Using alternating electric fields to increase permeability of blood brain barrier

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 hereby incorporated by reference in its entirety.

Background Art

[0002] Normally, cerebral microvessels tightly regulate the movement of substances between blood and brain tissue. This regulation by cerebral microvessels is called the blood - brain barrier (BBB), and is due to the intercellular tight junctions (TJs) formed between the capillary endothelial cells of the brain. 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 (claudin and occludin) and cytoplasmic accessory proteins (ZO - 1 and ZO - 2, singlyrin, AF - 6, and 7H6). By linking to the actin cytoskeleton, these proteins form strong intercellular connections. About 75 - 80% of the resistance of the BBB to substances is borne by the brain endothelial cells that form the endothelium of cerebral microvessels, and the remaining resistance is provided by other cells such as astrocytes and pericytes.

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

[0004] In healthy humans, the BBB plays a very important function by preventing harmful substances (e.g., bacteria, viruses, and potentially harmful large or hydrophilic molecules) from entering the brain. However, there are situations where the function of the BBB poses problems. 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 normally, these drugs are blocked from entering the brain by the BBB.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems 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, an alternating electric field is applied to the subject's brain for a period of time to deliver a related substance through the blood-brain barrier of the subject's brain. The application of the alternating electric field to the subject's brain for a period of time improves the permeability of the blood-brain barrier in the subject's brain. The substance is administered to the subject after a period of time has elapsed, and the improved permeability of the blood-brain barrier enables the substance to pass through the blood-brain barrier.

Means for Solving the Problems

[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 an electric field strength of at least 1 V / cm in at least a part 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 an electric field strength of at least 1 V / cm in at least a part 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 does not have a tumor.

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

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

[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, by applying an alternating electric field at a first frequency to the subject's brain for a certain time period, a related substance can be delivered through the blood-brain barrier of the subject's brain, where the first frequency is less than 190 kHz, the certain time period is at least 24 hours, and where the application of the alternating electric field at the first frequency to the subject's brain during the certain time period improves the permeability of the blood-brain barrier in the subject's brain. The substance is administered to the subject after the certain time period has elapsed, and the improved permeability of the blood-brain barrier enables 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 certain time period is at least 48 hours. In some cases of the second method, the alternating electric field has an electric field strength of at least 1 V / cm in at least a part 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 an electric field strength of at least 1 V / cm in at least a part of the subject's brain.

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

[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 situation, another aspect of the present invention is directed to a third method of delivering a substance through the blood-brain barrier of the brain of a subject. In this third method, by applying an alternating electric field to the brain of the subject at a first frequency for a period of time, a related substance can be delivered through the blood-brain barrier of the brain of a subject without a tumor. The application of the alternating electric field to the brain of the subject at the first frequency 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 enables 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 an electric field strength of at least 1 V / cm in at least a part of the brain of the subject. 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 an electric field strength of at least 1 V / cm in at least a part of the brain of the subject.

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

[0017] The methods described herein can be used to deliver substances across the blood-brain barrier of a subject having a brain tumor. In this context, another aspect of the invention is directed to a fourth method of treating a tumor in a subject's brain and delivering a substance across the blood-brain barrier of the subject's brain. In this fourth method, a first alternating electric field is applied to the subject's brain at a first frequency for a first time period. The application of the first alternating electric field at the first frequency to the subject's brain during the first time period improves the permeability of the blood-brain barrier in the subject's brain. The substance is administered to the subject after the first time period has elapsed, and the improved permeability of the blood-brain barrier enables the substance to cross the blood-brain barrier. A second alternating electric field at a second frequency is applied to the subject's brain for a second time period that is at least one week in length. The second frequency is different from the first frequency, and the second alternating electric field at the second frequency has an intensity 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 in length. In other cases of the fourth method, the second time period includes a plurality of discontinuous time intervals during which the second alternating electric field at the second frequency is applied to the subject's brain, and the plurality of discontinuous time intervals together total at least one week.

