observed using a 20× microscope objective 407 under illumination from a lamp 408. Pressure was measured using a digital manometer 409 connected to a computer 410 via an RS232 cable 411.

[0049] Using the micromanipulator 412, the micropipette 402 was positioned around a single cell. Upon closing the T-piece 406, negative pressure was applied to the cell under investigation, and just as the cell was observed to detach from the culture dish 401, the pressure was released by opening the T-piece 406. The negative pressure required to detach the cell was recorded on the computer as a pressure spike. The peak of the spike ("detachment negative pressure" (DNP)) was used as a measure of the adhesion of the cell. Using this technique, several recordings were taken from a single dish within a few minutes.

[0050] To simulate hypoxic conditions for the cells, hypoxia was chemically induced by application of hydrogen peroxide (1-500 μM) for the final 24 h prior to testing.

[0051] To test for the reversibility of a given effect, the pharmacological agent was washed away, fresh medium was added, and the plate was incubated for an additional 10 min before re-measurement. Each treatment was performed on at least two dishes of cells, at least 100 cells / dish were measured, and experiments were repeated three times (with corresponding controls).

[0052] Lateral motility assay This assay was used to demonstrate the "free" motility of cancer cells during localized spreading. Figure 5(a) shows a top plan view of a cell culture dish 501 with a semi-confluent layer of cells 502 on its surface, the cells being present in an aqueous medium 503.

[0053] To determine lateral motility, a "wound healing ("scratch") test was performed in which a scratch 504 of approximately 0.5 mm was made through the layer of cells, as shown in Figure 5(b), a cross-sectional side view of a cell culture dish.

[0054] During the 24 hour period following scratch formation, cells migrated into the gap.

[0055] 5(c) and 5(d) are graphs showing the time t=0, when the width of scratch 504 is w0, and the time t=1, when the width of scratch 504 is w 24 5 is a schematic plan view of cell culture dish 501 at a point in time, t=24 hours.

[0056] Transverse migration assay This assay was used to demonstrate the migration ability of cells as they intravasate / extravasate. Figure 6 shows a schematic side cross-sectional view of a migration chamber 601 with a Transwell® insert 602 that separates the chamber into two compartments, referred to for convenience as the upper compartment 603 and the lower compartment 604 of the chamber. The insert 602 has a migration filter membrane 605 with 8 μm pores 606 extending through its base.

[0057] Cells 607 were plated on filter membrane 605 at 2 × 104 The cells were plated at a density of 100 / ml and placed under growth medium 608 containing 1% fetal bovine serum (FBS). A chemotactic gradient was generated across the filter membrane 605 by placing growth medium 609 containing 10% FBS in the lower compartment 604 of the chamber.

[0058] The cells were allowed to migrate across the filter membrane 605 over a 24 hour period, whereupon the cells migrated to and adhered to the underside of the filter membrane 605 .

[0059] At the end of each assay, non-migrated cells were removed from the upper surface of the insert 602 with two separate wipes.

[0060] The number of cells that migrated to the underside of the insert 602 was determined using crystal violet staining. The migrated cells were fixed with ice-cold methanol for 15 minutes. 0.5% crystal violet (in 25% methanol) was then added for 15 minutes. The inserts were wiped again, then washed in water and dried. The cells were then counted using 12 separate fields (magnification x200) per insert.

[0061] Invasion assay This assay is an extension of the transverse migration assay described above. To "invade", cells need both (i) to migrate, as in the transverse migration assay, and (ii) to secrete proteolytic enzymes that digest their surroundings. Therefore, the ability of cells to invade adjacent tissues by secreting enzymes was assessed by using a layer of Matrigel™ (BD Biosciences) spread across the porous membrane of a Transwell® insert. Matrigel™ is composed of laminin, type IV collagen, nidogen / enactin, and proteoglycans (compositions comparable to basement membrane proteins).

[0062] 7 is a schematic cross-sectional side view of an invasion chamber 701 with a Transwell® insert 702 that separates the chamber into an upper compartment 703 and a lower compartment 704. The insert 702 has a transfer filter membrane 705 with 8 μm pores 706 extending through its base. A Matrigel™ layer 707 is shown covering the filter membrane 705.

[0063] Cells 708 were plated at 2×10 6 onto a Matrigel™ layer 707 in a 24-well plate (Becton-Dickinson) according to the manufacturer's instructions. 4 The inserts were plated at a density of 100 / ml. 50 μl of Matrigel™ was seeded onto the inserts at a 1:7 dilution (10 mg / ml stock) overnight. Prior to seeding the cells, the Matrigel™ was rehydrated using medium without supplements. This medium was removed before the cells were seeded.

[0064] Cells were plated overnight (12 hours) in a chemotaxis gradient of 1-5% FBS. The nutrient concentration in medium 709 in the upper compartment 703 of the chamber was lower than the concentration of nutrients in medium 710 in the lower compartment 704, inducing migration of cells through the Matrigel™ layer 707 and pores 706 to the underside of the filter membrane 705. At the end of each assay, non-invading / non-migrating cells were removed from the upper surface of the insert 702 with two separate wipes.

