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 Increase Permeability of the Blood-Brain Barrier”. TECHNICAL FIELD

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

[0003] Generally, the brain microvasculature strictly regulates the transfer of substances between the blood and brain tissue. This regulation of the brain microvasculature is known as the blood-brain barrier (BBB), and is due to the formation of intercellular tight junctions (TJs) between brain capillary endothelial cells. In the brain capillaries, the expression of TJ proteins is 50-100 times greater than in peripheral microvasculature. TJs are formed by a complex complex of transmembrane proteins (claudins and occludins) with cytoplasmic accessory proteins (ZO-1 and -2, cingulin, AF-6, and 7H6). Through linkage with the actin cytoskeleton, these proteins form a firm intercellular connection. Brain endothelial cells, which form the brain microvascular endothelium, are responsible for about 75-80% of the BBB’s resistance to substances, 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 generally limits the diffusion of microscopic objects and macromolecules or hydrophilic molecules into the brain while allowing the diffusion of hydrophobic molecules (transcellular, not paracellular transport).

[0005] In healthy humans, the BBB plays a very important role because it prevents harmful substances (e.g., bacteria, viruses, and potentially harmful macromolecules or hydrophilic molecules) from entering the brain. However, in certain situations, the role of the BBB can present difficulties. For example, it can be desirable to deliver a large or hydrophilic drug molecule to treat a disease in a patient’s brain. But when the BBB is working properly, the BBB prevents these drugs from entering the brain. SUMMARY

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

[0007] In some instances of the first method, the alternating electric field is applied at a frequency between 75 kHz and 125 kHz. In some instances of the first method, the period of time is at least 24 hours. In some instances of the first method, the period of time is at least 48 hours. In some instances 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 instances 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 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 instances of the first method, administering the substance is performed intravenously. In some instances of the first method, administering the substance is performed orally. In some instances of the first method, the subject does not have a tumor in the brain.

[0009] In some instances of the first method, the substance comprises a drug for treating a disease. Examples of these instances include a cancer treatment drug, an infectious disease treatment drug, a neurodegenerative disease treatment drug, or an autoimmune disease treatment drug, an anti-epileptic drug, a hydrocephalus drug, a stroke intervention drug, or a psychiatric drug. In some instances of the first method, the substance is used to monitor brain activity. Examples of these instances include a brain dye, a reporter molecule, or a marker.

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

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

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

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

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

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

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

[0017] The methods described herein can be used to deliver a substance across the blood brain barrier of a subject that does not have a tumor in the brain. In this case, another aspect of the application relates to a third method for delivering a substance across the blood brain barrier of a subject's brain. In this third method, a relevant substance can be delivered across the blood brain barrier of a subject's brain that does not include a tumor by applying an alternating electric field at a first frequency to the subject's brain for a first period of time. The application of the alternating electric field at the first frequency to the subject's brain for the first period of time increases the permeability of the blood brain barrier in the subject's brain. After a period of time has elapsed, a substance is administered to the subject and the increased permeability of the blood brain barrier allows the substance to cross the blood brain barrier.

[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 at least one week long. In other cases of the fourth method, the second time period comprises a plurality of discontinuous time intervals during which the second alternating electric field at the second frequency is applied to the subject's brain, wherein the plurality of discontinuous time intervals totals at least one week.

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

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

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

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

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

[0026] Some embodiments of the first device further comprise a set of electrodes configured to be attached to the subject's body; and a lead connecting an output of the alternating current voltage generator to the set of electrodes.

[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 7Depiction of the increase in BBB permeability induced by in vivo alternating electric fields at three different locations in the rat brain, as determined using contrast agent-enhanced MRI.

[0037] Figure 8 Depiction of the increase in BBB permeability induced by in vivo alternating electric fields in the rat cortex, as determined using contrast agent-enhanced MRI.

[0038] Figure 9 Depiction of the appropriate timing relationship between the application of an alternating electric field to a subject and the administration of a substance.

