Targeted osmotic lysis of malignant cancer cells using pulsed magnetic field gradients

Abstract

To provide a drug and method for cancer treatment.SOLUTION: Provided is use of a Na+, K+-ATPase inhibitor in manufacturing a drug for the treatment of a cancer in a mammal, the treatment including co-administering to tumor cells that over-express voltage-gated sodium channels a Na+, K+-ATPase inhibitor and a magnetic field capable of stimulating voltage-gated sodium channels to cause osmotic lysis of the tumor cells, the magnetic field including a pulsed magnetic field, and the pulsed magnetic field being a pulsed z-gradient magnetic field.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (Related Applications) This application claims priority to U.S. Provisional Application No. 62 / 534,947, filed July 20, 2017, and U.S. Provisional Application No. 62 / 696,702, filed July 11, 2018, both of which are incorporated by reference in their entireties for all purposes.

[0002] The present disclosure targets cancer cells that overexpress voltage-gated sodium channels (VGSCs, or "sodium channels") and induces the production of sodium, potassium-adenosine triphosphatase (Na + ,K + The present invention relates to a method for causing osmotic lysis of these cancer cells by inhibiting VGSCs (VIII-ATPase, i.e., the "sodium pump"), as well as a method for stimulating VGSCs with pulsed magnetic field gradients to allow sodium and water to enter the cancer cells. [Background technology]

[0003] Chemotherapy and radiation therapy for metastatic cancer present clinicians with a difficult challenge: attempting to kill the neoplastic disease before it kills the patient due to toxicity to both normal and abnormal tissues. All conventional cancer treatments are associated with toxicity, increased mortality, and reduced quality of life that can far exceed the treatment period, and the primary focus of current antitumor treatments is targeting treatments to cancer cells, for example, by targeting proteins expressed or overexpressed by cancer cells rather than normal tissues.

[0004] Many invasive cancer cell types overexpress VGSCs more than 1000 times more than normal cells. Cancer cells that overexpress VGSCs are epithelial cancers, including, but not limited to, highly invasive breast cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, neuroblastoma, and cervical cancer. Some sarcomas, notably rhabdomyosarcoma, mesothelioma, and osteosarcoma, also overexpress VGSCs. Mesotheliomas, which are not classified as epithelial cancers, are also known to overexpress VGSCs. When these sodium channels are activated, Na + are introduced into the cells. In these cancers, the degree of metastasis is directly related to increased expression of VGSCs. Physiologically, these cancer cells share certain cellular properties with normal excitable cells such as neurons and cardiomyocytes (e.g., conduction of action potentials). Patent document 1 discloses the use of inhibitors of VGSCs as a treatment for cancers, including breast cancer.

[0005] Of the 1.6 million people diagnosed with epithelial cell carcinoma each year in the United States, 40% are considered "highly invasive" and overexpress VGSCs. These patients diagnosed with malignant / metastatic cancer are currently treated using extensive and often disfiguring surgery, chemotherapy, and / or radiation. More than 400,000 people die from epithelial cell carcinoma each year in the United States, and an estimated 10 times that number worldwide. Furthermore, another 1.2 million U.S. patients diagnosed with invasive cancer are successfully treated using conventional surgery, chemotherapy, and / or radiation. Breast cancer is an example of a highly invasive cancer. More than 40,000 women die from breast cancer each year in the United States and 465,000 worldwide. More than 90% of these deaths are due to metastasis of the primary tumor. Furthermore, another 170,000 U.S. women diagnosed with invasive breast cancer are successfully treated using conventional mastectomy, lumpectomy, chemotherapy, and / or radiation. Of the 207,000 people who develop breast cancer each year, 40% of cancers are considered to be "highly aggressive" and overexpress VGSCs.