[0065] The number of cells that invaded the underside of the insert 702 was determined using crystal violet staining. The invaded cells were fixed with ice-cold methanol for 15 minutes. Then, 0.5% crystal violet (in 25% methanol) was added for 15 minutes. The inserts were wiped again, then washed in water and dried. The cells were then counted using 12 separate fields (magnification x200) per insert. If the difference in the average number of cells that invaded the two control inserts was higher than 40%, the experiment was rejected.

[0066] Cell viability assay Cells were plated at 5 × 10 in 35 mm Falcon tissue culture dishes. 4 Cells were seeded at a density of 10 ...

[0067] Cell growth (proliferation) assay Cells were plated at 2 × 10 4 Cells were plated at 1000 cells / ml and left overnight. The cells were then treated for the required incubation time (24 h+), with medium replaced every 24 h. At the end of treatment, the medium was removed, followed by a colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (Grimes et al., 1995). Briefly, 0.1 ml of MTT (made up to 5 mg / ml in growth medium) and 0.4 ml of growth medium were added to each well, and the plates were incubated at 37°C for 3–4 h. The medium was then removed from the chamber and replaced with 0.5 ml dimethyl sulfoxide (DMSO) and 0.063 ml glycine buffer (0.1 M glycine and 0.1 M NaCl; pH 10.5). 15 min after addition of glycine buffer, the absorbance at 570 nm was determined. Results were calculated as the average of nine replicate spectrophotometer readings of each treatment vs. control from individual infiltrated wells.

[0068] tissue culture Experiments were performed on two highly metastatic cell lines: (i) human metastatic breast cancer MDA-MB-231, and (ii) The highly metastatic rat prostate cancer Mat-LyLu. Cells were cultured using known methods (eg, Grimes et al., 1995; Fraser et al., 2005).

[0069] Normoxic and hypoxic conditions With the exception of the single cell adhesion studies discussed in the following paragraphs, experiments were performed under either: (i) normal normoxic conditions (95% oxygen, 5% carbon dioxide), or (ii) Pretreatment with hypoxia (2% O2, 5% CO2, 93% N2) for the next 24 h, which lasted throughout the assay.

[0070] In single cell adhesion experiments, hypoxia was chemically induced by application of hydrogen peroxide (1–500 μM) for 24 h.

[0071] (Example) Example 1 Effect of chemical hypoxia on single-cell adhesion of MDA-MB-231 cells Chemical hypoxia was induced by treating cells with different concentrations of hydrogen peroxide for 24 hours. Single cell adhesion was measured using the technique described above and shown in Figure 4. The change in detachment negative pressure (ΔDNP) was expressed as a percentage of the control population of untreated cells. As shown in Figure 8, hypoxia reduced cell adhesion, and increasing the hydrogen peroxide concentration (i.e., increasing the degree of hypoxia) reduced cell adhesion to a greater extent. In this figure, the vertical axis shows the change in detachment negative pressure (ΔDNP) increasing downwards, with more negative values ​​indicating less adhesion of cells, i.e., a tendency to detach. The horizontal axis is a logarithmic scale of hydrogen peroxide concentration increasing from left to right.

[0072] Human breast cancer cells derived from the MDA-MB-231 cell line were cultured at 2.5 × 10 4Cells were plated in cell culture dishes at a density of 100 cells / ml and allowed to rest for 48 hours before measurements. Cells were subjected to hydrogen peroxide concentrations of 1 μM, 10 μM and 100 μM and the negative pressure required to detach the cells from the bottom of the cell culture dish was measured. For each hydrogen peroxide concentration, measurements were taken on at least two dishes of cells with at least 100 cells / dish. Experiments were repeated three times. Measurements of the detachment negative pressure are shown as mean ± SEM in Figure 8.

[0073] In FIG. 8, data point 801 shows that cells exposed to hydrogen peroxide at a concentration of 1 μM had an average detachment negative pressure of about −9%, data point 802 shows that cells exposed to hydrogen peroxide at a concentration of 10 μM had an average detachment negative pressure of about −14%, and data point 803 shows that cells exposed to hydrogen peroxide at a concentration of 100 μM had an average detachment negative pressure of about −20%. Thus, increasing the hydrogen peroxide concentration reduced cell adhesion and facilitated cell detachment. In other words, increasing the severity of the hypoxic conditions led to increased cell detachability.

[0074] Example 2 Effect of ranolazine on single-cell adhesion of MDA-MB-231 cells under normoxic and hypoxic conditions Using the technique described above and shown in FIG. 4, single cell adhesion was measured for human MDA-MB-231 cells exposed to different concentrations of ranolazine under normoxic and hypoxic conditions. Ranolazine increased cell substrate adhesion under normoxia in a dose-dependent manner. The dose-dependent increase in cell adhesion was more pronounced under hypoxia. See FIG. 9. In this figure, the vertical axis shows a measure of cell adhesion. The horizontal axis is a logarithmic scale of ranolazine concentration, increasing from left to right. Data were collected from n=7 independent experiments for each condition and are shown in FIG. 9 as mean±SEM.

