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

Abstract

This invention relates to the use of an alternating electric field to increase the permeability of the blood-brain barrier. [Solution] Many drugs and other molecules cannot normally pass through the blood-brain barrier (BBB). However, when an alternating current field of a specific optimal frequency (e.g., 100 kHz) is applied to the brain, the BBB becomes permeable to those molecules. When the alternating current field is stopped, the BBB eventually returns to its original impermeable state. The BBB is then reopened by reapplying the alternating current field at the optimal frequency. Typically, many frequencies of the alternating current field (e.g., 200 kHz) are ineffective or less effective in inducing permeability than the optimal frequency. However, once the BBB has been opened by applying an alternating current field at the optimal frequency, alternating current fields at non-optimal frequencies can maintain the permeability of the previously opened BBB.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims the benefit of U.S. Provisional Application Nos. 63 / 015,099 (filed Apr. 24, 2020) and 63 / 071,748 (filed Aug. 28, 2020), each of which is incorporated herein by reference in its entirety.

Background Art

[0002] Normally, the brain's microvessels tightly regulate the movement of substances between blood and brain tissue. This regulation by the brain's microvessels is called the blood - brain barrier (BBB) and is due to the tight junctions (TJs) formed between brain capillary endothelial cells. In brain capillaries, TJ proteins are expressed 50 - 100 times more than in peripheral microvessels. TJs are formed by a complex of transmembrane proteins (claudin and occludin) and cytoplasmic accessory proteins (ZO - 1 and - 2, singlyrin, AF - 6, and 7H6). By binding to the actin cytoskeleton, these proteins form strong cell - to - cell connections. Brain endothelial cells that form the endothelium of the brain's microvessels account for about 75 - 80% of the BBB's resistance to substances, and other cells such as astrocytes and pericytes provide the remaining resistance.

[0003] The BBB is composed of tight junctions around the capillaries and usually limits the diffusion of fine objects and large or hydrophilic molecules into the brain while allowing the diffusion of hydrophobic molecules (transcellular transport rather than paracellular transport).

[0004] In healthy individuals, the BBB serves a very important function in preventing harmful substances (such as bacteria, viruses, potentially harmful large molecules, or hydrophilic molecules) from entering the brain. However, there are situations where the function of the BBB causes difficulties. For example, when it is desirable to deliver large or hydrophilic drug molecules to treat a patient's brain disease. However, when the BBB is functioning properly, these drugs are blocked from entering the brain by the BBB.

Prior Art Documents

[0005] [Patent Document 1] U.S. Patent No. 6868289 [Patent Document 2] U.S. Patent No. 7565205 [Overview of the project] [Means for solving the problem]

[0006] One aspect of the present invention relates to a first method for delivering a substance across the blood-brain barrier in a subject's brain. The first method comprises applying an alternating electric field to the subject's brain for a first period of time, thereby increasing the permeability of the blood-brain barrier in the subject's brain. The first method also comprises administering a first substance to the subject after the first period has elapsed, the increased permeability of the blood-brain barrier in the subject's brain allowing the first substance to pass through the blood-brain barrier. The first method also comprises stopping the application of the alternating electric field for a sufficient amount of time for the blood-brain barrier to recover. The first method also comprises applying an alternating electric field to the subject's brain for a second period of time after the blood-brain barrier has recovered, thereby increasing the permeability of the blood-brain barrier in the subject's brain. The first method also involves administering a second substance to a subject after a second period has elapsed, thereby increasing the permeability of the blood-brain barrier and allowing the second substance to cross the blood-brain barrier.

[0007] In some examples of the first method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz. In some examples of the first method, the alternating electric field is applied at a frequency of at least 50 kHz. In some examples of the first method, the second period is at least 24 hours. In some examples of the first method, the second period is at least 48 hours. In some examples of the first method, the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

[0008] In some examples of the first method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz, the second period is at least 24 hours, and the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

[0009] In some examples of the first method, the second substance is administered intravenously. In some examples of the first method, the second substance is administered orally. In some examples of the first method, the first and second substances are identical. In some examples of the first method, at least one of the first and second substances contains paclitaxel.

[0010] Another aspect of the present invention relates to a second method for delivering a substance across the blood-brain barrier in a subject's brain. The second method comprises applying a first alternating electric field at a first frequency to the subject's brain for a first period, thereby increasing the permeability of the blood-brain barrier in the subject's brain. The second method also comprises applying a second alternating electric field at a second frequency to the subject's brain for a second period, following the first period, wherein the second frequency is different from the first frequency, and the second alternating electric field at the second frequency acts to maintain the permeability of the blood-brain barrier. The second method also comprises administering the substance to the subject at least 24 hours after the first period has elapsed, so that the maintained permeability of the blood-brain barrier allows the substance to pass through the blood-brain barrier.

[0011] In some examples of the second method, the first frequency is between 75 kHz and 125 kHz.

[0012] In some examples of the second method, the first frequency is between 50 kHz and 190 kHz. Optionally, in these cases, the second frequency is at least 190 kHz.

[0013] In some examples of the second method, the first frequency is at least 50 kHz, and the second frequency is higher than the first frequency. In some examples of the second method, the first period is at least 24 hours. In some examples of the second method, the second period includes a single uninterrupted time interval that is at least one week long.

[0014] In some examples of the second method, the second period includes a series of discontinuous time intervals in which a second alternating electric field at a second frequency is applied to the subject's brain, with the series of discontinuous time intervals totaling at least one week.

[0015] Another aspect of the present invention relates to a third method for delivering a substance across the blood-brain barrier in a subject's brain. The third method comprises applying an alternating electric field to the subject's brain for a period of time, thereby increasing the permeability of the blood-brain barrier in the subject's brain. The third method also comprises administering paclitaxel to the subject after the period has elapsed, wherein the increased permeability of the blood-brain barrier allows paclitaxel or at least one metabolite to cross the blood-brain barrier.

