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

Abstract

By applying an alternating electric field to the brain for a period of time, certain substances (e.g., macromolecules) that would normally be unable to cross the blood-brain barrier can be introduced into the brain, where the frequency of the alternating electric field is chosen such that application of the alternating electric field increases the permeability of the blood-brain barrier. In some embodiments, the frequency of the alternating electric field is less than 190 kHz (e.g., 100 kHz). Once the permeability of the blood-brain barrier is increased, the substance is able to cross the blood-brain barrier.

Description

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

[0002] This application claims the benefit of U.S. Provisional Application 62 / 722,100, filed August 23, 2018, which is incorporated herein by reference in its entirety. Background Technology

[0003] Normally, brain microvessels tightly regulate the transfer of substances between the blood and brain tissue. This regulation of brain microvessels is called the blood-brain barrier (BBB) ​​and is due to the formation of tight junctions (TJs) between brain capillary endothelial cells. TJ protein expression in brain capillaries is 50-100 times higher than in peripheral microvessels. TJs are formed by a complex complex of transmembrane proteins (sealing and closing proteins) and cytoplasmic accessory proteins (ZO-1 and -2, cingulate proteins, AF-6 and 7H6). These proteins form strong intercellular junctions through their association with the actin cytoskeleton. Brain endothelial cells that form the brain microvascular endothelium are responsible for approximately 75-80% of the resistance to substances in the BBB, while other cells such as astrocytes and pericytes provide the remaining resistance.

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

[0005] In healthy individuals, the brain's white blood cell (BBB) ​​plays a vital role in preventing harmful substances (such as bacteria, viruses, and potentially harmful large or hydrophilic molecules) from entering the brain. However, in certain situations, the BBB's function can be challenging. For instance, it may be desirable to deliver large or hydrophilic drug molecules to treat a disease in a patient's brain. But when the BBB is functioning properly, it prevents these drugs from reaching the brain. Summary of the Invention

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

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

[0008] In some cases of the first method, the substance is administered intravenously. In some cases of the first method, the substance is administered orally. In some cases of the first method, the subject does not have a brain tumor.

[0009] In some instances of the first method, the substance includes a drug used to treat a disease. Examples of these include drugs for treating cancer, infectious diseases, neurodegenerative diseases, or autoimmune diseases, antiepileptic drugs, hydrocephalus medications, stroke interventions, or psychiatric medications. In some instances of the first method, the substance is used to monitor brain activity. Examples of these include brain dyes, reporter molecules, or markers.

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

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

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

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

[0014] The methods described herein can be used to deliver substances across the blood-brain barrier in a subject whose brain does not have a tumor. In this context, another aspect of the invention relates to a third method for delivering substances across the blood-brain barrier in a subject's brain. In this third method, the relevant substance can be delivered across the blood-brain barrier in a tumor-free subject's brain by applying an alternating electric field at a first frequency to the subject's brain for a first time period. Applying an alternating electric field at the first frequency to the subject's brain for the first time period increases the permeability of the blood-brain barrier in the subject's brain. After a period of time, the substance is applied to the subject, and the increased permeability of the blood-brain barrier allows the substance to cross the blood-brain barrier.

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

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

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

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

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

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

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

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

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

[0024] In some cases, any of the above methods are used to deliver a substance across the blood-brain barrier of a subject's brain, wherein the substance has at least one characteristic that normally prevents the substance from crossing a non-leaking BBB.

[0025] Another aspect of the invention relates to a first device for treating tumors in a subject's body and facilitating substance delivery across the blood-brain barrier in the subject's body. The first device includes an alternating current (AC) voltage generator capable of operating at a first frequency between 50 and 190 kHz and a second frequency between 50 and 500 kHz. The second frequency differs from the first frequency. The AC voltage generator has a control input and 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) place the control input in the second state such that the AC voltage generator outputs the second frequency, (b) accept a request to switch to the first frequency, (c) upon receiving the request, place the control input in the first state such that the AC voltage generator outputs the first frequency for a time interval, and (d) after the time interval has elapsed, place the control input in the second state such that the AC voltage generator outputs the second frequency.

