Use an alternating electric field to enhance the permeability of the blood-brain barrier

Abstract

By applying an alternating electric field to the brain for a period of time, certain substances (such as macromolecules) that normally cannot cross the blood-brain barrier can be introduced into the brain, where the frequency of the alternating electric field is selected such that applying the alternating electric field increases the permeability of the blood-brain barrier. In some embodiments, the frequency of the alternating electric field is less than 190 kHz (such as 100 kHz). Once the permeability of the blood-brain barrier is increased, the substances can cross the blood-brain barrier.

Description

[0001] This application is a divisional application of the following application: Application date: August 22, 2019; Application number: 201980039088.9; Invention title "Using an alternating electric field to enhance the permeability of the blood-brain barrier". Technical Field

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 722,100, filed on August 23, 2018, which is incorporated herein by reference in its entirety. Background Art

[0003] Generally, the cerebral microvasculature strictly regulates the transfer of substances between the blood and the brain tissue. This regulation of the cerebral microvasculature is called the blood-brain barrier (BBB), and it is due to the formation of intercellular tight junctions (TJs) between the cerebral capillary endothelial cells. In cerebral capillaries, the expression of TJ proteins is 50-100 times that in peripheral microvasculature. TJs are formed by a complex of transmembrane proteins (occludin and claudin) and cytoplasmic accessory proteins (ZO-1 and -2, cingulin, AF-6, and 7H6). Through their connection with the actin cytoskeleton, these proteins form strong intercellular junctions. The cerebral endothelial cells that form the cerebral microvascular endothelium are responsible for approximately 75-80% of the resistance of the BBB to substances, while other cells such as astrocytes and pericytes provide the remaining resistance.

[0004] The BBB is composed of tight junction proteins around the capillaries, and it generally restricts the diffusion of microscopic objects and large molecules or hydrophilic molecules into the brain, while allowing the diffusion of hydrophobic molecules (transcellular rather than paracellular transport).

[0005] In healthy humans, the BBB plays a very important role because it can prevent harmful substances (such as bacteria, viruses, and potentially harmful large molecules or hydrophilic molecules) from entering the brain. However, in some cases, the role of the BBB can cause difficulties. For example, it may be desirable to deliver large or hydrophilic drug molecules to treat diseases in a patient's brain. But when the BBB is functioning normally, the BBB prevents these drugs from entering the brain. Summary of the Invention

[0006] One aspect of the present invention relates to a first method for delivering a substance across the blood-brain barrier of a subject's brain. In this first method, by applying an alternating electric field to the subject's brain for a period of time, the relevant substance can be delivered across the blood-brain barrier of the subject's brain. Applying the alternating electric field to the subject's brain for a period of time increases the permeability of the blood-brain barrier of the subject's brain. After a period of time, the substance is administered to the subject, and the increased permeability of the blood-brain barrier allows the substance to cross the blood-brain barrier.

[0007] In some cases of the first method, an alternating electric field is applied at a frequency between 75 kHz and 125 kHz. In some cases of the first method, a period of time is at least 24 hours. In some cases of the first method, a period of time is at least 48 hours. In some cases of the first method, the field strength of the alternating electric field in at least a portion of the subject's brain is at least 1 V / cm. In some cases of the first method, an alternating electric field is applied at a frequency between 75 kHz and 125 kHz, for a period of time of at least 24 hours, and the field strength of the alternating electric field in at least a portion of the subject's brain is at least 1 V / cm.

[0008] In some cases of the first method, the administration of the substance is performed intravenously. In some cases of the first method, the administration of the substance is performed orally. In some cases of the first method, the subject's brain is tumor-free.

[0009] In some cases of the first method, the substance includes a drug for treating a disease. Examples of such cases include cancer treatment drugs, infectious disease treatment drugs, neurodegenerative disease treatment drugs, or autoimmune disease treatment drugs, anti-epileptic drugs, hydrocephalus drugs, stroke intervention drugs, or psychiatric drugs. In some cases of the first method, the substance is used to monitor brain activity. Examples of such cases include brain dyes, reporter molecules, or markers.

[0010] In any of the cases of the first method described above, the application of the alternating electric field can be stopped to allow the blood-brain barrier to recover.

[0011] Another aspect of the present invention relates to a second method for delivering a substance across the blood-brain barrier of a subject's brain. In this second method, by applying an alternating electric field at a first frequency to the subject's brain for a period of time, the relevant substance can be delivered across the blood-brain barrier of the subject's brain, where the first frequency is less than 190 kHz, and the period of time is at least 24 hours, and applying the alternating electric field at the first frequency to the subject's brain for a period of time increases the permeability of the blood-brain barrier in the subject's brain. After a period of time, the substance is administered to the subject, and the increased permeability of the blood-brain barrier allows the substance to cross the blood-brain barrier.

[0012] In some cases of the second method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz. In some cases of the second method, the period of time is at least 48 hours. In some cases of the second method, the field strength of the alternating electric field in at least a portion of the subject's brain is at least 1 V / cm. In some cases of the second method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz, and the field strength of the alternating electric field in at least a portion of the subject's brain is at least 1 V / cm.

