EP4712885A1 - Systems and methods for applying electrically based therapeutics - Google Patents

Abstract

Pulsed electric field methods and systems for applying a pulsed electric field therapy are provided. A system includes a control unit comprising a controller, an impedance measurement system in communication with the controller, and a pulse generator controlled by the controller. The system also includes a handle coupled to the control unit. The handle includes a radiation source controlled by the controller and a temperature measurement system in communication with the controller. The system further includes an electrode array cartridge removably coupled to the handle. The electrode array cartridge includes an array of independently addressable electrodes in communication with the impedance measurement system and the pulse generator, and a series of holes between the electrodes to enable heat from the radiation source in the handle to reach a target tissue and temperature measurements of the target tissue to be obtained by the temperature measurement system in the handle.

Description

SYSTEMS AND METHODS FOR APPLYING ELECTRICALLY BASED THERAPEUTICS

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 to United States Provisional Patent Application No. 63/502,804 filed on May 17, 2023, the entire contents of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 EB027497 2107- 1135-02 awarded by the National Institute of Health. The government has certain rights in the invention.

BACKGROUND

[0003] Pulsed electric fields (PEF) have been shown to directly influence cell membranes leading to a reversible or irreversible effect depending on parameters of the applied electric fields. The use of PEF has made tremendous progress since it was first utilized on isolated cells in the 1980s and then in preclinical models in the 1990s. More recently in the past 10-20 years, PEF technologies have been successfully used clinically, particularly for cancer therapy such as with electrochemotherapy (ECT) and irreversible electroporation (IRE). Gene delivery (gene electrotransfer; GET) has also been clinically tested for a variety of indications predominately for cancer and DNA vaccines. ECT and GET rely on reversible electroporation and the membrane recovers its integrity after delivery. The procedure typically involves microsecond (drugs) or millisecond (nucleic acid) pulses with electric fields generally between 0.1 - 2 kilovolts per centimeter (kV/cm). IRE typically uses microsecond pulses with higher field strengths and more pulses causing irreversible damage to the cell membrane. This approach has been used for directly inducing cell death and therefore tissue ablation. One advantage of IRE is that it does not cause significant temperature increase of the treated tissue and, thus, is generally regarded as a nonthermal method.

[0004] ECT has been a major local ablative approach utilizing PEF and has obtained acceptance in many European countries. In the mid-2000s, ablation of solid tumors was demonstrated with PEF without the addition of a chemotherapeutic. This was accepted as a major advantage to achieve tumor ablation in a non-thermal manner. IRE can ablate soft tissue including solid tumors and is performed utilizing direct current at high voltages of up to 3kV to induce nanopores in the cell membrane leading to permeability and subsequent cell death (e.g., by apoptosis or other mechanisms). IRE devices deliver high-voltage and high-frequency pulses through the electrodes and create an appropriate ablation area throughout the tumor. For example, one mechanism is related to stable pores forming in the cell membrane. Each pore is an open channel for ion exchange and subsequent irreversible damage dependent on the applied voltage and the resulting increase in cell transmembrane potential. Pulses applied to cell membrane lipid bilayers disrupt the internal environment of tumor cells, causing apoptosis and death.

[0005] The use of IRE has seen significant expansion in the last decade and a half. The approach has been used in multiple tumor types in both preclinical and clinical applications. Because there is not a significant temperature rise (e.g., above 45 degrees Celsius) with IRE, it is regarded as a safer means of ablation than with thermal approaches such as radiofrequency and cryotherapy. This enables performing ablation without damage to vital structures and minimal damage to normal surrounding tissue.

[0006] Although the use of PEF is being explored for multiple applications, there are still issues that need to be overcome to gain wider acceptance of the technology and advance its capabilities to a point where it can be a significant tool in treating solid tumors. Some of the issues that have plagued IRE particularly with respect to pancreatic cancer, for example, are the potential for local recurrence due to incomplete ablation as well as the need for applying high voltages to achieve the necessary field strengths. This limits the size of tumor that can be treated and also reduces the potential to achieve complete ablation and to provide the treatment without complications. These are areas that require improvement to further advance the technology and enhance the potential for effectively treating a variety of tumor types.