[0075] Human breast cancer cells derived from the MDA-MB-231 cell line were cultured at 2.5 × 10 4 The cells were plated in cell culture dishes at a density of 1000 cells / ml and left undisturbed for 48 hours before measurement.

[0076] In normoxia experiments (curve 901), cells plated in different dishes were treated with ranolazine at concentrations of 0.1 μM, 0.5 μM, 1 μM, 10 μM, 20 μM and 100 μM. At the lowest concentration of 0.1 μM, ranolazine had no effect on cell adhesion. At concentrations of 0.5 μM, 1 μM, 10 μM, 20 μM and 100 μM, adhesion increased in a dose-dependent manner. The increase in adhesion appeared to plateau at a concentration of 100 μM ranolazine.

[0077] In the hypoxic experiment (curve 902), hypoxia was chemically induced by treating cells with hydrogen peroxide (50 μmol) for 24 hours. Plated cells in different dishes were treated with ranolazine at concentrations of 0.1 μM, 1 μM, 10 μM and 100 μM. Even at the lowest concentration of 0.1 μM ranolazine, hypoxic cell adhesion and adhesion increased and continued to increase in a dose-dependent manner at ranolazine concentrations ranging from 1 μM, 10 μM and 100 μM. The amount of increase in adhesion appeared to plateau at a concentration of 100 μM ranolazine, and the curves from the normoxic and hypoxic experiments appeared to converge around this concentration.

[0078] A comparison of the effect of ranolazine on cell adhesion under normoxia (curve 901) and hypoxia (curve 902) showed that the effect of ranolazine was approximately 10-fold greater in hypoxia before converging at higher drug concentrations.

[0079] Example 3 Effects of ranolazine on lateral motility of MDA-MB-231 cells under normoxia and hypoxia The lateral motility of cells was measured using the techniques described above and shown in FIG.

[0080] Referring to Figure 10, the vertical axis shows the measured cell motility index, the reference point for normal motility being shown by a control sample of MDA-MB-231 cells under normoxia and without drug (block 1001 of the histogram), normalized to 100%. Across the horizontal axis, the set of results of the experiments carried out under normoxia is plotted on the left, and the set of results of the experiments carried out under hypoxia is plotted on the right. For each set of results, the concentrations of ranolazine used increase from left to right.

[0081] Block 1005 shows the results obtained with the control sample (no drug) for MDA-MB-231 cells under hypoxic conditions. Comparing blocks 1001 and 1005, it can be seen that hypoxia increased motility.

[0082] Human breast cancer cells derived from the MDA-MB-231 cell line were treated with different concentrations of ranolazine under normoxia and hypoxia. In normoxia experiments, cell motility was measured after treating cells with no drug (control) and with ranolazine at concentrations of 1 μM, 10 μM, and 100 μM. The results are shown in FIG. 10 as blocks 1001, 1002, 1003, and 1004, respectively. Increasing ranolazine concentrations reduced the lateral motility of cells, but only the reduction at a concentration of 100 μM ranolazine was statistically significant.

[0083] Similarly, in hypoxia experiments, cell motility was measured after treating cells with no drug (control) and with ranolazine at concentrations of 1 μM, 10 μM, and 100 μM. The results are shown in Figure 10 as blocks 1005, 1006, 1007, and 1008, respectively. Increasing ranolazine concentrations reduced lateral motility of cells, with each concentration of ranolazine tested giving a statistically significant decrease in lateral motility of cells.

[0084] Data are collected from n=3 independent experiments for each condition and are shown as mean±SEM. (*) indicates significance of P<0.05; (**) indicates significance of P<0.01.

[0085] Example 4 Effect of ranolazine on transverse migration of MDA-MB-231 cells under normoxia and hypoxia Using the techniques described above and shown in FIG. 6, transverse migration of cells was measured.

[0086] Referring to Figure 11, the vertical axis shows the relative values ​​for the measured transverse migration of cells, with the reference point for normal transverse migration being shown by a control sample of MDA-MB-231 cells under normoxia and without drug (block 1101), normalized to 100%. Across the horizontal axis, the set of results of experiments carried out under normoxia is plotted on the left, and the set of results of experiments carried out under hypoxia is plotted on the right. For each set of results, the concentrations of ranolazine used increase from left to right.

[0087] Block 1105 shows the results obtained with the control sample (no drug) for MDA-MB-231 cells under hypoxic conditions. Comparing blocks 1101 and 1105, it can be seen that hypoxia increases transverse migration.

[0088] Human breast cancer cells derived from the MDA-MB-231 cell line were treated with different concentrations of ranolazine under normoxia and hypoxia. In normoxia experiments, cell migration was measured after cells were treated with no drug (control) and with ranolazine at concentrations of 1 μM, 10 μM, and 100 μM. The results are shown in FIG. 11 as blocks 1101, 1102, 1103, and 1104, respectively. Increasing ranolazine concentration only slightly decreased the transverse migration of cells, and the results were not statistically significant.

