Use of alternating electric fields to increase blood-brain barrier permeability

Abstract

To enable certain substances (e.g., large molecules) that ordinarily cannot traverse the blood brain barrier to be introduced into the brain by applying an alternating electric field to the brain for a period of time, where the frequency of the alternating electric field is selected such that application of the alternating electric field increases permeability of the blood brain barrier.SOLUTION: In some embodiments, the frequency of the alternating electric field is less than 190 kHz (e.g., 100 kHz). Once the permeability of the blood brain barrier has been increased, the substance is able to cross the blood brain barrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 722,100, filed Aug. 23, 2018, which is incorporated by reference in its entirety. [Background technology]

[0002] Normally, cerebral microvessels tightly regulate the transfer of substances between blood and brain tissue. This regulation by cerebral microvessels is due to intercellular tight junctions (TJs), called the blood-brain barrier (BBB), which form between brain capillary endothelial cells. In cerebral capillaries, TJ proteins are expressed 50-100 times more than in peripheral microvessels. TJs are formed by an intricate complex of transmembrane proteins (claudins and occludin) and cytoplasmic accessory proteins (ZO-1 and ZO-2, cingulin, AF-6, and 7H6). By linking to the actin cytoskeleton, these proteins form strong intercellular connections. Approximately 75-80% of the resistance of the BBB to substances is borne by brain endothelial cells that form the endothelium of cerebral microvessels, while the remaining resistance is provided by other cells such as astrocytes and pericytes.

[0003] The BBB consists of tight junctions around the capillaries and normally restricts the diffusion of small objects and large or hydrophilic molecules into the brain, while allowing the diffusion of hydrophobic molecules (transcellular instead of paracellular transport).

[0004] In healthy humans, the BBB serves a very important function, since it prevents harmful substances (e.g., bacteria, viruses, and potentially harmful large or hydrophilic molecules) from entering the brain. However, there are situations in which the operation of the BBB poses challenges. For example, it may be desirable to deliver large or hydrophilic drug molecules to treat diseases in the brain of a patient. However, when the BBB is functioning normally, these drugs are blocked from entering the brain by the BBB. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,868,289 [Patent Document 2] U.S. Patent No. 7,565,205 Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect of the present invention is directed to a first method of delivering a substance through the blood-brain barrier of a subject's brain. In this first method, a relevant substance can be delivered through the blood-brain barrier of a subject's brain by applying an alternating electric field to the subject's brain for a period of time. The application of the alternating electric field to the subject's brain for a period of time increases the permeability of the blood-brain barrier in the subject's brain. The substance is administered to the subject after the period of time has elapsed, and the increased permeability of the blood-brain barrier allows the substance to pass through the blood-brain barrier. [Means for solving the problem]

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

[0008] In some cases of the first method, the step of administering the substance is performed intravenously. In some cases of the first method, the step of administering 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 comprises a drug for treating a disease. Examples of these cases include a cancer drug, an infectious disease drug, a neurodegenerative disease drug, an autoimmune disease drug, an anti-epileptic drug, a hydrocephalus drug, a stroke intervention drug, or a psychiatric drug. In some cases of the first method, the substance is used to monitor brain activity. Examples of these cases include a brain dye, a reporter, or a marker.

[0010] In either case of the first method described above, application of the AC electric field can be discontinued to allow the blood-brain barrier to recover.

[0011] Another aspect of the present invention is directed to a second method of delivering a substance through the blood-brain barrier of a subject's brain.In this second method, a relevant substance can be delivered through the blood-brain barrier of a subject's brain by applying an alternating electric field at a first frequency to the subject's brain for a period of time, where the first frequency is less than 190kHz, and the period of time is at least 24 hours, where applying the alternating electric field at the first frequency to the subject's brain during the period of time improves the permeability of the blood-brain barrier in the subject's brain.The substance is administered to the subject after the period of time has elapsed, and the improvement in the permeability of the blood-brain barrier allows the substance to pass through 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 alternating electric field has a field strength of at least 1 V / cm in at least a portion of the subject's brain. In some cases of the second method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz, and the alternating electric field has a field strength of at least 1 V / cm in at least a portion of the subject's brain.

[0013] In either case of the second method described above, application of the AC electric field can be discontinued to allow the blood-brain barrier to recover.