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

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

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

[0024] In some cases, any of the above methods is used to deliver a substance through the blood-brain barrier of the target brain, where the substance has at least one characteristic that normally impedes the substance from passing through 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 the delivery of a substance through the blood-brain barrier of the subject's body. The first device includes an AC voltage generator that can operate 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 to output the second frequency when the control input is in a second state. The first device also includes a controller programmed to (a) set the control input to the second state so that the AC voltage generator outputs the second frequency, (b) receive a request to switch to the first frequency, (c) upon receiving the request, set the control input to the first state so that the AC voltage generator outputs the first frequency for a period of time, and (d) after the period of time has elapsed, set the control input to the second state so that the AC voltage generator outputs the second frequency.

[0026] Some embodiments of the first device a set of electrodes configured for attachment to the subject's body; and wiring connecting the output of the AC voltage generator to the 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, a time interval is at least 24 hours. In some embodiments of the first device, a time interval is at least 72 hours. In some embodiments of the first device, the controller is further programmed to alternately switch the control input between a first state and a second state following receipt of a request.

[0028] In some embodiments of the first device, the AC voltage generator can operate at at least one additional frequency between 50 and 500 kHz, and the AC voltage generator is configured to output at least one additional frequency when the control input is in at least one further state, and the controller is programmed to cyclically repeat the second state and at least one additional state on the control input before receipt of a request, and after a time interval has elapsed, cyclically repeat the second state and at least one additional state on the control input.

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

Brief Description of the Drawings

[0030]

Figure 1

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Best Mode for Carrying Out the Invention

[0031] This application relates to a novel approach for temporarily improving the permeability of the blood-brain barrier (BBB) using an alternating electric field so that substances normally blocked by the BBB can pass through the BBB.

[0032] An in vitro experiment set was performed to create an artificial in vitro version of the BBB by growing immortalized mouse brain capillary endothelial cells (cerebEND) on cover glasses and Transwell inserts, and the setup of these experiments is described in Figure 1. Next, the cells were treated with an alternating electric field (100 - 300 kHz) for 24 hours, 48 hours, and 72 hours. The direction of the alternating electric field was switched every second between two perpendicular directions (i.e., one direction for 1 second, followed by the other direction for 1 second, in a repeating sequence). Next, the following effects were analyzed: (a) cell morphology (immunofluorescent staining of tight junction proteins claudin 5 and ZO - 1); (b) BBB integrity (using trans - endothelial 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, and visualization of the localization of stained proteins. This experiment was designed to confirm how the frequency of the alternating electric field affected the artificial BBB. Here, the cells were grown on cover glasses and an alternating electric field was applied for 72 hours at four different frequencies (100 kHz, 150 kHz, 200 kHz, and 300 kHz) at an electric field strength of 1.7 V / cm. The direction of the alternating electric field was switched every second between two perpendicular directions. There was also a control without the application of the alternating electric field. Next, cell morphology images were obtained that described the presence of claudin 5, ZO - 1, and 4,6 - diamidino - 2 - phenylindole (DAPI) (stained in different colors). Claudin 5 and ZO - 1 indicate the presence of an intact BBB. What was revealed by this set of cell morphology images was that the alternating electric field disrupted the artificial BBB by delocalization of tight junction proteins from the cell border into 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 confirm how the duration of application of an alternating electric field affected the artificial BBB. Endothelial cells were grown on cover glasses, and in addition to the control, an alternating electric field at a frequency of 100 kHz was applied for three different durations (24 hours, 48 hours, 72 hours). The direction of the alternating electric field was switched every second between two perpendicular directions. Subsequently, cell morphology images were obtained that described the presence of claudin 5 and DAPI (stained in different colors). What was revealed by this set of cell morphology images was that the phenomenon discussed above in relation to the first set of experiments was already visible after 24 hours, and its effect was most prominent after 72 hours.

[0035] The third set of experiments also included visualization of cell morphology. This experiment was the same as the second set of experiments, except that the endothelial cells were grown on transwell inserts instead of cover glasses. The results were the same as those of the second set of experiments. The delocalization of TJ proteins was visible after 24 hours, and its effect was most prominent after 72 hours. The above three experiments support the conclusion that an alternating electric field causes structural changes in cells, which may be the cause of the improvement in BBB permeability.