[0026] Some embodiments of the first device also include an electrode assembly configured to be attached to the subject's body; and a wire connecting the output of an AC voltage generator to the electrode assembly.

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

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

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

[0030] Figure 1 An exemplary setup for in vitro experiments is depicted, in which fixed rat brain capillary endothelial cells (cerebEND) are grown on coverslips and transwell inserts to produce an artificial in vitro form of BBB.

[0031] Figure 2A and 2B The results of integrity and permeability tests on the artificial BBB are described separately.

[0032] Figure 3A and 3B The data depicted showed that the increased permeability of the artificial BBB was not caused by cell death.

[0033] Figure 4 Describe the locations where rat brain slices were prepared for in vivo experiments.

[0034] Figure 5 The accumulation of EB in different parts of the rat brain during this in vivo experiment is depicted.

[0035] Figure 6 The average EB accumulation in the rat brain at all sites is depicted in this in vivo experiment.

[0036] Figure 7The study depicted the increased BBB permeability in three different locations of the rat brain induced by an alternating electric field in vivo, as determined by contrast-enhanced MRI.

[0037] Figure 8 The increased BBB permeability in the rat cortex induced by an alternating electric field in vivo was depicted, as determined by contrast-enhanced MRI.

[0038] Figure 9 Describe the appropriate timing relationship between applying an alternating electric field to the subject and the applied substance.

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

[0040] This application describes a novel method that uses an alternating electric field to temporarily increase the permeability of the BBB, allowing substances that are normally blocked by the BBB to pass through it.

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

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

[0043] The second set of experiments also involved visualization of cell morphology. This experiment was designed to determine how the duration of application of an alternating electric field affected the artificial BBB. Endothelial cells were grown on coverslips, and an alternating electric field was applied at a frequency of 100 kHz for three different durations (24 hours, 48 ​​hours, and 72 hours), with a control group present. The direction of the alternating electric field switched every second between two perpendicular directions. Cell morphology images depicting the presence of Claudin 5 and DAPI (each stained in a different color) were then obtained. This set of cell morphology images shows that the aforementioned phenomenon associated with the first set of experiments was visible after 24 hours, and the effect was most pronounced after 72 hours.

[0044] The third set of experiments also involved visualization of cell morphology. This experiment was similar to the second set, except that endothelial cells grew on the transporous insert instead of on a coverslip. The results were similar to those of the second set. The delocalization of the TJ protein was visible after 24 hours, with the most significant effect observed after 72 hours. These three experiments support the conclusion that alternating electric fields cause changes in cell structure, which may explain the increased BBB permeability.

[0045] Figure 2A and Figure 2B The results of integrity and permeability tests on an artificial BBB after being subjected to an alternating electric field at a frequency of 100 kHz (the direction of the alternating electric field switches between two perpendicular directions every 1 second) for 72 hours are described, with controls provided. More specifically, Figure 2A The results of the transthelial resistance (TEER) test are depicted, showing that the alternating electric field reduces the integrity of the artificial BBB to 35% of the control. Figure 2BThe results of fluorescein isothiocyanate (FITC) permeability tests demonstrate that an alternating electric field increases the permeability of artificial BBB to FITC-glucan with a molecular weight of 4 kDa to 110% of the control. These experiments further support the conclusion that an alternating electric field increases the permeability of BBB to molecules that normally cannot pass through non-leaking BBB.

[0046] In summary, these in vitro experiments demonstrate that applying an alternating electric field at a certain frequency for a sufficiently long duration induces delocalization of tight junction proteins (Claudin 5, ZO-1) from the cell boundary to the cytoplasm (with the most pronounced effect at 100 kHz) and increases BBB permeability. The effect of the alternating electric field was observed after 24 hours and was most pronounced after 72 hours. More specifically, after increasing BBB permeability using an alternating electric field, a 4 kDa molecule could cross the BBB.