[0006] The family of sodium channels, termed "voltage-gated sodium channels," is so named due to its sensitivity to small changes in voltage across the cell membrane (approximately 30 mV; from a resting membrane potential of -70 mV to a threshold of -40 mV). They have also been shown to be activated by many stimuli, including electrical currents, mechanical membrane disturbances, ultrasound, magnetic fields, and some drugs. The VGSC family has nine members and nine distinct isoforms. These are designated Nav1.X, where X represents 1 to 9. One distinct channel, NaX, does not respond to voltage shifts but does respond to extracellular sodium concentration.

[0007] Na + ,K + ATPase is a ubiquitous transmembrane protein in animal cells that functions to maintain an ionic imbalance across the cell membrane in favor of more charged ions, mostly sodium ions, outside the cell than inside the cell. This generates an electrochemical gradient that is in homeostatic balance. When the ionic imbalance shifts in the presence of a change in voltage, an action potential is generated, causing a temporary osmotic shift to a hypertonic state within the cell. Restoration of the sodium imbalance is achieved by the release of Na + ,K + This is an essential function performed by Na-ATPase. + ,K + When the ATPase does not function properly, water forces sodium into the cell to restore osmotic balance, thereby increasing cell volume. In normal cells, this change in cell volume is tolerated by membrane compliance. + ,K + Blocking the function of the sodium pump can result in loss of cellular excitability and an increase in cell volume. Many inhibitors are known, including cardiac glycosides. Isoenzymes vary in their sensitivity to each cardiac glycoside. Over 30 drugs have been shown to inhibit the activity of the sodium pump. These include ouabain, digitalis, and its active ingredients, digoxin and digitoxin.

[0008] U.S. Patent No. 5,999,623 discloses the use of cardiac glycosides (e.g., ouabain and proscillaridin) either alone or in combination with other standard cancer therapeutics to treat pancreatic cancer by inducing cell apoptosis. U.S. Patent No. 5,999,623 discloses the use of cardiac glycosides, including digoxin and ouabain, to induce cell apoptosis as a treatment for cancer.

[0009] US Patent No. 5,999,239 discloses a method for treating cancer in a mammal comprising co-administering a first agent and a second agent to tumor cells that overexpress voltage-gated sodium channels, wherein the first agent is a Na + ,K + The first agent inhibits the ATPase, and the second agent stimulates voltage-gated sodium channels, causing osmotic lysis of tumor cells, resulting in a substantial decrease in tumor cell viability within approximately one hour of coadministration. In this regard, by blocking the sodium pump and simultaneously stimulating the opening of sodium channels in these cells, highly malignant cancer cells can be selectively killed. The first agent may include ouabain or digitoxin, and the second agent may include an electric current, ultrasound, a magnetic field, or a drug compound such as veratridine.

[0010] There is a need for improved mechanisms and techniques for providing magnetic stimulation to achieve targeted osmotic lysis of malignant cancer cells. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 7,393,657 [Patent Document 2] U.S. Patent Application Publication No. 2007 / 0105790 [Patent Document 3] U.S. Patent Application Publication No. 2009 / 0018088 [Patent Document 4] U.S. Patent No. 8,921,320 [Patent Document 5] U.S. Patent No. 5,779,637 [Patent Document 6] U.S. Patent No. 5,666,056 [Patent Document 7] U.S. Patent No. 4,411,270 Summary of the Invention

[0012] Aspects and embodiments of the present disclosure provide methods for targeted osmotic lysis (TOL) of cancer cells that overexpress voltage-gated sodium channels (VGSCs or "sodium channels") and targeting of sodium, potassium-adenosine triphosphatase (Na + ,K + The present invention provides a method for causing osmotic lysis of cancer cells by inhibiting the VGSC (V-ATPase or "sodium pump") and stimulating the VGSC with a pulsed magnetic field gradient to allow sodium and water to enter the cancer cells.