[0036] Figures 2A and 2B describe the results of the integrity and permeability tests for an artificial BBB after being subjected to an alternating electric field at a frequency of 100 kHz for 72 hours (the direction of the alternating electric field was switched every second between two perpendicular directions), and for the subject, respectively, regarding the artificial BBB. More specifically, Figure 2A describes the results of the transendothelial electrical resistance (TEER) test, which reveals that the integrity of the artificial BBB was reduced to 35% of the control by the alternating electric field. Figure 2B describes the results of the fluorescein isothiocyanate (FITC) permeability test, which reveals that the permeability of the artificial BBB to FITC-dextran with a molecular weight of 4 kDa was increased to 110% of the control by the alternating electric field. These experiments further support the conclusion that the alternating electric field improves the permeability of the BBB to molecules that normally cannot pass through the non-leaky BBB.

[0037] In summary, what is clear from these in vitro experiments is that applying an alternating electric field at a specific frequency for a sufficient duration results in the delocalization of tight junction proteins (claudin 5, ZO-1) from the cell boundary to the cytoplasm (the most dramatic effect being at 100 kHz), improving the permeability of the BBB. The effect of the alternating electric field appears already after 24 hours and is most pronounced after 72 hours. More specifically, after using the alternating electric field to improve the permeability of the BBB, molecules of 4 kDa can pass through the BBB.

[0038] Subsequent in vitro experiments were then performed to determine what happens to the BBB after the AC electric field is turned off. In these experiments, visualization of cell morphology was used to show how the artificial BBB recovers after the AC electric field is stopped. In these experiments, endothelial cells were grown on cover glasses and treated with an AC electric field of 100 kHz at an electric field strength of 1.7 V / cm for 72 hours. The direction of the AC electric field was switched every second between two perpendicular directions. The AC electric field was then turned off, and the cells were followed up for 96 hours after the AC electric field was stopped. Cell morphology images representing 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 claudin between the cell boundary and the cytoplasm in the images at 24 hours, 48 hours, 72 hours, and 96 hours. Furthermore, comparison of these four images with each of the control images (no AC electric field was applied either during the first 72 hours or between any of the subsequent 96 hours) revealed that the morphology of the endothelial cells partially recovered 48 hours after the AC electric field was stopped, and that the BBB completely recovered (i.e., was equivalent to the control) 96 hours after the AC electric field was stopped.

[0039] Figures 3A and 3B show the results of in vitro experiments designed to determine whether the observed changes in the permeability of the artificial BBB could be due to cell death. In this experiment, cell division was tested by comparing the cell counts of (a) endothelial cells grown on coverslips and treated with an alternating current (AC) electric field at 1.7 V / cm and 100 kHz for 72 hours followed by 96 hours without the AC electric field applied, and (b) a control with no AC electric field applied at all. The direction of the AC electric field was switched every second between two perpendicular directions. Then, after turning off the AC electric field and stopping it, the cells were followed for 96 hours. The cell counts per milliliter for the AC electric field and the control are shown in Figures 3A and 3B (for the subject and the AC electric field, respectively). These results clearly show that there was no statistically significant increase in the cell count during or after the application of the AC electric field, indicating that the observed changes in BBB permeability are not due to cell death, as described above.

[0040] In another in vitro experiment, a TUNEL assay was used to determine whether the observed changes in the permeability of the artificial BBB could be due to cell death. In this experiment, endothelial cells were grown on coverslips and treated with an AC electric field at 1.7 V / cm and 100 kHz for 72 hours. The direction of the AC electric field was switched every second between two perpendicular directions. No AC electric field was applied to the control. Cell morphology images showing apoptosis (TUNEL) and nuclei (DAPI), stained in different colors, were obtained at 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 observed changes in BBB permeability were not due to cell death, as described above.

[0041] To quantify the improvement in vascular permeability caused by exposure to an alternating electric field, a set of in vivo experiments on rats was also conducted. In these experiments, Evans blue (EB) dye was used, which is an azo dye with a very high affinity for serum albumin (molecular size approximately 69 kDa). Due to its large molecular size, serum albumin normally cannot pass through the BBB. However, when the permeability of the BBB is sufficiently improved, a portion of the serum albumin molecules (along with the EB dye bound to them) will pass through the BBB and can then be detected by searching for EB in the rat's brain.