[0016] In some examples of the third method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz. In some examples of the third method, the duration is at least 24 hours. In some examples of the third method, the duration is at least 48 hours. In some examples of the third method, the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

[0017] In some examples of the third method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz for a period of at least 24 hours, and the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

[0018] Optionally, in any of the first, second, or third methods described above, the substance has a molecular weight of at least 4 kDa or at least 69 kDa.

[0019] Optionally, in any of the above first, second, or third methods, the substance has at least one characteristic that normally prevents the substance from passing through the non-leaky BBB.

Brief Description of the Drawings

[0020] [Figure 1] An exemplary setup for an in vitro experiment to grow undifferentiated mouse brain capillary endothelial cells (cerebEND) on cover glasses and Transwell inserts is shown to create an artificial in vitro version of the BBB. [Figure 2A] Results of integrity and permeability tests on the artificial BBB are shown. [Figure 2B] Results of integrity and permeability tests on the artificial BBB are shown. [Figure 3A] Data showing that the increased permeability of the artificial BBB was not caused by cell death are shown. [Figure 3B] Data showing that the increased permeability of the artificial BBB was not caused by cell death are shown. [Figure 4] The position where the rat brain was sliced for the in vivo experiment is shown. [Figure 5] EB accumulation in different sections of the rat brain in this in vivo experiment is shown. [Figure 6] The average EB accumulation in the rat brain for this in vivo experiment averaged over all sections is shown. [Figure 7] Increases in BBB permeability in three different sections of the rat brain induced by an alternating current electric field in vivo, determined using contrast MRI, are shown. [Figure 8] Increases in BBB permeability in the rat cortex induced by an alternating current electric field in vivo, determined using contrast MRI, are shown. [Figure 9]This shows the appropriate timing relationship between the application of an alternating electric field and the administration of a substance to a subject. [Figure 10] This graph shows the reduction in cell proliferation in GBM tumors treated with alternating electric field + paclitaxel therapy compared to a control group. [Figure 11] This plot shows the doubling of tumor volume in GBM tumors treated with alternating electric field + paclitaxel therapy compared to three controls. [Figure 12] This is a flowchart of a method for delivering substances across the blood-brain barrier in a subject's brain. [Figure 13] This is a block diagram of a dual-frequency device that generates a first frequency to induce BBB permeability and a second frequency to induce cytotoxicity. [Figure 14] This is a flowchart of an alternative method for delivering substances across the blood-brain barrier in a subject's brain. [Modes for carrying out the invention]

[0021] This application describes a novel approach to temporarily increase the permeability of a BBB using an alternating electric field, allowing materials that would normally be blocked by the BBB to pass through it.

[0022] A series of in vitro experiments were performed to create an artificial in vitro version of the blood-brain barrier (BBB), in which undifferentiated mouse brain capillary endothelial cells (cerebEND) were grown on coverslips and Transwell inserts. Figure 1 shows the setup for these experiments. The cells were then treated with an alternating electric field (100–300 kHz) for 24, 48, and 72 hours. The direction of the alternating electric field was switched every second between two perpendicular directions (i.e., in a repeating sequence, 1 second in one direction, then 1 second in the other direction). The following effects were then analyzed: (a) cell morphology (immunofluorescence staining for 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).

[0023] The first series of experiments involved visualization of cell morphology and orientation, and visualization of the localization of stained proteins. This experiment was designed to determine how the frequency of an alternating electric field affects the artificial blood-brain barrier (BBB). Here, cells were grown on coverslips and an alternating electric field was applied for 72 hours at an electric field intensity of 1.7 V / cm at four different frequencies (100 kHz, 150 kHz, 200 kHz, and 300 kHz). The direction of the alternating electric field was switched between two perpendicular directions every second. A control was also provided without the application of an alternating electric field. Next, cell morphological images (each stained in a different color) showing the presence of claudin 5, ZO-1, and 4,6-diamidino-2-phenylindole (DAPI) were obtained. Claudin 5 and ZO-1 indicate the presence of an intact BBB. This series of cell morphological images revealed that the alternating electric field disrupts the artificial BBB by delocalizing tight-junction proteins from the cell boundary to the cytoplasm, with the most dramatic effect at 100 kHz.

[0024] The second series of experiments also involved the visualization of cell morphology. This experiment was designed to determine how the duration of exposure to an alternating electric field affects the artificial blood-brain barrier (BBB). Endothelial cells were grown on coverslips, and an alternating electric field with a frequency of 100 kHz was applied for three different periods (24 hours, 48 ​​hours, and 72 hours) and a control. The direction of the alternating electric field was switched every second between two perpendicular directions. Cell morphological images showing the presence of claudin 5 and DAPI (stained in different colors) were then obtained. This series of cell morphological images revealed that the phenomenon described above in relation to the first series of experiments was already visible after 24 hours, and its effect was most pronounced after 72 hours.

[0025] A third series of experiments also involved the visualization of cell morphology. This experiment was similar to the second series, except that endothelial cells were grown on Transwell inserts instead of coverslips. The results were similar to those of the second series. Delocalization of TJ proteins was observed after 24 hours, and its effect was most pronounced after 72 hours. The three experiments described above support the conclusion that alternating electric fields may induce structural changes in cells and be responsible for increased blood-brain barrier (BBB) ​​permeability.

[0026] Figures 2A and 2B show the results of integrity and permeability tests, respectively, after exposing an artificial BBB to an alternating electric field at a frequency of 100 kHz for 72 hours (switching the direction of the alternating electric field between two perpendicular directions every second), and against a control. More specifically, Figure 2A shows the results of a transendothelial resistance (TEER) test, which reveals that the alternating electric field reduced the integrity of the artificial BBB to 35% of that of the control. Figure 2B shows the results of a fluorescein isothiocyanate (FITC) permeability test, which reveals that the alternating electric field increased the permeability of the artificial BBB to FITC-dextran with a molecular weight of 4 kDa to 110% of that of the control. These experiments further support the conclusion that an alternating electric field increases the permeability of the BBB to molecules that would normally not be able to pass through a non-leakable BBB.

