Oncomagnetic Therapy Vibration Patterns

Abstract

A method for disrupting mitochondrial function in a cell includes vibrating one or more magnets along at least a first axis of rotation and a second axis of rotation substantially perpendicular to the first axis of rotation to generate an oscillating magnetic field. To this end, the method includes selecting a first axis of rotation, applying a vibration pattern along the first axis of rotation, selecting a second axis of rotation, and applying a vibration pattern along the second axis of rotation. The vibration includes a period of increasing vibration frequency, a period of decreasing vibration frequency, and a period of inactivity following the period of decreasing vibration. The method further includes applying the oscillating magnetic field to a tissue containing cancer cells to induce apoptosis of the cancer cells.

Description

[Technical field]

[0001] The present disclosure relates generally to cancer treatment methods and devices, and more particularly to non-invasive cancer treatment utilizing oscillating magnetic fields. [Background technology]

[0002] Cancer is one of the greatest health problems facing modern society, yet progress in improving active treatments for some cancers, including glioblastoma (GBM), remains low. Common cancer treatments include chemotherapy and radiation therapy, which can be devastating to the patient's body and cause severe physical and psychological trauma. Due to the heavy burden on patients, they may decide not to complete the proposed chemotherapy or radiation therapy cycles. Therefore, there is a need for cancer treatments with better life prognosis and less toxicity.

[0003] Recently, a treatment called Tumor Treating Field (TTF) therapy or Optune® therapy, which applies an alternating electric field (AEF) to the scalp, has shown therapeutic benefits for GBM patients. TTF therapy has been approved by the U.S. Food and Drug Administration (FDA) as a monotherapy for GBM and in combination with other therapies for newly diagnosed GBM. TTF therapy is typically administered by attaching electrodes to the patient's head, which requires shaving the patient's head at the electrode site. In addition, the electrodes often cause skin lesions, rashes, and other dermatological side effects, and in some cases the applied current can even cause burns.

[0004] Cancer cells are known to possess high levels of reactive oxygen species (ROS). Intracellular ROS levels play diverse roles in normal cellular processes, such as proliferation and differentiation of developmental cells, programmed cell death, cell motility, immune defense mechanisms, inflammation, and neuronal activity and plasticity. They are also involved in cancer cell proliferation and tissue invasion on the one hand, and in cellular aging and neurodegeneration as mediators of oxidative stress on the other. Cancer cells are known to possess high levels of ROS due to enhanced oxidative metabolism and mitochondrial dysfunction. Previous studies have shown that abnormally high levels of ROS induce apoptosis. Summary of the Invention

[0005] In general, the disclosed system or device applies an oscillating magnetic field (sOMF) to tissue to induce cell death (e.g., apoptosis) of cancer cells (e.g., GBM). The system may include one or more magnetic assemblies and control hardware, such as a non-transitory computer-readable medium storing instructions executable by the control hardware and one or more processors. Each magnetic assembly, hereinafter also referred to as an "oncosilator," may include a motor configured to impart rotational motion to a magnet radially magnetized relative to an axis of rotation according to a specific pattern. The system is configured to generate, sequentially or simultaneously, an oscillating magnetic field about an orthogonal axis for a specific tissue. In some embodiments, the system includes multiple magnetic assemblies arranged around the tissue, or a single magnetic assembly configured to change the axis of rotation, for example, by rotating the axis about which the corresponding magnet rotates by about 90 degrees in one plane and / or about 90 degrees in another plane.

[0006] Certain amplitude, frequency, and timing parameters of the fast rotating magnetic field result in maximum rise in superoxide in glioma cells. In particular, certain ranges of dynamic magnetic field parameters show peak effectiveness in inducing ROS and inhibiting cancer cell colony formation. In an exemplary embodiment, both the magnetic field oscillation and axial rotation are related to the efficacy of the treatment.

[0007] According to some exemplary configurations, the system stimulates the brain tissue intermittently for 1-4 hours at a time, 2-3 times per day. The system rotates the axis of one magnetic assembly or uses multiple magnetic assemblies such that the axis about which the magnet rotates is orthogonal to the axis of the other magnetic assembly or orthogonal to the axes of both of the other two magnetic assemblies. The system can activate the magnetic assemblies sequentially or simultaneously, and thus the system can irradiate the tissue with orthogonal sOMFs sequentially or simultaneously. Each magnet assembly generates an oscillating magnetic field. The system forms an effective magnetic field in the tissue with a strength in the range of 1-200 mT. At each magnetic assembly, the system generates pulse trains for 250-500 milliseconds, with an OFF period between the stimulation pulse trains being 250-2,750 milliseconds. The system can ramp the pulse train from zero to a peak frequency in a time of 50-100 milliseconds and ramp the pulse train down to a frequency below 50 Hertz in a time of 50-200 milliseconds. The peak frequency may be in the range of 100-300 Hertz.

[0008] The system, which may be referred to as an "oncomagnetic" system or device, operates in a non-invasive manner: in particular, the system may apply sOMF to the subject's scalp when used for brain tumors, or without direct contact with the subject's skin when used for other parts of the body.

[0009] In operation, the system increases reactive oxygen species (ROS) levels in oxidatively stressed cancer cells, leading to macromolecular damage and cell death as part of an anti-cancer therapeutic strategy. The system consistently increases ROS to levels that are cytotoxic in patient-derived glioblastoma cells but have no effect on normal brain cells.

[0010] These techniques advantageously do not require, for example, high currents through coils or solenoids, since the system may include a stimulator with a permanent magnet and an electric motor configured to impart an oscillatory motion (e.g., rotation, translational vibration) to the permanent magnet. In some implementations, the stimulators are miniaturized and are called "microstimulators." When the system includes multiple stimulators, the control hardware can operate the stimulators for different times, at different frequencies, at different pulse rates, etc. to define specific stimulation patterns that can be subject-specific.

[0011] One embodiment of these techniques is a method for disrupting mitochondrial function in cells. The method includes vibrating one or more magnets along (i) a first axis and (ii) a second axis substantially orthogonal to the first axis to generate an oscillating magnetic field by controlling hardware, and applying the oscillating magnetic field to a tissue containing cells with mitochondrial disorders to induce apoptosis in the cells with mitochondrial disorders. In a related embodiment, vibrating one or more magnets further includes vibrating one or more magnets along a third axis substantially orthogonal to the first axis and the second axis. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a block diagram of a system for disrupting mitochondrial function in specific cells by applying an oscillating magnetic field, according to one embodiment.

[0013] [Diagram 2] FIG. 2 illustrates a schematic of a magnetic assembly operable in the system of FIG.

[0014] [Diagram 3] FIG. 3 shows an example of an oscillating magnetic field output generated by the apparatus of FIG.

[0015] [Figure 4] FIG. 4 illustrates the sequential actuation of different magnetic assemblies along different axes.

[0016] [Diagram 5] FIG. 5 shows a schematic of the arrangement of the magnetic assembly on a sphere.

[0017] [Figure 6] FIG. 6 is a flow diagram of an example method for applying an oscillating magnetic field to tissue for oncomagnetic therapy that may be performed by the system or oncomagnetic device of FIG.

[0018] [Figure 7] Figure 7 shows a schematic of the cell culture sOMF stimulation setup used in the lab, with a close-up of the cell culture dish placed on top of each oncostimulator displayed on the right.

[0019] [Figure 8A] FIG. 8A shows a scheme for stimulating cultured cells with a static magnetic field produced by one fixed magnet (left), three fixed magnets on three orthogonal axes (center), and an oscillating magnetic field produced by one rotating magnet (right).

[0020] [Figure 8B] FIG. 8B shows the fluorescence intensity quantification of hydroethidine in GBM (GBM115) cells.

[0021] [Figure 8C] Figure 8C shows the fluorescence intensity quantification of hydroethidium in DIPG cells using microscopy images. In particular, for each of the three time periods, the Ctrl element corresponds to the case of no stimulation, the 3DSt element corresponds to stimulation by the magnetic fields of three fixed magnets in orthogonal axes, the 1Dst element corresponds to one fixed magnet, and the 1Dsp element corresponds to one rotating magnet.