[0013] Certain embodiments of the present disclosure relate to a method for the translocation of cancer cells overexpressing VGSCs by stimulating the VGSCs with a z-gradient magnetic field in the presence of a static magnetic field, with a ramp from -90 mV to -30 mV over a rise time of approximately 0-20 ms, an inter-pulse interval of approximately 7.5-30 ms, and a pulse cycle frequency of approximately 20-60 pps. In a preferred embodiment, the pulsed magnetic field is a z-gradient magnetic field with a ramp from -90 mV to -30 mV over a rise time of approximately 10 ms, a return to base over 2.5 ms, an inter-pulse interval of 7.5 ms, and a pulse cycle frequency of 25 pps. The pulsed magnetic field reaches a stimulation intensity of 80 mT, causing near 100% cell lysis. [Brief explanation of the drawings]

[0014] [Figure 1] Stimulus intensity-response curves to determine the intensity of pulsed magnetic field required to produce cytolysis of MDA-MB-231 cells in vitro ( p < .01). [Figure 2]TOL is dependent on the presence of sodium. This bar graph shows that cell lysis occurs in the presence of sodium, digoxin, and PMF (black bars, Dig-PMF), but not in the absence of sodium and TOL (white bars, Dig-PMF) (p<0.01). [Figure 3] Example of H&E stained sections of MDA-MB-231 xenografts showing representative sections from tumors graded 1-5 for tumor necrosis level (1 = normal tumor morphology, 5 = complete destruction). [Figure 4] The median tumor necrosis score of xenografts treated with TOL was significantly greater than the median tumor necrosis score of xenografts treated with control, drug only, stimulus only, or vehicle (control scores combined, ρ < .05). [Figure 5] TOL does not damage non-cancerous tissue. Photomicrographs illustrate sections taken from non-cancerous organs exposed to TOL treatment during the processing of MDA-MB-231 xenografts. Tissue from each organ showed no signs of damage associated with TOL treatment. [Figure 6] The growth rate of the tumor is significantly slowed after treatment with TOL(Dig-PMF) when compared to the control. [Figure 7] (7A) Mice treated with TOL reached the humane endpoint euthanasia criteria significantly longer than mice in the control group. Arrows indicate days of treatment. (7B) Bar graphs show the average time for 50% of each treatment group to reach the humane endpoint euthanasia criteria. Thus, the life expectancy of mice treated with TOL exceeds that of controls by approximately one week. DETAILED DESCRIPTION OF THE INVENTION

[0015] All patents and other publications identified are incorporated herein by reference for the purpose of describing and disclosing, for example, methodologies described in such publications that might be used in connection with the present invention, but are not intended to provide definitions of terms that contradict those provided herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute an admission as to the accuracy of the dates or contents of these documents.

[0016] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Throughout this specification, unless otherwise indicated, "comprise," "comprises," and "comprising" are used inclusively rather than exclusively, such that a stated integer or group of integers may include one or more other unstated integers or groups of integers. The term "or" is inclusive unless modified, for example, by "either." Other than in the operational examples, or where expressly stated or otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about."

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. The terms "male" and "female" may be used interchangeably to describe corresponding components or complementary aspects thereof and are not a limitation to a particular structure unless the context clearly indicates otherwise.

[0018] Headings are provided for convenience only and should not be construed as limiting the invention in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined solely by the claims. In order to make this disclosure more readily understandable, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0019] Targeted osmotic lysis (TOL) has many advantages over conventional cancer therapies. Chemotherapy typically damages not only cancerous tissue but also healthy tissue, leading to long-term recovery and chronic morbidity. In contrast, TOL destroys only cells that overexpress VGSCs. Therefore, more selective damage to diseased tissue is expected. This contributes to reducing the long-term adverse effects of treatment. Radiation therapy is usually aimed at killing healthy tissue surrounding cancerous tissue. Like chemotherapy, this often leads to long-term recovery and chronic morbidity. Due to the selectivity of TOL for cells that overexpress VGSCs, there is little or no damage around the neoplasm.