[0027] In summary, these in vitro experiments demonstrate that applying an alternating electric field at a specific frequency for a sufficient duration causes delocalization of tight junction proteins (claudin 5, ZO-1) from the cell boundary to the cytoplasm (with the most dramatic effect at 100 kHz), thereby increasing blood-brain barrier (BBB) ​​permeability. The AC field effect is already apparent after 24 hours and becomes most pronounced after 72 hours. More specifically, after using an AC field to increase BBB permeability, molecules as small as 4 kDa can pass through the BBB.

[0028] Next, additional in vitro experiments were conducted to determine what happens to the blood-brain barrier (BBB) ​​after the AC electric field is turned off. These experiments used cell morphology visualization to show how the artificial BBB recovers after the AC electric field is stopped. In these experiments, endothelial cells were grown on coverslips and treated with a 100 kHz AC electric field at an electric 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 tracked for 96 hours after the AC electric field was stopped. Cell morphology images showing the presence of (stained) claudin 5 were acquired at 24, 48, 72, and 96 hours. These images revealed a gradual change in the localization of claudin between the cell boundary and the cytoplasm in the images at 24, 48, 72, and 96 hours. Furthermore, comparing these four images with the respective control images (in which no alternating electric field was applied during either the first 72 hours or the last 96 hours), it became clear that endothelial cell morphology partially recovered 48 hours after the cessation of the alternating electric field, and the blood-brain barrier (BBB) ​​was fully recovered (i.e., equivalent to the control) 96 hours after the cessation of the alternating electric field.

[0029] Figures 3A and 3B show the results of an in vitro experiment designed to determine whether the observed changes in the permeability of the artificial BBB described above could be due to cell death. In this experiment, cell division was tested by comparing the number of cells in (a) a case where an AC electric field was applied for 72 hours followed by a 96-hour period without application of the AC electric field, and (b) a control with no application of the AC electric field. Endothelial cells were grown on coverslips and treated with a 100 kHz AC electric field at an electric 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 in the AC electric field and the control was counted, and the results are shown in Figures 3A and 3B (control and AC electric field, respectively). These results reveal 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 could not be due to cell death.

[0030] In another in vitro experiment, the TUNEL assay for apoptosis was used to determine whether the observed changes in the permeability of the artificial BBB described above could be attributable to cell death. In this experiment, endothelial cells were grown on coverslips and treated with a 100 kHz alternating electric field at an electric field strength of 1.7 V / cm for 72 hours. The direction of the alternating electric field was switched every second between two perpendicular directions. No alternating electric field was applied to the control group. Cell morphological images showing apoptosis (TUNEL) and nucleus (DAPI) (stained in different colors, respectively) were acquired at 24, 48, and 72 hours. None of these images revealed additional evidence of apoptosis, indicating that the alternating electric field did not induce cell death. This confirms that the changes in BBB permeability described above were not attributable to cell death.

[0031] A series of in vivo experiments in rats were also performed to quantify the increase in vascular permeability caused by exposure to an alternating electric field. These experiments used Evans blue (EB) dye, an azo dye with very high affinity for serum albumin (molecular size approximately 69 kDa). Due to its large molecular size, serum albumin normally cannot cross the blood-brain barrier (BBB). However, if BBB permeability is sufficiently increased, some serum albumin molecules (along with the EB dye bound to them) can cross the BBB and be detected by searching for EB in the rat brain.

[0032] In this series of experiments, a 100 kHz AC electric field was applied to the brains of rats for 72 hours, with the direction of the AC electric field switched between two vertical directions every second. This was achieved by shaving the heads of each rat 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. Next, a 100 kHz AC voltage was applied between the upper and lower electrodes for 1 second, followed by repeated application of a 100 kHz AC voltage between the left and right electrodes for 1 second each.

[0033] Under the conditions and for the times shown in Table 1, EB was intravenously injected into the tail vein under anesthesia (upon injection, EB immediately binds to albumin), and in all cases, EB was circulated for 2 hours. Next, the following steps were performed: (a) intracardiac perfusion with saline; (b) the brain was sliced ​​into four sections with a brain slicer; (c) the sections were photographed and weighted to identify stains; (d) EB extraction after homogenization of the tissue with 50% TCA (1:3) and centrifugation; and (e) EB quantification at 610 nm. Results are shown as EB μg per gram of tissue.

[0034] [Table 1]

[0035] During the experiment, two animals from group 2 and one animal from group 4 were excluded (due to interruption of treatment and failure of EB injection 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 pseudo-thermia animals and the control animals (groups 3 and 4), and therefore these animals were grouped together.

[0036] The rat brain was sliced ​​into four sections using a brain slicer at the locations shown in Figure 4. EB accumulation was then measured in these four specific sections. Furthermore, computer simulations were performed to determine the electric field strength for each of these four sections. Table 2 shows the electric field strengths obtained from the simulations for each of these four sections, all values ​​expressed in V / cm RMS.

[0037] [Table 2]

[0038] The results of EB accumulation in sections 1 to 4 are shown in Figure 5. A summary of these results is as follows: (1) A statistically significant increase was observed in sections 1 and 2 (frontal cerebrum), where the electric field strength was highest, and a smaller increase (not statistically significant) was observed in the more posterior sections (3 and 4), where the electric field strength was lower.

[0039] Figure 6 shows the average EB accumulation in rat brains, averaged across all four sections 1-4. This result revealed a higher accumulation of EB in the brains of rats treated with an AC electric field for 72 hours, and this result was statistically significant (p<0.05).