[0039] Figure 10 Block diagram of a dual-frequency device that generates a first frequency for inducing BBB permeability and a second frequency for inducing cytotoxicity. DETAILED DESCRIPTION

[0040] The present application describes novel methods that use alternating electric fields to temporarily increase the permeability of the BBB, enabling substances that are normally blocked by the BBB to cross the BBB.

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

[0042] The first set of experiments involves visualization of cell morphology and orientation, as well as visualization of stained protein localization. This experiment was designed to determine how the frequency of the alternating electric field affects the 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 was switched between two perpendicular directions every 1 second. There was also a control where no alternating electric field was applied. Cell morphology images were then obtained depicting the presence of Claudin 5, ZO-1, and 4,6-diamidino-2-phenylindole (DAPI), each of which was stained in a different color. Claudin 5 and ZO-1 indicate the presence of an intact BBB. This set of cell morphology images demonstrates that the alternating electric field interferes with the artificial BBB by dislodging tight junction proteins from the cell border to the cytoplasm, with the effect being most pronounced at 100 kHz.

[0043] The second set of experiments also involves visualization of cell morphology. This experiment was designed to determine how the duration of time during which the alternating electric field is applied affects 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 of time (24 hours, 48 hours, 72 hours), with a control. The direction of the alternating electric field was switched between two perpendicular directions every 1 second. Cell morphology images were then obtained depicting the presence of Claudin 5 and DAPI, each of which was stained in a different color. This set of cell morphology images demonstrates that the above-mentioned phenomena related to the first set of experiments are visible after 24 hours, and the effect is most pronounced after 72 hours.

[0044] The third set of experiments also involves visualization of cell morphology. This experiment is similar to the second set of experiments, except that the endothelial cells were grown on transwell inserts instead of coverslips. The results were similar to those of the second set of experiments. Delocalization of TJ proteins was visible after 24 hours, while the effect was most pronounced after 72 hours. The above three experiments support the conclusion that the alternating electric field causes changes in cell structure, which can be the cause of increased BBB permeability.

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

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

[0047] Additional in vitro experiments were then performed to determine what happens to the BBB after the alternating electric field is turned off. These experiments used visualization of cell morphology to show how the artificial BBB recovers after the alternating electric field is stopped. In these experiments, endothelial cells were grown on coverslips and treated with a 100 kHz alternating electric field at a field strength of 1.7 V / cm for 72 hours. The direction of the alternating electric field was switched between the 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 hours, 48 hours, 72 hours, and 96 hours. These images show that the localization of Claudin between the cell border and the cytoplasm changes gradually over the images at 24 hours, 48 hours, 72 hours, and 96 hours. In addition, comparing these four images to the corresponding images of the control (where no alternating electric field was applied during the initial 72 hours or the final 96 hours) shows that the endothelial cell morphology has partially recovered 48 hours after the alternating electric field was stopped and the BBB has completely recovered (i.e., is comparable to the control) 96 hours after the alternating electric field was stopped.

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

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

[0050] A set of in vivo experiments were also performed on rats to quantify the increase in vascular permeability due to exposure to an alternating electric field. These experiments used Evans Blue (EB) dye, which is an azo dye that has a very high affinity for serum albumin (molecular size of approximately 69 kDa). Due to its large molecular size, serum albumin would normally be unable to pass through the BBB. However, if the permeability of the BBB has been sufficiently increased, some of the serum albumin molecules (along with the EB dye bound to them) will make it through the BBB, and then can be detected by looking for EB in the rat brain.

[0051] In this set of experiments, an alternating electric field of 100 kHz was applied to the rat brain for 72 hours, and the direction of the alternating electric field was switched 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 on the top and bottom of the rat's head, and placing 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 alternating voltage was applied between the top and bottom electrodes for 1 second, followed by a 100 kHz alternating voltage between the left and right electrodes for 1 second.

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

[0053] Group Number Treatment Rat Number EB Injection Time 1 72 hour 100 kHz electric field 3 2 hours before end of 72 hour period 2 72 hour 100 kHz electric field + rest for 2 hours 3 2 hours after end of 72 hour period 3 Heated for 72 hours using dummy electrodes 3 2 hours before end of heating 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 (interruption of treatment, failure to inject EBs into the tail vein). There was no difference between the animals treated with alternating electric fields (Group 1 and Group 2), and therefore these animals were grouped together. Likewise, there was no difference between the sham and control animals (Group 3 and Group 4), and therefore these animals were grouped together.