[0042] In this set of experiments, an alternating electric field of 100 kHz was applied to the rat's brain for 72 hours, and the direction of the alternating electric field was switched every second between two perpendicular directions. This was done by shaving the head of each rat and placing the first pair of capacitive coupling electrodes on the upper and lower parts of the rat's head, and the second pair of capacitive coupling electrodes on the left and right sides of the rat's head. Then, in a repeating sequence, an AC voltage of 100 kHz was applied between the upper and lower electrodes for 1 second, followed by applying an AC voltage of 100 kHz between the left and right electrodes for 1 second.

[0043] Under the conditions shown in Table 1 and for the time shown in Table 1, EB was intravenously injected into the tail vein under anesthesia (once injected, EB immediately binds to albumin), and EB was circulated for 2 hours in all cases. Then, the following steps were performed: (a) intracardiac perfusion with physiological saline; (b) slicing the brain into 4 pieces with a brain slicer; (c) taking pictures of each piece to localize the staining and make a comparative study; (d) EB extraction when the tissue was homogenized and centrifuged with 50% TCA (1:3), and (e) quantification of EB at 610 nm. The results are given as μg of EB per gram of tissue.

[0044]

Table 1

[0045] During the experiment, two animals from group 2 and one animal from group 4 were excluded (interruption of treatment, failure of EB injection into the tail vein). Since there was no difference between the animals treated with the alternating electric field (groups 1 and 2), these animals were grouped together. Similarly, since there was no difference between the sham-heated animals and the control animals (groups 3 and 4), these animals were grouped together.

[0046] At the positions shown in Figure 4, the rat brain was sliced into four pieces using a brain slicer. Next, the EB accumulation in specific sections of these four pieces was measured. In addition, computer simulations were performed to determine the electric field strength of each of these four sections. Table 2 explicitly shows the electric field strength obtained from the simulations for 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 described in Figure 5. A summary of these results is as follows: (1) Statistically significant increases were observed in sections 1 and 2 (frontal cerebrum) where the electric field strength was the highest; and smaller increases (not statistically significant) were observed in the more posterior sections (3 and 4) where the electric field strength was lower.

[0049] Figure 6 depicts the average EB accumulation in the rat brain averaged over all four sections 1 to 4. This result reveals that the accumulation of EB in the brains of rats treated with the alternating electric field for 72 hours was higher, and this result was statistically significant (p < 0.05).

[0050] What is confirmed by the above in vivo experiments is: (1) the application of an alternating electric field enables the passage of molecules with an average molecular size of approximately 69 kDa across the BBB into brain tissue; (2) the improvement in BBB permeability is maintained for 2 hours after the application of the alternating electric field is terminated; and (3) the improvement in BBB permeability varies among different sections of the brain, which can be a result of different electric field strengths applied to various sections of the brain. These experiments further support our conclusion that an alternating electric field improves the permeability of the BBB to molecules that normally cannot pass through the non-leaky BBB.

[0051] In another set of in vivo experiments, five rats were treated with an alternating electric field at 100 kHz for 72 hours, and four control rats were not treated with an alternating electric field for the same time period. At the end of the 72-hour period, a 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 that the accumulation level of fluorescent 4 kDa TRITC-dextran in the brain tissue of rats subjected to the alternating electric field was significantly higher (compared to the control), and as a result, it was also confirmed that the permeability of the BBB was improved by the alternating electric field.

[0052] Another set of in vivo experiments was performed using dynamic contrast-enhanced MRI (DCE-MRI) with intravenous injection of a gadolinium-based contrast agent (Gd-DTPA, Magnetol, MW 547). In these experiments, test rats were treated with an alternating electric field at 100 kHz for 72 hours, and 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 T1w MRI scans (each scan having a duration of 28 seconds) were collected. The gadolinium-based contrast agent was injected into the tail vein of the rats at the 7th of these 60 scans.