[0013] In any of the cases of the second method described above, the application of the alternating electric field can be stopped to allow the restoration of the blood-brain barrier.

[0014] The methods described herein can be used to deliver a substance across the blood-brain barrier of a subject without a brain tumor. In this case, another aspect of the present invention relates to a third method for delivering a substance across the blood-brain barrier of a subject's brain. In this third method, by applying an alternating electric field at a first frequency to the subject's brain for a first period of time, the relevant substance can be delivered across the blood-brain barrier of the subject's brain that does not include a tumor. Applying the alternating electric field at the first frequency to the subject's brain for the first period of time increases the permeability of the blood-brain barrier in the subject's brain. After a period of time, the substance is administered to the subject, and the increased permeability of the blood-brain barrier allows the substance to cross the blood-brain barrier.

[0015] In some cases of the third method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz. In some cases of the third method, the period of time is at least 24 hours. In some cases of the third method, the period of time is at least 48 hours. In some cases of the third method, the field strength of the alternating electric field in at least a portion of the subject's brain is at least 1 V / cm. In some cases of the third method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz, the period of time is at least 24 hours, and the field strength of the alternating electric field in at least a portion of the subject's brain is at least 1 V / cm.

[0016] In any of the cases of the third method described above, the application of the alternating electric field can be stopped to allow the restoration of the blood-brain barrier.

[0017] The methods described herein can be used to deliver a substance across the blood-brain barrier of a subject with a brain tumor. In this case, another aspect of the present invention relates to a fourth method for treating a tumor in a subject's brain and delivering a substance across the blood-brain barrier of the subject's brain. In this fourth method, a first alternating electric field at a first frequency is applied to the subject's brain for a first period of time. Applying the first alternating electric field at the first frequency to the subject's brain for the first period of time increases the permeability of the blood-brain barrier in the subject's brain. After the first period of time, the substance is administered to the subject, and the increased permeability of the blood-brain barrier allows the substance to cross the blood-brain barrier. A second alternating electric field at a second frequency is applied to the subject's brain for a second period of time that is at least one week long. The second frequency is different from the first frequency, and the second alternating electric field at the second frequency has a sufficient intensity to inhibit the tumor.

[0018] In some cases of the fourth method, the first frequency is between 75 kHz and 125 kHz.

[0019] In some cases of the fourth method, the first frequency is between 50 kHz and 190 kHz. In some of these cases, the second frequency is between 190 kHz and 210 kHz.

[0020] In some cases of the fourth method, the first time period is at least 24 hours. In some cases of the fourth method, the second time period includes a single uninterrupted time interval that is at least one week long. In other cases of the fourth method, the second time period includes a plurality of discontinuous time intervals during which a second alternating electric field at the second frequency is applied to the subject's brain, where the plurality of discontinuous time intervals total at least one week.

[0021] In any of the cases of the fourth method described above, the application of the alternating electric field can be stopped to allow the blood-brain barrier to recover.

[0022] In some cases, any of the above methods is used to deliver a substance having a molecular weight of at least 4 kDa across the blood-brain barrier of a subject's brain.

[0023] In some cases, any of the above methods is used to deliver a substance having a molecular weight of at least 69 kDa across the blood-brain barrier of a subject's brain.

[0024] In some cases, any of the above methods is used to deliver a substance across the blood-brain barrier of a subject's brain, where the substance has at least one characteristic that normally impedes the passage of substances across a non-leaky BBB.

[0025] Another aspect of the present invention relates to a first device for treating a tumor in a subject's body and promoting the delivery of a substance across the blood-brain barrier of the subject's body. The first device includes an alternating voltage generator that is capable of operating at a first frequency between 50 and 190 kHz and a second frequency between 50 and 500 kHz. The second frequency is different from the first frequency. The alternating voltage generator has a control input, and the alternating voltage generator is configured to output the first frequency when the control input is in a first state and to output the second frequency when the control input is in a second state. The first device further includes a controller that is programmed to (a) place the control input in the second state such that the alternating voltage generator outputs the second frequency, (b) receive a request to switch to the first frequency, (c) after receiving the request, place the control input in the first state such that the alternating voltage generator outputs the first frequency for a time interval, and (d) after the time interval has elapsed, place the control input in the second state such that the alternating voltage generator outputs the second frequency.

[0026] Some embodiments of the first device further include an electrode set that is configured to be attached to the subject's body; and a wire that connects the output terminal of the alternating voltage generator to the electrode set.

[0027] In some embodiments of the first device, the first frequency is between 75 kHz and 125 kHz, and the second frequency is between 150 kHz and 250 kHz. In some embodiments of the first device, the time interval is at least 24 hours. In some embodiments of the first device, the time interval is at least 72 hours. In some embodiments of the first device, the controller is further programmed to switch the control input back and forth between a first state and a second state after receiving a request.

[0028] In some embodiments of the first device, the AC voltage generator is capable of operating at at least one additional frequency between 50 and 500 kHz, and the AC voltage generator is configured to output at least one additional frequency when the control input is at the at least one additional frequency, and the controller is programmed to cycle the control input through the second state and at least one additional state before receiving a request, and to cycle the control input through the second state and at least one additional state after an elapsed time interval.