[0007] Another limitation is the current requirement to empirically develop protocols by manipulating multiple variables regarding electrical treatment. More specifically, from the inception of each of the PEF technologies (ECT, IRE and GET), protocols were established utilizing pre-clinical in vivo studies through a trial-and-error process. A range of electrical parameters would be tested to find the best combination that could achieve the desired effect. This approach to find appropriate parameters for the therapy can be cumbersome and a daunting task and essentially is the current state of the art for PEF technology. [0008] There is also a need to better control how the electric field is applied and targeted and facilitate a successful therapy. In light of all of the above, there is a need for next-generation instrumentation and methods to better administer electrically based therapeutics.

SUMMARY

[0009] Some embodiments provide a pulsed electric field method. The method includes applying an electroporation pulse to a target tissue, measuring an impedance of the target tissue, and continuing to apply the electroporation pulse and measure the impedance until a steady state impedance is reached. The method also includes further applying an additional electroporation pulse to the target tissue after the steady state impedance is reached.

[0010] Some embodiments provide a pulsed electric field method. The method includes injecting a target tissue with a first molecule and a second molecule and treating the target tissue with a multi el ectrode array comprising a first sector and a second sector. The method further includes, for each of the first sector and the second sector, applying an electroporation pulse to the target tissue adapted for a respective molecule of the first molecule or the second molecule, measuring an impedance of the target tissue adjacent a respective sector, and continuing to apply the electroporation pulse and measure the impedance until a desired impedance is reached.

[0011] Some embodiments provide a system for applying a pulsed electric field therapy. The system includes a control unit comprising a controller, an impedance measurement system in communication with the controller, and a pulse generator controlled by the controller. The system also includes a handle coupled to the control unit. The handle includes a radiation source controlled by the controller and a temperature measurement system in communication with the controller. The system further includes an electrode array cartridge removably coupled to the handle. The electrode array cartridge includes an array of independently addressable electrodes in communication with the impedance measurement system and the pulse generator, and a series of holes between the electrodes to enable heat from the radiation source in the handle to reach a target tissue and temperature measurements of the target tissue to be obtained by the temperature measurement system in the handle.

DESCRIPTION OF THE DRAWINGS [0012] FIG. 1 is a schematic view of a system for applying a pulsed electric field therapy, according to some embodiments.

[0013] FIG. 2 is a flowchart of an example process for heat delivery during pulsed electric field therapy.

[0014] FIG. 3 is a flowchart of another example process for impedance-based feedback during pulsed electric field therapy.

[0015] FIG. 4 is a flowchart of yet another example process for combining tissue heating and impedance feedback during pulsed electric field therapy.

[0016] FIG. 5 is schematic cross-sectional view of an example handle, according to some embodiments, for use with the system of FIG. 1.

[0017] FIG. 6A is an isometric view of example electrode array for use with the handle of FIG. 5.

[0018] FIG. 6B is a schematic representation of electrodes of the electrode array of FIG. 6A.

[0019] FIG. 7A is a front view of an example electrode array for use with the handle of FIG. 5.

[0020] FIG. 7B is a schematic representation of electrodes of the electrode array of FIG. 7A.

[0021] FIG. 8 is a flowchart of yet another example process for combining tissue heating and impedance feedback during pulsed electric field therapy.

[0022] FIG. 9 is a graph illustrating results of an experiment testing delivery of plasmid encoding IL-12 and/or a plasmid encoding PD1 extracellular domain (pPDlex) with electroporation according to processes of some embodiments.

[0023] FIG. 10 is a graph illustrating results of an experiment testing irreversible electroporation using tissue heating and impedance feedback processes of some embodiments. [0024] FIG. 11 is an isometric view of an example handle, according to some embodiments, for use with the system of FIG. 1.

[0025] FIG. 12A is an isometric view of a removable multiple electrode array cartridge for use with the handle of FIG. 11.

[0026] FIG. 12B is an underside view of the cartridge of FIG. 12A.

[0027] FIG. 13 is a schematic representation of an example electrode array according to some embodiments.

[0028] FIG. 14 is another schematic representation of an example electrode array according to some embodiments.

DETAILED DESCRIPTION

[0029] Some embodiments provide devices and methods for applying electrically based therapeutics, including pulsed electric field (PEF) therapies such as irreversible electroporation (IRE), electrochemotherapy (ECT), and gene electrotherapy (GET), among others, that address the current shortcomings to standard practices. The basic principles of solutions to these shortcomings will initially be described, followed by further enhancements according to some embodiments. For example, one shortcoming of standard practices is the historical need to apply high voltages to achieve the necessary field strengths. This can limit the size of a treatment area and increase both pain and other adverse events. There is also a need for a means to determine when successful delivery has been accomplished and for better control of the delivery of therapy.