[0056] The rat brain was cut into four pieces using a brain slicer at the locations shown in Figure 4 The four specific sites were then measured for EB accumulation. In addition, computer simulations were performed to determine the field strength in each of the four sites. Table 2 specifies the field strength obtained by simulation in each of the four sites, all values given in V / cm RMS.

[0057] Region 1 2 3 4 Average Field Strength 2.7 V / cm 3 V / cm 2.6 V / cm 1.6 V / cm Medium Field Strength 2.5 V / cm 2.6 V / cm 2.4 V / cm 1.6 V / cm

[0058] Figure 5 The results of EB accumulation in Groups 1-4 are depicted in Table 3. A summary of these results is as follows: (1) a statistically significant increase was observed in Groups 1 and 2 (frontal lobe brain), which are the sites with the highest field strength; and a smaller increase (not statistically significant) was observed in Groups 3 and 4, which are the more posterior sites with lower field strength.

[0059] Figure 6The average EB accumulation in rat brain averaged over all four locations 1-4 is depicted. This result shows higher accumulation of EBs in rat brain treated with alternating electric field for 72 hours, and this result is statistically significant (p < 0.05).

[0060] The above in vivo experiments demonstrate that: (1) application of alternating electric field allows molecules of average molecular size of about 69 kDa to cross the BBB to reach brain tissue; (2) the permeability of the BBB remains elevated 2 hours after termination of the alternating electric field application; and (3) the increase in permeability of the BBB varies between different locations in the brain. The latter can be a result of different field strengths applied at various locations in the brain. These experiments further support our conclusion that alternating electric field increases the permeability of the BBB to molecules that normally cannot cross the non-leaking BBB.

[0061] In another set of in vivo experiments, 5 rats were treated with alternating electric field at 100 kHz for 72 hours, while 4 control rats were not treated with alternating electric field for the same time. At the end of the 72 hour period, 4 kDa fluorescent compound TRITC-dextran was injected intravenously into the tail vein under anesthesia and allowed to circulate for 2 minutes in all cases. The brain was then removed, frozen, sectioned and scanned with a fluorescence scanner. All slides were scanned under the same conditions. The resulting images show significantly higher levels of fluorescent 4 kDa TRITC-dextran accumulation in the brain tissue of rats subjected to alternating electric field (compared to controls), again demonstrating that alternating electric field can increase the permeability of the 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, MW 547). In these experiments, test rats were treated with 100 kHz alternating electric field for 72 hours, while control rats were not treated with alternating electric field for the same period of time. After this 72 hour period, the alternating electric field was turned off, the rats were anesthetized, and a series of 60 Tlw MRI scans was acquired (each scan lasting 28 seconds). During the 7th scan of the 60 scans, the gadolinium contrast agent was injected into the tail vein of the rat.

[0063] Image analysis for each rat included: (1) determination of the baseline for each voxel by calculating the average of the first six Tlw MRI scans (i.e., scans before injection of gadolinium) for each voxel; (2) voxel-by- voxel calculation of the percent signal change over time relative to baseline (i.e., gadolinium accumulation); (3) division of the brain into anterior, middle and posterior segments; (4) generation of the average percent signal change relative to baseline over all voxels in the respective segment for each of the three segments, and then (5) averaging of 4 consecutive time points (i.e., 4 scans). Finally, data from all rats in any given group was averaged.

[0064] Figure 7 The results of this DCE-MRI experiment on each of the three segments of the brain (i.e., anterior, middle, and posterior) are depicted in FIG. 6. This data shows that the contrast agent accumulation in rat brain tissue treated with alternating electric fields (Tracer-Tagged TTFields; n=6) was significantly higher than in control rats (Tracer-Tagged Control; n=3). Moreover, this distinction was most pronounced in the posterior brain, the part of the brain where the alternating electric fields have the highest field strength. From this we conclude that the alternating electric fields successfully increased the permeability of the BBB in vivo.