[0047] Additional in vitro experiments were then performed to determine what happened to the BBB after the alternating electric field was turned off. These experiments used visualization of cell morphology to show how the artificial BBB recovered after the alternating electric field was stopped. In these experiments, endothelial cells were grown on coverslips and treated with an alternating electric field of 1.7 V / cm at 100 kHz for 72 hours. The direction of the alternating electric field switched between two perpendicular directions every 1 second. The alternating electric field was then turned off, and the cells were tracked for 96 hours after the alternating electric field was stopped. Cell morphology images depicting the presence of Claudin 5 (stained) were obtained at 24, 48, 72, and 96 hours. These images show the gradual change in the localization of Claudin between the cell boundary and the cytoplasm on images at 24, 48, 72, and 96 hours. Furthermore, comparing these four images with corresponding images of the control (without an alternating electric field applied during the initial 72 hours or the last 96 hours) showed that endothelial cell morphology partially recovered 48 hours after the alternating electric field was stopped, and that BBB fully recovered 96 hours after the alternating electric field was stopped (i.e., comparable to the control).

[0048] Figure 3A and 3BThe results of an in vitro experiment designed to determine whether the observed changes in permeability of the aforementioned artificial BBB were attributable to cell death are depicted. This experiment tested cell division by comparing (a) cell counts with an alternating electric field applied for 72 hours followed by 96 hours without the alternating electric field, and (b) cell counts of a control group never subjected to an alternating electric field. Endothelial cells were grown on coverslips and treated with an alternating electric field of 1.7 V / cm at 100 kHz for 72 hours. The direction of the alternating electric field was switched every 1 second between two perpendicular directions. The alternating electric field was then turned off, and cells were tracked for 96 hours after the alternating electric field was stopped. Cell counts per milliliter were performed for both the alternating electric field and the control groups, and the results are depicted in [the table / image]. Figure 3A and 3B (For control and alternating electric field, respectively). These results show that there was no statistically significant increase in cell number during or after the application of the alternating electric field, indicating that the aforementioned changes in BBB permeability are not attributable to cell death.

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

[0050] A series of in vivo experiments were also conducted in rats to quantify the increased vascular permeability induced by exposure to an alternating electric field. These experiments used Evans blue (EB) dye, an azo dye with a high affinity for serum albumin (approximately 69 kDa). Due to its large molecular size, serum albumin typically cannot pass through the blood-brain barrier (BBB). However, if BBB permeability is sufficiently increased, some of the serum albumin molecules (along with the EB dye bound to them) will allow it to pass through the BBB, and this can then be detected by searching for EB in the rat brain.

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

[0052] Under the conditions and timeframes indicated in Table 1, EB was intravenously injected into the tail vein under anesthesia (EB immediately binds to albumin upon injection), and EB circulation was allowed for 2 hours in all cases. The following steps were then performed: (a) intracardiac perfusion with saline; (b) quadrupling of the brain using a brain slicer; (c) photographing the sections for localization staining and weighting; (d) EB extraction after homogenization of the tissue with 50% TCA (1:3) and centrifugation; and (e) EB quantification at 610 nm. Results are given in micrograms of EB per gram of tissue.

[0053] Group number deal with Rat Number EB injection time 1 72 hours 100kHz electric field 3 2 hours before the end of the 72-hour cycle 2 72 hours of 100kHz electric field + 2 hours of rest 3 2 hours after the 72-hour cycle ends 3 Heating with dummy electrodes for 72 hours 3 2 hours before heating ends 4 Control (No electric field + No heating) 3 Simultaneous with Group 1

[0054] Table 1

[0055] During the experiment, two animals from group 2 and one animal from group 4 were excluded (treatment was interrupted, and EB injection failed to be administered into the tail vein). There were no differences between the animals treated with alternating electric fields (groups 1 and 2), and these animals were therefore grouped together. Similarly, there were no differences between the sham-heat and control animals (groups 3 and 4), and these animals were therefore grouped together.

[0056] Using a brain slicer Figure 4 The rat brain was cut into four sections at the locations shown. The accumulated electric field (EB) in each of these four specific sites was then measured. Additionally, computer simulations were performed to determine the field strength in each of the four sites. Table 2 details the field strength obtained through simulation in each of the four sites; all values ​​are given in V / cm RMS.