[0020] The present disclosure provides a method for the treatment of sodium, potassium adenosine triphosphatase (Na + ,K +The present invention provides a method for targeting cancer cells that overexpress voltage-gated sodium channels (VGSCs or "sodium channels"), causing osmotic lysis of these cancer cells, by inhibiting voltage-gated sodium channels (VGSCs or "sodium channels") and stimulating voltage-gated sodium channels (VGSCs or "sodium channels") with a pulsed magnetic field gradient to allow sodium and water to enter the cancer cells. For example, magnetic stimulation may be used to produce a depolarization of between about -90 mV and -30 mV in vitro.

[0021] Sodium channels are responsible for the upstroke of the neuronal action potential. The average resting membrane potential of neurons is -70 mV, that of skeletal muscle is -90 mV, and that of epithelial cells is -50 mV. The action potential peaks at +40 mV. After the peak, the channel closes and there is a variable period of recovery during which another action potential cannot be initiated—the refractory period. Channel opening, closing, and reopening appear to be subtype-specific rather than cell-type-specific. Greater depolarization is associated with more rapid opening and slower inactivation of sodium channels, resulting in greater sodium influx. Sodium influx initially flows down its charge gradient to 0 mV, then down its concentration gradient to +40 mV.

[0022] There are nine known isoforms of voltage-gated sodium channels. The opening of these isoforms varies between -90 mV and -30 mV. Some isoforms open slowly at subthreshold levels, between -80 mV and -60 mV, and then close slowly, providing a leak current, a mechanism for stimulus summation. Other isoforms open rapidly at threshold potentials. Channel isoforms reprime or recover impulses at different rates, allowing for rapid firing (nerve impulses) or constant, regular firing (cardiac muscle). The average time for depolarization is 10-20 ms. Full recovery from inactivation is approximately 45 ms.

[0023] When electrical stimulation was used in targeted osmotic lysis, parameters included: (A) in vitro—1.0 V DC, 1 ms pulse, 15 pps for up to 5 min, and (b) in vivo—10 V DC, 1 ms pulse, 15 pps for 2.5 min, then rotate the electrode pair 90° and stimulate again for 2.5 min.

[0024] The present disclosure provides an embodiment using magnetic stimulation for targeted osmotic lysis. Parameters include a ramp rise time from -90 mV to -30 mV over 10 ms, a 2.5 ms return to base, and a 7.5 ms inter-pulse interval, delivered at various gradient planes, with a pulse cycle frequency of 25 pps, i.e., 20 ms per pulse cycle. The ramp may range from about 0 to about 20 ms, and the inter-pulse interval may range from about 15 to about 30 ms. In some embodiments, the pulse cycle frequency may be about 20-60 pps. In one embodiment, parameters include a waveform with a ramp rise time of 12.5 ms, a 7.5 ms plateau, and a 12.5 ms return to baseline, where the inter-pulse interval is 7.5 ms, and the pulse frequency range is about 25-50 pps.

[0025] As used herein, "magnetic field" refers to the magnetic effect of electric current and magnetic materials. A magnetic field can be generated when charge carriers, such as electrons, move through space or within an electrical conductor. The geometry of the magnetic flux lines generated by the movement of charge carriers (current) is similar to that of the magnetic flux lines in an electrostatic field. Magnetic fields can be generated by a variety of devices, including magnetic resonance imaging devices. Devices and systems for magnetic resonance imaging have been described. See, for example, U.S. Patent Nos. 5,629,997, ... and 5,629,997. For example, a donut-shaped superconducting magnet or a copper-wound room-temperature electromagnet can provide the initial static magnetic field.

[0026] Suitable devices and systems for generating magnetic field stimulation include a device consisting of toroidal or solenoidal winding hardware and enclosure, current drive stage, and power supply, along with a microcontroller and circuit board design, software drivers, and user interface, as well as mechanisms for adjusting the magnetic pulses and waveforms developed above. The pulse waveform is controlled using a microcontroller, which generates a pulse-width modulated (PWM) signal that is used to drive a Class D-style electronic amplifier, which in turn drives current through the toroidal winding. The device generates engineered electric field pulses in the empty bore, delivering field strengths of 0.2 to 0.9 Tesla.