[0014] The methods described herein can be used to deliver substances through the blood-brain barrier of the brain of a subject without a tumor. In this context, another aspect of the present invention is directed to a third method of delivering substances through the blood-brain barrier of the brain of a subject. In this third method, the relevant substances can be delivered through the blood-brain barrier of the brain of a subject without a tumor by applying an alternating electric field at a first frequency to the brain of the subject for a period of time. The application of an alternating electric field at a first frequency to the brain of the subject for a period of time improves the permeability of the blood-brain barrier in the brain of the subject. The substance is administered to the subject after a period of time has elapsed, and the improved permeability of the blood-brain barrier allows the substance to pass through 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 alternating electric field has a field strength of at least 1 V / cm in at least a portion of the subject's brain. 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 alternating electric field has a field strength of at least 1 V / cm in at least a portion of the subject's brain.

[0016] In either case of the third method described above, application of the AC electric field can be discontinued to allow the blood-brain barrier to recover.

[0017] The methods described herein can be used to deliver a substance through the blood-brain barrier of a subject with a brain tumor. In this context, another aspect of the present invention is directed to a fourth method of treating a tumor in the brain of a subject and delivering a substance through the blood-brain barrier of the brain of the subject. In this fourth method, a first alternating electric field is applied to the brain of the subject at a first frequency for a first period of time. The application of the first alternating electric field at the first frequency to the brain of the subject during the first period of time increases the permeability of the blood-brain barrier in the brain of the subject. The substance is administered to the subject after the first period of time has elapsed, and the increased permeability of the blood-brain barrier allows the substance to pass through the blood-brain barrier. A second alternating electric field at a second frequency is applied to the brain of the subject 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 magnitude that is large enough 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 multiple discrete time intervals during which the second alternating electric field at the second frequency is applied to the subject's brain, the multiple discrete time intervals collectively totaling at least one week.

[0021] In either case of the fourth method described above, application of the AC electric field can be discontinued to allow the blood-brain barrier to recover.

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

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

[0024] In some cases, any of the above methods are 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 prevents the substance from crossing 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 facilitating delivery of a substance through a blood-brain barrier in the subject's body. The first device includes an AC voltage generator 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 AC voltage generator has a control input, and the AC voltage generator is configured to output the first frequency when the control input is in a first state and output the second frequency when the control input is in a second state. The first device also includes a controller programmed to: (a) place the control input in a second state such that the AC voltage generator outputs the second frequency; (b) receive a request to switch to the first frequency; (c) upon receiving the request, place the control input in the first state such that the AC voltage generator outputs the first frequency for a time interval; and (d) place the control input in the second state such that the AC voltage generator outputs the second frequency after the time interval has elapsed.

[0026] Some embodiments of the first apparatus include: A set of electrodes configured for attachment to a body of a subject; and Wiring connecting the output of the AC voltage generator to a set of electrodes, Further includes:

[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 alternate the control input between the first state and the second state following receipt of the request.

[0028] In some embodiments of the first apparatus, the AC voltage generator is capable of operating at at least one additional frequency between 50 and 500 kHz, the AC voltage generator is configured to output the at least one additional frequency when the control input is in the at least one further state, and the controller is programmed to cause the control input to periodically cycle through the second state and the at least one additional state prior to receipt of the request, and to cause the control input to periodically cycle through the second state and the at least one additional state after a time interval has elapsed.

[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). [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 describes an exemplary in vitro experimental setup in which immortalized mouse brain capillary endothelial cells (cerebEND) are grown on cover slips and transwell inserts to create an artificial in vitro version of the BBB. [Figure 2A] FIG. 1 describes the results of integrity tests on the artificial BBB. [Figure 2B] FIG. 1 describes the results of permeability studies on artificial BBB. [Figure 3A]FIG. 1 describes data showing that the increased permeability of the artificial BBB is not caused by cell death. [Figure 3B] FIG. 1 describes data showing that the increased permeability of the artificial BBB is not caused by cell death. [Figure 4] FIG. 1 depicts the locations where rat brains were sliced ​​for in vivo experiments. [Diagram 5] FIG. 14 describes this in vivo experiment EB accumulation in different sections of the rat brain. [Figure 6] FIG. 10 describes the mean EB accumulation in rat brains from this in vivo experiment, averaged across all sections. [Figure 7] FIG. 10 describes the increase in BBB permeability in three different sections of the rat brain induced by an alternating electric field in vivo as determined using contrast-enhanced MRI. [Figure 8] FIG. 1 depicts the increased BBB permeability in rat cortex induced by alternating electric fields in vivo as determined using contrast-enhanced MRI. [Figure 9] FIG. 13 is a diagram describing an appropriate timing relationship between application of an alternating electric field and administration of a substance to a subject. [Figure 10] FIG. 1 shows a schematic diagram of a dual frequency device generating a first frequency to induce BBB permeability and a second frequency to induce cytotoxicity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present application relates to a novel approach that uses an alternating electric field to temporarily increase the permeability of the BBB, allowing substances normally blocked by the BBB to pass through the BBB.