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

[0058] Figure 5 The results of EB accumulation in sites 1 through 4 are described below. These results are summarized as follows: (1) Statistically significant increases were observed in sites 1 and 2 (frontal lobes) with the highest field strength; and smaller increases (not statistically significant) were observed in more posterior sites (3 and 4) with lower field strength.

[0059] Figure 6The mean EB accumulation in the rat brain is plotted across all four sites 1–4. This result shows higher EB accumulation in the rat brain treated with an alternating electric field for 72 hours, and this result is statistically significant (p<0.05).

[0060] The in vivo experiments described above confirmed that: (1) applying an alternating electric field allows molecules with an average molecular size of approximately 69 kDa to pass through the BBB to reach brain tissue; (2) the permeability of the BBB remains increased for 2 hours after the application of the alternating electric field is terminated; and (3) the increase in BBB permeability varies among different brain regions. The latter may be due to the different field strengths applied to different brain regions. These experiments further support our conclusion that an alternating electric field increases the permeability of the BBB to molecules that normally cannot pass through a non-leaking BBB.

[0061] In another in vivo experiment, five rats were treated with an alternating electric field at 100 kHz for 72 hours, while four control rats were not treated with an alternating electric field for the same period. At the end of the 72-hour period, a 4 kDa fluorescent compound, TRITC-glucan, was injected intravenously into the tail vein under anesthesia, with circulation allowed for 2 minutes in all cases. The brain was then removed, frozen, sectioned, and scanned using a fluorescence scanner. All slides were scanned under the same conditions. The resulting images showed significantly higher levels of accumulated 4 kDa fluorescent TRITC-glucan in the brain tissue of rats treated with the alternating electric field (compared to controls), further confirming that the alternating electric field can increase the permeability of the blood-brain barrier (BBB).

[0062] Another set of in vivo experiments was 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 alternating electric field of 100 kHz for 72 hours, while control rats were not treated with an alternating electric field for the same period. After this 72-hour period, the alternating electric field was turned off, the rats were anesthetized, and a series of 60 T1w MRI scans (each scan lasting 28 seconds) were acquired. During the 7th scan of these 60 scans, the gadolinium contrast agent was injected into the tail vein of the rat.

[0063] Image analysis for each rat included: (1) determining the baseline for each voxel by calculating the average of the first six Tlw MRI scans (i.e., scans prior to gadolinium injection); (2) calculating the percentage change in signal over time relative to baseline (i.e., gadolinium accumulation) for each voxel; (3) dividing the brain into anterior, middle, and posterior segments; (4) generating the average percentage change in signal relative to baseline across all voxels in the corresponding segment for each of the three segments; and then (5) averaging the four consecutive time points (i.e., four scans). Finally, the data from all rats in any given group were averaged.

[0064] Figure 7 The results of this DCE-MRI experiment are depicted for each of the three segments of the brain (i.e., anterior, middle, and posterior). These data show that contrast agent accumulation in rat brain tissue treated with an alternating electric field (tracer-labeled TTFields; n=6) was significantly higher than in control rats (tracer-labeled controls; n=3). Furthermore, this difference was most pronounced in the posterior brain, the brain region where the alternating electric field had the highest field strength. From this, we can conclude that the alternating electric field successfully increases the permeability of the brain brain (BBB) ​​in vivo.

[0065] To test whether this increase in BBB permeability was temporary, the same test conditions were repeated, but followed by an additional 96-hour test without an alternating electric field. After this 96-hour period, a series of 60 Tlw MRI scans (each scan lasting 28 seconds) were acquired using the same procedure as described above (including gadolinium injection). Figure 8 The results of this part of the DCE-MRI experiment on each of the three segments of the brain are also described. The data show that contrast agent accumulation in rat brain tissue treated with an alternating electric field for 72 hours, followed by 96 hours without the alternating electric field (tracer-labeled TTFields + 96 h; n = 7) was not significantly different from that in control rats (tracer-labeled control + 96 h; n = 3). From this, we can conclude that the permeability of the BBB returns to normal after the alternating electric field is removed.