[0053] Image analysis of each rat included the following: (1) determining the baseline of each voxel by calculating the average of the first six Tlw MRI scans of each voxel (i.e., scans before gadolinium injection); (2) calculating the percent signal change over time (i.e., gadolinium accumulation) for each voxel relative to the baseline; (3) dividing the brain into anterior, middle, and posterior segments; (4) creating the average percent signal change relative to the baseline across all voxels of each respective segment for each of the three segments; then, (5) averaging together four consecutive time points (i.e., four scans). 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 segments of the brain (i.e., anterior, middle, and posterior) are presented in Figure 7. What is revealed by this data is that the accumulation of the contrast agent in the brain tissue of rats treated with an alternating current electric field (trajectory shown by TT field; n = 6) was significantly higher than that in control rats (trajectory shown by control; n = 3). Furthermore, this specificity was most prominent in the posterior part of the brain, which was the part of the brain where the alternating current electric field had the highest field strength. From this, it can be concluded that the alternating current electric field successfully improved the permeability of the BBB in vivo.

[0055] To test whether this improvement in BBB permeability was transient, the same test conditions were repeated, to which an additional 96 hours without an alternating electric field was added. 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 segments of the brain are also described in Figure 7. What is clear from this data is that the accumulation of the contrast agent in the brain tissue of rats (trajectory indicated by TT field + 96h; n = 7) treated with an alternating electric field for 72 hours followed by 96 hours without an alternating electric field was not significantly different from that of control rats (trajectory indicated by control + 96h; n = 3). From this, it can be concluded that after stopping the alternating electric field, the permeability of the BBB returns to normal.

[0056] Prior to applying the alternating electric field to the rats (n = 2), an additional series of 60 Tlw MRI scans (each scan having a duration of 28 seconds) were also collected using the same procedure. The results of this part of the DCE-MRI experiment for each of the three segments of the brain (i.e., front, middle, and rear) are also described in Figure 7 (see the trajectory indicated by "before").

[0057] Figure 8 shows the averages for all three segments of the brain (i.e., front, middle, and rear) for 72 hours of TT field (n = 6) and controls without TT field for 72 hours (n = 3), along with standard deviation bars. A paired t-test was used to compare the two groups, and p < 0.0001.

[0058] Note that the upper size limit of the molecules that can pass through the BBB after applying the alternating electric field has not yet been determined. However, 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 would seem to be at least about 69 kDa, most certainly at least 4 kDa.

[0059] The implications of being able to reversibly improve the permeability of BBB at will are far-reaching, because in this case it is possible to deliver many substances through the target BB, despite the fact that the substance has at least one characteristic that normally impedes it from passing through the non-leaky BBB. Many of these implications mean delivering substances, including but not limited to therapeutic and diagnostic agents, through the blood-brain barrier of the target brain.

[0060] Examples include, but are not limited to: Delivering chemotherapeutic agents through the BBB to treat cancer (in this context, it may be possible to reduce the dosage of drugs for treating brain tumors and metastases, which have severe side effects in other parts of the body, based on improved drug permeability to the brain); delivering antibodies and / or cell-based therapies through the BBB for immunotherapy; delivering contrast agent dyes, reporters, and markers through the BBB for diagnostic and research purposes (e.g., monitoring brain activity); delivering antibacterial agents through the BBB to treat infectious diseases; delivering antiviral agents or virus-neutralizing antibodies through the BBB to treat viral infections; delivering antiparasitic agents through the BBB to treat parasites; delivering drugs through the BBB to treat neurodegenerative and autoimmune diseases; delivering psychotropic drugs; delivering anti-epileptic drugs; delivering hydrocephalus drugs; delivering cerebrovascular intervention and recovery drugs; delivering compounds lacking in the brain through the BBB to treat the state of deficiency of those compounds (e.g., treating Parkinson's disease, etc.).

[0061] The above tests were conducted in vitro and in live rats, but similar results are expected to be obtained in other animals and in humans as well.

[0062] The methods 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 improves the permeability of the BBB, allowing molecules that are normally blocked or impeded by the BBB to pass through. This can be achieved, for example, by placing electrodes on or under the subject's skin, such that an alternating electric field is applied to the subject's brain when an AC voltage is applied between a selected subset of those electrodes.