[0022] [Figure 8D] FIG. 8D shows the fluorescence intensity quantification of hydroethidine in GBM (GBM115) cells.

[0023] [Figure 8E] Figure 8E shows the fluorescence intensity quantification of hydroethidine in DIPG cells using microscopy images. In particular, for each of the three time periods, the Cont element corresponds to a magnet rotating continuously, the Int element corresponds to a magnet rotating intermittently for 250 ms (Ton) and stopped for 250 ms (Toff), and the Ctrl element corresponds to the control. The scatter plot with bars represents the average normalized fluorescence from three independent experiments with each data point shown as a dot (n=24). Error bars indicate SEM.

[0024] [Figure 9A] Figures 9A and 9B are bar graphs with scatter plots showing ROS levels in GBM115 cells (Figure 9A) and DIPG cells (Figure 9B) normalized to pre-stimulation control baseline during stimulation (2 and 4 hours) and 2 hours after stimulation had ceased with a single oncosilator with a Helmholtz coil and magnet rotating in a two-dimensional plane. In these cases, stimulation lasted for 4 hours with a PPA of approximately 137 Hz. Error bars indicate SEM. [Figure 9B] Figures 9A and 9B are bar graphs with scatter plots showing ROS levels in GBM115 cells (Figure 9A) and DIPG cells (Figure 9B) normalized to pre-stimulation control baseline during stimulation (2 and 4 hours) and 2 hours after stimulation had ceased with a single oncosilator with a Helmholtz coil and magnet rotating in a two-dimensional plane. In these cases, stimulation lasted for 4 hours with a PPA of approximately 137 Hz. Error bars indicate SEM.

[0025] [Figure 10A] FIG. 10A shows the stimulation scheme for cultured cells with sOMFs of one rotating magnet (1Dsp) with a Ton of 250 ms and a Toff of 250 ms or three rotating magnets (3Dsp) sequentially turned on and off with a Ton of 250 ms and a Toff of 250 ms.

[0026] [Figure 10B]Figure 10B and Figure 10C show fluorescence intensity quantification of hydroethidine in GBM (GBM115) cells or DIPG cells using microscopy images. Ctrl elements correspond to no stimulation, 3DSt elements correspond to stimulation with the magnetic fields of three fixed magnets in orthogonal axes, 1Dst elements correspond to one fixed magnet, and 1Dsp elements correspond to one rotating magnet. Scatter plots with bars represent the average normalized fluorescence intensity from three independent experiments with each data point shown as a dot (n=24). Error bars indicate SEM. [Figure 10C] Figure 10B and Figure 10C show fluorescence intensity quantification of hydroethidine in GBM (GBM115) cells or DIPG cells using microscopy images. Ctrl elements correspond to no stimulation, 3DSt elements correspond to stimulation with the magnetic fields of three fixed magnets in orthogonal axes, 1Dst elements correspond to one fixed magnet, and 1Dsp elements correspond to one rotating magnet. Scatter plots with bars represent the average normalized fluorescence intensity from three independent experiments with each data point shown as a dot (n=24). Error bars indicate SEM.

[0027] [Figure 11] Figure 11A shows the position of the cell culture dish relative to the oncosilator to obtain three different values ​​of peak-to-peak amplitude (PPA) or magnetic field strength. In particular, the bar graphs with scatter plots show the effect of varying PPA (Figures 11B and 11C) and peak frequency (PF) (Figures 11D and 5E). Data points and asterisks are as shown in Figure 1 (n=24 and n=40, respectively).

[0028] [Figure 12A] FIG. 12A shows a scheme for sOMF stimulation of cultured cells with constant Ton (250 ms) and Toff (250 ms, 750 ms, 2750 ms).

[0029] [Figure 12B]Figure 12B and Figure 12C show fluorescence intensity quantification of hydroethidine in GBM (GBM115) cells and DIPG cells using microscopy images. Scatter plots with bars represent the average normalized fluorescence intensity from three independent experiments with each data point shown as a dot (n=24). Error bars indicate SEM. [Figure 12C] Figure 12B and Figure 12C show fluorescence intensity quantification of hydroethidine in GBM (GBM115) cells and DIPG cells using microscopy images. Scatter plots with bars represent the average normalized fluorescence intensity from three independent experiments with each data point shown as a dot (n=24). Error bars indicate SEM.

[0030] [Figure 13A] Figures 13A and 13B show fluorescence intensity quantification of hydroethidine in GBM (GBM115) and DIPG cells using microscopy images. Scatter plots with bars represent the average normalized fluorescence intensity from three independent experiments with each data point shown as a dot (n=32). Data points and asterisks are as above. Error bars indicate SEM. [Figure 13B] Figures 13A and 13B show fluorescence intensity quantification of hydroethidine in GBM (GBM115) and DIPG cells using microscopy images. Scatter plots with bars represent the average normalized fluorescence intensity from three independent experiments with each data point shown as a dot (n=32). Data points and asterisks are as above. Error bars indicate SEM.

[0031] [Figure 14A] Figure 14A and Figure 14B are scatter plots with bar graphs showing viability in clonogenic cell survival assays of GBM (GBM115) and DIPG cells from independent experiments, with each data point represented by a dot (n=12). Error bars indicate SEM. Representative images of increased caspase-3 activity in GBM and DIPG cells 12 hours after the end of a 4-hour sOMF exposure. [Figure 14B]Figure 14A and Figure 14B are scatter plots with bar graphs showing viability in clonogenic cell survival assays of GBM (GBM115) and DIPG cells from independent experiments, with each data point represented by a dot (n=12). Error bars indicate SEM. Representative images of increased caspase-3 activity in GBM and DIPG cells 12 hours after the end of a 4-hour sOMF exposure.

[0032] [Figure 15A] Figures 15A and 15B show the fluorescence intensity measurements of hydroethidine-stained GBM (BT115) and DIPG cells, respectively, when exposed to different magnetic field strengths (in mT). Scatter plots with bars show the mean of n=4, error bars show SEM. [Figure 15B] Figures 15A and 15B show the fluorescence intensity measurements of hydroethidine-stained GBM (BT115) and DIPG cells, respectively, when exposed to different magnetic field strengths (in mT). Scatter plots with bars show the mean of n=4, error bars show SEM.

[0033] [Figure 16A] FIG. 16A shows the effect of a magnetic field on electron spin.

[0034] [Figure 16B] FIG. 16B shows the hypothetical effect as a function of magnetic field strength.

[0035] [Figure 17] FIG. 17 shows a schematic of the interaction of the system of FIG. 1 with mitochondrial function in cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] overview In general, electromagnetic field (EMF) stimulation can increase ROS levels in cancer cells in vitro and induce cell death in malignant tumor cells. However, it is difficult to develop devices that are both safe and effective, and it is also difficult to ascertain the exact range of EMF physical parameters that will result in a therapeutic increase in ROS levels.

[0037] The disclosed non-invasive EMF device addresses these limitations because its stimulation parameters can be better and more precisely controlled and targeted. The device generates an oscillating magnetic field (sOMF) by rapidly rotating a powerful neodymium permanent magnet. Experimental data suggest that the device, which can be called an "oncomagnetic" device, substantially and consistently elevates ROS in patient-derived glioblastoma (GBM) to levels that are selectively cytotoxic to these cells while sparing normal cells. Evidence is also available demonstrating the safety and efficacy of the device in mice implanted with orthotopic GBM xenografts and in patients with recurrent GBM for whom standard treatment options are unavailable.

[0038] The sOMF-induced increase in ROS may be due to perturbations in the electron transport process in the mitochondrial electron transport chain (ETC). The magneto-electron perturbation (MEP) hypothesis was proposed based on the known effects of weak (<1 mT) and moderate magnetic fields (1 mT-10 mT) on the mixing of the spins of the unpaired electrons of free radical intermediates in a spin-correlated electron pairing process called the radical pair mechanism (RPM).