[0066] Before applying the alternating electric field to the rats (n=2), a series of additional 60 Tlw MRI scans (each scan lasting 28 seconds) were obtained using the same procedure. Figure 8 The results of this part of the DCE-MRI experiment for each of the three segments of the brain (i.e., anterior, middle and posterior) are also described (see “Pre-” tracer markers).

[0067] Figure 8 The mean values ​​of all three brain segments (i.e., anterior, middle, and posterior) are shown for 72 hours of TTFields (n=6) and 72 hours of controls without TTFields (n=3), with standard deviation bars. Paired t-tests were used to compare the two groups, and p < 0.0001.

[0068] We note that the upper limit of molecular size that can pass through the BBB after an alternating electric field has not yet been determined. However, based on (a) the in vitro experiments described herein using FITC-glucan with a molecular weight of 4 kDa, and (b) the in vivo experiments described herein using EB (which binds to serum albumin with a molecular size of approximately 69 kDa), the upper limit appears to be at least approximately 69 kDa, and most certainly at least 4 kDa.

[0069] The implications of being able to arbitrarily and reversibly increase the permeability of the blood-brain barrier (BBB) ​​are profound, as it now becomes possible to deliver many substances across a subject's BBB, even those substances that possess at least one characteristic that typically prevents substances from passing through a non-leaking BBB. Many of these implications involve delivering substances (including, but not limited to, therapeutic and diagnostic agents) across the blood-brain barrier in a subject's brain.

[0070] Examples include, but are not limited to, the following: delivering chemotherapy agents across the BBB to treat cancer (in which case it is possible to reduce the dosage of drugs used to treat brain tumors and metastases that have serious side effects in other parts of the body based on increased drug permeability to the brain); delivering antibody- and / or cell-based therapies across the BBB for immunotherapy; delivering contrast dyes, reporter molecules, and biomarkers across the BBB for diagnostic and research purposes (e.g., monitoring brain activity); delivering antibacterial agents across the BBB to treat infectious diseases; delivering antiviral agents or virus-neutralizing antibodies across the BBB to treat viral infections; delivering antiparasitic agents across the BBB to treat parasites; delivering drugs to treat neurodegenerative and autoimmune diseases across the BBB; delivering psychotropic drugs; delivering antiepileptic drugs; delivering hydrocephalus drugs; delivering stroke intervention and rehabilitation drugs; and delivering compounds lacking in the brain across the BBB to treat diseases lacking these compounds (e.g., for treating Parkinson's disease, etc.).

[0071] Although the above tests were conducted in vitro and in live rats, similar results are expected for other animals and humans.

[0072] 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 will increase 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 applying an alternating voltage between a selected subset of these electrodes will apply an alternating electric field to the subject's brain.

[0073] For example, one pair of electrodes may be placed on the front and back sides of the subject's head, while a second pair of electrodes may be placed on the left and right sides of the subject's head. In some embodiments, the electrodes are capacitively coupled to the subject's body (e.g., by using electrodes that include a conductive plate and also have a dielectric layer disposed between the conductive plate and the subject's body). However, in alternative embodiments, the dielectric layer may be omitted, in which case the conductive plate directly contacts the subject's body. In another embodiment, the electrodes may be inserted subcutaneously into the patient's skin.

[0074] An AC voltage generator applies an AC voltage between the left and right electrodes at a selected frequency (e.g., 100 kHz, or between 50 and 190 kHz) for a first time period (e.g., 1 second), inducing an alternating electric field parallel to the transverse axis of the subject's head, with the most significant component of the field line in the field. The AC voltage generator then applies an AC voltage between the front and rear electrodes at the same (or different) frequency for a second time period (e.g., 1 second), inducing an alternating electric field parallel to the sagittal axis of the subject's head, with the most significant component of the field line in the field. These two steps are then repeated during treatment. Optionally, thermal sensors may be included at the electrodes, and the AC voltage generator may 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 may be added and included in the sequence. In alternative embodiments, only one pair of electrodes is used, in which case the direction of the field line is not switched. Note that any parameters of this in vivo embodiment (e.g., frequency, field strength, duration, rate of direction switching, and electrode placement) may vary as described above in conjunction with the in vitro embodiment. However, care must be taken in the in vivo environment to ensure that the electric field remains safe for the subject at all times.