[0040] The above in vivo experiments demonstrate that: (1) the application of an alternating electric field allows molecules with an average molecular size of approximately 69 kDa to pass through the BBB into brain tissue; (2) the increased permeability of the BBB is maintained for 2 hours after the discontinuation of the application of the alternating electric field; and (3) the increased permeability of the BBB differs among different sections of the brain. The latter may be a result of the different electric field intensities applied to different sections of the brain. These experiments further support our conclusion that alternating electric fields increase the permeability of the BBB to molecules that would normally not be able to pass through a non-leakable BBB.

[0041] In another series of in vivo experiments, five rats were treated with a 100 kHz alternating electric field for 72 hours, while four control rats were not treated with the alternating electric field for the same amount of time. At the end of the 72 hours, the 4 kDa fluorescent compound TRITC-dextran was intravenously injected into the tail vein under anesthesia and circulated for 2 minutes in all cases. The brains were then removed, frozen, sectioned, and scanned with a fluorescence scanner. All slides were scanned under the same conditions. The resulting images revealed significantly higher levels of accumulation of fluorescent 4 kDa TRITC-dextran in the brain tissue of rats exposed to the alternating electric field (compared to controls), again confirming that the alternating electric field enhances blood-brain barrier permeability.

[0042] A further series of in vivo experiments were performed using dynamic contrast-enhanced MRI (DCE-MRI) with intravenous injection of gadolinium contrast agent (Gd-DTPA, Magnetol, MW547). In these experiments, test rats were treated with an AC electric field at 100 kHz for 72 hours, while control rats were not treated with the AC electric field for the same amount of time. After these 72 hours, the AC electric field was turned off, the rats were anesthetized, and a series of 60 T1wMRI scans (each scan lasting 28 seconds) were acquired. The gadolinium contrast agent was injected into the tail vein of the rats during the 7th of these 60 scans.

[0043] Image analysis for each rat involved: (1) determining the baseline for each voxel by calculating the mean of the first six T1wMRI scans (i.e., scans before gadolinium injection) for each voxel; (2) calculating the rate of signal change over time relative to the baseline (i.e., gadolinium accumulation) on a voxel basis; (3) dividing the brain into anterior, middle, and posterior segments; (4) for each of the three segments, generating the mean rate of signal change relative to the baseline across all voxels in each segment; and (5) averaging four consecutive time points (i.e., four scans) together. Finally, data from all rats in any given group were averaged together.

[0044] Figure 7 shows the results of this DCE-MRI experiment for each of the three brain segments (i.e., anterior, middle, and posterior). The data reveals that contrast agent accumulation (downward-pointing triangular traces labeled TTFields) in rat brain tissue treated with an alternating electric field was significantly higher than in control rats (square traces labeled control). Furthermore, this distinction was most pronounced in the hindbrain, the part of the brain where the alternating electric field had the highest field intensity. From this, we can conclude that the alternating electric field successfully increased blood-brain barrier (BBB) ​​permeability in vivo.

[0045] To test whether this increase in BBB permeability was temporary, the same test conditions were repeated, but without the alternating electric field for another 96 hours. After this 96-hour period, a series of 60T1wMRI scans (each scan lasting 28 seconds) were acquired 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 shown in Figure 8. This data shows that the accumulation of contrast agent in the brain tissue of rats treated with an alternating electric field for 72 hours, followed by 96 hours without the alternating electric field (diamond traces labeled TTFields+96h) was not significantly different from that of control rats (upward triangle traces labeled control+96h). From this, it can be concluded that BBB permeability returned to normal after the alternating electric field was discontinued.

[0046] Before applying the alternating electric field to the rats (n=2), an additional series of 60 T1wMRI scans (each scan lasting 28 seconds) were also acquired using the same procedure. The results of this portion of the DCE-MRI experiment for each of the three brain segments (i.e., anterior, middle, and posterior) are also shown in Figure 8 (see trace labeled “anterior”).

[0047] Figure 8 shows the mean values ​​for all three brain segments (i.e., anterior, middle, and posterior) of the 72-hour TTFields (n=6) compared to the 72-hour TTFields-free control (n=3), along with standard deviation bars. A paired t-test was used to compare the two groups, with p<0.0001.

[0048] It should be noted that the upper limit of the size of molecules that can pass through the blood-brain barrier (BBB) ​​after an AC electric field has been applied has not yet been determined. However, based on (a) in vitro experiments described herein using FITC-dextran with a molecular weight of 4 kDa, and (b) in vitro 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 certainly at least 4 kDa.

[0049] The ability to freely and reversibly increase BBB permeability has far-reaching implications, as it allows for the delivery of many substances across a subject's BBB, even though these substances normally possess at least one characteristic that prevents them from passing through non-leakable BBB. Much of this implies the delivery of substances, including but not limited to therapeutic and diagnostic agents, across the blood-brain barrier of a subject's brain.

[0050] Examples include, but are not limited to, the following: delivering chemotherapy agents across the blood-brain barrier (BBB) ​​to treat cancer (in this situation, increased drug permeability to the brain may allow for reduced dosages of drugs used to treat brain tumors and metastases that have strong 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 research (e.g., monitoring brain activity); delivering antimicrobial agents across the BBB to treat infections; delivering antiviral agents or antiviral antibodies across the BBB to treat viral infections; delivering anthelmintics across the BBB to treat parasitic infections; delivering drugs across the BBB to treat neurodegenerative and autoimmune diseases; delivering psychiatric medications; delivering antiepileptic drugs; delivering hydrocephalus medications; delivering stroke intervention and recovery drugs; delivering compounds deficient in the brain across the BBB to treat conditions in which those compounds are deficient (e.g., to treat Parkinson's disease, etc.).

[0051] The above tests were conducted in vitro and in live rats, but similar results are expected in other animals and humans.

[0052] The method described herein can also be applied in vivo by applying an alternating electric field to the brain of a living subject. Applying an electric field to the subject's brain increases the permeability of the blood-brain barrier (BBB), allowing molecules that would normally be blocked or obstructed by the BBB to pass through. This can be achieved, for example, by placing electrodes on or under the subject's skin, and consequently applying an alternating electric field to the subject's brain by applying an AC voltage between a selected subset of these electrodes.