[0029] Some embodiments of the first device further include a user interface, and the request is accepted via the user interface. In some embodiments of the first device, the request is accepted via radio frequency (RF). Description of the Drawings

[0030] Figure 1 Depicts an exemplary setup for in vitro experiments, where fixed murine brain capillary endothelial cells (cerebEND) are grown on cover slips and transwell inserts to create an artificial in vitro form of the BBB.

[0031] Figure 2A and 2B Respectively depict the results of integrity and permeability tests on the artificial BBB.

[0032] Figure 3A and 3B Depicts data showing that the increased permeability of the artificial BBB is not caused by cell death.

[0033] Figure 4 Depicts the location for preparing rat brain slices for in vivo experiments.

[0034] Figure 5 Depicts the EB accumulation in different parts of the rat brain in this in vivo experiment.

[0035] Figure 6 Depicts the average EB accumulation in the rat brain averaged over all parts in this in vivo experiment.

[0036] Figure 7Depicting the enhancement of BBB permeability in three different regions of the rat brain induced by an in vivo alternating electric field, as determined using contrast-enhanced MRI.

[0037] Figure 8 Depicting the enhancement of BBB permeability in the rat cortex induced by an in vivo alternating electric field, as determined using contrast-enhanced MRI.

[0038] Figure 9 Depicting a suitable timing relationship between applying an alternating electric field to a subject and administering a substance.

[0039] Figure 10 Is a block diagram of a dual-frequency device that generates a first frequency for inducing BBB permeability and a second frequency for inducing cytotoxicity. Detailed Description

[0040] This application describes a novel method that uses an alternating electric field to temporarily enhance the permeability of the BBB, enabling substances that are normally blocked by the BBB to cross the BBB.

[0041] A set of in vitro experiments were conducted in which fixed rat brain capillary endothelial cells (cerebEND) were grown on cover slips and transwell inserts to create an artificial in vitro form of the BBB, and Figure 1 The setup of these experiments is depicted. The cells were then treated with an alternating electric field (100 - 300 kHz) for 24 hours, 48 hours, and 72 hours. The direction of the alternating electric field was switched every 1 second between two perpendicular directions (i.e., in a repeating sequence, 1 second in one direction followed by 1 second in the other direction). The following utilities were then analyzed: (a) cell morphology (immunofluorescent staining of tight junction proteins Claudin 5 and ZO-1); (b) BBB integrity (using transendothelial electrical resistance (TEER)); and (c) BBB permeability (using fluorescein isothiocyanate conjugated to dextran (FITC) for flow cytometry).

[0042] The first set of experiments involved visualizing cell morphology and orientation, as well as the localization of stained proteins. This experiment was designed to determine how the frequency of the alternating electric field affects the artificial BBB. Here, the cells were grown on cover slips, and the alternating electric field was applied at four different frequencies (100 kHz, 150 kHz, 200 kHz to 300 kHz) for up to 72 hours at a field strength of 1.7 V / cm. The direction of the alternating electric field was switched every 1 second between two perpendicular directions. There was also a control in which no alternating electric field was applied. Then, cell morphology images depicting the presence of Claudin 5, ZO-1, and 4,6-diamidino-2-phenylindole (DAPI) (each of which was stained a different color) were obtained. Claudin 5 and ZO-1 indicate the presence of an intact BBB. This set of cell morphology images showed that the alternating electric field disrupts the artificial BBB through the dislocation of tight junction proteins from the cell boundary to the cytoplasm, with the most pronounced effect at 100 kHz.

[0043] The second set of experiments also involved visualizing cell morphology. This experiment was designed to determine how the duration during which the alternating electric field is applied affects the artificial BBB. Endothelial cells were grown on cover slips, and the alternating electric field was applied at a frequency of 100 kHz for three different durations (24 hours, 48 hours, 72 hours), with a control. The direction of the alternating electric field was switched every 1 second between two perpendicular directions. Then, cell morphology images depicting the presence of Claudin 5 and DAPI (each of which was stained a different color) were obtained. This set of cell morphology images showed that the above-mentioned phenomenon associated with the first set of experiments was visible after 24 hours and had the most pronounced effect after 72 hours.

[0044] The third set of experiments also involved visualizing cell morphology. This experiment was similar to the second set of experiments, except that the endothelial cells were grown on transwell inserts instead of cover slips. The results were similar to those of the second set of experiments. The delocalization of TJ proteins was visible after 24 hours and had the most pronounced effect after 72 hours. The above three experiments support the conclusion that the alternating electric field causes changes in cell structure, which may be the reason for the increased permeability of the BBB.

[0045] Figure 2A and Figure 2B respectively depict the results of integrity and permeability tests on the artificial BBB after subjecting it to an alternating electric field at a frequency of 100 kHz (the direction of the alternating electric field was switched every 1 second between two perpendicular directions) for 72 hours, with a control. More specifically, Figure 2A depicts the results of the transendothelial electrical resistance (TEER) test, which shows that the alternating electric field reduces the integrity of the artificial BBB to 35% of the control. Figure 2BDepict the results of fluorescein isothiocyanate (FITC) permeability tests, which show that the alternating electric field increases the permeability of the artificial BBB to FITC-dextran with a molecular weight of 4 kDa to 110% of the control. These experiments further support the conclusion that the alternating electric field increases the permeability of the BBB to molecules that normally cannot cross the non-leaky BBB.