[0039] The device is programmable, allowing precise control of all physical parameters of sOMF exposure (stimulation) both in vitro and in vivo, including magnetic field strength, frequency of magnetic field oscillation, rotation angle of magnetic field axis, on and off intervals of intermittent stimulation, duration and frequency of stimulation. Thus, the device allows us to determine the exact range of parameters that consistently achieves optimal increase in ROS. Furthermore, we can also investigate in more detail the relationship between the aspects of the field generated by the device and their interaction with the physical properties of RPM manifested in mitochondrial ETC protein complexes. For example, since these complexes have a transmembrane orientation fixed in three dimensions within the time frame of the sOMF pulse train, we can verify the possibility that changes in the magnetic field axis angle in three dimensions, only possible with a rotating magnet device, may be effective in inducing ROS, as opposed to a static magnetic field distributed along one axis.

[0040] Based on the effect of varying each of the physical parameters that define the sOMF produced by the active component of the device, called the "Oncosilator", in patient-derived GBM and diffuse intrinsic cavernous glioma (DIPG) cells, the device can be configured and the corresponding procedure parameterized. The effectiveness of various stimulation strengths, frequencies, and on-off durations to induce ROS was tested. Furthermore, it was investigated whether a static magnetic field with orthogonal axes in three dimensions and a sOMF generated by a non-rotating Helmholtz coil could induce ROS in the same range as a rotating magnetic field. This was to identify whether the change in the angle of the magnetic field axis or the magnetic field oscillation caused by the rotating magnet is more important for the ROS-inducing effect. The latter may, in addition, be important in establishing a resonance with the periodic and cyclic nature of the electron transport process. Furthermore, the anticancer effect of the optimized ROS-inducing stimulation parameters was verified in clonogenic cell survival and caspase-3 activation assays of GBM and DIPG cells.

[0041] System Overview 1 shows a system of the present disclosure that applies an oscillating magnetic field, more specifically a rapidly changing magnetic field, to tissue to target cancer cells, which generally have altered bioenergetics, mitochondrial function, and reduced numbers of mitochondria due to uncontrolled cell division.

[0042] The disclosed system uses electromagnetic fields (EMF) to modulate cellular metabolism. Prolonged exposure to repeated EMF pulses, radiating radio frequency, or non-radiating local magnetic field oscillations has varying degrees of effectiveness against cancer cells in culture. Cancer cells are subject to oxidative stress and have higher levels of reactive oxygen species (ROS) than normal cells of the same tissue, which, when further amplified, leads to apoptosis of the cancer cells. Application of EMF pulses to cells causes an increase in intracellular levels of ROS leading to cell apoptosis. Disclosed herein are methods and systems for generating oscillating magnetic fields (OMF) for treating tumors and cancer tissues in vitro and in vivo by inducing the generation of ROS and causing apoptosis of cancer cells.

[0043] As described herein, a "magnetic stimulator," "oncosilator," or "microstimulator" generates OMFs by the high speed rotation of a permanent magnet. The microstimulator has been modified to create a pattern of magnetic field oscillations that induces selective apoptosis in cultured GBM cells, but not in normal astrocytes. The lack of lethality in normal cells is due to the abundance of mitochondria, lack of or reduced oxidative stress, and a much lower demand for ATP compared to rapidly dividing malignant cells. In fact, repetitive transcranial magnetic stimulation has been shown to reduce apoptosis in non-cancer cells. The system applies OMFs of a range of frequencies in a defined pulse pattern, disrupting the flow of electrons in the mitochondrial electron transport chain (ETC), resulting in the generation of ROS. The ROS cause the opening of the mitochondrial permeability transition pore (MPTP), resulting in depolarization of the mitochondrial membrane and extrusion of cytochrome C, inducing caspase-dependent apoptosis or apoptosis by another mechanism in cancer cells. The microstimulator may also be described herein as an oncosilator due to the tumor tissue selective nature of the therapeutic methods and systems described herein. In various embodiments, the system may include a microstimulator probe, a wearable instrument having multiple oncosillators, and a fixture for surrounding a patient or tissue body for treatment.

[0044] The described OMF treatment method and system does not have the limitations of chemotherapeutic agents such as the need for sufficient blood supply to all sites of malignancy, the ability to penetrate the blood-brain barrier in case of brain tumors, sufficiently high bioavailability, favorably tailored pharmacokinetic profile, and sufficiently large therapeutic index. In addition, various OMF treatment delivery mechanisms are available with great flexibility and versatility to have a significant impact on all types of primary and metastatic solid neoplasms, and possibly even systemic malignancies. Furthermore, OMF therapy requires only 1-3 hours of administration per day, whereas TTF therapy requires 18-20 hours of treatment each day. The proposed OMF treatment method and system is much less costly than TTF therapy. The specific OMF frequency and amplitude selectively kills cancer cells in any stage of the cell cycle, and is independent of cell division, independent of being in a dividing state, and independent of any other state. In fact, the disclosed method and system can selectively kill cancer cells even in the G0 phase of the cell cycle. The oncomagnetic therapy methods and systems described herein can be drug-free, ionizing radiation-free, and non-invasive, or oncomagnetic therapy can be performed in conjunction with other forms of therapy, such as chemotherapy, other forms of radiation therapy, drugs and prescriptions, and the like.

[0045] According to at least some of the techniques of the present disclosure, the system generates an oscillating magnetic field to induce changes in the flow of electrons in tissue. However, it is believed that at least some of these techniques can also be used in conjunction with AEF techniques, for example to eliminate the need for electrodes directly on the patient's skin. More specifically, it may be possible to use an oscillating magnet to generate an AEF with similar characteristics to those used in TTF therapy.

[0046] An example of a system that disrupts mitochondrial function in cells FIG. 1 illustrates an exemplary embodiment of a system 100 for disrupting mitochondrial function in specific cells by applying a rapidly changing magnetic field. The system 100 includes a computing device 102 that controls magnetic stimulators 104-1, 104-2, ... 104-N attached to a stimulator support platform 106, which may be a probe, helmet, brace, belt, mechanical frame, room, bed, cubicle, etc. The magnetic stimulators 104 are hereinafter described as microstimulators 104 because the stimulators 104 may be miniaturized, for example, 500 millimeters, 1 cm, 2 cm, etc. along their longest dimension. Furthermore, for the specific application of the magnetic field discussed herein, the magnetic stimulators 104 may be referred to as "oncostimulators." The computing device 102 includes control hardware 110, which may include one or more processing units 112 coupled to a non-transitory computer readable memory 114 that stores a control application 120 and stimulation parameters 122.

[0047] During operation, the control hardware 110 causes the microstimulator 104 to generate an oscillating magnetic field 130 and apply the oscillating magnetic field 130 to the tissue 140 using the stimulator support platform 106. The microstimulator 104 generates the oscillating magnetic field 130 in a manner that causes disruption of mitochondrial function in cells of the tissue 140. As described in more detail below, the oscillating magnetic field 130 inhibits the flow of electrons within the cells such that apoptosis is induced in cancer cells but not in healthy cells of the tissue 140.

[0048] In addition to being able to alter the flow of electrons in cellular tissue without direct contact with the skin of the subject's scalp or other parts of the body, system 100 offers the further advantage of being configurable for a variety of different solid tumors. As described below, various stimulation parameters and stimulation parameters can generate different configurations of OMFs, which can be tailored to specific cell types and / or specific subjects to implement personalized treatment protocols. Furthermore, the system has the advantage that it can be an imperceptible sham treatment, serving as a placebo control in double-blind studies. In particular, the high field strength magnets of the microstimulators described below can be replaced with degaussing magnets. Because subjects cannot sense the magnetic field, study subjects and investigators cannot distinguish between an operating instance of system 100 and a sham stimulation system by examination or during a treatment session.

[0049] The computing device 102 may be a general-purpose computing device such as a desktop computer, a laptop computer, a table computer, a smartphone, a wearable device such as a smart watch, etc. The processing unit or units 112 in these embodiments may be a central processing unit (CPU), and the memory 114 may include non-persistent elements (e.g., random access memory (RAM)) as well as persistent elements (e.g., flash drive, disk). In other embodiments, the computing device 102 is a special-purpose medical device that is specifically configured to control one or more microstimulators 104. The processing unit 112 in this case may include a field programmable gate array (FPGA), an application specific integrated circuit (ASIC), or any other type of dedicated hardware. Additionally, the control hardware 110 in some embodiments is integrated into the stimulator support platform 106.