[0027] Na + ,K + Non-limiting examples of pharmaceutical compounds that can be used to block -ATPase include ouabain (g-strophanthin), dihydroouabain, ouabain octahydrate, ouabagenin, digoxin, digitoxin, digitalis, acetyldigitoxin, acetyldigoxin, lanatoside C, deslanoside, methyldigoxin, gitoformate, oleandelin, oleandrigenin, bufotoxin, bufotalin, marinobufageni (3,5-dihydroxy-14,15-epoxybufodienolide), palytoxin, oligomycins A, B, C, E, F, and G, rutamycin (oligomycin D), rutamycin B, strophanthin (g-strophanthin, acocanthelin), k-β-strophanthin, strophanthidin, k-strophantoside, cymarin, elisimoside (cardenolide), helveticoside, pervoside, hypothalamic Na + ,K + -ATPase inhibitors (HIF), HIF aglycones, arenobufagin, cinobufagin, marinobufagin, proscillaridin, sciliroside, diglemmotianin, 3,4,5,6, tetrahydroxyxanthone, and Na + ,K + -All other inhibitors of ATPase, their respective combinations and derivatives.

[0028] Na + ,K + ATPase blockers can be delivered to a single tumor via direct or intravenous administration, to a single organ or region via intravenous or intraluminal administration, or systemically via intravenous, subcutaneous, intramuscular, or oral administration. Magnetic field stimulation of sodium channels can be delivered to a single tumor, a single organ, a body part, or the entire body. All types and subtypes of the VGSC family should be equally susceptible to this technology. For example, cell lines overexpressing Nav1.1, Nav1.2, Nav1.5, Nav1.5a, and Nav1.7 are susceptible to targeted lysis mediated by Nav1. [Example]

[0029] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light thereof, and are within the spirit and scope of the present application and the appended claims. All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0030] Example 1. MRI in vitro studies in vials containing pellets of live and alcohol-killed malignant breast cancer cells Baseline images of cultured normal control and alcohol-killed malignant breast cancer cells (MDA-MB-231) were obtained using a magnetic resonance imaging (MRI) device and system to assess any differences between live and dead cells. For this study, three vials of cells were used to provide enough cells to form a pellet that was easily visualized during imaging. The number of cells in a single vial was insufficient to form a pellet large enough to clearly discern differences. The cells were placed in a microcentrifuge tube and centrifuged to form a pellet. The tubes were then placed side-by-side in the center of the MRI's computed field and scanned. Clear differences were observed between the tubes.

[0031] The parameters of the magnetic stimulation set were selected to include the generation of a magnetic field pulse with a z-gradient using a 10 ms rise time to achieve an amplitude of 187.5973, followed by a 2.5 ms return to baseline and an inter-stimulus interval of -17.5 ms. The gradient field was pulsed for 5 min.

[0032] Example 2. Results of an in vitro study on ouabain-treated (TOL) malignant breast cancer cells exposed to pulsed magnetic field stimulation in a z-gradient Two tubes of MDA-MB-231 cells were prepared. To block the sodium pump, the medium in one tube was replaced with medium containing 100 nM ouabain. Both tubes were placed 317 mm offset from the center in the z-gradient field in an MRI and exposed to a pulsed magnetic field. The samples were then repositioned and imaged. Magnetic resonance imaging of vials containing pellets of control MDA-MB-231 cells and ouabain-treated (TOL) MDA-MB-231 cells exposed to pulsed magnetic field stimulation in a z-gradient for 5 minutes displayed observable differences.

[0033] After imaging, cells in each vial were stained with trypan blue to estimate the number of cells lysed by each treatment: (A) magnetic stimulation alone or (B) ouabain and magnetic stimulation. Virtually all cells exposed to pulsed magnetic field stimulation alone remained viable (excluded trypan blue stain), while 10-15% of cells treated simultaneously with ouabain and pulsed magnetic field stimulation were dead (shrunk in appearance and retained stain).