[0032] One set of in vitro experiments was performed in which immortalized mouse brain capillary endothelial cells (cerebEND) were grown on glass coverslips and transwell inserts to create an artificial in vitro version of the BBB; the experimental setup is described in Figure 1. The cells were then treated with an alternating electric field (100-300 kHz) for 24, 48, and 72 h. The direction of the alternating electric field was switched every 1 s between two perpendicular directions (i.e., 1 s in one direction followed by 1 s in the other direction in a repeating sequence). The following effects were then analyzed: (a) cell morphology (immunofluorescent staining for the tight junction proteins claudin-5 and ZO-1); (b) BBB integrity (using transendothelial electrical resistance (TEER)); and (c) BBB permeability (using fluorescein isothiocyanate-labeled dextran (FITC) for flow cytometry).

[0033] The first set of experiments included visualization of cell morphology and orientation, as well as visualization of stained protein localization. This experiment was designed to see how the frequency of the AC electric field affected the artificial BBB. Here, cells were grown on glass coverslips and an AC electric field was applied for 72 hours at four different frequencies (100 kHz, 150 kHz, 200 kHz, and 300 kHz) with a field strength of 1.7 V / cm. The direction of the AC electric field was switched every second between two perpendicular directions. There was also a control where no AC electric field was applied. Cell morphology images were then obtained, documenting the presence of claudin-5, ZO-1, and 4,6-diamidino-2-phenylindole (DAPI), each stained in a different color. Claudin-5 and ZO-1 indicate the presence of an intact BBB. This set of cell morphology images revealed that AC electric fields disrupted the artificial BBB by delocalizing tight junction proteins from the cell border to the cytoplasm, with the most dramatic effect at 100 kHz.

[0034] The second set of experiments also included visualization of cell morphology. This experiment was designed to determine how the duration of the applied AC electric field affected the artificial BBB. Endothelial cells were grown on glass coverslips and, in addition to the control, an AC electric field at a frequency of 100 kHz was applied for three different durations (24 h, 48 h, and 72 h). The direction of the AC electric field was switched every second between two perpendicular directions. Cell morphology images were then obtained, documenting the presence of claudin 5 and DAPI (each stained in a different color). This set of cell morphology images revealed that the phenomenon discussed above in relation to the first set of experiments was already visible after 24 h, and the effect was most pronounced after 72 h.

[0035] The third set of experiments also included visualization of cell morphology. This experiment was similar to the second set of experiments, except that endothelial cells were grown on transwell inserts instead of coverslips. The results were similar to those of the second set of experiments. Delocalization of TJ proteins was visible after 24 hours, and the effect was most pronounced after 72 hours. The above three experiments support the conclusion that AC electric fields induce structural changes in cells that may be responsible for the increased BBB permeability.

[0036] 2A and 2B show the results of integrity and permeability tests, respectively, for the artificial BBB after it was subjected to an AC electric field at a frequency of 100 kHz for 72 hours (with the direction of the AC electric field switching between two perpendicular directions every second) and for the control. More specifically, FIG. 2A shows the results of a transendothelial electrical resistance (TEER) test, which reveals that the AC electric field reduced the integrity of the artificial BBB to 35% of the control. FIG. 2B shows the results of a fluorescein isothiocyanate (FITC) permeability test, which reveals that the AC electric field improved 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 AC electric field improves the permeability of the BBB to molecules that normally cannot pass through a non-leaky BBB.

[0037] Taken together, these in vitro experiments show that the application of an AC electric field at a certain frequency for a sufficient duration leads to delocalization of tight junction proteins (claudin 5, ZO-1) from the cell border to the cytoplasm (the most dramatic effect at 100 kHz), improving the permeability of the BBB. The effect of the AC electric field is already apparent after 24 hours and is most pronounced after 72 hours. More specifically, after using an AC electric field to improve the permeability of the BBB, molecules of 4 kDa can pass through the BBB.