[0053] For example, one pair of electrodes can be placed on the front and back of the subject's head, and a second pair of electrodes can 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 electrodes that include a conductive plate with a dielectric layer placed 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 can be subcutaneously inserted under the patient's skin.

[0054] The AC voltage generator applies an AC voltage between the left and right electrodes for a first period (e.g., 1 second) at a selected frequency (e.g., 100 kHz, or between 50 and 190 kHz), thereby inducing an alternating electric field where the largest component of the force lines is parallel to the transverse axis of the subject's head. Next, the AC voltage generator applies an AC voltage between the front and rear electrodes for a second period (e.g., 1 second) at the same frequency (or a different frequency), thereby inducing an alternating electric field where the largest component of the force lines is parallel to the sagittal axis of the subject's head. This sequence of two steps is then repeated throughout the treatment period. Optionally, thermal sensors may be included in the electrodes, and the AC voltage generator may be configured to reduce the magnitude of the AC voltage applied to the electrodes if the temperature detected on the electrodes becomes too high. In some embodiments, one or more additional electrode pairs may be added and included in the sequence. In alternative embodiments, only a pair of electrodes is used, in which case the direction of the force lines is not switched. It should be noted that any of the parameters of this in vivo embodiment (e.g., frequency, electric field strength, duration, direction switching speed, and electrode placement) may be modified as described above in relation to the in vivo embodiment. However, care must be taken in the in vivo setting to ensure that the electric field remains safe for the subject at all times.

[0055] A wide variety of applications for increasing blood-brain barrier (BBB) ​​permeability can be easily imagined in biological contexts. For 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 before or during the administration of chemotherapy or other antitumor agents (e.g., 72 hours, or at least 24 hours).

[0056] Figure 9 illustrates the appropriate timing relationship between the application of an alternating electric field and the administration of a substance to a living patient. Based on the data above, assuming that the substance is introduced or administered at a given time t=0, the alternating 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., from t=0 to 12 hours later). In this situation, the permeability of the blood-brain barrier (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 case of chemotherapy, this corresponds to starting the application of the alternating electric field, administering the chemotherapeutic agent 72 hours later, and then applying the alternating electric field for an additional time interval (e.g., from the time the chemotherapeutic agent was administered to 12 hours later).

[0057] It should be noted that the time intervals discussed above in relation to Figure 9 may be uninterrupted or may include short breaks. For example, a 12-hour interval can be filled in a single, uninterrupted 12-hour block. Alternatively, a 12-hour interval can be filled by applying an alternating electric field for 6 hours, followed by a 1-hour break, followed by another 6 hours of application of the alternating electric field. Similar breaks may optionally interrupt the 72-hour interval prior to the administration of the substance. It should also be noted that, in the situation of Figure 9, if the substance is administered to a living patient, the administration of the substance may be carried out using any of a variety of approaches, including but not limited to intravenous, oral, subcutaneous, intrathecal, intraventricular, and intraperitoneal.

[0058] In some preferred embodiments, the frequency of the AC electric field is less than 190 kHz (e.g., between 50 and 190 kHz, or between 25 and 190 kHz). Based on the experiments described above, using frequencies below 190 kHz in combination with a period of at least 24 hours increases the change in transmittance (compared to operation outside these ranges).

[0059] Another series of in vivo experiments were conducted to determine whether paclitaxel (PTX, a drug that normally cannot cross the blood-brain barrier) could cross the blood-brain barrier (BBB) ​​when subjects were treated with an alternating electric field. In one of these experiments, glioblastoma was induced in rats by orthotopic injection of GBM F98 cells on day 0 of the experiment. On day 6, a first set of MRI scans of the brain of each rat was taken. From day 7 to day 10, an alternating electric field of 100 kHz was applied to all rats. On day 10 (i.e., after 72 hours of electric field application), some rats were intraperitoneally injected with 25 mg / kg of PTX, but the others were not. Subsequently, on day 13 (i.e., 3 days after PTX administration), a second set of MRI scans of the brain of each rat was taken. Ki67 (red) and DAPI (blue) staining were evaluated in test rats and control rats. Quantification of the observed Ki67 / DAPI ratio revealed a significant reduction in cell proliferation in GBM tumors with the combination of AC electric field + PTX treatment compared to the control (AC electric field only), as shown in Figure 10.

[0060] Similar experiments were conducted using a 15 mg / kg dose of PTX, except that instead of quantifying the Ki67 / DAPI ratio on day 13, MRI scans and postmortem tissue sections were used to determine the doubling of tumor volume on day 15. Tumor volumes from these experiments are shown in Figure 11. Here again, the experiments revealed a significant reduction in the tumor volume increase factor of GBM tumors with the combination of AC electric field + PTX treatment compared to all three controls (i.e., heat alone, heat + PTX, and AC electric field alone). In summary, these two experiments demonstrate that paclitaxel affects GBM cell proliferation and tumor growth only in combination with an AC electric field (100 kHz). The inventors concluded that the AC electric field allows PTX (or its metabolites) to cross the blood-brain barrier (BBB).

[0061] Experiments were also conducted to determine the relationship between the strength of the alternating electric field and the effects of these fields on the integrity and permeability of the blood-brain barrier (BBB). In in vitro experiments, the effects of a 100 kHz alternating electric field on cell integrity and permeability were evaluated by TEER measurements and FITC-dextran permeability assays. This experiment was repeated three times, and statistical significance was assessed using an unpaired two-sided Student's t-test. In this experiment, artificial BBBs created using mouse cerebEND cells were treated with a 100 kHz alternating electric field for 24–72 hours at electric field intensities of 1.62, 0.97, and 0.76 V / cm RMS, respectively. These cells were stained with AlexaFluor488 (green)-bound claudin-5 antibody immunofluorescence, and the nuclei were stained with DAPI (blue).