[0030] First, in such therapies, the applied voltage can be reduced by moderately raising tissue temperature to, for example, 43 degrees Celsius. That is, a moderate increase in tissue temperature can allow for a reduction in the applied voltage and the ability to treat a larger area with the same voltage. Second, tissue impedance can be used as a measured parameter to monitor the reaction of the tissue to the applied voltage. Changes in the tissue impedance can be utilized as an indicator for when the therapy has been successfully completed. Third, a user- friendly electrode array can be used to administer the electric fields. The array contains a multitude of independently addressable electrodes and a means to apply moderate heat and impedance monitoring to specific sections of the target tissue. This addresses the issue of tissue heterogeneity which can affect the effectiveness of electrically based therapies.

[0031] Any of these principles can be used alone or in combination to improve PEF therapies. For example, heat application alone can be used, impedance measurements alone can be used, or a combination of heat application and impedance measurements can be used, as described in the following paragraphs. To assist such discussion, FIG. 1 illustrates a general schematic of a system 10 for treating tissues, according to some embodiments. As shown in FIG. 1, the system 10 can include a control unit 12 with an impedance measurement system 14, a pulse generator 16, and a controller 18 including a memory 20 and a processor 22. The system 10 also includes a handle 24 coupled to the control unit 12. The handle 24 can include an electrode array 26 comprising a plurality of electrodes, a radiation source 28, and a temperature measurement system 30. While components of the system 10 will be referenced with regard to the example processes described below in FIGS. 2-4 and 8, it should be noted that different systems 10 comprising more or fewer components may be used in such processes.

[0032] Generally, the pulse generator 16 can be configured to generate electromagnetic energy in the form of a plurality of voltage pulses. The electrode array 26 can be coupled to the pulse generator 16 and arranged to apply the plurality of voltage pulses to a target tissue in order to generate an electric field between electrodes in the target tissue. The radiation source 28 can be configured to generate non-ionizing radiation (e.g., heat) toward the target tissue. The temperature measurement system 30 can be configured to measure or determine a temperature of the target tissue. The impedance measurement system 14 can be configured to measure an impedance of the target tissue between electrodes. The controller 18 can be configured to control the pulse generator 16, control the radiation source 28, analyze temperature measurements from the temperature measurement system 30, and/or analyze impedance measurements from the impedance measurement system 14. That is, the memory 20 may be a non-transitory computer-readable storage medium comprising instructions stored thereon that can be executed by the processor 22 to cause the processor 22 to perform any of the above actions of the controller 18 or certain steps associated with any of the processes described herein.

[0033] Turning now to FIG. 2, an example process 40 for heat delivery using PEF (also known as electroporation) is illustrated. As shown in FIG. 2, at step 42, a target tissue is injected with a molecule (e.g., vaccine, therapeutic drug, gene, protein, nucleic acid sequences, plasmid DNA, etc.), electrodes of the electrode array 26 are placed adjacent the target tissue (e.g., on or within the tissue), and heat is applied. At step 44, an electroporation pulse generator 16 is controlled to emit voltage pulses. For example, the processor 22 of the controller 18 controls the pulse generator 16 to emit voltage pulses based on pulse parameters stored in the memory 20 (e.g., by executing computer-readable instructions stored in the memory 20). At step 46, the voltage pulses are applied to the target tissue via the electrode array 26. At step 48, the target tissue exhibits a biological response (e.g., allowing delivery of the molecule into cells of the target tissue). At step 50, another section of target tissue is treated or the procedure is stopped.

[0034] Initial experiments demonstrating the effect of moderate temperature increases to enhance PEF approaches for GET were performed in guinea pig skin and showed a significant increase in gene expression when delivering plasmid encoding luciferase. For example, in response to the heating and pulse application, the injected molecule can enter cells of the treated tissue and be expressed, and expressed proteins are then excreted from the cells and can be measured. Results demonstrated that expression could be increased almost 10-fold when delivering plasmid encoding luciferase and using the same GET parameters plus or minus heat, or obtained the same expression pattern when applying half the voltage. Additional experiments were conducted to evaluate the potential clinical application of this approach for DNA vaccines and showed a significant increase in antibody production when delivering a plasmid encoding Hepatitis B Surface Antigen with moderate heat. According to these experiments, antibody production was significantly elevated when delivery was performed with moderate heat. In particular, antibody production was 20-fold greater than standard GET and 300-fold greater than injection only. There was also no damage observed in the treated tissue. These results suggest that this moderate heating approach can enhance gene delivery with reduced or no impact on the treated tissue.