[0038] Further in vitro experiments were then performed to determine what happens to the BBB after the AC field is turned off. These experiments used visualization of cell morphology to show how the artificial BBB recovers after the AC field is stopped. In these experiments, endothelial cells were grown on glass coverslips and treated with a 100 kHz AC field at a field strength of 1.7 V / cm for 72 hours. The direction of the AC field was switched between two perpendicular directions every second. The AC field was then turned off and the cells were followed for 96 hours after the AC field was stopped. Cell morphology images showing the presence of claudin 5 (staining) were acquired at 24 hours, 48 ​​hours, 72 hours, and 96 hours. These images revealed progressive changes in the localization of claudins between the cell border and the cytoplasm in the 24 hour, 48 hour, 72 hour, and 96 hour images. Furthermore, comparison of these four images with the respective control images (where no AC field was applied for either the first 72 hours or the subsequent 96 hours) revealed that endothelial cell morphology had partially recovered 48 hours after the AC field was stopped, and that the BBB had fully recovered (i.e., was comparable to the control) 96 hours after the AC field was stopped.

[0039] Figures 3A and 3B show the results of an in vitro experiment designed to determine whether the observed changes in artificial BBB permeability described above could be due to cell death. In this experiment, cell division was tested by comparing cell counts between (a) 72 hours of AC electric field application followed by 96 hours of no AC electric field application and (b) a control in which no AC electric field was applied at all. Endothelial cells were grown on glass coverslips and treated with a 100 kHz AC electric field at a field strength of 1.7 V / cm for 72 hours. The direction of the AC electric field was switched between two perpendicular directions every second. The AC electric field was then turned off and the cells were followed for 96 hours after the AC electric field was stopped. The number of cells per ml was counted for the AC electric field and the control, and the results are shown in Figures 3A and 3B (for control and AC electric field, respectively). These results revealed that there was no statistically significant increase in cell number during or after the application of the AC electric field, indicating that the changes in BBB permeability described above cannot be attributed to cell death.

[0040] In another in vitro experiment, a TUNEL assay for apoptosis was used to determine whether the observed changes in the permeability of the artificial BBB described above could be due to cell death. In this experiment, endothelial cells were grown on glass coverslips and treated with a 100 kHz AC electric field at a field strength of 1.7 V / cm for 72 hours. The direction of the AC electric field was switched between two perpendicular directions every second. In the control, no AC electric field was applied. Cell morphology images documenting apoptosis (TUNEL) and nuclei (DAPI), each stained with a different color, were acquired after 24, 48, and 72 hours. None of these images revealed further evidence of apoptosis, indicating that the AC electric field did not cause cell death. This confirms that the changes in BBB permeability described above were not due to cell death.

[0041] A set of in vivo experiments on rats was also performed to quantify the increased vascular permeability caused by exposure to an AC electric field. These experiments used Evans Blue (EB) dye, an azo dye that has a very high affinity for serum albumin (molecular size approximately 69 kDa). Due to its large molecular size, serum albumin would not normally be able to cross the BBB. However, if the BBB permeability is sufficiently increased, some of the serum albumin molecules (along with the EB dye bound to it) will cross the BBB and can then be detected by looking for EB in the rat brain.

[0042] In this set of experiments, a 100 kHz AC electric field was applied to rats' brains for 72 hours, with the direction of the AC field switched between two perpendicular orientations every 1 second. This was done by shaving each rat's head and 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. A 100 kHz AC voltage was then applied between the top and bottom electrodes for 1 second, followed by a 100 kHz AC voltage between the left and right electrodes in a repeating sequence.

[0043] EBs were injected intravenously into the tail vein under anesthesia under the conditions and for the times shown in Table 1 (EBs bind albumin immediately upon injection) and were allowed to circulate for 2 hours in all cases. The following steps were then performed: (a) intracardiac perfusion with saline; (b) slicing the brains into 4 pieces with a brain slicer; (c) photographing the pieces to localize and compare the staining; (d) EB extraction where the tissue was homogenized and centrifuged in TCA 50% (1:3), and (e) EB quantification at 610 nm. Results are given as μg EBs per gram of tissue.

[0044] [Table 1]

[0045] During the experiment, two animals from group 2 and one from group 4 were excluded (interruption of treatment, failure of EB injection into the tail vein). There were no differences between the animals treated with AC electric fields (groups 1 and 2), so these animals were grouped together. Similarly, there were no differences between the sham-heated and control animals (groups 3 and 4), so these animals were grouped together.