[0063] 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 and also have a dielectric layer provided 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 will be in direct contact with the subject's body. In another embodiment, the electrodes can be subcutaneously inserted under 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 time period (e.g., 1 second), thereby inducing an alternating electric field in which the most important component of the lines of force is parallel to the transverse axis of the subject's head. Next, the AC voltage generator applies an AC voltage at the same frequency (or a different frequency) between the front and rear electrodes for a second time period (e.g., 1 second), thereby inducing an alternating electric field in which the most important component of the lines of force is parallel to the sagittal axis of the subject's head. Then, these two process sequences are repeated for the duration of the treatment. Optionally, a temperature sensor can be included at 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 alternative embodiments, only a single pair of electrodes is used, in which case the direction of the lines of force does not change. Note that any of the parameters of this in vivo embodiment (e.g., frequency, electric 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 always remains safe for the subject.

[0065] Regarding the various applications for improving the permeability of the BBB, it can be easily envisioned in the in vivo situation. In one example, a 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 time period (e.g., 72 hours or at least 24 hours) before or during the administration of chemotherapy or other anti-tumor agents.

[0066] Figure 9 depicts an appropriate relationship in terms of timing between the application of an alternating electric field and the administration of a substance to a living patient. Based on the above data, assuming that the substance is introduced or administered at a predetermined time t = 0, the alternating electric field can start before the predetermined time (e.g., 72 hours before t = 0) and continue for a certain time interval after the predetermined 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 enables the substance to pass through the BBB immediately upon arrival. In the context of chemotherapy, this would correspond to starting the application of the alternating electric field, administering the chemotherapeutic agent 72 hours later, and subsequently applying the alternating electric field for a further time interval (e.g., up to 12 hours after the time when the chemotherapeutic agent was administered).

[0067] It should be noted that in relation to Figure 9, the time intervals discussed above may be either non - interrupted or may include short breaks, whichever is preferred. For example, the 12 - hour interval can be filled by a single non - interrupted block of 12 hours. Alternatively, the 12 - hour interval can be filled by applying the alternating electric field for 6 hours, followed by a 1 - hour break, and then applying the alternating electric field for a further 6 hours. Similarly, by the same breaks, optionally, the 72 - hour interval prior to the administration of the substance may be interrupted. In relation to Figure 9, when the substance is administered to a living patient, the administration of the substance can be carried out 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, using a frequency less than 190 kHz in combination with a time period of at least 24 hours results in an increased change in permeability (compared to operation outside these ranges).

[0069] The methods described herein can be used to deliver a substance through the blood-brain barrier of a subject's brain when the subject's brain has a tumor. One of the existing approaches 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. In the case of glioblastoma, 200 kHz is the most preferred frequency. The 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 hereby incorporated by reference in its entirety. Briefly, in these two applications, it is described that dividing cells are destroyed during mitosis. The effectiveness of TT fields is improved when the direction of the electric field is periodically switched, when the intensity of the electric field in at least a portion of the tumor is at least 1 V / cm, 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.

[0070] In patients with brain tumors, situations may arise where it is desirable to treat the tumor with TT fields and also deliver a substance through the blood-brain barrier of the same patient (e.g., to help obtain a therapeutically effective amount of a chemotherapeutic agent across the BBB, thereby providing an additional line of attack on the tumor). In some situations, it may be possible to use a single frequency of alternating electric field to both treat the tumor and improve the permeability of the BBB. In other situations, it may be desirable to use alternating electric fields having different frequencies: a first frequency selected to provide an improved result with respect to improving the permeability of the BBB, and a second frequency selected to provide an improved result with respect to the anti-tumor effect of the TT field.

[0071] FIG. 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 is similar to a conventional Optune® electric field generator unit, but includes an AC voltage generator 44 having 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 a first set of components or a second set of components into 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 a first state, the AC voltage generator 44 outputs the first frequency, and when the control input is in a 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 can arrive via a user interface 40 that can be implemented using any of a variety of conventional approaches, including but not limited to push buttons, touch screens, etc. In an alternative embodiment, the request can arrive via RF (e.g., Bluetooth, WiFi, etc.) from a tablet, smartphone, etc.