[0062] Visual examination of the obtained images revealed the following results: At 0.97 V / cm, regardless of the duration of AC field application (24-72 hours), the images revealed several morphological deformations. Cells lost their spindle shape and transformed into larger cells with frayed borders. The corresponding effect at 1.62 V / cm was more dramatic than at 0.97 V / cm. On the other hand, cells treated with an electric field of 0.76 V / cm appeared more similar to the control, being long and narrow cells tapering towards the ends, regardless of the duration of AC field administration. The presence of frayed membrane contours was more pronounced at 0.76 V / cm, and the presence of larger cells was also evident, although these effects were less pronounced compared to the 0.97 V / cm case. These findings indicate that the cellular response to an AC electric field depends on the electric field strength.

[0063] The relationship between the intensity of alternating electric fields and their effects on the integrity and permeability of the brain barrier (BBB) ​​was also evident from in vivo experiments in rats using continuous dynamic contrast (DCE) MRI to measure gadolinium (Gd) uptake after applying a 100 kHz alternating electric field for 72 hours. The rat brain was divided into three regions (anterior, posterior, and central), and simulations of alternating electric field delivery to these three regions yielded electric field intensities of 1.5 ± 0.6 V / cm RMS, 2.1 ± 1.2 V / cm RMS, and 2.7 ± 1.7 V / cm RMS. The results of continuous DCE MRI showed a significantly increased signal enhancement in the midbrain and hindbrain approximately 10 minutes after Gd injection (compared to control rats), indicating increased Gd accumulation. On the other hand, no significant Gd accumulation was observed in the anterior brain, which is consistent with the lower intensity in this region. These findings also indicate that the cellular response to alternating electric fields is dependent on the electric field intensity.

[0064] Other findings in this experiment were as follows: No significant Gd accumulation was observed in control rats (not treated with an alternating electric field). Furthermore, no significant Gd accumulation was observed when Gd injection was delayed up to 96 hours after the alternating electric field was turned off. The latter finding leads to the conclusion that the blood-brain barrier (BBB) ​​of those rats was restored to its original state. Minimal enhancement was observed in the brains of control rats and in the brains of rats in which Gd injection was delayed up to 96 hours after the alternating electric field was turned on, whereas an analysis of the spatial distribution of Gd accumulation 20 to 23 minutes after contrast agent administration showed that Gd enhancement was distributed throughout the brains of the test rats.

[0065] Another series of experiments was conducted to determine whether a blood-brain barrier (BBB) ​​that had previously been opened by applying an alternating electric field (by stopping the alternating electric field for a sufficient amount of time for the BBB to recover) could be reopened by applying an alternating electric field to the BBB again. In this experiment, artificial BBBs created using mouse cerebEND cells stained with claudin-5 antibody immunofluorescence were tested, and the nuclei were stained with DAPI. The BBBs were treated with an alternating electric field of 100 kHz at an electric field strength of 1.62 V / cm for 72 hours, with the direction of the electric field switched between two perpendicular directions every second. This initial 72-hour interval was followed by a 96-hour recovery period during which no alternating electric field was applied. Subsequently, an alternating electric field of 100 kHz was applied for a second 96-hour interval at an electric field strength of 1.62 V / cm. No alternating electric field was applied to the control.

[0066] As explained above, Claudin 5 indicates the presence of an intact BBB. Visual examination of images obtained after the initial 72 hours revealed that the alternating electric field interfered with the artificial BBB by delocalizing tight junction proteins from the cell boundary to the cytoplasm, indicating that the artificial BBB was no longer intact. Visual examination of images obtained after a 96-hour recovery period revealed that the artificial BBB had returned to its original intact state, and its appearance was similar to that of the control. In particular, visual examination of images obtained during and at the end of the second 72-hour interval revealed that the alternating electric field interfered with the artificial BBB again by delocalizing tight junction proteins from the cell boundary to the cytoplasm, indicating that the artificial BBB was no longer intact. These findings indicate that a previously opened and subsequently closed BBB can indeed be reopened by applying an alternating electric field to the BBB. This could be particularly advantageous for repeated administration of drugs for the treatment of CNS diseases.

[0067] Taking this into consideration, the following method for delivering a substance across the blood-brain barrier in the subject's brain, as shown in Figure 12, can be used. Firstly, in step S10, an alternating electric field (e.g., with a frequency between 75 kHz and 125 kHz) is applied to the subject's brain for a first period. Preferably, the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain. Applying an alternating electric field to the subject's brain for a first period increases the permeability of the blood-brain barrier in the subject's brain. In some cases, the first period is at least 24 hours, and in some cases, the first period is at least 48 hours.

[0068] Next, in step S20, after the first period has elapsed, the first substance is administered to the subject (for example, intravenously or orally). The increased permeability of the blood-brain barrier allows the first substance to pass through the blood-brain barrier. Then, in step S30, the application of the alternating electric field is stopped for a sufficient amount of time for the blood-brain barrier to recover.

[0069] In step S50, after the blood-brain barrier has been restored, an alternating electric field (e.g., with a frequency between 75 kHz and 125 kHz) is applied to the subject's brain for a second period. Preferably, the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain. Applying the alternating electric field to the subject's brain for a second period increases the permeability of the blood-brain barrier in the subject's brain. In some cases, the second period is at least 24 hours, and in some cases, the second period is at least 48 hours.

[0070] Next, in step S60, after the second period has elapsed, a second substance (which may, for example, be, optionally identical to, the first substance) is administered to the subject (for example, intravenously or orally). The increased permeability of the blood-brain barrier allows the second substance to cross the blood-brain barrier. Optionally, at least one of the first and second substances is paclitaxel.