[0046] A brain slicer was used to slice the rat brain into four pieces at the positions shown in Figure 4. EB accumulation in specific sections of these four pieces was then measured. In addition, computer simulations were performed to determine the electric field strength in each of these four sections. Table 2 shows the electric field strength obtained from the simulation in each of these four sections, with all values ​​given in V / cm RMS.

[0047] [Table 2]

[0048] The results of EB accumulation in sections 1 to 4 are shown in Figure 5. The results are summarized as follows: (1) A statistically significant increase was observed in sections 1, 2 (anterior cerebrum), where the field intensity was highest; and a smaller increase (not statistically significant) was observed in the more posterior sections (3, 4), where the field intensity was lower.

[0049] Figure 6 shows the average EB accumulation in rat brains, averaged across all four sections 1 to 4. The results revealed that rats treated with an AC electric field for 72 hours had higher EB accumulation in their brains, and this result was statistically significant (p < 0.05).

[0050] The above in vivo experiments confirm that: (1) application of an AC electric field allows passage of molecules with an average molecular size of approximately 69 kDa through the BBB into brain tissue; (2) the increase in BBB permeability is maintained for 2 hours after the end of application of the AC electric field; and (3) the increase in BBB permeability differs among different sections of the brain. The latter can be a result of different electric field strengths applied to different sections of the brain. These experiments further support our conclusion that an AC electric field increases the permeability of the BBB to molecules that would not normally be able to pass through a non-leaky BBB.

[0051] In another set of in vivo experiments, five rats were treated with AC electric fields at 100 kHz for 72 hours, while four control rats were not treated with AC electric fields for the same time period. At the end of the 72-hour period, the 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. The brains were then removed, frozen, sectioned, and scanned with a fluorescent scanner. All slides were scanned under the same conditions. The resulting images revealed significantly higher accumulation levels of the fluorescent 4 kDa TRITC-dextran in the brain tissue of rats subjected to AC electric fields (compared to controls), again confirming that AC electric fields enhance the permeability of the BBB.

[0052] A further set of in vivo experiments was performed using dynamic contrast-enhanced MRI (DCE-MRI) with intravenous injection of gadolinium contrast agent (Gd-DTPA, Magnetol, MW 547). In these experiments, test rats were treated with an alternating electric field of 100 kHz for 72 hours, while control rats were not treated with an alternating electric field for the same time period. After this 72 hour period, the alternating electric field was turned off, the rats were anesthetized, and a series of 60 Tlw MRI scans (each scan having a duration of 28 seconds) were collected. Gadolinium contrast agent was injected into the rat's tail vein during the seventh of these 60 scans.

[0053] Image analysis for each rat included: (1) determining the baseline for each voxel by calculating the average of the first six Tlw MRI scans (i.e., scans before gadolinium injection) for each voxel; (2) calculating the percent signal change over time relative to baseline (i.e., gadolinium accumulation) for each voxel; (3) dividing the brain into anterior, middle, and posterior segments; (4) generating an average percent signal change relative to baseline for each of the three segments across all voxels in each segment; and then (5) averaging the four consecutive time points (i.e., the four scans) together. Finally, data from all rats within any given group were averaged overall.

[0054] The results of this DCE-MRI experiment for each of the three brain segments (i.e., anterior, middle, and posterior) are depicted in Figure 7. The data reveal that the accumulation of contrast agent in the brain tissue of rats treated with AC electric fields (TT field traces; n=6) was significantly higher than in control rats (control traces; n=3). Moreover, this specificity was most evident in the posterior brain, the part of the brain where the AC electric field had the highest field strength. From this, it can be concluded that AC electric fields successfully improved the permeability of the BBB in vivo.

[0055] To test whether this increase in BBB permeability was temporary, the same test conditions were repeated, but followed by another 96 hours without AC electric field. After this 96-hour period, a series of 60 Tlw MRI scans (each scan having a duration of 28 seconds) were collected using the same procedure as above (including gadolinium injection). The results of this part of the DCE-MRI experiment for each of the three brain segments are also depicted in FIG. 7. The data reveal that the accumulation of contrast agent in the brain tissue of rats treated with AC electric field for 72 hours followed by 96 hours without AC electric field (trajectories shown for TT field + 96h; n = 7) was not significantly different from that of control rats (trajectories shown for control + 96h; n = 3). From this, it can be concluded that the BBB permeability returns to normal after the AC electric field is stopped.