[0073] Upon receiving a request, the controller 42 sets the control input to a first state, and as a result, the AC voltage generator 44 will output a first frequency for a certain time period (e.g., 72 hours). After a certain time period has elapsed, the controller 42 sets the control input to a second state, and as a result, the AC voltage generator 44 returns to outputting a 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 periodically repeat the state of causing the AC voltage generator 44 to output a second frequency and one or more additional frequencies to the control input 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 a first state, and as a result, the AC voltage generator 44 will output a first frequency for a certain time period (e.g., 72 hours). After a certain time period has elapsed, the controller 42 will return to periodically repeating the state of causing the AC voltage generator 44 to output a second frequency and one or more additional frequencies to the control input.

[0075] The system described in FIG. 10 is particularly useful when a patient has a tumor being treated by a combination therapy including a TT field and chemotherapy. In this situation, the system operates at the second frequency most of the time to provide the maximum cytotoxic effect. However, before a human visits a chemotherapy clinic for a chemotherapy dose, a healthcare provider (or user) activates the user interface 40 to switch the system to the first frequency that promotes BBB permeability. In this situation, the activation of the user interface can be performed, 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 a first frequency for a period of time (e.g., one hour), and then alternates between a second frequency and the first frequency (e.g., switches every hour). Eventually (e.g., when the relevant substance has been depleted 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.

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

[0078] In connection with any of the above methods, it should be noted that the BBB needs to return to its initial low permeability state after an appropriate amount of time has elapsed following the termination of the alternating electric field. This can be important in many situations for the safety of the subject.

[0079] The present invention has been disclosed with reference to certain embodiments, but 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. Accordingly, the present invention is not intended to be limited to the described embodiments, but rather is intended to have the full scope defined by the description of the following claims and their equivalents.

Description of the Reference Numerals

[0080] 40 User interface 42 Controller 44 AC voltage generator

Claims

1. A method for delivering a substance through the blood-brain barrier of a subject, comprising: applying an alternating electric field to the subject's brain for a period of time, wherein the application of the alternating electric field to the subject's brain for a period of time improves the permeability of the blood-brain barrier in the subject's brain; and administering a substance to the subject after a period of time has elapsed, wherein the improvement in the permeability of the blood-brain barrier enables the substance to pass through the blood-brain barrier. A method comprising the above steps.

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 period of time is at least 24 hours.

4. The method according to claim 1, wherein the period of time is at least 48 hours.

5. 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 part of the subject's brain.

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

7. The method according to claim 1, wherein the step of administering the substance is performed intravenously.

8. The method according to claim 1, wherein the step of administering the substance is performed orally.

9. The method according to claim 1, wherein the subject's brain does not have a tumor.

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

11. The method according to claim 10, wherein the drug comprises an anti-cancer drug, an anti-infective drug, a neuro-degenerative disease treatment drug, an autoimmune disease treatment drug, an anti-epileptic drug, a hydrocephalus drug, a stroke intervention drug, or a psychotropic drug.

12. The method according to claim 1, wherein the substance is used to monitor brain activity.

13. The method according to claim 12, wherein the substance is a brain dye, a reporter, or a marker.

14. The method according to claim 1, further comprising the step of stopping the application of the alternating electric field to restore the blood-brain barrier.

15. A method for delivering a substance through the blood-brain barrier of a subject, comprising: A step of applying an alternating current electric field to the brain of a subject at a first frequency during a certain time period, wherein the first frequency is less than 190 kHz, the certain time period is at least 24 hours, and the application of the alternating current electric field to the brain of the subject at the first frequency during the certain time period improves the permeability of the blood-brain barrier in the brain of the subject; and A step of administering a substance to the subject after a certain time period has elapsed, wherein the improvement in the permeability of the blood-brain barrier enables the substance to pass through the blood-brain barrier, A method comprising the above steps.

16. The method according to claim 15, wherein the alternating current electric field is applied at a frequency between 75 kHz and 125 kHz.

17. The method according to claim 15, wherein the certain time period is at least 48 hours.

18. The method according to claim 15, wherein the alternating current electric field has an electric field strength of at least 1 V / cm in at least a part of the brain of the subject.

19. The method according to claim 15, wherein the alternating current electric field is applied at a frequency between 75 kHz and 125 kHz and the alternating current electric field has an electric field strength of at least 1 V / cm in at least a part of the brain of the subject.