[0050] The control application 120 can specify one or more sets of stimulation parameters 122 for a stimulation session, which can specify at least some of the duration of the session, the pulse width (length) of the stimulation pulses, the duration of the stimulation pulse interval, and the oscillation frequency of the magnet. In some cases, the stimulation parameters 122 can define separate sets of values ​​for the different microstimulators 104-1, 104-2, ... 104-N to define a particular stimulation pattern. More specifically, the stimulation pattern can specify a particular activation time and operating parameters (frequency, pulse parameters) for the microstimulator 104-1, a different activation time and different operating parameters for the microstimulator 104-2, etc. These parameters can in some cases depend on the relative positioning of the microstimulators 104, e.g., microstimulators 104-i and 104-j positioned at a fixed distance and angle from each other can generate stimulation pulses with a fixed phase offset to generate an oscillating magnetic field with certain desired characteristics.

[0051] In the example of FIG. 1, the computing device 102 includes a peripheral interface 150 and a user interface 152 in addition to one or more processors 112 and memory 114. The peripheral interface 152 may be any suitable wireless interface, such as a serial peripheral interface (SPI), a universal serial bus (USB), or a wireless personal area network (WPAN) interface (e.g., Bluetooth), a wireless local area network (WLAN) interface (e.g., Wi-Fi), etc. The computing device 102 may use the peripheral interface 150 to send commands to the microstimulator 104. More specifically, the computing device 102 of the exemplary implementation of FIG. 1 provides commands to the microstimulator 104 via the peripheral interface 150 and a control circuit 154 of the stimulator support platform 106.

[0052] The control circuitry 154 may be configured to receive commands for the individual microstimulators 104-1, 104-02, etc., turn the motors of the microstimulators 104-1, 104-02, etc., on or off, vary the rotational speed or other type of vibration of the motors, etc. In this manner, the control circuitry 154 may act as a demultiplexer. Furthermore, as indicated above, in some implementations, the control hardware 110 is incorporated into the stimulator support platform 106, and thus, all of the control functionality of the system 100 may be provided to the control circuitry 154. More generally, the control functionality of the system 100, such as control logic for operating the microstimulators 104 according to various stimulation patterns, may be distributed between the computing device 102 and the control circuitry 154 in any suitable manner, including providing the entire control functionality entirely within the computing device 102 or entirely within the control circuitry 154.

[0053] The user interface 152 may include a touch screen configured to receive input and display output, or the input (e.g., keyboard, pointing device) and output (e.g., display) components may be separate. An operator may use the user interface 152 to provide commands to select a particular desired stimulation parameter 122 for a particular microstimulator, or a stimulation pattern including multiple stimulation parameters 122 for each microstimulator. Additionally, the system 100 in some embodiments may include one or more magnetic sensors, such as microelectromechanical system (MEMS) sensors, and the user device 102 may provide readings from these magnetic sensors via the user interface 152.

[0054] The microstimulator platform 106 may be a harness, helmet, brace, etc. Additionally, the microstimulator platform 106 may be part of a hospital bed, and in some cases, the system 100 may apply stimulation to a sleeping subject. Additionally, in some implementations, the microstimulator platform 106 may be an intraoperative probe that may be manually guided by a clinician. In some implementations, the microstimulator platform 106 includes one or more magnetic sensors to provide sensor measurements to an operator as described above and / or to provide feedback signals to the control hardware 110, which may adjust certain operating parameters to achieve a desired strength of the magnetic field.

[0055] The microstimulator platform 106 in some embodiments may include a power storage device, such as a battery, to power the microstimulator 104. The battery may be, for example, a Tenergy NiMH 9.6V, 5,000 mAh rechargeable battery. In other embodiments, the computing device 102 may be configured to provide power to the microstimulator platform 106 via the peripheral interface 150.

[0056] The system 100 in various scenarios can generate intermittent or continuous stimulation sessions for a period of between 1 minute and 20 hours for a given treatment session, depending on the amount of stimulation desired as determined by the treatment plan. In various embodiments, the oscillating magnetic field is generated intermittently or continuously for a period of between about 20-30 hours. The frequency of each vibration stimulus, i.e., the vibration motion of the motor, can range from 5 Hertz to 400 Hertz. In various aspects, the frequency of each vibration stimulus, i.e., the vibration motion of the motor, is between 30-300 Hertz. In various aspects, the frequency of each vibration stimulus, i.e., the vibration motion of the motor, is about 300 Hertz. The microstimulator 104 can provide the vibration stimulation as a series of one or more pulses. In some embodiments, the pulse length ranges from about 10 milliseconds to about 5 seconds, and the inter-stimulus pulse interval ranges from about 10 milliseconds to about 10 minutes, resulting in a duty cycle of the pulse train ranging from 0.001% to 50%. The oscillation frequency, timing, pulse duration, and inter-stimulus pulse interval of the oscillatory stimulation provided by any of the microstimulators 104 can be kept the same, varied together, and / or varied independently for each microstimulator 104. The oscillatory stimulation pulses can vary in time length and stimulation pulse interval from pulse to pulse as necessary to provide the appropriate electric field strength at the target site or volume. The stimulation pulses can have amplitude envelopes shaped like square, Gaussian, sinusoidal, ramped, sawtooth, etc., to provide the appropriate electric field amplitude at the target site or volume. Additionally, the system 100 can ramp up and / or down the oscillation frequency up to a maximum desired frequency or between frequencies for a given treatment session.

[0057] In some embodiments, the control hardware 110 can activate the microstimulators 104 one at a time, simultaneously, sequentially, in pairs, in groups, or according to any combination thereof (e.g., sequentially in pairs, one group at a time). Indeed, the control hardware 110 can actuate any number of microstimulators 104 in any order that can deliver the desired OMF to the target site or volume.

[0058] The control hardware 110 may, in some cases, perform beat frequency stimulation when generating OMF at the target site or volume. According to the beat frequency stimulation approach, the control hardware 110 causes magnets in the two microstimulators to oscillate at different frequencies to generate a beat frequency at the target site or volume. For example, the microstimulator 104-1 may oscillate at 320 Hz and the microstimulator 104-2 may oscillate at 400 Hz. Due to electromagnetic interference, the microstimulators 104-1 and 104-2 generate a beat frequency of 80 Hz at the target site or volume, which is the difference between the frequencies of 320 Hz and 400 Hz. The amplitude of the OMF at this beat frequency is twice the amplitude of the OMF that the microstimulators 104-1 or 104-02 would induce independently. The system 100 may perform beat frequency stimulation when a high amplitude low frequency electric field is required or desired in the treatment.

[0059] The small size of the magnets and motors of the microstimulator 104 and its battery powered operation make the system 100 portable, so that treatment can be performed anytime, anywhere. Additionally, the control application 120 can be installed on a smart phone, tablet computer, or handheld computing device, as described above.

[0060] As described above, the control application 120 can operate according to stimulation parameters 122 and various stimulation patterns or preprogrammed therapy sessions. The control application 120 can further implement safety features, such as limiting the amount of therapy a patient can receive daily, weekly, or monthly, limiting how often or when a patient can receive therapy (e.g., only on weekdays, only on certain days of the week, only during a four-week period), etc.

[0061] FIG. 2 illustrates an example of a magnetic assembly or oncosilator 200 operable as the microstimulator 104 in the system of FIG. 1. The oncosilator 200 includes an electric motor 202 and a magnet 206 attached to a shaft 204. In this embodiment, the magnet 206 is radially magnetized with a semicircular pole arrangement, and the motor 202 imparts a rotational motion to the magnet 206 in the direction indicated by the arrow (or the opposite direction). The motor 202 may be a high-speed motor capable of generating 21,000 revolutions per minute (RPM) in unloaded mode. The oncosilator 200 may optionally include a housing or sleeve 210 made of a material (e.g., plastic) that is permeable to magnetic fields so as not to affect the OMF generated by the oncosilator 200.