[0046] Overall, these in vitro experiments show that an alternating electric field applied at a certain frequency for a sufficient duration causes the delocalization of tight junction proteins (Claudin 5, ZO-1) from the cell boundary to the cytoplasm (with the most obvious effect at 100 kHz) and increases the permeability of the BBB. The effect of the alternating electric field appears after 24 hours and is most obvious after 72 hours. More specifically, after using the alternating electric field to increase the permeability of the BBB, 4 kDa molecules can cross the BBB.

[0047] Additional in vitro experiments were then performed to determine what happens to the BBB after turning off the alternating electric field. These experiments used visualization of cell morphology to show how the artificial BBB recovers after the alternating electric field is stopped. In these experiments, endothelial cells were grown on coverslips and treated with an alternating electric field of 1.7 V / cm at 100 kHz for 72 hours. The direction of the alternating electric field was switched every 1 second between two perpendicular directions. Then the alternating electric field was turned off, and the cells were tracked for 96 hours after the alternating electric field was stopped. Images of cell morphology depicting the presence of Claudin 5 (stained) were obtained at 24 hours, 48 hours, 72 hours, and 96 hours. These images show a progressive change in the localization of Claudin between the cell boundary and the cytoplasm in the images at 24 hours, 48 hours, 72 hours, and 96 hours. In addition, comparing these four images with the corresponding images of the control (no alternating electric field applied during the initial 72 hours or the last 96 hours) shows that the endothelial cell morphology has partially recovered 48 hours after the alternating electric field is stopped, and the BBB has fully recovered (i.e., comparable to the control) 96 hours after the alternating electric field is stopped.

[0048] Figure 3A and 3BDepict the results of in vitro experiments designed to determine whether the observed permeability changes of the above artificial BBB can be attributed to cell death. This experiment tested cell division by comparing: (a) the cell count when an alternating electric field was applied for 72 hours followed by 96 hours without the alternating electric field, and (b) the cell count of a control that never had the alternating electric field applied. Endothelial cells were grown on coverslips and treated with an alternating electric field at a field strength of 1.7 V / cm at 100 kHz for 72 hours. The direction of the alternating electric field was switched every 1 second between two perpendicular directions. Then, the alternating electric field was turned off, and the cells were tracked for 96 hours after the alternating electric field was stopped. The number of cells per milliliter for the alternating electric field and the control was counted, and the results are depicted in Figure 3A and 3B (for the control and the alternating electric field respectively). These results show that there was no statistically significant increase in the number of cells during or after the application of the alternating electric field, indicating that the above-mentioned changes in BBB permeability cannot be attributed to cell death.

[0049] Another in vitro experiment used the TUNEL method to detect apoptosis to determine whether the observed permeability changes of the above artificial BBB can be attributed to cell death. In this experiment, endothelial cells were grown on coverslips and treated with an alternating electric field at a field strength of 1.7 V / cm at 100 kHz for 72 hours. The direction of the alternating electric field was switched every 1 second between two perpendicular directions. In the control, no alternating electric field was applied. After 24, 48, and 72 hours, images of cell morphology depicting apoptosis (TUNEL) and cell nuclei (DAPI) (each of which was stained a different color) were obtained. None of these images showed additional evidence of apoptosis, indicating that the alternating electric field does not cause cell death. This confirms that the above-mentioned changes in BBB permeability are not attributed to cell death.

[0050] A group of in vivo experiments were also conducted on rats to quantify the increase in vascular permeability due to exposure to an alternating electric field. These experiments used Evans blue (EB) dye, which is an azo dye with a high affinity for serum albumin (molecular size approximately 69 kDa). Due to its large molecular size, serum albumin generally will not be able to pass through the BBB. However, if the permeability of the BBB has been sufficiently increased, some of the serum albumin molecules (along with the EB dye bound to them) will pass through the BBB and can then be detected by looking for EB in the rat brain.

[0051] In this set of experiments, an alternating electric field of 100 kHz was applied to the rat brain for 72 hours, and the direction of the alternating electric field was switched every 1 second between two perpendicular directions. This was achieved by shaving each rat's head, placing a first pair of capacitively coupled electrodes on the top and bottom of the rat's head, and a second pair of capacitively coupled electrodes on the left and right sides of the rat's head. Then, in a repeating sequence, an AC voltage of 100 kHz was applied between the top and bottom electrodes for 1 second, followed by an AC voltage of 100 kHz being applied between the left and right electrodes for 1 second.

[0052] Under the conditions indicated in Table 1 and for the times indicated in Table 1, EB was injected intravenously into the tail vein under anesthesia (once injected, EB immediately binds to albumin), and in all cases, EB was allowed to circulate for 2 hours. Then the following steps were performed: (a) intracardiac perfusion with saline; (b) the brain was cut into four pieces using a brain slicer; (c) the slices were photographed to localize the staining and weighted; (d) EB extraction was performed after homogenizing the tissue with 50% TCA (1:3) and a centrifuge, and (e) EB quantification was performed at 610 nm. The results are given as micrograms of EB per gram of tissue.