[0034] In this example, cells were exposed to ouabain for 60 minutes while preparing for magnetic field stimulation. Because exposure to ouabain alone may result in lysis in a small percentage of cells, studies were conducted in which vials of cells were exposed to ouabain without pulsed magnetic field stimulation for longer than 90 minutes. Less than 1% of cells exposed to ouabain alone were found to die during this period, reflecting a similar killing rate as when cells were exposed to ouabain for 10 minutes.

[0035] The duration, amplitude, and frequency of the pulsed z-gradient field were varied. All targeted osmotic lysis (TOL) vials (ouabain and pulsed magnetic field stimulation) were exposed to 100 nM ouabain for 10 min. Ouabain-treated and normal MDA-MB-231 control vials were exposed to the same pulsed magnetic field and then stained with trypan blue to estimate the amount of cell lysis in each tube. Two trials were performed using the maximum available magnetic field strength (289.1689 amps). The first pulse stimulus of these trials was delivered with a rise time of 10 ms, a return to baseline of 2.5 ms, and an inter-stimulus interval of 17.5 ms. In the second trial, the stimulus was delivered with a rise time of 10 ms, a return to baseline of 2.5 ms, and an inter-stimulus interval of 7.5 ms. An additional trial was performed with a stimulation amplitude of 187.5973 amps, but unlike the other trials, using a 10 ms rise time, a 2.5 ms return to baseline, and a 7.5 ms inter-stimulus interval to further evaluate the contribution of stimulation frequency to cell lysis. We found good cell lysis, e.g., 80-90% kill rates, when the magnetic field amplitude was 289.1689 amps and the inter-pulse interval was 7.5 ms (50 Hz).

[0036] Example 3. Results of an in vitro study on digoxin-treated (TOL) malignant breast cancer cells exposed to pulsed magnetic field stimulation in a z-gradient MDA-MB-231 breast cancer cells cultured in DMEM or MCF-10a normal breast epithelial cells cultured in mammary epithelial growth medium (MEGM; Lonza, www.lonza.com) were dissociated using a Corning Cellstripper (Corning, NY) and resuspended in 1.5 ml microcentrifuge tubes using DMEM with or without 100 nM digoxin (Sigma-Aldrich). The tubes were placed in an 8-inch diameter, 2.5-inch, solenoid with 697 turns (500 feet) of 12 ga copper wire. 5 VDC current from an AE Techron 7224 DC-enhanced AC amplifier was pulsed at 25 Hz with a 10-msec ramp and fall, generating an 80 mT magnetic pulse in the solenoid. The current was controlled by a Techtronics AFG 3021B waveform generator. Digoxin-treated MDA-MB-231 cells were 97.5% lysed within 10 minutes, whereas only 4.5% of drug-free controls were nonviable. Neither digoxin-treated nor untreated MCF-10a cells lysed upon stimulation.

[0037] Example 4. Simultaneous Pulsing of x, y, and z Gradient Fields for Targeted Osmotic Lysis A study was conducted to evaluate the effect of simultaneous x-, y-, and z-gradient field pulsing on targeted osmotic lysis. Cell vials were prepared as described above. The waveforms presented were identical for each gradient field, and all pulses were administered for 5 minutes, although there was some variation in amplitude and frequency between trials. The amplitude used was the maximum allowed by the system to deliver a 5-minute pulse train. The system automatically shuts down at higher amplitudes. One trial had an amplitude of 262.4531, a rise time of 10 ms, a return to baseline of 2.5 ms, and an interpulse interval of 17.5 ms. Another trial included an amplitude of 224.9519, a rise time of 10 ms, a return to baseline of 2.5 ms, and an interpulse interval of 7.5 ms. Yet another trial included an amplitude of 206.2014, a rise time of 10 ms, a return to baseline of 2.0 ms, and an interpulse interval of 3.0 ms. Cell lysis was significantly lower, 80–90% lethal, when the three gradient fields were pulsed together than when the z-gradient field was pulsed alone.