[0062] The magnet 206 may have a cylindrical shape with a bore configured to receive the shaft 204. The diameter of the magnet 206, which is particularly suitable for GBM treatment, is approximately 3 / 4 inch. The height of the cylinder defined by the magnet 206 is also 3 / 4 inch in the exemplary embodiment. The magnet 206 may be a relatively strong rare earth magnet having a strength of over 1.2 Tesla, for example, a neodymium N42 magnet.

[0063] FIG. 3 shows an example of an output 300 of the oncosilator 200. To generate the output 300, the oncosilator 200 RAMP UP The frequency is increased from about 0 Hz to a peak frequency of about 150 Hz over a period of time (e.g., 50-100 ms). The oncoiler 200 thenRAMP DOWN Over a period of time (e.g., 50 to 200 ms), the frequency can be ramped down from the peak frequency to less than 50 Hz, or even down to 0 Hz. In the example of FIG. 3, T RAMP DOWN is T RAMP UP The Oncosilator 200 then RAMP UP T, which can be between 250 ms and 2750 ms, until a new cycle begins again with a period of INACTIVE The inactive state can be maintained for a period of time.

[0064] More generally, the oncosilator 200 can increase the frequency to a peak frequency of between 100 Hz and 300 Hz, depending on the particular implementation.

[0065] As shown in Figure 4, the oncomagnetic device of the present disclosure can sequentially activate multiple oncosilators 104-1, 104-2, 104-3 to affect the same tissue. Each of these oncosilators can be implemented as shown in Figure 2. The axis about which oncosilator 104-1 rotates its associated magnet is substantially orthogonal to the axis about which oncosilator 104-2 rotates its associated magnet, and the axes of oncosilators 104-1, 104-2 are orthogonal to the axis about which oncosilator 104-3 rotates its associated magnet. The pulse train is shown in a simplified manner but can be similar to output 300 described above.

[0066] Figure 5 shows an example of three oncosillators arranged on the surface of a sphere similar to a patient's head for the purpose of GBM treatment. Each oncosillator 104-1, 104-2, and 104-3 may be spaced 10-12 cm apart in an arc relative to the other oncosillators. The magnetic flux penetrates to a depth of approximately 12 cm.

[0067] In the exemplary embodiment, oncosillators 104-1 and 104-2 are attached to supporting headgear on either side in the front, and oncosillator 104-3 is attached to the back at the midline.

[0068] FIG. 6 illustrates an example of a method that may be implemented in the device of claim 1. In block 602, the oncomagnetic device ramps up the frequency to a peak frequency (e.g., 100-300 Hz) over a first period, e.g., 50-100 ms. In block 604, the oncomagnetic device ramps down the frequency from the peak frequency over a second period, e.g., 50-200 ms. In block 606, the oncomagnetic device applies no OMF for an off period, e.g., 250-2750 ms. In block 608, the oncomagnetic device changes the axis about which the magnet rotates if a single magnet is used. Alternatively, the oncomagnetic device selects another magnetic assembly having an axis orthogonal to the axis of the magnetic assembly that was active in blocks 602 and 604 (see FIG. 4). Flow then returns to block 602.

[0069] For further clarity, several experiments related to the oncomagnetic system of the present disclosure are described below.

[0070] Cell lines and reagents At least some of the examples below relate to GBM115, a temozolomide-resistant cell line derived from GBM patient tissue excised by DSB, and the DIPG cell line was purchased from Sigma-Aldrich. Both cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 11 mM glucose, and penicillin and streptomycin antibiotics at 37°C in a humidified incubator with 5% CO2. All sOMF irradiations and control sets were performed in a humidified incubator with 37°C and 5% CO2. Dihydroethidium (FisherScientific) was dissolved in DMSO at a concentration of 10 mM (stored at -20°C) and diluted to 5 μM in culture medium immediately before use.

[0071] Exposure to static and oscillating magnetic fields As shown in Figure 7, sOMF stimulation produced by magnet rotation was performed using an oncosilator held in a custom-made non-magnetic wooden frame with aluminum clamps and a plastic holder made by a 3D printer. The oncosilator was positioned at different distances from the cell culture plate to vary the peak-to-peak amplitude (PPA) of the sOMF as seen by the cells to approximately 0.4, approximately 1, and approximately 5 mT. The magnetic flux density values ​​at various distances from the axis of the axially magnetized cylindrical neodymium (N52) magnet used in the oncosilator were measured using a Homend handheld digital WT10A Gaussmeter. Three values ​​of peak frequency (PF) of magnet rotation (sOMF oscillation frequency) were used: approximately 77 Hz, approximately 137 Hz, and approximately 277 Hz. For intermittent stimulation, the on-time (T on or sOMF pulse train duration) was kept constant at 250 ms. off ) were set to 250, 750, and 2750 ms.

[0072] All frequency and timing values ​​were programmed into the microprocessor that controlled the oncosilator (see Figure 1). The sOMF effects produced by each parameter set were compared with the other two sets and with a no-stimulation control. They were also compared with the sOMF produced by exposure to a static magnetic field of one non-rotating magnet and three orthogonal non-rotating magnets, and by rotating the latter three magnets. Stimulation with sOMF produced by a non-rotating Helmholtz coil was also performed by passing a sinusoidal current of sufficient strength to produce a PPA of approximately 5 mT between two solenoids on which the cell culture plate was placed. The sinusoidal current was generated by a function generator (Wavetek, San Diego, CA) and amplified to the desired current amplitude by a high-current amplifier (Taidacent, Shenzhen Taida Century Technology Co., Ltd., China). Cells were stimulated once in each experiment for a total of 4 h. In one experiment, three 2-h stimulations were performed, with two 2-h intervals between the three stimulation periods, to examine the effect of repetitive stimulation. For the same PF and PPA, we compared 4 hours of intermittent stimulation with the same duration of continuous stimulation.

[0073] ROS detection and caspase-3 activation assay Cells grown in glass-bottom 4-chamber dishes (one cell line per 2 chambers) were incubated with 5 μM dihydroethidium (hydroethidine) for 30 min in the dark at 37 °C in a humidified incubator with 5% CO2. Three to four fluorescent images from each chamber were randomly taken at each time point for each treatment group using a CarlZeiss microscope. Images were taken before the start of static magnetic field or sOMF exposure (0 h), 2 h and 4 h after sOMF exposure. The last image was taken 2 h after the end of sOMF exposure (2 h). To detect caspase-3 activity, cells were incubated with NucView488 dye for 30 min 12 h after the end of 4 h sOMF exposure. Cells were then fixed with 4% PFA, permeabilized with Triton-X100, and incubated with DAPI for 10 min before imaging. Exposure times and magnifications were the same for all images from all experiments. Unmodified fluorescent micrographs stained with hydroethidine at 10x resolution were digitized on a desktop computer connected to the microscope and imported into the MATLAB programming environment (Mathworks, Natick, MA). RGB-encoded red blood cell pixels with fluorescence intensity above a uniformly set threshold were automatically counted using a MATLAB script written in-house. The resulting intensity values ​​were normalized to T0, i.e., the pre-stimulation time point in each stimulation condition, and to the first time point in the unstimulated condition, or the average of all T0 values. The normalized values ​​at each time point in the stimulated condition were then re-normalized to the normalized values ​​at the corresponding time point in the unstimulated condition. Data were pooled from three replicates of each experiment, and a two-tailed Student's pooled t-test with false discovery rate test for multiple comparisons was used to assess statistical significance at the p = 0.05 level.

[0074] Clonogenic cell survival assay BT-115 or DIPG cells were seeded at 200 cells per dish for clonogenic assay and kept in a humidified incubator at 37°C with 5% CO2. After 8–10 h, these dishes were transferred to an incubator with the sOMF device running. After sOMF exposure, the dishes were transferred to a cell culture incubator and colonies were grown for 10 days for DIPG cells and 14 days for BT-115 cells. Colonies were fixed and stained with crystal violet stain (0.05% crystal violet, 1% formaldehyde, 1% methanol) for 15–30 min. Colonies were washed and dried and counted manually. Viability was calculated by dividing the number of colonies by the number of cells seeded and normalized by the average viability of the controls in each experiment. Viability experiments were repeated and the averages were plotted in a scatter plot with bar graphs using the Prism program. Each dot in the scatter plot represents the normalized viability of one dish.