[0053]

[0054] Table 1

[0055] During the experiment, two animals from Group 2 and one animal from Group 4 were excluded (interrupted treatment, failure to inject EB into the tail vein). There was no difference between the animals treated with the alternating electric field (Groups 1 and 2), and therefore these animals were grouped together. Similarly, there was no difference between the sham-heated and control animals (Groups 3 and 4), and therefore these animals were grouped together.

[0056] Using a brain slicer, the rat brain was cut into four pieces at the positions shown in Figure 4 . Then the EB accumulation in these four specific sites was measured. Additionally, computer simulations were performed to determine the field strength in each of these four sites. Table 2 details the field strengths obtained by simulation in each of these four sites, all values are given in units of V / cm RMS.

[0057] Part 1 2 3 4 Average field strength 2.7 V / cm 3 V / cm 2.6 V / cm 1.6 V / cm Dielectric field strength 2.5 V / cm 2.6 V / cm 2.4 V / cm 1.6 V / cm

[0058] Table 2

[0059] Figure 5 depicts the results of EB accumulation in Sites 1 to 4. A summary of these results is as follows: (1) Statistically significant increases were observed in Sites 1 and 2 (frontal lobe of the brain) where the field strength was highest; and smaller increases (not statistically significant) were observed in the more posterior sites (3 and 4) where the field strength was lower.

[0060] Figure 6 Depicts the average EB accumulation in the rat brain averaged over all four regions 1 - 4. This result shows a higher accumulation of EB in the rat brain treated with an alternating electric field for 72 hours, and this result is statistically significant (p < 0.05).

[0061] The above in - vivo experiments confirm that: (1) Applying an alternating electric field allows molecules with an average molecular size of approximately 69 kDa to pass through the BBB to reach the brain tissue; (2) Two hours after terminating the application of the alternating electric field, the permeability of the BBB remains elevated; and (3) The increase in the permeability of the BBB varies among different regions of the brain. The latter may be due to the different field strengths applied to various regions of the brain. These experiments further support our conclusion that an alternating electric field increases the permeability of the BBB to molecules that normally cannot cross an intact BBB.

[0062] In another set of in - vivo experiments, five rats were treated with an alternating electric field at 100 kHz for 72 hours, while four control rats were not treated with an alternating electric field for the same time. At the end of the 72 - hour period, a 4 kDa fluorescent compound, TRITC - dextran, was injected intravenously into the tail vein under anesthesia and allowed to circulate for 2 minutes in all cases. Then the brains were removed, frozen, sectioned, and scanned with a fluorescence scanner. All slides were scanned under the same conditions. The resulting images show a significantly higher level of accumulation of fluorescent 4 kDa TRITC - dextran in the brain tissue of rats subjected to the alternating electric field (compared to controls), again confirming that the alternating electric field can increase the permeability of the BBB.

[0063] Another set of in - vivo experiments was performed using dynamic contrast - enhanced MRI (DCE - MRI) with intravenous injection of a gadolinium - based contrast agent (Gd - DTPA, Magnetol, MW 547). In these experiments, test rats were treated with an alternating electric field at 100 kHz for 72 hours, while control rats were not treated with an alternating electric field for the same period. After this 72 - hour period, the alternating electric field was turned off, the rats were anesthetized, and a series of 60 T1w MRI scans (each scan lasting 28 seconds) were acquired. During the 7th scan of these 60 scans, the gadolinium - based contrast agent was injected into the tail vein of the rats.

[0064] Image analysis for each rat included: (1) determining the baseline for each voxel by calculating the mean of the first six Tlw MRI scans for each voxel (i.e., scans before gadolinium injection); (2) calculating the percentage change in signal over time relative to the baseline on a voxel-by-voxel basis (i.e., gadolinium accumulation); (3) dividing the brain into anterior, middle, and posterior segments; (4) generating for each of the three segments the mean percentage change in signal relative to the baseline over all voxels in the respective segment, and then (5) averaging four consecutive time points (i.e., four scans). Finally, the data from all rats in any given group were averaged.

[0065] Figure 7 depicts the results of this DCE-MRI experiment for each of the three segments of the brain (i.e., anterior, middle, and posterior). This data shows that contrast agent accumulation in the rat brain tissue treated with alternating electric fields (TTFields with tracer label; n = 6) was significantly higher than that in control rats (control with tracer label; n = 3). In addition, this difference was most obvious in the posterior brain, which is the part of the brain where the alternating electric field has the highest field strength. From this, we can conclude that the alternating electric field successfully increased the permeability of the BBB in vivo.

[0066] To test whether this increase in BBB permeability was temporary, the same test conditions were repeated, but then an additional 96-hour test was conducted without the alternating electric field. After these 96 hours, a series of 60 Tlw MRI scans (each scan lasting 28 seconds) were acquired using the same procedure as above (including gadolinium injection). Figure 8 also depicts the results of this part of the DCE-MRI experiment for each of the three segments of the brain. This data shows that there was no significant difference in contrast agent accumulation between the rat brain tissue treated with alternating electric fields for 72 hours and then without the alternating electric field for 96 hours (TTFields + 96h with tracer label; n = 7) and the control rats (control + 96h with tracer label; n = 3). From this, we can conclude that after stopping the alternating electric field, the permeability of the BBB returned to normal.