[0089] In hypoxia experiments, cell migration was measured after treating cells with no drug (control) and with ranolazine at concentrations of 1 μM, 10 μM, and 100 μM. The results are shown in FIG. 11 as blocks 1105, 1106, 1107, and 1108, respectively. Ranolazine at concentrations of 1 μM and 10 μM statistically significantly reduced the transverse migration of cells; ranolazine at a concentration of 100 μM also reduced the transverse migration of cells, but the reduction was not statistically significant.

[0090] Data are from n=3 independent experiments for each condition and are shown as mean±SEM. (*) indicates significance at P<0.05.

[0091] Example 5 Effect of ranolazine on the invasiveness of MDA-MB-231 cells under normoxia and hypoxia (without pretreatment) The invasiveness of the cells was measured using the techniques described above and shown in FIG.

[0092] Referring to Figure 12, the vertical axis shows the relative values ​​for the measured cellular invasiveness, with the reference point for normal invasiveness being indicated by a control sample of MDA-MB-231 cells under normoxia and without drug (block 1201), normalized to 100%. Across the horizontal axis, the set of results of experiments carried out under normoxia is plotted on the left, and the set of results of experiments carried out under hypoxia is plotted on the right. For each set of results, the concentrations of ranolazine used increase from left to right.

[0093] FIG. 12 also includes results for further paired controls, displayed in the figure as blocks 1202 and 1208. In these further controls, cells were exposed to the toxin tetrodotoxin (TTX), whose binding site is located at the pore opening of the VGSC. The TTX measurements are a positive control that confirms that sodium channel activity actually contributes (promotes) the metastatic cell behavior under investigation. It should be noted that at the concentration of TTX used here (10 μmol), the blockade of the VGSC is not complete. Thus, the drug (ranolazine) appears to be more potent than the toxin (TTX), especially at the higher concentrations tested.

[0094] Block 1207 shows the results obtained with the control sample (no drug) for MDA-MB-231 cells under hypoxic conditions. Comparing blocks 1201 and 1207, it can be seen that hypoxia increased invasiveness.

[0095] Human breast cancer cells derived from MDA-MB-231 cell line were treated with different concentrations of ranolazine under normoxia and hypoxia. In normoxia experiments, the invasiveness of cells was measured after treatment with no drug (control) and with ranolazine at concentrations of 10 μM, 20 μM, 50 μM and 300 μM. The results are shown in Figure 12 as blocks 1201, 1203, 1204, 1205 and 1206, respectively. Treatment with ranolazine reduced cell migration at concentrations of 10 μM, 20 μM and 300 μM, but the effect of ranolazine at a concentration of 50 μM was not statistically significant.

[0096] In hypoxia experiments, cell migration was measured after treatment of cells with no drug (control) and with ranolazine at concentrations of 5 μM, 10 μM, 20 μM, 50 μM and 300 μM. The results are shown in Figure 12 as blocks 1207, 1209, 1210, 1211, 1212 and 1213, respectively. Treatment with ranolazine reduced cell migration at concentrations of 20 μM, 50 μM and 300 μM, while the effect of ranolazine at concentrations of 5 μM and 10 μM was not statistically significant.

[0097] Data are collected from n=3 independent experiments for each condition and are presented as mean±SEM. (*) indicates significance of P<0.05; (**) indicates significance of P<0.01.

[0098] Example 6 Effect of ranolazine on the invasiveness of MDA-MB-231 cells under normoxia and hypoxia with pretreatment of cells Since the decrease in invasiveness at low concentrations of ranolazine under hypoxia was not statistically significant, a modified invasion assay was performed in which cells were pretreated with ranolazine for different periods of time compared to the corresponding control condition (i.e., no ranolazine application).

[0099] Figures 13(a) and (b) show the effect of 5 μM ranolazine (ran) on MDA-MB-231 cell invasion under hypoxic conditions in vitro. The histograms show the number of invading MDA-MB-231 cells after treatment with 5 μM ranolazine for different periods of time compared to the corresponding control condition (i.e., no ranolazine application).

[0100] In Figure 13(a), block 1301 shows invasiveness results obtained under control conditions (no drug) for invading cells (i.e., virtually no pretreatment used) over a 24 hour assay duration. Block 1302 shows invasiveness results for invading cells treated with 5 μM ranolazine (no pretreatment) over a 24 hour assay duration. The effect of 5 μM ranolazine treatment on the invasiveness of cells was not statistically significant.

[0101] In Figure 13(b), block 1303 shows invasiveness results obtained under control conditions (no drug) for invading cells (i.e., effectively no pretreatment) over a 24 hour assay duration. Block 1304 shows invasiveness results over a 24 hour assay duration for invading cells pretreated with 5 μM ranolazine for 72 hours. The invasiveness of cells pretreated with 5 μM ranolazine for 72 hours was very significantly reduced.