[0075] Data were pooled from three replicates of each experiment, and a two-tailed Student's pooled t test with false discovery rate test for multiple comparisons was used to assess statistical significance at the p = 0.05 level.

[0076] Effect of static magnetic fields compared to sOMF It was hypothesized that weak and moderate magnetic fields could perturb the electron transport process (EMP hypothesis) and generate superoxide by interacting with the RPM machinery of the mitochondrial ETC. ETC membrane complex molecules, unlike those in solution, are omnidirectional and do not roll around. It was therefore predicted that a rotating magnet should induce more ROS than a non-rotating magnet oriented along one fixed axis (1Dst). Furthermore, a static magnetic field of three non-rotating magnets aligned along three orthogonal axes in three-dimensional space (3Dst) should also generate an amount of ROS comparable to that of a rotating magnet (1Dsp).

[0077] These predictions were verified by stimulating GBM and DIPG cells side-by-side under three conditions (Figure 8A). sOMF was generated by an oncocytometer equipped with a rotating magnet, with a PF of approximately 272 Hz and a T on and T off The duration of stimulation was 250 ms, respectively. Stimulation was performed for 4 h. The ROS generated by sOMF(1Dsp) was observed to be significantly higher than that generated by 1Dst and 3Dst static magnetic fields at 2 h (during stimulation), 4 h (end of stimulation), and 6 h (after stimulation) in both GBM and DIPG cells (Figures 8C and 8C). The higher effect of the rotating magnetic field compared to the static magnetic field along one dimension supports the predictions of the MEP hypothesis, while the lack of a significant ROS-inducing effect of the static magnetic field oriented in all three dimensions suggests that the magnetic field oscillation itself is important for this effect.

[0078] Intermittent vs. continuous sOMF If the total amount of exposure to peak frequency vibration is important for ROS induction, continuous stimulation is expected to increase ROS more. Therefore, we examined whether there was a difference between continuous and intermittent stimulation of sOMF with oncolytic agents. Intermittent stimulation was performed at T on and T off and sOMF stimulation for 250 ms each. The PPA was approximately 277 Hz for both types of stimulation. Analysis showed that ROS levels in cells exposed to continuous sOMF stimulation were not significantly higher than in cells exposed to intermittent sOMF stimulation in both GBM and DIPG cells. These data indicate that repeated pulse trains of increasing and decreasing peak frequencies, separated by pauses, are sufficient to achieve a near-maximal increase in ROS levels.

[0079] Effect of sOMF along the fixed axis The magnetic field oscillation induced by the rotating magnet is characterized by two distinct components: the sine wave of the magnetic field and the angle of the magnetic field axis that changes periodically. The above results indicate that the magnetic field oscillation plays an important role in inducing ROS. To verify whether the oscillation is sufficient to produce the ROS effect, the effect of the magnetic field oscillation produced by a Helmholtz coil, whose axis remains fixed in one direction, was compared with that produced by the rotating magnet of the oncosilator.

[0080] The current passing through the coil alternated at approximately 137 Hz, which was sufficient to generate a PPA of approximately 5 mT. These values ​​were the same as those generated by the rotating magnet of the Oncosilator used in this study. Both devices provided 4 hours of continuous stimulation. It can be seen that the Helmholtz coil did not produce a significant increase in ROS in GBM and DIPG cells at 2 and 4 hours during stimulation, or 2 hours after the end of stimulation (Figures 9A and 9B). In contrast, the Oncosilator significantly increased ROS levels at all three time points in both cancer cell types. These observations indicate that the oscillating magnetic field alone may not be sufficient to induce ROS, and that a change in the angle of the magnetic axis may be necessary to achieve this effect.

[0081] Three-axis rotation of the magnet increased ROS levels Because repeatedly changing the angle of the magnetic field axis in three dimensions may have a greater effect on ETC complexes oriented in any direction in space, we further investigated whether rotating the magnet along all three orthogonal axes in three-dimensional space would result in a greater increase in ROS than rotating the magnet along only one axis. Three oncosillators (3DSeq, Figure 10A) were positioned at right angles to each other and actuated in repeated sequential or alternating cycles compared to intermittent stimulation with one oncosillator (1DSp). This experiment showed that the increase in ROS after 2 hours with 3DSeq was greater in both GBM and DIPG cells compared to 1DSp stimulation. However, this difference was not statistically significant. The significant increase in ROS levels relative to the control seen at 4 hours and 2 hours after the end of stimulation was also not significantly different between 3DSeq and 1DSeq (Figures 10B and 10C). This suggests that 1DSp stimulation may be sufficient to result in the greatest ROS increase, given that one active oncosillator sweeps all angles in a two-dimensional plane.

[0082] Changes in stimulation parameters Oncolytic stimulation has three physical parameters that can be varied along a continuous scale. The relevant parameters are (1) the strength of the magnetic field or PPA of the stimulation, (2) the PF of the stimulation, and (3) T off It is time. on was kept constant at 250 ms, and the effect of varying the value of each of the other parameters to three different levels was determined. The effects of approximately 0.42 mT, approximately 1.2 mT, approximately 5.5 mT, and 58.3 mT were examined by placing the cell culture dish at distances of 7 cm, 5 cm, 3 cm, and 1.4 cm, respectively, from the oncosilator (Figure 11A; see also Figures 15A and 15B). All field strengths tested showed a significant increase in ROS levels at 4 hours in GBM and DIPG cells (Figures 11B and 11C). With regard to the change in PF between approximately 77 Hz, approximately 135 Hz, and approximately 277 Hz, the latter two frequencies were significantly more effective than approximately 77 Hz (Figures 11D and 11E). Ton While maintaining 250ms, three different T off Comparing the T values ​​(250, 750, and 2750 ms), the T values ​​of 250 ms and 750 ms were significantly higher in GBM cells. off had a slightly more favorable effect on ROS generation than 2750ms (Figures 12A and 12B). In contrast, in DIPG cells, T off The greatest amount of ROS was generated by (Figure 12C).

[0083] The effect of repeated stimulation To determine whether repeated sOMF stimulation would result in a cumulative increase or other effect on ROS levels, cells were stimulated three times for 2 h with 2-h intervals between each stimulation. off This stimulation pattern with PF and PF resulted in a similar increase in ROS levels with each subsequent repetition compared to the first stimulation session in both GBM and DIPG cells (Figures 13A and 13B), suggesting that the ROS-inducing mechanisms underlying the sOMF effects produced by the rotating magnet are neither potentiated nor desensitized.

[0084] Effect of sOMF on cancer cell clonogenesis and caspase-3 activation By examining various stimulation parameters, the most effective range for maximal induction of ROS in GBM and DIPG cells can be identified. As higher ROS can cause oxidative damage of macromolecules and induce cell death, optimally effective parameters were used to stimulate GBM and DIPG cells and evaluate clonogenic cell survival and caspase 3 activation. The optimal range of parameters used was a PPA of approximately 5 mT, a PF of approximately 137 Hz, and a T of 250 ms. on , T of 750ms off The stimulation time was 4 h with intermittent stimulation mode. A standard clonogenic cell survival assay was performed with the optimized parameters to determine the extent to which viability was reduced by 2 and 4 h of sOMF stimulation under single cell conditions. offThe duration of stimulation was 250 ms and 740 ms for 4 h. This corresponds to 28,800 pulse trains for 2 h and 19,200 pulse trains for 4 h. A reduction of >60% and >40% in DIPG and GBM viability by sOMF was observed at 2 h stimulation (Fig. 14A). At 4 h stimulation, the values ​​were >80% and >60% (Fig. 8B). This result may be due to the fact that short sOMF stimulation may produce an immediate cytotoxic effect on single cancer cells and may also produce a delayed cytotoxic effect to stop cell proliferation. Moreover, in both DIPG and GBM cells, caspase 3 activity was slightly increased 12 h after 4 h stimulation with optimal parameters. [Industrial Applicability]

[0085] In patients with late-stage recurrent GBM, a first-in-human compassionate use monotherapy using the Oncomagnetic device was shown to induce tumor size reduction with evidence of therapeutic efficacy in brain autopsies, and the treatment was well tolerated. Furthermore, sOMF generated by the Oncomagnetic device was shown to be selectively cytotoxic to several cancer cell lines, including human GBM cells, U87 cells, A549 lung cancer cells, and GL261 mouse GBM cells. Furthermore, sOMF was shown to be non-toxic to normal human astrocytes and cortical neurons in culture.