[0075] In the in vivo environment, various applications for enhancing the permeability of the brain brain (BBB) ​​are readily apparent. In one instance, by applying an alternating electric field to the brain for a period of time (e.g., 72 hours or at least 24 hours) before and during the administration of chemotherapeutic agents or other antitumor agents, a local enhancement of drug uptake by tumor cells (e.g., glioblastoma cells) in the brain can be induced.

[0076] Figure 9 This describes a suitable timing relationship between the application of an alternating electric field to a living patient and the administration of a substance. Based on the data above, and assuming that a substance is introduced or administered at a given time t=0, the alternating electric field may begin before the given time (e.g., 72 hours before t=0) and continue for a certain time interval after the given time (e.g., until 12 hours after t=0). In this case, the permeability of the BBB will begin to increase before the substance is administered and before the substance reaches the BBB. This will allow the substance to pass through the BBB immediately after reaching it. In the context of chemotherapy, this would correspond to the start of the application of the alternating electric field, administration of the chemotherapeutic agent 72 hours later, followed by the application of the alternating electric field for an additional time interval (e.g., until 12 hours after the administration of the chemotherapeutic agent).

[0077] Note the above combination Figure 9The time intervals discussed can be continuous or include shorter intervals. For example, a 12-hour interval can be satisfied by a single uninterrupted block of 12 hours. Alternatively, a 12-hour interval can be satisfied by applying an alternating electric field for 6 hours, followed by a 1-hour interval, and then applying an alternating electric field for another 6 hours. A similar interval can also be optionally a 72-hour interval before the interruption of the application of the substance. It should also be noted that... Figure 9 In the context of this study, when a substance is administered to a living patient, it can be administered using any of a variety of methods, including but not limited to intravenous, oral, subcutaneous, intrathecal, intracardiac, and intraperitoneal administration.

[0078] 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 combined with a time period of at least 24 hours will improve the variation in transparency (compared to working outside these ranges).

[0079] The methods described herein can be used to deliver substances across the blood-brain barrier in a subject's brain when the brain includes a tumor. An existing method for treating brain tumors (e.g., glioblastoma) involves applying an alternating electric field at a frequency between 50 and 500 kHz, preferably between 100 and 300 kHz, to the tumor. For glioblastoma, 200 kHz is the most preferred frequency. 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 its entirety. In short, both applications describe the disruption of dividing cells 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 a portion of the tumor is at least 1 V / cm, and when the electric field is applied for extended periods (e.g., weeks or months) with as few interruptions as possible.

[0080] In patients with brain tumors, situations may arise where TTFields are needed to treat the tumor, and there is also a need to deliver the substance across the same patient's blood-brain barrier (e.g., to help obtain a therapeutically effective amount of chemotherapy drug across the BBB to provide an additional attack route against the tumor). In some cases, a single-frequency alternating electric field can be used to treat the tumor and improve BBB permeability. In other cases, it may be desirable to use alternating electric fields with 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 in the tumor-targeting effect of TTFields.

[0081] Figure 10This is a block diagram of a device for generating a first frequency for inducing BBB permeability and a second frequency for inducing cytotoxicity. The device includes an AC voltage generator 44, which is similar to a conventional one. The field generator unit 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.

[0082] The system can operate at two different frequencies; for example, by using a relay to switch either the first or second set of components into a conventional circuit that generates AC voltage, and to adjust 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 place the control input in the second state, causing the AC voltage generator 44 to output the second frequency. The controller 42 is also programmed to accept requests to switch to the first frequency. Figure 10 In the illustrated embodiment, the request arrives via a user interface 40, which can be implemented using any of a variety of conventional methods, including but not limited to buttons, touchscreens, etc. In an alternative embodiment, the request may arrive via RF (e.g., Bluetooth, WiFi, etc.) from a tablet, smartphone, etc.