[0056] An additional series of 60 Tlw MRI scans (each scan having a duration of 28 seconds) was also collected using the same procedure before applying the AC electric field to the rats (n=2). The results of this part of the DCE-MRI experiment for each of the three brain segments (i.e., anterior, middle, and posterior) are also depicted in Figure 7 (see the traces marked "before").

[0057] The averages for all three brain segments (i.e., anterior, middle, and posterior) for 72 hour TT fields (n=6) and 72 hour no TT fields control (n=3) are shown with standard deviation bars in Figure 8. Comparisons between the two groups were made using a paired t-test with p<0.0001.

[0058] It should be noted that the upper size limit for molecules that can cross the BBB after application of an AC electric field has not yet been determined, but based on (a) the in vitro experiments described herein using FITC-dextran with a molecular weight of 4 kDa, and (b) the 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 about 69 kDa, and most likely at least 4 kDa.

[0059] The implications of being able to reversibly increase the permeability of the BBB at will are far-reaching, since it now becomes possible to deliver many substances through the BB of a subject, despite the fact that the substance has at least one characteristic that normally prevents it from passing through a non-leaky BBB. Many of these implications include delivering substances, including but not limited to therapeutic and diagnostic agents, through the blood-brain barrier of the brain of a subject.

[0060] Examples include, but are not limited to: delivering chemotherapeutic agents across the BBB to treat cancer (in this regard, based on the increased permeability of drugs to the brain, it may be possible to reduce the dosage of drugs for treating brain tumors and metastases that have severe side effects in other parts of the body); delivering antibodies and / or cell-based therapies across the BBB for immunotherapy; delivering contrast dyes, reporters, and markers across the BBB for diagnostic purposes and for 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 agents to treat neurodegenerative and autoimmune diseases across the BBB; delivering psychotropic drugs; delivering antiepileptic drugs; delivering hydrocephalus drugs; delivering stroke intervention and recovery drugs; delivering compounds that are deficient in the brain across the BBB to treat conditions in which those compounds are deficient (e.g., treating Parkinson's disease, etc.).

[0061] Although the above studies were performed in vitro and in live rats, it is expected that similar results will be obtained in other animals and in humans.

[0062] The methods described herein can also be applied in an in vivo context by applying an AC electric field to the brain of a living subject. Applying an electric field to the brain of a subject increases the permeability of the BBB, allowing molecules that are normally blocked or impeded by the BBB to pass through. This can be accomplished, for example, by placing electrodes on or under the skin of a subject, such that applying an AC voltage between a selected subset of the electrodes applies an AC electric field to the brain of the subject.

[0063] For example, one pair of electrodes may be placed on the front and back of the subject's head, and a second pair of electrodes may be placed on the right and left sides of the subject's head. In some embodiments, the electrodes are capacitively coupled to the subject's body (e.g., by using an electrode that includes a conductive plate and also has 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 would be in direct contact with the subject's body. In another embodiment, the electrodes may be inserted subcutaneously beneath the patient's skin.

[0064] The AC voltage generator applies an AC voltage at a selected frequency (e.g., 100 kHz, or between 50 and 190 kHz) between the right and left electrodes for a first period of time (e.g., 1 second), which induces an alternating electric field with the most significant component of the field lines parallel to the transverse axis of the subject's head. The AC voltage generator then applies an AC voltage at the same frequency (or a different frequency) between the anterior and posterior electrodes for a second period of time (e.g., 1 second), which induces an alternating electric field with the most significant component of the field lines parallel to the sagittal axis of the subject's head. This two-step sequence is then repeated for the duration of the treatment. Optionally, a temperature sensor can be included in the electrodes, and the AC voltage generator can be configured to reduce the amplitude of the AC voltage applied to the electrodes if the temperature sensed at the electrodes becomes too high. In some embodiments, one or more additional pairs of electrodes can be added and included in the above sequence. In an alternative embodiment, only a single pair of electrodes is used, in which case the direction of the field lines is not switched. It should be noted that any of the parameters of this in vivo embodiment (e.g., frequency, field strength, duration, direction switching rate, and electrode placement) may be varied as described above in relation to the in vitro embodiment, however care should be taken in the in vivo situation to ensure that the electric field remains safe for the subject at all times.

[0065] A wide variety of applications for improving BBB permeability can be easily envisioned in in vivo situations. In one example, localized enhancement of drug uptake by brain tumor cells (e.g., glioblastoma cells) 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 or during administration of chemotherapy or other antitumor agents.