[0065] To test whether this increase in permeability of the BBB was temporary, the same test conditions were repeated, but followed by an additional 96 hours of testing without alternating electric fields. After these 96 hours, a series of 60 Tlw MRI scans (each scan lasting 28 seconds) were taken 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 depicted in FIG. 6. This data shows that the contrast agent accumulation in rat brain tissue treated with alternating electric fields for 72 hours, followed by 96 hours without alternating electric fields (Tracer-Tagged TTFields + 96h; n=7) was not significantly different from control rats (Tracer-Tagged Control + 96h; n=3). From this we conclude that after stopping the alternating electric fields, the permeability of the BBB returned to normal.

[0066] A series of additional 60 Tlw MRI scans (each scan lasting 28 seconds) were also taken using the same procedure before applying the alternating electric fields to the rats (n=2). Figure 8 The results of this part of the DCE-MRI experiment on each of the three segments of the brain (i.e., anterior, middle, and posterior) are also depicted in FIG. 6 (see "Before" Tracer-Tagged).

[0067] Figure 8 The average values for all 3 segments of the brain (i.e., anterior, middle, and posterior) are shown for 72 hours of TTFields (n=6) and 72 hours of control without TTFields (n=3) with standard deviation bars. A paired t-test was used to compare the two groups, and p<0.0001.

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

[0069] The ability to reversibly increase the permeability of the BBB at will is profound, as it now becomes possible to deliver many substances across the BBB of a subject, despite the fact that these substances have at least one characteristic that would normally prevent them from crossing a non-leaking BBB. Many of these implications relate to the delivery of substances (including but not limited to therapeutic and diagnostic agents) across the blood-brain barrier of a subject's brain.

[0070] Examples include but are not limited to the following: delivery of chemotherapeutic agents across the BBB to treat cancer (in this case, it is possible to reduce the dosage of a drug used to treat brain tumors and metastases that have severe side effects elsewhere in the body, based on the increased permeability of the drug to the brain); delivery of antibody- and / or cell-based therapies across the BBB for immunotherapy; delivery of contrast dyes, reporter molecules, and markers across the BBB for diagnosis and research (e.g., monitoring brain activity); delivery of antibacterial agents across the BBB to treat infectious diseases; delivery of antiviral agents or virus-neutralizing antibodies across the BBB to treat viral infections; delivery of antiparasitic agents across the BBB to treat parasites; delivery of drugs to treat neurodegenerative and autoimmune diseases; delivery of psychotropic drugs; delivery of anti-epileptic drugs; delivery of hydrocephalus drugs; delivery of stroke intervention and recovery drugs; delivery of compounds that are deficient in the brain across the BBB to treat diseases that are caused by a deficiency in these compounds (e.g., for treating Parkinson's disease, etc.).

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

[0072] The methods described herein can also be applied in vivo by applying an alternating electric field to the brain of a live subject. Applying an electric field to the brain of a subject will increase the permeability of the BBB, which will enable molecules that are normally prevented or impeded by the BBB to pass through. This can be accomplished, for example, by placing electrodes on or under the skin of the subject, such that applying an alternating voltage between a selected subset of these electrodes will apply an alternating electric field to the brain of the subject.

[0073] For example, one pair of electrodes can be placed on the front and back of the subject's head, while a second pair of electrodes can 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). But in alternative embodiments, the dielectric layer can be omitted, in which case the conductive plate directly contacts the subject's body. In another embodiment, the electrodes can be inserted subcutaneously into the patient's skin.