[0071] In some examples of the method shown in Figure 12, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz, the second period is at least 24 hours, and the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

[0072] The methods described herein may be used to deliver substances across the blood-brain barrier in a subject's brain when the subject's brain contains a tumor. One existing approach to treating brain tumors (e.g., glioblastoma) is to apply an alternating electric field to the tumor at a frequency between 50 and 500 kHz, preferably between 100 and 300 kHz. In the case of glioblastoma, 200 kHz is the most preferred frequency. Alternating electric fields at these frequencies are called TTFields and are described in U.S. Patents 6,868,289 and 7,565,205, each of which is incorporated herein by reference in whole. Briefly, these two applications describe the destruction of cells that are dividing during mitosis. The effectiveness of TTFields is enhanced when the direction of the electric field is periodically switched, when the electric field strength in at least part of the tumor is 1 V / cm or greater, and when the electric field is applied for a long period of time (e.g., several weeks or months) with as few breaks as possible.

[0073] In patients with brain tumors, situations may arise where it is desirable to treat the tumor with TTFields and deliver substances across the blood-brain barrier in the same patient (e.g., to help therapeutically effective amounts of chemotherapy drugs cross the BBB to provide an additional line of attack against the tumor). In some situations, a single frequency of the alternating current field may be used for both tumor treatment and increasing BBB permeability. In other situations, it may be desirable to use alternating current fields of different frequencies: a first frequency is chosen to provide improved results in increasing BBB permeability, and a second frequency is chosen to provide improved results for the antitumor effect of TTFields. In the latter situation, the second frequency is usually higher than the first frequency.

[0074] 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 AC voltage generator 44 similar to a conventional Optune® field generator unit, but has the capability 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.

[0075] The ability to operate at two different frequencies can be achieved, for example, by using a relay to switch either a first set of components or a 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 a first frequency or a second frequency depending on the state of the 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 put the control input to the second state so that the AC voltage generator 44 outputs the second frequency. The controller 42 is also programmed to accept requests to switch to the first frequency. In the embodiment shown in Figure 10, requests arrive via a user interface 40 which can be implemented using any of a variety of conventional approaches, including but not limited to push buttons and touchscreens. In an alternative embodiment, requests may arrive via RF (e.g., Bluetooth, WiFi, etc.) from a tablet, smartphone, etc.

[0076] Upon receiving a request, the controller 42 sets the control input to a first state, causing the AC voltage generator 44 to output a first frequency for a certain time interval (e.g., 72 hours). After this time interval has elapsed, the controller 42 sets the control input to a second state, causing the AC voltage generator 44 to return to outputting a second frequency.

[0077] Optionally, the AC 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 AC voltage generator 44 to output a 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. Upon receiving a request, the controller 42 sets the control input to the first state, causing the AC voltage generator 44 to output the first frequency for a certain time interval (e.g., 72 hours). After this time interval has elapsed, the controller 42 returns to cycling the control input through states that cause the AC voltage generator 44 to output a second frequency and one or more additional frequencies.

[0078] The system shown in Figure 13 is particularly useful when a person has a tumor being treated with combination therapy including TTFields and chemotherapy. In this situation, the system operates at the second frequency in most cases, providing the maximum cytotoxic effect. However, before a person visits a chemotherapy clinic for chemotherapy administration, a healthcare professional (or user) activates the user interface 40 to switch the system to the first frequency, which promotes blood-brain barrier (BBB) ​​permeability. In this situation, the activation of the user interface may occur, for example, 72 hours before the expected start of chemotherapy.

[0079] Alternatively, upon receiving a request (for example, from the user interface 40), the controller 42 may control the control input so that the AC voltage generator 44 outputs a first frequency at regular time intervals (for example, 1 hour), and then alternates between the second frequency and the first frequency (for example, switching every hour). Finally (for example, when the relevant substance has been removed from the patient's bloodstream), the controller 42 controls the control input so that the AC voltage generator 44 returns to outputting the second frequency.

[0080] A set of electrodes (not shown) similar to conventional electrodes used in Optune is connected to the output of the AC voltage generator 44.

[0081] Additional experiments were conducted to examine the effect on BBB transparency when the BBB is opened by applying an AC electric field at one frequency optimized for opening the BBB (e.g., 100 kHz), and then switching the frequency of the AC electric field to another frequency (e.g., 200 kHz).

[0082] More specifically, these experiments tested artificial blood-brain barriers (BBBs) created using mouse cerebEND cells stained with claudin-5 antibody immunofluorescence, with the nuclei stained with DAPI. These BBBs were treated with a 100 kHz alternating electric field at a field strength of 1.62 V / cm for 72 hours, with the direction of the field switching between two vertical directions every second. After this initial 72-hour interval, the 100 kHz alternating electric field was discontinued, and a 200 kHz alternating electric field at a field strength of 1.62 V / cm was applied for a second interval of 5 days. No alternating electric field was applied to the control.

[0083] As explained above, Claudin 5 indicates the presence of an open BBB. Visual examination of images obtained after the first 72 hours revealed that the 100 kHz AC electric field disrupted the artificial BBB by delocalizing the tight junction protein from the cell boundary to the cytoplasm, indicating that the artificial BBB was open. In particular, visual examination of images acquired over the second interval (i.e., 5-day interval) revealed that the artificial BBB remained open even though the original 200 kHz AC electric field was no longer applied. These findings indicate that a BBB previously opened at one frequency optimized for opening (e.g., 100 kHz) will remain open when an AC electric field of a different frequency is applied, even if the different frequency is not effective in switching it from a closed to an open state. In other words, different frequencies act to maintain the permeability of a previously opened BBB.

[0084] Taking this into consideration, the following method, shown in Figure 14, can be used to deliver a substance across the blood-brain barrier in a subject's brain. Firstly, in step S120, a first alternating electric field at a first frequency (e.g., 100 kHz, 75–125 kHz, or 50–190 kHz) is applied to the subject's brain for a first period (e.g., at least 24 hours). Applying the first alternating electric field at the first frequency to the subject's brain for a first period increases the permeability of the blood-brain barrier in the subject's brain. Subsequently, in step S130, a second alternating electric field at a second frequency (e.g., 200 kHz or 190–210 kHz) is applied to the subject's brain for a second period. The second frequency is different from the first frequency, and the second alternating electric field at the second frequency acts to maintain the permeability of the blood-brain barrier.