[0102] In each of Figures 13(a) and (b), the number of "invading cells" represents the average number of cells counted in 24 randomly selected fields from two separate inserts (experiments).

[0103] Figure 13(c)-(e) shows the effect of 25 μM ranolazine (ran) on MDA-MB-231 cell invasion under hypoxic conditions in vitro. The histograms show the number of invading MDA-MB-231 cells after treatment with 25 μM ranolazine for different periods of time compared to the corresponding control condition (i.e., no application of ranolazine).

[0104] In Figure 13(c), block 1305 shows invasiveness results obtained under control conditions (no drug) for invading cells (i.e., effectively no pretreatment) over a 24 hour assay duration. Block 1306 shows invasiveness results for invading cells treated with 25 μM ranolazine (i.e., no pretreatment) over a 24 hour assay duration. Cells treated with 25 μM ranolazine were statistically significantly less invasive than untreated cells.

[0105] In Figure 13(d), block 1307 shows invasiveness results obtained under control conditions for cells kept drug-free for 24 hours and allowed to invade for the subsequent 24 hours before measurement. Block 1308 shows invasiveness results for cells pre-treated with 25 μM ranolazine for 24 hours and allowed to invade for the subsequent 24 hours before measurement. Cells pre-treated with 25 μM ranolazine for 24 hours before starting the 24 hour long-term assay were significantly less invasive than untreated cells.

[0106] In Figure 13(e), block 1309 shows invasiveness results obtained under control conditions for cells kept drug-free for 48 hours and allowed to invade for the subsequent 24 hours before measurement. Block 1310 shows invasiveness results for cells pre-treated with 25 μM ranolazine for 48 hours and allowed to invade for the subsequent 24 hours before measurement. Cells pre-treated with 25 μM ranolazine for 48 hours before starting the 24 hour long-term assay were also significantly less invasive than untreated cells.

[0107] As above, the number of "invading cells" in the histograms in Figures 13(c)-(e) represents the average number of cells counted in 24 randomly selected fields from two separate experiments.

[0108] Pretreatment of cells with ranolazine at both concentrations tested (5 μM and 25 μM) results in a statistically significant decrease in cell invasiveness. Such pretreatment of cells with drug in in vitro studies is considered to represent in vivo treatment, where patients receive continuous therapeutic doses of the drug.

[0109] Example 7 Effect of ranolazine on the growth of MDA-MB-231 cells under normoxia and hypoxia Cell growth was measured using the techniques described above under the heading "Cell Growth (Proliferation) Assay."

[0110] Referring to Figure 14, the vertical axis shows the total cell number in the plated samples, and the reference point for normal growth is shown by control samples of MDA-MB-231 cells (blocks 1401, 1405 and 1409) under normoxia and without drug at the start, after 24 and 48 hours, respectively. Across the horizontal axis, three sets of results are shown for measurements taken at the start, after 24 and 48 hours of growth, where the term "growth" includes cell proliferation and cell death, and there was an overall increase in cell number over time in this assay both with and without drug. For each of the three sets of results, the concentration of ranolazine used increases from left to right.

[0111] Human breast cancer cells derived from the MDA-MB-231 cell line were treated with different concentrations of ranolazine under normoxia.

[0112] At the beginning (time 0), cell number was measured after treatment of cells with no drug (control) and with ranolazine at concentrations of 1 μM, 10 μM and 100 μM. The results are shown in FIG. 14 as blocks 1401, 1402, 1403 and 1404, respectively. Increasing ranolazine concentrations had no effect on the initial cell number.

[0113] After 24 hours, the cell numbers at each concentration of ranolazine were counted again. The results are shown in Figure 14 as blocks 1405, 1406, 1407 and 1408. There was no significant difference between the cell numbers in the control sample (block 1405) and the cell numbers in the samples treated with ranolazine at concentrations of 1 μM, 10 μM and 100 μM (blocks 1406, 1407 and 1408, respectively).

[0114] After an additional 24 hours (48 hours total), the cell numbers at each concentration of ranolazine were counted again. The results are shown in Figure 14 as blocks 1409, 1410, 1411 and 1412. There was no statistically significant difference between the cell numbers in the control sample (block 1409) and the cell numbers in the samples treated with ranolazine at concentrations of 1 μM, 10 μM and 100 μM (blocks 1410, 1411 and 1412, respectively).

[0115] Similarly, in hypoxia experiments, cell motility was measured after treating cells with no drug (control) and with ranolazine at concentrations of 1 μM, 10 μM, and 100 μM. The results are shown in FIG. 10 as blocks 1005, 1006, 1007, and 1008, respectively. Increasing ranolazine concentrations reduced the lateral motility of cells, and the results were statistically significant for each concentration of ranolazine.

[0116] Data are collected from n=3 independent experiments for each condition and are shown as mean±SEM.

[0117] The same concentrations of ranolazine under hypoxia also had no effect on the growth of MDA-MB-231 cells (results not shown in FIG. 14).