[0038] Example 5. Studies in cells pulsed with a z-gradient magnetic field in the absence of a static magnet. In a study in which cells were pulsed with a z-gradient magnetic field in the absence of a static magnet, cytological assessment of the amount of cytolysis of MDA-MB-231 cells obtained with the static field was significantly greater than the amount obtained without the field.

[0039] Example 6. Stimulus Intensity Studies - Response Curves to Determine the Intensity of Pulsed Magnetic Fields Required to Produce Cytolysis of MDA-MB-231 Cells In Vitro To determine the strength of the pulsed magnetic field required to produce cytolysis of MDA-MB-231 cells in vitro, a study was conducted in which cells were pulsed at increasing stimulation intensities. Cells were dispensed into microcentrifuge tubes with or without 500 nM digoxin. All tubes were exposed to PMFs ranging from 0 to 80 mT. Figure 1 shows that a stimulation intensity of 80 mT produced nearly 100% cytolysis of MDA-MB-231 cells.

[0040] Example 7. Study of the relationship between the presence of sodium and the effectiveness of TOL A study was performed in which the culture medium containing MDA-MB-231 cells was replaced with Ringer's solution with or without sodium, followed by stimulation with a pulsed magnetic field of 80 mT for 10 minutes. + or Ringer-Na + The cells were transferred to microcentrifuge tubes containing either Ringer vehicle and no stimulus (VEH-No PMF), vehicle and 80mT PMF (Veh-PMF), 500nM digoxin and no stimulus (Dig-No PMF), and digoxin with PMF stimulation (Dig-PMF), no digoxin, with stimulus (Veh-PMF), or without stimulus (Veh-No PMF). Figure 2 shows the results of Ringer + Na+ treatment. + TOL cells (Dig-PMF) showed approximately 70% non-viability, while pulses without sodium showed less than 20% non-viability, indicating that sodium is an essential participant in the TOL process (p<0.01).

[0041] Example 8. Results of in vivo studies using pulsed magnetic field (PMF) stimulation of voltage-gated sodium channels For in vivo validation of the pulsed magnetic field to induce osmotic lysis, four groups of J / Nu mice (n = 8) bearing MDA-MB-231 xenografts (0.7-1.2 cm diameter, sublumbar) were injected (subcutaneously, behind the back) with 5 mg / kg of digoxin or saline five times at 1-hour intervals. This protocol establishes steady-state pharmacokinetics even in poorly vascularized tissues. These mice were exposed to a pulsed magnetic field for 15 minutes, beginning 30 minutes after the last injection, using the same parameters as for cultured cells. This treatment was repeated two and four days after the first treatment. Mice were sacrificed and fixed in 4% paraformaldehyde 24 hours after the third treatment. Tissues from these animals were sectioned and evaluated by a pathologist blinded to the treatment. Figure 3 shows examples of H&E-stained sections of MDA-MB-231 xenografts, showing representative sections from tumors graded 1 to 5 for the level of tumor necrosis. Figure 4 shows that the median tumor necrosis score of xenografts treated with TOL was significantly greater than that of control xenografts treated with drug only, stimulation only, or vehicle. Tumors in mice treated with digoxin and magnetic stimulation (TOL) showed 80-100% tumor lysis. No lysis was observed in normal muscle, kidney, brain, or heart (Figure 5). Drug-only and stimulation-only controls were no different from untreated controls. Three additional immunocompromised nude mice bearing xenografted mesothelioma tumors showed no signs of tumor growth after treatment with TOL. Figure 6 shows that the tumor growth rate after treatment with TOL (Dig-PMF) was significantly slower compared to controls.