[0067] Before applying the alternating electric field to the rats (n = 2), a series of an additional 60 Tlw MRI scans (each scan lasting 28 seconds) were also acquired using the same procedure. Figure 8 also depicts the results of this part of the DCE-MRI experiment for each of the three segments of the brain (i.e., anterior, middle, and posterior) (refer to the "before" tracer label).

[0068] Figure 8Mean values of all three segments of the brain (i.e., front, middle, and back) for 72 hours of TTFields (n = 6) and 72 hours of control without TTFields (n = 3), with standard deviation bars. A paired t-test was used to compare the two groups, and p < 0.0001.

[0069] We note that the upper molecular size limit that can pass through the BBB after application of an alternating electric field has not been determined. However, based on (a) in vitro experiments described herein using FITC-dextran with a molecular weight of 4 kDa, and (b) in vivo experiments described herein using EB (which binds to serum albumin with a molecular size of approximately 69 kDa), the upper limit appears to be at least approximately 69 kDa, and most definitively at least 4 kDa.

[0070] The significance of being able to reversibly and optionally increase the permeability of the BBB is profound, as it is now possible to deliver many substances across the BBB of a subject, even though these substances have at least one characteristic that would normally impede the passage of substances across an intact BBB. Many of these implications involve delivering substances (including but not limited to therapeutic and diagnostic agents) across the blood-brain barrier of a subject's brain.

[0071] Examples include, but are not limited to, the following: delivering chemotherapeutic agents across the BBB to treat cancer (in which case, it may be possible to reduce the drug dose used to treat brain tumors and metastases with severe side effects in other parts of the body, based on the increased permeability of the drug to the brain); delivering antibody- and / or cell-based therapies across the BBB for immunotherapy; delivering contrast agent dyes, reporter molecules, and markers across the BBB for diagnosis and research (e.g., monitoring brain activity); delivering antibacterial agents across the BBB to treat infectious diseases; delivering antiviral agents or virus-neutralizing antibodies across the BBB to treat viral infections; delivering antiparasitic agents across the BBB to treat parasites; delivering drugs for treating neurodegenerative and autoimmune diseases across the BBB; delivering psychiatric drugs; delivering antiepileptic drugs; delivering drugs for hydrocephalus; delivering drugs for stroke intervention and recovery; delivering compounds lacking in the brain across the BBB to treat diseases lacking these compounds (e.g., for treating Parkinson's disease, etc.).

[0072] Although the above tests were conducted in vitro and in live rats, similar results are expected for other animals and humans.

[0073] The methods described herein can also be applied in vivo by applying an alternating electric field to the brain of a live subject. Applying an electric field to the subject's brain will increase the permeability of the BBB, which will enable molecules that are normally blocked or impeded by the BBB to pass through. For example, this can be achieved by, for example, placing electrodes on or under the skin of the subject such that applying an alternating voltage between a selected subset of these electrodes will apply an alternating electric field in the subject's brain.

[0074] For example, a pair of electrodes may be placed on the front and back sides of the subject's head, while a second pair of electrodes may be placed on the left and right sides of the subject's head. In some embodiments, the electrodes are capacitively coupled to the subject's body (e.g., by using electrodes that include a conductive plate and also have a dielectric layer disposed between the conductive plate and the subject's body). However, in alternative embodiments, the dielectric layer may be omitted, in which case the conductive plate directly contacts the subject's body. In another embodiment, the electrodes may be inserted subcutaneously into the patient's skin.

[0075] An AC voltage generator applies an AC voltage at a selected frequency (e.g., 100 kHz, or between 50 and 190 kHz) between the left and right electrodes for a first time period (e.g., 1 second), which induces an alternating electric field whose most important component of the field lines is parallel to the transverse axis of the subject's head. Then, the AC voltage generator applies an AC voltage at the same frequency (or a different frequency) between the front and back electrodes for a second time period (e.g., 1 second), which induces an alternating electric field whose most important component of the field lines is parallel to the sagittal axis of the subject's head. Then these two steps are repeated during the treatment. Optionally, a thermal sensor may be included at the electrodes, and if the temperature sensed at the electrodes becomes too high, the AC voltage generator may be configured to reduce the amplitude of the AC voltage applied to the electrodes. In some embodiments, one or more additional pairs of electrodes may be added and included in the sequence. In alternative embodiments, only a single pair of electrodes is used, in which case the direction of the field lines is not switched. Note that any parameters of this in-vivo embodiment (e.g., frequency, field strength, duration, direction change rate, and placement of the electrodes) may vary as described above in connection with the in-vitro embodiment. However, care must be taken in the in-vivo environment to ensure that the electric field always remains safe for the subject.

[0076] In the in-vivo environment, various applications for enhancing the permeability of the BBB can readily be envisioned. In one example, local enhancement of drug uptake by tumor cells (e.g., glioblastoma cells) in the brain can be induced by applying an alternating electric field to the brain for a period of time (e.g., 72 hours or at least 24 hours) before and during the administration of a chemotherapeutic agent or other anti-tumor agent.