[0066] Figure 9 describes a suitable relationship in timing between the application of an AC electric field and the administration of a substance to a living patient. Based on the data above, assuming that a substance is introduced or administered at a given time t=0, the AC electric field can begin before the given time (e.g., 72 hours before t=0) and continue for a time interval after the given time (e.g., up to 12 hours after t=0). In this situation, the permeability of the BBB begins to increase before the substance is administered and before the substance reaches the BBB. This allows the substance to pass through the BBB as soon as it arrives. In the context of chemotherapy, this would be consistent with beginning the application of an AC electric field, administering the chemotherapy agent 72 hours later, followed by application of the AC electric field for a further time interval (e.g., up to 12 hours after the time the chemotherapy agent is administered).

[0067] It should be noted that the time intervals discussed above in connection with FIG. 9 may be either uninterrupted or include breaks, preferably short. For example, a 12 hour interval may be filled by a single uninterrupted block of 12 hours. Alternatively, a 12 hour interval may be filled by applying an AC electric field for 6 hours, followed by a break for 1 hour, followed by application of an AC electric field for an additional 6 hours. A similar break may optionally interrupt a 72 hour interval prior to administration of the substance. In connection with FIG. 9, when a substance is administered to a living patient, administration of the substance may be performed using any of a variety of approaches, including, but not limited to, intravenous, oral, subcutaneous, intrathecal, intraventricular, and intraperitoneal.

[0068] 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, the use of frequencies less than 190 kHz in combination with time periods of at least 24 hours enhances the change in permeability (compared to operation outside these ranges).

[0069] The methods described herein can be used to deliver substances through the blood-brain barrier of a subject's brain when the subject's brain has a tumor. One existing approach to treating brain tumors (e.g., glioblastoma) is by applying an alternating electric field to the tumor at a frequency between 50 and 500 kHz, preferably between 100 and 300 kHz. For glioblastoma, 200 kHz is the most preferred frequency. Alternating electric fields at these frequencies are called TT fields and are described in U.S. Patent Nos. 6,868,289 and 7,565,205, each of which is incorporated herein by reference in its entirety. In summary, these two applications describe destroying dividing cells during mitosis. The effectiveness of TT fields improves if the direction of the electric field is periodically switched, if the strength of the electric field in at least a portion of the tumor is at least 1 V / cm, and if the electric field is applied for an extended period of time (e.g., weeks or months) with as few disruptions as possible.

[0070] Situations may arise in patients with brain tumors where it would be desirable to both treat the tumor with TTFields and deliver a substance across the blood-brain barrier in the same patient (e.g., to help get a therapeutically effective amount of a chemotherapy drug across the BBB, thus providing an additional line of attack to the tumor). In some situations, it may be possible to both treat the tumor and improve the permeability of the BBB using a single frequency of an alternating electric field. In other situations, it may be desirable to use alternating electric fields having different frequencies: a first frequency selected to provide improved results for improving the permeability of the BBB, and a second frequency selected to provide improved results for the anti-tumor action of the TTFields.

[0071] 10 is a schematic diagram of a device that generates a first frequency to induce BBB permeability and a second frequency to induce cytotoxicity. The device includes an AC voltage generator 44 similar to a conventional Optune® field generator unit, but with 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.

[0072] The ability to operate at two different frequencies can be implemented, for example, using a relay to switch either the first set of components or the second set of components to a conventional circuit that generates an AC voltage, thereby adjusting the operating frequency of the oscillator. The AC voltage generator 44 is configured to output either the first frequency or the second frequency depending on the state of a control input. When the control input is in the first state, the AC voltage generator 44 outputs the first frequency, and when the control input is in the second state, the AC voltage generator 44 outputs the second frequency. The controller 42 is programmed to place the control input in the second state, such that the AC voltage generator 44 outputs the second frequency. The controller 42 is also programmed to accept a request to switch to the first frequency. In the embodiment depicted in FIG. 10, the request arrives via a user interface 40, which can be implemented using any of a variety of conventional approaches, including, but not limited to, push buttons, touch screens, and the like. In alternative embodiments, the request may arrive via RF (e.g., Bluetooth, WiFi, and the like) from a tablet, smartphone, and the like.

[0073] Upon receiving the request, the controller 42 places the control input in a first state, which causes the AC voltage generator 44 to output the first frequency for a period of time (e.g., 72 hours). After the period of time has elapsed, the controller 42 places the control input in a second state, which causes the AC voltage generator 44 to revert to outputting the second frequency.