[0074] The 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), which induces an alternating electric field with the most important component of the field lines parallel to the transverse axis of the subject's head. Then, the AC voltage generator applies an AC voltage between the front and back electrodes at the same frequency (or a different frequency) for a second time period (e.g., 1 second), which induces an alternating electric field with the most important component of the field lines parallel to the sagittal axis of the subject's head. Then the two steps are repeatedly performed during the treatment. Optionally, thermal sensors can be included at the electrodes, and if the temperature sensed at the electrodes becomes too high, the AC voltage generator can be configured to reduce the amplitude of the AC voltage applied to the electrodes. In some embodiments, one or more additional pairs of electrodes can 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 lines is not switched. Note that any of the parameters of this in vivo embodiment (e.g., frequency, field strength, duration, rate of direction switching, and placement of the electrodes) can be varied as described above in connection with the in vitro embodiment. But in the in vivo context, care must be taken to ensure that the electric field remains safe for the subject at all times.

[0075] In the in vivo context, various applications of increasing the permeability of the BBB can be readily envisioned. In one example, prior to and during administration of a chemotherapeutic or other anti-tumor agent, a local increase in uptake of the drug by tumor cells (e.g., glioblastoma cells) in the brain can be induced by applying an alternating electric field to the brain for a period of time (e.g., 72 hours or at least 24 hours).

[0076] Figure 9 A suitable timing relationship between applying an alternating electric field to a living patient and administering a substance is depicted. Based on the data described above, and assuming that the substance is introduced or administered at a given time t = 0, the alternating electric field can be started before the given time (e.g., 72 hours before t = 0), and continued for a time interval after the given time (e.g., until 12 hours after t = 0). In this case, the permeability of the BBB will start to increase before the substance is administered and before the substance reaches the BBB. This will allow the substance to cross the BBB immediately after it reaches the BBB. In the context of chemotherapy, this would correspond to starting to apply the alternating electric field, administering the chemotherapeutic agent 72 hours later, and then applying the alternating electric field for an additional time interval (e.g., until 12 hours after the administration of the chemotherapeutic agent).

[0077] Note that the above, in connection with Figure 9The time interval discussed can be uninterrupted, or can 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 an interval of 1 hour, followed by applying an alternating electric field for another 6 hours. Similar intervals can also optionally interrupt the 72 hour interval prior to administering the substance. Note also that, in the context of Figure 9 administering the substance to a living patient, the substance can be administered using any of a variety of methods, including but not limited to intravenous, oral, subcutaneous, intrathecal, intraventricular, and intraperitoneal.

[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 in conjunction with a time period of at least 24 hours will improve the change in permeability (as compared to operating outside of these ranges).

[0079] The methods described herein can be used to deliver a substance across the blood brain barrier of a subject's brain when the subject's brain includes a tumor. One existing method of treating brain tumors (e.g., glioblastoma) is by 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 referred to as 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. Briefly, these two applications describe destroying dividing cells during mitosis. The effectiveness of TTFields is improved when the electric field strength in at least a portion of the tumor is at least 1 V / cm, when the electric field is applied for a long period of time (e.g., weeks or months) with as few intervals as possible, and when the direction of the electric field is switched periodically.

[0080] In patients with brain tumors, it can occur that the tumor needs to be treated with TTFields, and that a substance also needs to be delivered across the blood brain barrier of the same patient (e.g., to help obtain a therapeutically effective amount of a chemotherapeutic drug across the BBB, to provide an additional line of attack against the tumor). In some cases, a single frequency of alternating electric field can be used to treat the tumor and to increase the permeability of the BBB. In other cases, it can be desirable to use alternating electric fields with different frequencies: a first frequency is selected to provide an improved result in increasing the permeability of the BBB, and a second frequency is selected to provide an improved result in the tumor effect of TTFields.

[0081] Figure 10is a block diagram of an apparatus that generates a first frequency for inducing permeability of the BBB and a second frequency for inducing cytotoxicity. The apparatus includes an alternating current voltage generator 44, which is similar to a conventional field generator unit, but has the ability to operate at two different frequencies. The first frequency is between 50 and 190 kHz, and the second frequency is between 50 and 500 kHz. In some embodiments, the first frequency is between 75 kHz and 125 kHz, and the second frequency is between 150 kHz and 250 kHz.