[0118] Example 8 Effect of ranolazine on viability of MDA-MB-231 cells under normoxia Cell viability was measured using the techniques described above under the heading "Cell Viability Assay."

[0119] 15, the vertical axis represents the relative viability of cells in the plated samples, with a reference point for normal viability provided by a normoxia and drug-free control sample of MDA-MB-231 cells (Block 1501). The concentrations of ranolazine with which the cells of the different samples were treated are plotted increasing from left to right across the horizontal axis.

[0120] Human breast cancer cells derived from MDA-MB-231 cell line were treated with different concentrations of ranolazine under normal oxygen. After treating cells for 48 hours with no drug (control) and with ranolazine at concentrations of 1 μM, 10 μM and 100 μM, cell viability was measured. The results are shown in Figure 15 as blocks 1501, 1502, 1503 and 1504, respectively. Ranolazine had no effect on cell viability.

[0121] Data are collected from n=3 independent experiments for each condition and are shown as mean±SEM.

[0122] Example 9 Effect of ranolazine on lateral migration of Mat-LyLu cells under normoxia and hypoxia Using the techniques described above and shown in FIG. 6, transverse migration of cells was measured.

[0123] Referring to Figure 16, the vertical axis shows the relative values ​​for the measured transverse migration of cells, with the reference point for normal transverse migration being shown by a control sample of Mat-LyLu cells under normoxia and without drug (block 1601), normalized to 100%. Across the horizontal axis, the set of results of experiments carried out under normoxia is plotted on the left, and the set of results of experiments carried out under hypoxia is plotted on the right. For each set of results, the concentrations of ranolazine used increase from left to right.

[0124] FIG. 16 also includes results for further paired controls, displayed in the figure as blocks 1602 and 1607. In these further controls, cells were exposed to the toxin tetrodotoxin (TTX), whose binding site is located at the pore opening of the VGSC. The TTX measurements are a positive control that confirms that sodium channel activity actually contributes (promotes) the metastatic cell behavior under investigation. It should be noted that at the concentration of TTX used here (1 μmol), the blockade of the VGSC is not complete. Thus, the drug (ranolazine) appears to be more potent than the toxin (TTX), especially at the higher concentrations tested.

[0125] Block 1606 shows the results obtained with a control sample (no drug) for Mat-LyLu cells under hypoxic conditions. Comparing blocks 1601 and 1606 it can be seen that hypoxia increases transverse migration.

[0126] Rat prostate cancer cells derived from the Mat-LyLu cell line were treated with different concentrations of ranolazine under normoxia and hypoxia. In normoxia experiments, cell migration was measured after cells were treated with no drug (control) and with ranolazine at concentrations of 20 μM, 50 μM and 300 μM. The results are shown in FIG. 16 as blocks 1601, 1603, 1604 and 1605, respectively. Increasing the concentration of ranolazine reduced the transverse migration of cells at concentrations of 20 μM and 50 μM. The effect of ranolazine at a concentration of 300 μM on transverse migration was not statistically significant.

[0127] In hypoxia experiments, cell migration was measured after treating cells with no drug (control) and with ranolazine at concentrations of 20 μM, 50 μM, and 300 μM. The results are shown in FIG. 16 as blocks 1606, 1608, 1609, and 1610, respectively. Ranolazine at concentrations of 20 μM, 50 μM, and 300 μM, respectively, statistically significantly reduced transverse cell migration.

[0128] Data are from n=3 independent experiments per condition and are shown as mean±SEM (*) indicates significance at P<0.05 compared to control.

[0129] Example 10 Effect of ranolazine on invasiveness of Mat-LyLu cells under normoxia and hypoxia The invasiveness of the cells was measured using the techniques described above and shown in FIG.

[0130] Referring to Figure 17, the vertical axis shows the relative values ​​for the measured cellular invasiveness, with the reference point for normal invasiveness being shown by the control sample of Mat-LyLu cells under normoxia and without drug (block 1701), normalized to 100%. Across the horizontal axis, the set of results of the experiments carried out under normoxia is plotted on the left, and the set of results of the experiments carried out under hypoxia is plotted on the right. For each set of results, the concentrations of ranolazine used increase from left to right.

[0131] FIG. 17 also includes results for further paired controls, displayed in the figure as blocks 1702 and 1707. In these further controls, cells were exposed to the toxin tetrodotoxin (TTX), whose binding site is located at the pore opening of the VGSC. The TTX measurements are a positive control that confirms that sodium channel activity actually contributes (promotes) the metastatic cell behavior under investigation. It should be noted that at the concentration of TTX used here (1 μM), the blockade of the VGSC is not complete. Thus, the drug (ranolazine) appears to be more potent than the toxin (TTX), especially at the higher concentrations tested.

[0132] Block 1706 shows the results obtained with the control sample (no drug) for Mat-LyLu cells under hypoxic conditions. Comparing blocks 1701 and 1706, it appears that hypoxia does not affect the invasiveness of the cells.