[0074] Optionally, the AC voltage generator 44 can 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 cause the control input to cycle through states that cause the AC voltage generator 44 to output the second frequency and the 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. Upon receiving the request, the controller 42 places the control input in a first state, such that the AC voltage generator 44 outputs the first frequency for a period of time (e.g., 72 hours). After the period of time has elapsed, the controller 42 will revert to causing the control input to cycle through states that cause the AC voltage generator 44 to output the second frequency and the one or more additional frequencies.

[0075] The system described in FIG. 10 is particularly useful when a patient has a tumor that is being treated with a combination therapy that includes TTFields and chemotherapy. In this situation, the system operates at the second frequency most of the time to provide maximum cytotoxic effect. However, before the person presents to a chemotherapy clinic for a dose of chemotherapy, a medical professional (or user) activates the user interface 40 to switch the system to the first frequency that promotes BBB permeability. In this situation, activation of the user interface can occur, for example, 72 hours before the expected start of chemotherapy.

[0076] Alternatively, upon receiving a request (e.g., from the user interface 40), the controller 42 can control the control input such that the AC voltage generator 44 outputs the first frequency for a period of time (e.g., one hour) and then alternates between the second frequency and the first frequency (e.g., switching every hour). Eventually (e.g., when the relevant substance has been depleted from the patient's bloodstream), the controller 42 controls the control input such that the AC voltage generator 44 reverts to outputting the second frequency.

[0077] A set of electrodes (not shown), similar to conventional electrodes used with Optune®, are connected to the output of AC voltage generator 44.

[0078] It should be noted that in connection with any of the above methods, the BBB must recover to its initial hypopermeable state after a suitable amount of time following termination of the AC electric field, which may be important in many situations for the safety of the subject.

[0079] Although the present invention has been disclosed with reference to certain specific embodiments, numerous modifications, changes, and variations to the described embodiments are possible without departing from the scope of the invention, as defined in the appended claims. It is therefore intended that the present invention not be limited to the described embodiments, but rather that it have its full scope as defined by the following claims and their equivalents. [Explanation of symbols]

[0080] 40 User Interface 42 Controller 44 AC Voltage Generator

Claims

1. 1. An apparatus for controlling the permeability of the blood-brain barrier to facilitate delivery of a substance across the blood-brain barrier in a subject, comprising: an AC voltage generator having a control input and configured to generate an alternating electric field at a frequency between 75 kHz and 190 kHz in the subject's brain in response to a request on the control input; and 1. An apparatus comprising: a controller programmed to receive a request on a control input of an AC voltage generator, the controller placing the control input in a first state upon receiving the request, whereby the AC voltage generator outputs an alternating electric field for a time period of at least 24 hours in response to the request on the control input of the AC voltage generator.

2. The device of claim 1 , wherein the period of time is at least 48 hours.

3. The device of claim 1 , wherein the period of time is at least 72 hours.

4. The apparatus of claim 1 , further comprising a user interface, wherein the request is accepted via the user interface.

5. The apparatus of claim 1 , wherein the request is accepted over RF.

6. 10. The apparatus of claim 1, wherein the alternating electric field has a field strength of at least 1 V / cm in at least a portion of the subject's brain.

7. 10. The device of claim 1, wherein the frequency is between 100 kHz and 125 kHz.

8. 10. The device of claim 1, wherein the frequency is 100 kHz.

9. The apparatus of claim 1 , wherein the time period is uninterrupted.

10. The apparatus of claim 1 , wherein the time period is interrupted by a break.

11. The device of claim 1 , wherein the substance has a molecular weight of at least 4 kDa.

12. The device of claim 1 , wherein the substance has a molecular weight of at least 69 kDa.

13. A set of electrodes configured for attachment to a body of a subject; and Wiring connecting the output of the AC voltage generator to a set of electrodes, The apparatus of claim 1 further comprising:

14. The device of claim 1 , wherein the substance comprises a drug for treating a disease.

15. 10. The device of claim 1, wherein the substance comprises a cancer treatment, an infectious disease treatment, a neurodegenerative disease treatment, an autoimmune disease treatment, an anti-epileptic drug, a hydrocephalus drug, a stroke intervention drug, or a psychiatric drug.

16. The device of claim 1 , wherein the substance is used to monitor brain activity.

17. The device of claim 1 , wherein the substance is a brain dye, reporter, or marker.

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