[0082] The ability to operate at two different frequencies can be implemented, for example, using relays to switch either the first set of components or the second set of components into a conventional circuit that generates an alternating current voltage, and adjusting the operating frequency of the oscillator. The alternating current 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 alternating current voltage generator 44 outputs the first frequency, and when the control input is in a second state, the alternating current voltage generator 44 outputs the second frequency. The controller 42 is programmed to place the control input in the second state so that the alternating current voltage generator 44 outputs the second frequency. The controller 42 is also programmed to accept a request to switch to the first frequency. In Figure 10 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, and the like. In alternative embodiments, the request can arrive via RF (e.g., Bluetooth, WiFi, etc.) from a tablet, smartphone, or the like.

[0083] After receiving the request, the controller 42 places the control input in the first state so that the alternating current voltage generator 44 will output the first frequency for a period of time (e.g., 72 hours). After the time interval has elapsed, the controller 42 places the control input in the second state so that the alternating current voltage generator 44 reverts to outputting the second frequency.

[0084] Optionally, the alternating 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 the control input through states that cause the alternating voltage generator 44 to output the second frequency and one or more additional frequencies until a request is received. The controller 42 is also programmed to accept a request to switch to the first frequency. After receiving the request, the controller 42 places the control input in the first state so that the alternating voltage generator 44 will output the first frequency for a period of time (e.g., 72 hours). After the time interval has elapsed, the controller 42 will revert to cycling the control input through states that cause the alternating voltage generator 44 to output the second frequency and one or more additional frequencies.

[0085] When a person has a tumor that is being treated with a combination therapy that includes TTFields and chemotherapy, Figure 10 The system depicted in FIG. 4B is particularly useful. In this case, the system is operated at the second frequency most of the time to provide maximum cytotoxicity utility. However, before a person goes to a chemotherapy clinic for a dose of chemotherapy, a medical professional (or user) initiates the user interface 40 to switch the system to the first frequency that promotes BBB permeability. In this case, the initiation of the user interface can be done, for example, 72 hours before the anticipated start of chemotherapy.

[0086] Alternatively, after receiving a request (e.g., from the user interface 40), the controller 42 can control the control input so that the alternating voltage generator 44 will output the first frequency for a time interval (e.g., 1 hour) and then switch back and forth between the second frequency and the first frequency (e.g., once per hour). Eventually (e.g., when the relevant substance has been flushed from the patient's bloodstream), the controller 42 controls the control input so that the alternating voltage generator 44 reverts to outputting the second frequency.

[0087] A set of electrodes (not shown) similar to the conventional electrodes used The electrodes (not shown) similar to the conventional electrodes used

[0088] Note that, in connection with any of the above methods, after a sufficient time has elapsed after the alternating electric field is terminated, the BBB should revert to its original low permeability state. This can be important for the safety of the subject in many cases.

[0089] While the application has been disclosed with reference to certain embodiments, many modifications, alterations, and variations will be apparent to those skilled in the art in light of the foregoing disclosure. Accordingly, it is intended that the present application not be limited to the described embodiments, but that it be construed as having the entire scope of the language of the appended claims and their equivalents.

Claims

1. A combination of a substance for delivery across the blood-brain barrier of a subject and a blood-brain barrier permeability control device for facilitating delivery of the substance when the substance is administered across the blood-brain barrier, wherein the device comprises an alternating voltage generator configured to generate an alternating electric field in the brain of the subject at a frequency of less than 190 kHz for a time interval of at least 24 hours.

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

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

4. The combination 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 brain of the subject.

5. The combination of claim 1, wherein the frequency is between 25 kHz and 190 kHz.

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

7. The combination of claim 1, wherein the frequency is between 75 kHz and 125 kHz.

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

9. The combination of claim 1, wherein the time interval is uninterrupted.

10. The combination of claim 1, wherein the time interval is interrupted.

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

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

13. The combination of claim 1, further comprising: a set of electrodes configured to be attached to the body of the subject; and a wire connecting an output of the alternating voltage generator to the set of electrodes.

14. The combination of claim 1, wherein the substance is administered intravenously, orally, subcutaneously, intrathecally, intraventricularly, or intraperitoneally.

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

16. The combination 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 anti-epileptic drug, a hydrocephalus drug, a stroke intervention drug, or a psychiatric drug.

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

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

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