[0042] Example 9. Results of an in vivo study using pulsed magnetic field (PMF) stimulation of voltage-gated sodium channels in immunocompetent BALBc mice For in vivo validation of the pulsed magnetic field to induce osmotic lysis, four groups of female immunocompetent BALBc mice (n = 8) bearing xenografts (0.7-1.2 cm diameter below the waist) established after injection of 500 K of the highly malignant murine breast cancer, 4T1, were injected with 7 mg / kg digoxin or saline (subcutaneously, behind the neck) five times at 1-hour intervals. This protocol establishes steady-state pharmacokinetics even in poorly vascularized tissues. Mice were exposed to the pulsed magnetic field for 30 min, starting 15 min after the last injection, using the same parameters as for cultured cells. This treatment was administered on days 0 (the first day of treatment), 2, and 4. Mice were monitored for tumor growth and sacrificed when they met the NIH criteria for humane endpoint euthanasia. Figure 7 shows that mice treated with TOL reached the humane endpoint euthanasia criteria significantly longer than mice in the control group.

Claims

1. Na in the manufacture of a medicament for treating cancer in mammals ＋ , K ＋ - Use of ATPase inhibitors, where the treatment is to induce Na+ in tumor cells that overexpress voltage-gated sodium channels. ＋ , K ＋ - co-administration of an ATPase inhibitor with a magnetic field capable of stimulating voltage-gated sodium channels to cause osmotic lysis of said tumor cells, The Na ＋ , K ＋ the ATPase inhibitor is dihydroouabain, ouabain octahydrate, ouabagenin, digitoxin, digitalis, acetyldigitoxin, or acetyldigoxin; The magnetic field includes a pulsed magnetic field, and the pulsed magnetic field is a pulsed z-gradient magnetic field.

2. The use according to claim 1 , wherein the pulsed magnetic field is a pulsed z-gradient magnetic field in the presence of a static magnetic field magnet.

3. 2. The use of claim 1, wherein the pulsed magnetic field is a pulsed z-gradient magnetic field with a ramp rising from -90 mV to -30 mV over a rise time of about 0 to 20 milliseconds, an inter-pulse interval of about 7.5 to about 30 milliseconds, and a pulse cycle frequency of about 20-60 PPS.

4. 2. The use of claim 1, wherein the pulsed magnetic field is a pulsed z-gradient magnetic field with a ramp rising from -90 mV to -30 mV over a rise time of about 12.5 ms, a plateau of 7.5 ms, and a return to baseline of 12.5 ms, with an inter-pulse interval of 7.5 ms and a pulse cycle frequency of about 25-50 PPS.

5. 2. The use of claim 1, wherein the pulsed magnetic field is a pulsed z-gradient magnetic field with a ramp rising from -90 mV to -30 mV over a rise time of about 10 ms, returning to base over 2.5 ms, an inter-pulse interval of 7.5 ms, and a pulse cycle frequency of 25 PPS.

6. 2. The use of claim 1, wherein the pulsed z-field reaches a stimulatory intensity of 80 mT, resulting in nearly 100% cell lysis.

7. The use according to claim 1, wherein the mammal is a human.

8. 2. The use of claim 1, wherein the tumor cells are associated with a cancer selected from the group consisting of breast cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, lymphoma, mesothelioma, neuroblastoma, glioma, neuroma, liver cancer, ovarian cancer, bladder cancer, pancreatic cancer, thyroid cancer, splenic cancer, gastric cancer, cervical cancer, skin cancer, testicular cancer, kidney cancer, oral cancer, and cervical cancer.

9. The use according to claim 1 , wherein the tumor cells are associated with breast cancer.

10. The use of claim 1 , wherein the tumor cells are associated with prostate cancer.

11. The use of claim 1 , wherein the tumor cells are associated with colon cancer.

12. The use of claim 1 , wherein the tumor cells are associated with small cell lung cancer.

13. The use of claim 1 , wherein the tumor cells are associated with non-small cell lung cancer.

14. The use of claim 1 , wherein the tumor cells are associated with mesothelioma.

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