[0133] Rat prostate cancer cells derived from Mat-LyLu cell line were treated with different concentrations of ranolazine under normoxia and hypoxia. In normoxia experiments, the invasiveness of cells was measured after treatment with no drug (control) and with 20 μM, 50 μM and 300 μM concentrations of ranolazine. The results are shown in Figure 17 as blocks 1701, 1703, 1704 and 1705, respectively. Treatment with ranolazine statistically significantly reduced cell migration at each concentration of ranolazine tested.

[0134] In hypoxia experiments, cell migration was measured after treatment of cells with no drug (control) and with ranolazine at concentrations of 20 μM, 50 μM, and 300 μM. The results are shown in Figure 17 as blocks 1706, 1708, 1709, and 1710, respectively. Treatment with ranolazine statistically significantly reduced cell migration at each concentration of ranolazine tested.

[0135] Data are from n=3 independent experiments for each condition and are shown as mean±SEM. (*) indicates significance of P<0.05 compared to normoxic control; (*) indicates significance of P<0.05 compared to both normoxic and hypoxic controls.

[0136] Example 11 Effect of ranolazine on the growth of Mat-LyLu cells Cell growth was measured using the techniques described above under the heading "Cell Growth (Proliferation) Assay."

[0137] Referring to Figure 18, the vertical axis shows the total cell number in the plated samples, with the reference point for normal growth being indicated by a control sample of Mat-LyLu cells under normoxia and without drug (block 1801), normalized to 100%. Across the horizontal axis, the set of results of experiments carried out under normoxia is plotted on the left, and the set of results of experiments carried out under hypoxia is plotted on the right. For each set of results, the concentration of ranolazine used increases from left to right. Note that the term "growth" includes cell proliferation and cell death.

[0138] Rat prostate cancer cells derived from Mat-LyLu cell line were treated with different concentrations of ranolazine under normoxia and hypoxia. In normoxia experiments, cells were treated with no drug (control) and with concentrations of 20 μM, 50 μM and 300 μM of ranolazine for 24 hours, and then cell number was measured. The results are shown in Figure 18 as blocks 1801, 1802, 1803 and 1804, respectively. Increasing ranolazine concentration did not produce a statistically significant effect on cell number.

[0139] Similarly, in hypoxia experiments, cells were treated with no drug (control) and with 20μM, 50μM and 300μM concentrations of ranolazine for 24 hours, and then cell number was measured. The results are shown in Figure 18 as blocks 1805, 1806, 1807 and 1808, respectively. At 20μM and 50μM concentrations of ranolazine, increasing ranolazine concentration did not produce a statistically significant effect on cell number. At 300μM concentration of ranolazine, cell number was reduced.

[0140] In summary, Mat-LyLu cell growth was unchanged under all conditions tested, except for 300 μM ranolazine under hypoxia. Data are collected from n=3 independent experiments per condition and are shown as mean ± SEM. (*) indicates significance at P<0.05 compared to both normoxic and hypoxic controls, and (x) indicates no statistical difference.

[0141] Example 12 Effect of ranolazine on viability of Mat-LyLu cells Cells were treated with different concentrations of ranolazine, [20 μM], [50 μM] and [300 μM], for 24 hours under normoxic or hypoxic (2% O2) conditions. The results are shown in Figure 19. Regardless of the concentration and whether the cells were under normoxic or hypoxic conditions, ranolazine had no effect on cell viability. Data were collected from n=3 independent experiments for each condition and are presented as mean ± SEM.

[0142] (Example 13) Effect of riluzole on lateral motility of MDA-MB-231 cells under normoxia and hypoxia The lateral motility of cells was measured using the technique described above and shown in Figure 5. Referring to Figure 20, block 2001 of the histogram shows the motility index for a control sample of MDA-MB-231 cells under normoxia and without drugs, normalized to 100%. Block 2005 is the corresponding control sample (without drugs) for MDA-MB-231 cells under hypoxic conditions. From the comparison between blocks 2001 and 2005, it can be seen that hypoxia significantly increased motility.

[0143] Cells were treated with different concentrations of riluzole ([1 μM], [10 μM] and [100 μM]) under normoxia and hypoxia. Increasing concentrations of riluzole reduced the lateral motility of cells, and this effect was greater under hypoxia. Data are collected from n=5 independent experiments for each condition and are shown as mean ± SEM. (*) indicates significance at P<0.05; (**) indicates significance at P<0.01.

[0144] Example 14 Effect of riluzole on transverse migration of MDA-MB-231 cells under normoxia and hypoxia Using the technique described above and shown in Figure 6, the transverse migration of cells was measured. Referring to Figure 21, block 2101 shows the percentage of transverse migration observed for a control sample of MDA-MB-231 cells under normoxia and no drug, normalized to 100%. Block 2105 is the corresponding control sample (no drug) for MDA-MB-231 cells under hypoxic conditions. Comparing blocks 2101 and 2105, hypoxia appeared to increase transverse migration, but this increase was not statistically significant for the number of test runs completed.