[0077] Figure 9Depict a suitable timing relationship between applying an alternating electric field to a living patient and administering a substance. Based on the above data, and assuming that the substance is introduced or administered at a given time t = 0, the alternating electric field can start before the given time (e.g., 72 hours before t = 0) and continue for a certain time interval after the given time (e.g., until 12 hours after t = 0). In this case, the permeability of the BBB will start to increase before the substance is administered and before the substance reaches the BBB. This will enable the substance to cross the BBB immediately after it reaches the BBB. In the context of chemotherapy, this will correspond to starting to apply the alternating electric field, administering the chemotherapeutic agent after 72 hours, and then applying the alternating electric field for an additional time interval (e.g., until 12 hours after the chemotherapeutic agent is administered).

[0078] Note that the time intervals discussed above in connection with Figure 9 may be continuous or may include shorter breaks. For example, the 12-hour interval can be met by a single continuous block of 12 hours. Alternatively, the 12-hour interval can be met by applying the alternating electric field for 6 hours, then having a 1-hour break, and then applying the alternating electric field for an additional 6 hours. Similar break times can also optionally interrupt the 72-hour interval before the substance is administered. Also note that in the Figure 9 context, when administering a substance to a living patient, any of a variety of methods can be used to administer the substance, including but not limited to intravenous, oral, subcutaneous, intrathecal, intraventricular, and intraperitoneal.

[0079] In some preferred embodiments, the frequency of the alternating electric field is less than 190 kHz (e.g., between 50 and 190 kHz or between 25 and 190 kHz). Based on the experiments discussed above, using a frequency less than 190 kHz and in combination with a time period of at least 24 hours will increase the change in permeability (compared to operating outside of these ranges).

[0080] The methods described herein can be used to deliver a substance across the blood-brain barrier of a subject's brain when the subject's brain includes a tumor. One existing method of treating brain tumors (e.g., glioblastoma) is to apply an alternating electric field at a frequency between 50 and 500 kHz, preferably between 100 and 300 kHz, to the tumor. For glioblastoma, 200 kHz is the most preferred frequency. The alternating electric field at these frequencies is called TTFields and is described in U.S. Patents 6,868,289 and 7,565,205, each of which is incorporated herein by reference in its entirety. Briefly, these two applications describe disrupting dividing cells during mitosis. The effectiveness of TTFields is enhanced when the direction of the electric field is periodically switched, when the electric field intensity in at least a portion of the tumor is at least 1 V / cm, and when the electric field is applied for a long time (e.g., weeks or months) with as few breaks as possible.

[0081] In patients with a brain tumor, the following situation may occur: there is a need to treat the tumor with TTFields and also to deliver a substance across the blood-brain barrier of the same patient (e.g., to help get a therapeutically effective amount of a chemotherapeutic drug across the BBB to provide an additional line of attack against the tumor). In some cases, an alternating electric field of a single frequency can be used to treat the tumor and increase the permeability of the BBB. In other cases, it may be desirable to use alternating electric fields having different frequencies: a first frequency is selected to provide an improved result of increasing the permeability of the BBB, and a second frequency is selected to provide an improved result of the tumor effect of TTFields.

[0082] Figure 10 is a block diagram of an apparatus that generates a first frequency for inducing BBB permeability and a second frequency for inducing cytotoxicity. The apparatus includes an alternating voltage generator 44, which is similar to a conventional field generator unit, but has the ability to operate at two different frequencies. The first frequency is between 50 and 190 kHz, and the second frequency is between 50 and 500 kHz. In some embodiments, the first frequency is between 75 kHz and 125 kHz, and the second frequency is between 150 kHz and 250 kHz.

[0083] The ability to operate at two different frequencies can be implemented, for example, by using a relay to switch a first set of components or a second set of components into a conventional circuit that generates an alternating voltage and by adjusting the operating frequency of the oscillator. The alternating voltage generator 44 is configured to output the first frequency or the second frequency depending on the state of a control input. When the control input is in a first state, the alternating voltage generator 44 outputs the first frequency, and when the control input is in a second state, the alternating voltage generator 44 outputs the second frequency. The controller 42 is programmed to place the control input in the second state such that the alternating voltage generator 44 outputs the second frequency. The controller 42 is also programmed to accept a request to switch to the first frequency. In Figure 10 the illustrated embodiment, the request arrives via the user interface 40, which can be implemented using any of a variety of conventional methods, including but not limited to buttons, touchscreens, etc. In an alternative embodiment, the request can arrive via RF (e.g., Bluetooth, WiFi, etc.) from a tablet computer, a smart phone, etc.

[0084] After receiving the request, the controller 42 places the control input in the first state such that the alternating voltage generator 44 will output the first frequency for a period of time (e.g., 72 hours). After that time interval has elapsed, the controller 42 places the control input in the second state such that the alternating voltage generator 44 reverts to outputting the second frequency.