20. A method for delivering a substance through the blood-brain barrier of the brain of a subject, comprising: A step of applying an alternating current electric field to the brain of the subject at a first frequency during a certain time period, wherein the brain of the subject does not have a tumor, and the application of the alternating current electric field to the brain of the subject at the first frequency during the certain time period improves the permeability of the blood-brain barrier in the brain of the subject; and A step of administering a substance to the subject after a certain time period has elapsed, wherein the improvement in the permeability of the blood-brain barrier enables the substance to pass through the blood-brain barrier, A method comprising the above steps.

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

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

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

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

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

26. A method for treating a tumor in a subject's brain and delivering a substance through the blood-brain barrier of the subject's brain, comprising: applying a first alternating electric field at a first frequency to the subject's brain during a first time period, wherein the application of the first alternating electric field at the first frequency to the subject's brain during the first time period improves the permeability of the blood-brain barrier in the subject's brain; administering a substance to the subject after the first time period has elapsed, wherein the improvement in the permeability of the blood-brain barrier enables the substance to pass through the blood-brain barrier; and applying a second alternating electric field at a second frequency to the subject's brain during a second time period having a length of at least one week, wherein the second frequency is different from the first frequency and the second alternating electric field at the second frequency has an intensity sufficient to inhibit the tumor. A method comprising the above steps.

27. The method according to claim 26, wherein the first frequency is between 75 kHz and 125 kHz.

28. The method according to claim 26, wherein the first frequency is between 50 kHz and 190 kHz.

29. The method according to claim 28, wherein the second frequency is between 190 kHz and 210 kHz.

30. The method according to claim 26, wherein the first time period is at least 24 hours.

31. The method according to claim 26, wherein the second time period includes a single non-interrupted time interval having a length of at least one week.

32. The method according to claim 26, wherein the second time period includes a plurality of discontinuous time intervals during which the second alternating electric field at the second frequency is applied to the subject's brain, and the plurality of discontinuous time intervals together total at least one week.

33. The method according to any one of claims 1 to 33, wherein the substance has a molecular weight of at least 4 kDa.

34. The method according to any one of claims 1 to 33, wherein the substance has a molecular weight of at least 69 kDa.

35. The method according to any one of claims 1 to 33, wherein the substance has at least one characteristic that normally impedes the substance from passing through the non-leaky BBB.

36. An apparatus for treating a tumor in a subject's body and facilitating the delivery of a substance through the blood-brain barrier of the subject's body, comprising: An AC voltage generator operable at a first frequency between 50 and 190 kHz and a second frequency between 50 and 500 kHz, the second frequency being different from the first frequency, the AC voltage generator having a control input and being configured to output the first frequency when the control input is in a first state and to output the second frequency when the control input is in a second state; and (a) setting the control input to the second state so that the AC voltage generator outputs the second frequency, (b) receiving a request to switch to the first frequency, (c) upon receiving the request, setting the control input to the first state so that the AC voltage generator outputs the first frequency for a time interval, and, (d) after a time interval has elapsed, setting the control input to the second state so that the AC voltage generator outputs the second frequency, a controller programmed to do so, An apparatus comprising.

37. A set of electrodes configured for attachment to a body of interest; and Wiring connecting the output of the AC voltage generator to the set of electrodes, The apparatus according to claim 36, further comprising.

38. The apparatus according to claim 36, wherein the first frequency is between 75 kHz and 125 kHz and the second frequency is between 150 kHz and 250 kHz.

39. The apparatus according to claim 36, wherein the time interval is at least 24 hours.

40. The apparatus according to claim 36, wherein the time interval is at least 72 hours.

41. The apparatus according to claim 36, wherein the controller is further programmed to alternately switch the control input between the first state and the second state following receipt of the request.

42. The AC voltage generator can operate at at least one additional frequency between 50 and 500 kHz, the AC voltage generator being configured to output at least one additional frequency when the control input is in at least one additional state, The controller is programmed to periodically repeat the second state and at least one additional state on the control input before receiving the request and to periodically repeat the second state and at least one additional state on the control input after a time interval has elapsed, The apparatus according to claim 36.

43. The apparatus according to claim 36, further comprising a user interface, wherein the request is received via the user interface.

44. The apparatus according to claim 36, wherein the request is received via RF.

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