[0086] The applicants tested the above MEP hypothesis and determined the effectiveness of several sets of sOMF stimulation parameters to induce the superoxide component of ROS. The applicants further tested whether sOMF stimulation with an optimized set of parameters exhibited high anti-cancer activity in a standardized assay. The results showed that simultaneous exposure to magnetic field oscillations generated by the rotation of the axis of a two-dimensional plane maximally increased the superoxide component of ROS in human GBM, human DIPG and GL261 mouse GBM cells.

[0087] International Telecommunication Union 44The narrow range of magnet rotation frequencies (100-300 Hz) in the extremely low frequency region defined by is effective in raising ROS to high levels. The magnetic field strength that shows the ROS-inducing effect is in the intermediate region of approximately 1 to approximately 5 mT. Increasing the strength to approximately 58 mT does not significantly increase the effect, but decreasing it to approximately 0.4 mT shows a decreasing tendency. Intermittent sOMF patterns do not seem to differ significantly in their effects from continuous sOMF stimulation. The combination of on-off timing sequences chosen for intermittent stimulation showed only slight differences in the observed effects. The set of values ​​selected from the optimal range of these parameters for the 2- and 4-h stimulation sessions is effective in killing GBM and DIPG cells and reducing the percentage of viable colonies in clonogenic cell survival assays. Although the total number of pulse train stimuli of the same duration (250 ms) delivered over 4 hours (19,200) was less than that delivered over 2 hours (28,800), 4 hours of stimulation more significantly inhibited the growth of GBM and DIPG cell colonies. This indicates that the total amount of energy delivered to the cancer cells is not clearly the determining factor in the efficacy of stimulation. Rather, the T between stimulations. off The longer duration of stimulation (750 ms for 4 h compared to 250 ms for 2 h) seems to be the key variable. Alternatively, longer intermittent stimulation may have produced a stronger effect. Although mitochondria are the main site of ROS production and extramitochondrial sources such as cryptochromes cannot be excluded, T is required for the generation of superoxide, which triggers the mitochondrial permeability transition (MPT). off This biological effect cannot be explained by stimulus-induced hyperthermia, since exposure to oncosilator-generated sOMF did not result in a temperature increase at the distance between the magnet and stimulated cell cultures used in this experiment.

[0088] It should be noted that the duration of TTF treatment showing an increase in H2O2 is reported to be 24 hours. Similarly, in contrast to 4 hours of sOMF stimulation with the disclosed device, which reduces cell viability by more than 60% and more than 80% in GBM and DIPG cells, respectively, TTF stimulation lasting as long as 3 days results in a reduction of approximately 10% to approximately 50% in three different types of GBM cell lines. TTF also results in a paradoxical increase in clonogenic viability in the U251 GBM cell line. These differences indicate that the underlying mechanisms of action between TTF and sOMF stimulation are different from each other.

[0089] Moreover, the induction of ROS by sOMF indicates that a direct target of its action is the intracellular redox machinery. Magnetic fields above 1 mT are known to alter the kinetics and yields of certain chemical reactions involving free radical intermediates that exchange unpaired electrons. These effects are now recognized in the context of RPM to arise from the conservation of electron spin in radical recombination reactions and the long-lasting spin coherence of spin-correlated electrons in radical pairs. The characteristics of RPM affected by magnetic flux densities in the range of 1–10 mT are as follows: (1) reactants that produce radical pairs whose electron spins are in the singlet configuration, i.e., with antiparallel spins; (2) spin-selective reactions of singlet pairs to produce singlet products, or nonselective back reactions; (3) competition between these reactions and interconversion of singlet pairs and triplet (parallel spin) pairs under the influence of an externally applied magnetic field; (4) triplet pairs also react spin-selectively or back-react nonselectively to give triplet products; (5) in many cases, the nonselective products regenerate the initial reactants, starting the cycle again; and (6) singlet and triplet states influenced by electron-nuclear hyperfine interactions.

[0090] According to the MEP hypothesis, the electron transport process in ETC complexes may also have such properties and therefore be optimally perturbed by sOMF in the observed range of 1 to 5 mT (Figure 10). The observation that increasing the magnetic flux density by an order of magnitude (5 mT to 58 mT) does not result in a significant increase in the ROS induction effect is consistent with the observation in the field of spin chemistry that the decrease in product yield in RPM reactions plateaus at magnetic flux densities in the range of 1 mT to >1 T. Thus, the dependence of the magnetic field oscillation and the orientation of the magnetic field axis on the maximum induction of ROS in these experiments supports the hypothesis that RPM is the target of sOMF. This magnetic stimulation method is particularly favorable because both can be achieved by rotating a permanent magnet. As experimental confirmation that electron flow in the ETC is perturbed by sOMF, oxygen electrode measurements have separately shown that sOMF stops or slows the consumption of oxygen by isolated rat liver mitochondria and permeabilized GBM and DIPG cells. It has also been observed that sOMF induces mitochondrial MPT, which is inhibited by bongkrekic acid. Superoxide opens the MPT pore in the inner mitochondrial membrane.

[0091] Thus, a combination of sinusoidal magnetic field oscillations and rapid changes in the angle of the magnetic field axis at low mT magnetic flux density with defined frequency and timing patterns causes a rapid increase in the superoxide component of ROS, inducing cell death and impeding colony growth in GBM and DIPG cells in culture. The bridge between these biological effects and the physical effects of the magnetic field appears to be the quantum effects involved in the RPM underlying the electron transfer reactions in the mitochondrial respiratory chain. Our experimental results provide a rationale for the use of sOMF generated by rotating permanent magnets for the non-invasive treatment of GBM, DIPG and other solid malignancies.

[0092] Effects of vibration patterns on GBM and DIPG cells Thus, as shown in Figures 8A-8E, sOMF with rotating magnets generates higher cellular ROS than static magnetic fields. Figures 9A and 9B show the advantages of magnetic field oscillation and axial rotation for ROS induction. Figures 10A and 10B show that one rotating magnet or three rotating magnets generate similar ROS levels. Figures 11A-11E show the effect of changing magnetic field strength, peak frequency, and stimulation repetition. Figures 12A-12C show the change in OFF period between stimulation trains. Figures 13A and 13B show that sOMF with optimal parameters induces a consistent increase in ROS in GBM and DIPG cells. Figures 14A and 14B show that sOMF exposure causes significant cell death in GBM and DIPG cells. Figures 15A and 15B show that increasing the magnetic field strength from approximately 5 mT to approximately 58 mT does not result in any further increase in ROS.

[0093] FIG. 16A illustrates the magnetic field affecting the interconversion of electron spins from singlet to triplet states. In particular, FIG. 16A illustrates the vector representation of spins. FIG. 16B illustrates that the effective range of magnetic field strengths used in the experiments described in this disclosure (approximately 1 mT to approximately 58 mT) falls within the plateau phase of the biological effect. The arrows point in the direction of increasing biological effect (i.e., increasing superoxide).

[0094] 17 shows a schematic of the interaction between the system 100, here represented by an exemplary microstimulator 104, and the mitochondrial function of an exemplary GBM cell 200. The microstimulator 104 (or some microstimulators as described above) generates OMF, which in the GBM cell reacts with the mitochondrial function [1,2- 13 It reduces mitochondrial glucose oxidation in the tricarboxylic acid (TCA) cycle (or "citric acid cycle") and increases metabolic flux in glycolysis through the production of [C]acetyl-CoA (Fig. 1b). 1 HNMR spectrum of lactic acid 13C enrichment), disrupts ETC electron flow, increases the generation of superoxide, peroxide, and other reactive oxygen species, opens MPTP, and activates the caspase-3-mediated apoptotic pathway or an alternative apoptotic pathway.