[0085] Optionally, the alternating voltage generator 44 may be configured to output one or more additional frequencies (e.g., a third frequency, a fourth frequency, etc.) depending on the state of the control input. Preferably, each of these additional frequencies is selected to induce cytotoxicity. In these embodiments, the controller 42 is programmed to cycle the control input through states that cause the alternating voltage generator 44 to output the second frequency and one or more additional frequencies before a request arrives. The controller 42 is also programmed to accept a request to switch to the first frequency. After receiving the request, the controller 42 places the control input in a first state such that the alternating voltage generator 44 will output the first frequency for a period of time (e.g., 72 hours). After the time interval has elapsed, the controller 42 will revert to cycling the control input through states that cause the alternating voltage generator 44 to output the second frequency and one or more additional frequencies.

[0086] When a person has a tumor that is undergoing combined treatment including TTFields and chemotherapy, Figure 10 the system depicted in is particularly useful. In such cases, the system operates at the second frequency most of the time to provide maximum cytotoxic utility. However, before a person goes to a chemotherapy clinic for a certain dose of chemotherapy, a healthcare provider (or the user) activates the user interface 40 to switch the system to the first frequency that promotes BBB permeability. In such cases, the activation of the user interface may be completed, for example, 72 hours before the expected start of chemotherapy.

[0087] Alternatively, after receiving a request (e.g., from the user interface 40), the controller 42 may control the control input such that the alternating voltage generator 44 will output the first frequency for a certain time interval (e.g., 1 hour), and then switch back and forth between the second frequency and the first frequency (e.g., once per hour). Eventually (e.g., when the relevant substance has been cleared from the patient's bloodstream), the controller 42 controls the control input such that the alternating voltage generator 44 reverts to outputting the second frequency.

[0088] A set of electrodes (not shown) similar to the conventional electrodes used are connected to the output of the alternating voltage generator 44.

[0089] Note that, in relation to any of the above methods, after sufficient time has elapsed after the termination of the alternating electric field, the BBB should return to its original low permeability state. This can be important for the safety of the subject in many cases.

[0090] Although the present invention has been disclosed with reference to certain embodiments, many modifications, variations and changes to the described embodiments are possible without departing from the scope of the present invention as defined by the appended claims. Accordingly, it is intended that the present invention not be limited to the described embodiments, but have the full scope defined by the language of the appended claims and their equivalents.

Claims

1. A device for treating tumors in a subject's body and facilitating the delivery of substances across the blood-brain barrier of the subject's body, characterized in that, The device comprises: an alternating voltage generator capable of generating an alternating electric field at a first frequency between 50 kHz and 190 kHz and at a second frequency between 50 kHz and 500 kHz, and having a control input, the second frequency being different from the first frequency, wherein the alternating voltage generator is configured to output the alternating electric field at the first frequency when the control input is in a first state, and to output the alternating electric field at the second frequency when the control input is in a second state; and a controller programmed to (a) place the control input in the second state such that the alternating voltage generator outputs the alternating electric field at the second frequency, (b) receive a request to switch the alternating electric field to the first frequency, (c) after receiving the request, place the control input in the first state such that the alternating voltage generator outputs the alternating electric field at the first frequency for a time interval, and (d) after the time interval has elapsed, place the control input in the second state such that the alternating voltage generator outputs the alternating electric field at the second frequency.

2. The device according to claim 1, further comprising: an electrode set configured to be attached to the body of the subject; and a wire connecting the output of the alternating voltage generator to the electrode set.

3. The device according to claim 1, wherein the first frequency is between 75 kHz and 125 kHz, and the second frequency is between 150 kHz and 250 kHz.

4. The device according to claim 1, wherein the first frequency is between 75 kHz and 125 kHz.

5. The device according to claim 1, wherein the second frequency is between 190 kHz and 210 kHz.

6. The device according to claim 1, wherein the field strength of the alternating electric field in at least a part of the subject's brain is at least 1 V / cm.

7. The device according to claim 1, wherein the time interval is at least 24 hours.

8. The device according to claim 1, wherein the time interval comprises a single uninterrupted time interval of at least one week in length.

9. The device according to claim 1, wherein the controller is further programmed to, after receiving the request, switch the control input back and forth between the first state and the second state.

10. The device according to claim 1, wherein the alternating voltage generator is capable of generating an alternating electric field at at least one additional frequency between 50 kHz and 500 kHz, and is configured to output the at least one additional frequency when the control input is in at least one additional state, and the controller is programmed to cycle the control input through the second state and the at least one additional state before receiving the request, and to cycle the control input through the second state and the at least one additional state after the time interval has elapsed.

11. The device according to claim 1, further comprising: a user interface, wherein the request is received via the user interface; or wherein the request is received via RF.

12. The device according to claim 1, wherein the molecular weight of the substance is at least 4 kDa.

13. The device according to claim 1, wherein the substance comprises a drug for treating a disease.

14. The device according to claim 1, wherein the substance is used for monitoring brain activity.

15. The device according to claim 1, wherein the time interval is at least 48 hours.

16. The device according to claim 1, wherein the molecular weight of the substance is at least 69 kDa.

17. The device according to claim 13, wherein the drug comprises a cancer treatment drug, an infectious disease treatment drug, a neurodegenerative disease treatment drug, an autoimmune disease treatment drug, an anti-epileptic drug, a hydrocephalus drug, a stroke intervention drug, or a psychiatric drug.

18. The device according to claim 1, wherein the substance is a brain dye, a reporter molecule, or a marker.

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