[0083] Upon receiving a request, controller 42 sets the control input to a first state, causing AC voltage generator 44 to output a first frequency for a period of time (e.g., 72 hours). After this time interval, controller 42 sets the control input to a second state, causing AC voltage generator 44 to return to outputting a second frequency.

[0084] 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 cycle through a state before a request arrives, which causes the AC voltage generator 44 to output a second frequency and one or more additional frequencies. The controller 42 is also programmed to accept a request to switch to a first frequency. Upon receiving the request, the controller 42 places the control input in a first state such that the AC voltage generator 44 outputs the first frequency for a period of time (e.g., 72 hours). After the time interval has elapsed, the controller 42 returns to the state of cycling through the control input, which causes the AC voltage generator 44 to output a second frequency and one or more additional frequencies.

[0085] When a person has a tumor that is being treated with a combination of TTFields and chemotherapy, Figure 10 The system described in the example is particularly useful. In this case, the system operates at the second frequency most of the time to provide maximum cytotoxic efficacy. However, before a person goes to a chemotherapy clinic for a dose of chemotherapy, the healthcare professional (or user) activates the user interface 40 to switch the system to the first frequency, which promotes BBB permeability. In this case, the activation of the user interface can, for example, be completed 72 hours before the expected start of chemotherapy.

[0086] Alternatively, upon receiving a request (e.g., from user interface 40), controller 42 may control the control input to cause AC voltage generator 44 to output a first frequency for a certain time interval (e.g., 1 hour), and then switch back and forth between a second frequency and the first frequency (e.g., once per hour). Finally (e.g., when the relevant substance has been removed from the patient's bloodstream), controller 42 controls the control input to cause AC voltage generator 44 to return to outputting the second frequency.

[0087] A group with The conventional electrode (not shown) is connected to the output of AC voltage generator 44.

[0088] Note that, in any of the methods described above, the BBB should return to its original low-permeability state after a sufficient period of time following the termination of the alternating electric field. This can be important for the safety of the subject in many cases.

[0089] Although the invention has been disclosed with reference to certain embodiments, many modifications, alterations, and variations of the described embodiments are possible without departing from the scope of the invention as defined by the appended claims. Therefore, it is intended that the invention be limited to the described embodiments, but have the full scope defined by the language of the appended claims and their equivalents.

Claims

1. A blood-brain barrier permeability control device for facilitating substance delivery across the blood-brain barrier of a subject, the device comprising: An alternating voltage generator is configured to generate an alternating electric field in the subject's brain at frequencies between 25 kHz and 190 kHz. and A controller is programmed to control the alternating voltage generator to output the alternating electric field at time intervals of at least 24 hours to induce delocalization of tight junction proteins from the cell boundary to the cytoplasm.

2. The apparatus of claim 1, wherein the time interval is at least 48 hours.

3. The apparatus of claim 1, wherein the time interval is at least 72 hours.

4. The apparatus of claim 1, further comprising: A user interface through which requests are accepted.

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

6. The apparatus of 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 apparatus of claim 1, wherein the frequency is between 50 kHz and 190 kHz.

8. The apparatus of claim 1, wherein the frequency is between 75 kHz and 125 kHz.

9. The apparatus of claim 1, wherein the frequency is 100 kHz.

10. The apparatus of claim 1, wherein the time interval is uninterrupted.

11. The apparatus of claim 1, wherein the time interval is interrupted.

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

13. The apparatus of claim 1, wherein the molecular weight of the substance is at least 69 kDa.

14. The apparatus of claim 1, further comprising: Electrode sets, which are configured to be attached to the subject's body; and A wire connects the output of the AC voltage generator to the electrode group.

15. The apparatus of claim 1, wherein the substance comprises a drug for treating a disease.

16. The device of claim 15, wherein the drug comprises a cancer treatment drug, an infectious disease treatment drug, a neurodegenerative disease treatment drug, an autoimmune disease treatment drug, an antiepileptic drug, a hydrocephalus drug, a stroke intervention drug, or a psychiatric drug.

17. The apparatus of claim 1, wherein the substance is used to monitor brain activity.

18. The apparatus of claim 17, wherein the substance is a brain dye, a reporter molecule, or a marker.

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