[0085] Next, in step S140, the substance is administered to the subject at least 24 hours after the first period has elapsed. Due to the maintained permeability of the blood-brain barrier, the substance can cross the blood-brain barrier.

[0086] In some examples of the method shown in Figure 14, the second period includes a single uninterrupted time interval that is at least one week long. In other examples of the method shown in Figure 14, the second period includes a series of discontinuous time intervals in which a second alternating electric field is applied to the subject's brain at a second frequency, and these discontinuous time intervals are collectively added up to at least one week.

[0087] In relation to any of the above methods, it should be noted that after the AC electric field has ceased and a sufficient amount of time has elapsed, the BBB should return to its original low-permeability state. This can be important in many situations for the safety of the subject.

[0088] While the above in vitro data was obtained using mouse cells, it should be noted that preliminary data shows similar effects in human cells in vitro.

[0089] While the present invention has been disclosed with reference to specific embodiments, numerous modifications, changes, and alterations to the embodiments described are possible without departing from the scope and scope of the invention, as defined in the appended claims. Therefore, the present invention is not limited to the embodiments described, but is intended to encompass the entire scope defined by the following claims and their equivalents.

Claims

1. A method for delivering a substance across the blood-brain barrier in the brain of a subject, A step of applying an alternating electric field to the brain of the subject for a first period of time, wherein applying the alternating electric field to the brain of the subject for a first period of time increases the permeability of the blood-brain barrier in the brain of the subject. A step of administering a first substance to the subject after the first period has elapsed, wherein the increased permeability of the blood-brain barrier allows the first substance to pass through the blood-brain barrier. The steps include stopping the application of the alternating electric field for a sufficient time for the blood-brain barrier to recover, The steps include: applying an alternating electric field to the subject's brain for a second period after the blood-brain barrier has been restored, wherein applying the alternating electric field to the subject's brain for the second period increases the permeability of the blood-brain barrier in the subject's brain; A step of administering a second substance to the subject after the second period has elapsed, wherein the increased permeability of the blood-brain barrier allows the second substance to pass through the blood-brain barrier. A method that includes this.

2. The method according to claim 1, wherein the alternating electric field is applied at a frequency between 75 kHz and 125 kHz.

3. The method according to claim 1, wherein the alternating electric field is applied at a frequency of at least 50 kHz.

4. The method according to claim 1, wherein the second period is at least 24 hours.

5. The method according to claim 1, wherein the second period is at least 48 hours.

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

7. The method according to claim 1, wherein the alternating electric field is applied at a frequency between 75 kHz and 125 kHz, the second period is at least 24 hours, and the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

8. The method according to claim 1, wherein the administration of the second substance is performed intravenously.

9. The method according to claim 1, wherein the administration of the second substance is performed orally.

10. The method according to claim 1, wherein the first substance and the second substance are the same.

11. The method according to claim 1, wherein at least one of the first substance and the second substance comprises paclitaxel.

12. A method for delivering a substance across the blood-brain barrier in the brain of a subject, A step of applying a first alternating electric field at a first frequency to the brain of the subject for a first period of time, wherein applying the first alternating electric field at a first frequency to the brain of the subject for a first period of time increases the permeability of the blood-brain barrier of the subject's brain. The steps include applying a second alternating electric field at a second frequency to the subject's brain for a second period, following the first period, wherein the second frequency is different from the first frequency, and the second alternating electric field at the second frequency acts to maintain permeability of the blood-brain barrier, A step of administering the substance to the subject at least 24 hours after the first period has elapsed, wherein the maintained permeability of the blood-brain barrier allows the substance to pass through the blood-brain barrier. A method that includes this.

13. The method according to claim 12, wherein the first frequency is between 75 kHz and 125 kHz.

14. The method according to claim 12, wherein the first frequency is between 50 kHz and 190 kHz.

15. The method according to claim 14, wherein the second frequency is at least 190 kHz.

16. The method according to claim 12, wherein the first frequency is at least 50 kHz and the second frequency is higher than the first frequency.

17. The method according to claim 12, wherein the first period is at least 24 hours.

18. The method according to claim 12, wherein the second period includes a single uninterrupted time interval having a length of at least one week.

19. The method according to claim 12, wherein the second period includes a plurality of discontinuous time intervals in which the second alternating electric field at the second frequency is applied to the brain of the subject, and the plurality of discontinuous intervals total at least one week.

20. A method for delivering a substance across the blood-brain barrier in the brain of a subject, A step of applying an alternating electric field to the brain of a subject for a certain period of time, wherein applying the alternating electric field to the brain of the subject for the said period of time increases the permeability of the blood-brain barrier of the subject's brain. A step of administering paclitaxel to the subject after the aforementioned period has elapsed, wherein the increased permeability of the blood-brain barrier allows paclitaxel or at least one metabolite to cross the blood-brain barrier. A method that includes this.

21. The method according to claim 20, wherein the alternating electric field is applied at a frequency between 75 kHz and 125 kHz.

22. The method according to claim 20, wherein the period is at least 24 hours.

23. The method according to claim 20, wherein the period is at least 48 hours.

24. The method according to claim 20, wherein the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

25. The method according to claim 20, wherein the alternating electric field is applied at a frequency between 75 kHz and 125 kHz, the period is at least 24 hours, and the alternating electric field has an electric field strength of at least 1 V / cm in at least a portion of the subject's brain.

26. The method according to any one of claims 1 to 25, wherein the substance has a molecular weight of at least 4 kDa.

27. The method according to any one of claims 1 to 25, wherein the substance has a molecular weight of at least 69 kDa.

28. The method according to any one of claims 1 to 25, wherein the substance has at least one feature that normally prevents the substance from passing through a non-leakable BBB.

Images