[0145] Cells were treated with different concentrations of riluzole ([1 μM], [10 μM] and [100 μM]) under normoxia and hypoxia. Under normoxia, treatment with riluzole at concentrations of 1 μM and 100 μM statistically significantly decreased the transverse migration of cells. Under hypoxia, increasing concentrations of riluzole statistically significantly decreased the transverse migration. Data are from n=3 independent experiments per condition and are presented as mean ± SEM. (*) indicates significance at P<0.05.

[0146] Example 15 Effect of riluzole on the invasiveness of MDA-MB-231 cells under normoxia and hypoxia (without pretreatment) Cell invasiveness was measured using the technique described above and shown in Figure 7. The results are shown in Figure 22. Riluzole at a concentration of 5 μM significantly inhibited invasiveness even without pretreatment. The number of "invading cells" represents the average number of cells counted in 24 randomly selected fields from two separate inserts (experiments).

[0147] Example 16 Effect of riluzole on invasiveness of MDA-MB-231 cells under normoxia and hypoxia (72 h pretreatment) The invasiveness of cells was measured using the same technique as described above for Example 6, and cells were pretreated with riluzole at a concentration of 5 μM for 72 hours. The results are shown in Figure 23. Cells pretreated with riluzole at a concentration of 5 μM for 72 hours also showed significantly inhibited invasiveness. As mentioned above, the number of "invading cells" represents the average number of cells counted in 24 randomly selected fields from two separate inserts.

[0148] (Example 18) Effect of riluzole on the growth of MDA-MB-231 cells under normoxia and hypoxia Cells were treated with different concentrations of riluzole ([1 μM], [10 μM] and [100 μM]) for 48 hours. The results obtained are shown in FIG. 24. Riluzole at a concentration of 100 μM statistically significantly reduced cell proliferation at 24 and 48 hours. Data were collected from n=3 independent experiments for each condition and are presented as mean±SEM. (*) indicates a significance level of P<0.05.

[0149] (Example 19) Effect of riluzole on viability of MDA-MB-231 cells under normoxia and hypoxia Cells were treated with different concentrations of riluzole ([1 μM], [10 μM] and [100 μM]) for 48 hours. The results are shown in FIG. 25. Riluzole at concentrations of 10 μM and 100 μM reduced cell viability. Data were collected from n=3 independent experiments for each condition and are presented as mean±SEM. (*) indicates a significance level of P<0.05.

[0150] The same concentrations of ranolazine under hypoxia also had no effect on the growth of MDA-MB-231 cells (results not shown in FIG. 25).

[0151] (Example 20) Effect of Riluzole on Matrigel™ Invasion of Mat-LyLu Cells under Normoxia and Hypoxia (without Pretreatment) Using the technique described above and shown in Figure 7, the invasiveness of cells was measured. Referring to Figure 26, block 2601 shows the invasion index for control samples of Mat-LyLu cells under normoxia and without drugs, normalized to 100%. Block 2604 is the corresponding control sample (without drugs) for Mat-LyLu cells under hypoxic conditions. Comparing blocks 2601 and 2604, it can be seen that hypoxia statistically significantly increased invasion.

[0152] Riluzole at a concentration of 1 μM significantly inhibited invasion under normoxia and hypoxia, even without pretreatment. Data are shown as mean ± SEM from n > 3 independent experiments for each condition. (*) indicates significance at P < 0.05 compared to control.

[0153] Example 21 Effect of riluzole on the growth of Mat-LyLu cells Cells were treated with different concentrations of riluzole, [3 μM], [5 μM], [10 μM] and [30 μM], for 24 hours under normoxic or hypoxic (2% O2) conditions. The results obtained are shown in Figure 27. Riluzole dose-dependently reduced cell proliferation under both normoxic and hypoxic conditions; the effect was greater under hypoxic conditions. Data were collected from n=3 independent experiments for each condition and are shown as mean ± SEM. (*) indicates significance of P<0.05 compared to control, and (x) indicates no statistical difference.

[0154] Example 22 Effect of riluzole on viability of Mat-LyLu cells Cells were treated with different concentrations of riluzole, 10 μM, 30 μM, and 100 μM, for 24 hours under normoxic or hypoxic (2% O2) conditions. The results are shown in Figure 28. Riluzole (10-100 μM) had no effect on cell viability. Data were collected from n=3 independent experiments for each condition and are presented as mean ± SEM.

[0155] Although the invention has been described primarily in relation to ranolazine and riluzole, other substances which have the effect of reducing sustained VGSC currents without eliminating transient VGSC currents may also be used, such as valproate, flecainide, lidocaine, mexiletine or F15845.

[0156] Furthermore, although the invention has been described primarily in relation to breast and prostate cancer, the invention is applicable to all metastatic cancers that express voltage-gated sodium channels.

[0157] (References) TIFF2025010391000005.tif17170TIFF2025010391000006.tif61170

Claims

[Claim 1] Ranolazine to reduce or prevent metastatic behavior in VGSC-expressing cancers.

Images