[0095] In particular, system 100 generates a rapidly changing magnetic field in one or more stimulators 104, which disrupts the flow of electrons in cells 200 present in tissue exposed to the magnetic field. As shown diagrammatically in Figure 2, system 100 creates electrical perturbations in mitochondria, the intracellular energy producing components, resulting in the localized release of harmful chemicals (reactive oxygen species and cytochrome C) in cells with impaired mitochondrial function, including but not limited to cancer cells. This localized release of harmful chemicals triggers apoptosis, a molecular cell death process.

[0096] More specifically, cancer cells are under high stress because uncontrolled cell division requires more energy in the form of adenosine triphosphate (ATP) produced by mitochondria. The system generates OMF, which results in rapid fluctuations and intermittent depolarization of the mitochondrial membrane potential (MMP) in the tissue. This process disrupts the mitochondrial network and inhibits ATP-generating proton flux / electron transport in individual mitochondria. This process also leads to leakage of cytochrome C and reactive oxygen species (ROS), which further depolarizes the MMP and causes mitochondrial destruction. Collectively, these events trigger molecular pathways that lead to DNA damage and apoptosis in cancer cells. In normal cells (e.g., healthy cells), the amount of mitochondria is higher, the demand for ATP is lower, and they are not under stress, so in normal cells, the inhibition of electron flow and the formation of small amounts of ROS and depolarization of MMP do not induce apoptosis. The absence of apoptosis may be due to the operation of antioxidant mechanisms that counteract the increase in ROS.

[0097] In some of the tests described below, the system 100 in one embodiment generated an oscillating magnetic field that, when applied to GBM cells, caused disruption of the mitochondrial network, mitochondrial collapse with MMP depolarization, and a decrease in citric acid cycle metabolites. The system 100 in this embodiment included a single microstimulator with a neodymium magnet magnetized at 1.48 Tesla. The motor rotated the magnet at about 350 Hz. The system 100 generated stimulation pulses of about 500 ms duration with a 1000 ms interstimulus interval (measured from the start of one pulse to the start of the next pulse). The microstimulator was placed at a distance of about 1 cm from the cells in the slide chamber. These 500 ms pulses were generated intermittently at 1000 ms intervals for 60 to 90 minutes.

[0098] In some embodiments, the microstimulator 104 oscillates at a frequency in the range of 250-350 Hz to generate an OMF having a frequency of 250-350 Hz. In some cases, the frequency at which the microstimulator 104 oscillates includes sub-harmonic and super-harmonic frequencies. The system 100 may apply the OMF, for example, as approximately 250 millisecond pulses (i.e., having an ON sub-cycle or pulse length of approximately 250 ms, followed by an OFF sub-cycle or inter-stimulus pulse interval of approximately 250 ms) with a duty cycle of 50 percent. The system 100 may apply these pulses (or other suitable pulses) for an ON period P ON and the subsequent OFF period P OFF In some embodiments, P ON lasts for 5-900 seconds, P OFF The period may last from 1 to 300 seconds. Additionally, the system 100 may increase the OMF frequency to a peak frequency over a period of 75 to 100 milliseconds, and then decrease the OMF frequency from the peak frequency over a period of 250 milliseconds.

[0099] Mitochondria in the experiments outlined below were stained with MitoTracker® and MitoSox® dyes. Live GBM cells were repeatedly stimulated for 10 and 90 minutes, and real-time imaging was performed to show changes in the fluorescence of these dyes in fixed cells 3 hours after stimulation, confirming the disruption of the mitochondrial network by the system 100 generating OMF, mitochondrial collapse due to MMP depolarization, and a decrease in citric acid cycle metabolites. Furthermore, using nuclear magnetic resonance spectroscopy, it was observed that mitochondrial acetyl-CoA formation was reduced by approximately 10%, and lactate (a by-product of glycolysis in the cytoplasm) was increased as well.

[0100] The following list of examples reflects various embodiments expressly contemplated by this disclosure.

[0101] Example 1 A method for disrupting mitochondrial function in a cell, comprising controlling hardware to vibrate one or more magnets aligned along multiple, substantially orthogonal axes of rotation to generate an oscillating magnetic field, and applying the oscillating magnetic field to tissue containing cancer cells to induce apoptosis of the cancer cells.

[0102] Example 2 The method of example 1, wherein the peak-to-peak amplitude (PPA) of the oscillating magnetic field is about 0.4 mT.

[0103] Example 3 The method of example 1, wherein the peak-to-peak amplitude (PPA) of the oscillating magnetic field is about 1 mT.

[0104] The method of example 1, wherein the peak-to-peak amplitude (PPA) of the oscillating magnetic field is about 5 mT.

[0105] Example 5 The method of example 1, wherein the peak frequency (PF) of the rotation of the one or more magnets is about 77 Hz.

[0106] Example 6 The method of example 1, wherein the peak frequency (PF) of the rotation of the one or more magnets is about 137 Hz.

[0107] Example 7 The method of example 1, wherein the peak frequency (PF) of the rotation of the one or more magnets is about 277 Hz.

[0108] Example 8 The method of Example 1, further comprising generating intermittent stimulation using one or more magnets, the intermittent stimulation comprising generating a pulse train having an ON time of about 250 ms.

[0109] Example 9 The method of Example 8, wherein the OFF time is about 250 ms.

[0110] Example 10 The method of Example 8, wherein the OFF time is about 750 ms.

[0111] Example 11 The method of Example 8, wherein the OFF time is about 2750 ms.

Claims

1. 1. A method for disrupting mitochondrial function in a cell, said method comprising: controlling hardware to oscillate the plurality of magnets along at least a first axis of rotation to generate an oscillating magnetic field; applying a vibration pattern along the first axis of rotation using a first magnet of the plurality of magnets, the vibration pattern including a period of increasing vibration frequency, a period of decreasing vibration frequency, and a period of inactivity following the period of decreasing vibration frequency; and applying the vibration pattern with a second magnet of the plurality of magnets, including activating the first magnet of the plurality of magnets and the second magnet of the plurality of magnets at different times to sequentially apply the vibration pattern with different magnets; and applying the oscillating magnetic field to tissue containing cancer cells to induce apoptosis of the cancer cells.

2. selecting a first axis of rotation for a first magnet of the plurality of magnets; selecting a second axis of rotation for a second magnet of the plurality of magnets; The method of claim 1 , wherein the first axis of rotation is substantially perpendicular to the second axis of rotation.

3. selecting a third axis of rotation that is orthogonal to the first axis and the second axis; and The method of claim 1 , further comprising applying the vibration pattern along the third axis of rotation with a third magnet of the plurality of magnets.

4. The method of claim 1 , wherein the ramp-up period has a duration of 50 to 100 ms.

5. The method of claim 1 , wherein the fall period has a duration of 50 to 200 ms.

6. The method of claim 1 , wherein the inactive period has a duration of 250 to 2750 ms.

7. vibrating one or more magnets to generate the oscillating magnetic field includes: The method of claim 1 , comprising causing the one or more magnets to reach a peak frequency of 50 to 300 Hz.

8. applying the oscillating magnetic field to the tissue comprises:

10. The method of claim 1, comprising producing a peak-to-peak amplitude of the oscillating magnetic field in the tissue in the range of 1-200 mT.

9. 10. The method of claim 1, comprising applying the oscillating magnetic field to the tissue for 1-4 hours at a time, repeated 2-3 times per day.

10. 2. The method of claim 1, comprising generating a train of pulses during the rising and falling periods with a duty cycle between 0.001% and 50%.

11. The method of claim 10 , wherein the pulse train includes a square amplitude envelope.

12. The method of claim 11 , wherein the pulse train includes a Gaussian amplitude envelope.

13. The method of claim 11 , wherein the pulse train includes a sinusoidal amplitude envelope.

14. The method of claim 11 , wherein the pulse train includes a ramp amplitude envelope.

15. The method of claim 11 , wherein the pulse train includes a sawtooth amplitude envelope.

16. at least one magnetic assembly configured to rotate the radial magnet about one or more axes; and 10. An apparatus comprising control hardware configured to operate the at least one magnetic assembly according to the method of claim 1.

Images