[0035] As noted above, historically, high voltages must be applied to achieve the necessary field strengths for therapeutic effectiveness in PEF therapies. As voltage is based on a distance between electrodes, the size of the treatment area may be limited. Furthermore, higher voltages increase the potential for discomfort and there is the potential for fringe effects and unwanted cellular damage at higher voltages. By adding moderate heat to the treatment process, these unwanted effects can be reduced by allowing for a reduction in applied voltage, or treatment area may be expanded for each pulse application using the same voltage level. [0036] Looking now to FIG. 3, another example process 52 for measuring impedance during PEF is illustrated. General PEF processes include applying a train of DC pulses using an experimentally derived “optimal” set of parameters (e.g., electric field intensity, pulse width, pulse duration, pulse polarity, number of pulses, period, etc.). These optimal pulse parameters are then used for all subsequent treatments and often translated to different animal models and from animal models to human tissues. The process 52 of FIG. 3, however, uses an impedancebased feedback model to control at least the number of applied pulses in an individual treatment. For example, impedance measurements can be used as an indicator of permeability of cell membranes and, thus, treatment effectiveness.

[0037] As shown in FIG. 3, to start, at step 54, a target tissue is injected with a molecule, electrodes are placed adjacent the target tissue (e.g., on or within the target tissue), and tissue impedance is measured. For example, the impedance measurement system 14 can be coupled to the electrode array 26 in order to measure impedance of the target tissue using the electrodes. At step 56, an electroporation pulse generator 16 is controlled to emit voltage pulses. For example, the processor 22 of the controller 18 controls the pulse generator 16 to emit voltage pulses based on pulse parameters stored in the memory 20 (e.g., by executing computer- readable instructions stored in the memory 20). At step 58, the voltage pulses are applied to the target tissue by the electrodes.

[0038] Furthermore, the process 52 can include closed-loop feedback to adjust the output of the electroporation pulse generator 16. That is, following step 58 when voltage pulses are applied to the target tissue, tissue impedance is again measured by the impedance measurement system 14 at step 60. The tissue impedance measurement from step 60 is then compared to the pre-pulse impedance measurement from step 54 at step 62. If tissue impedance has not yet been reduced by a set amount (e.g., a predefined amount stored in memory 20), the process 52 returns to step 56 to continue applying pulses to the target tissue. This predefined amount stored in memory may be, for example, a predefined percentage. If tissue impedance has been reduced by a set amount, the process 52 proceeds to step 64 to treat another section of tissue, if treatment of another section is required, or the process 52 is completed. For example, after step 64, the process 52 can revert back to step 54, including additional injections and/or new baseline prepulse impedance measurements (e.g., as applied pulses to one section may affect the impedance of another section). Alternatively, after step 64, the process 52 can revert back to step 56 and begin pulsing the new section. For example, whether the process 52 reverts back to step 54 or step 56 may be application-specific.

[0039] Impedance feedback control was investigated for its potential benefits to GET. This concept was tested by delivering a plasmid encoding luciferase using 200 volts per centimeter (V/cm) pulses that were 150 milliseconds (ms) in duration (500 ms apart). One approach had been to apply pulses using an electrode-array type device with sixteen electrodes and divided into nine sections of eight pulses administered in a four pulse-by-four pulse process (i.e., 72 total pulses). To evaluate the use of impedance monitoring, impedance was measured after each pulse and compared to the pre-pulse impedance of that sector. If the impedance was reduced by a prescribed percentage, the pulse generator was programmed to move on and begin pulsing the next sector. Delivery was continued until mean impedance was reduced or until a maximum of twenty sets of two pulses were applied to a section.

[0040] The three experimental groups were (1) pulsing until impedance was reduced to 80% of the pre-pulse value in each sector, (2) pulsing until an impedance reduction of 95% was achieved, and (3) standard 4X4 pulsing. These were compared to two control groups: no treatment and injection of plasmid DNA without pulsing over 14 days. The results showed that feedback-based pulsing to 80% and 95% reductions in impedance produced higher peak and total expression than standard pulsing. Both the 80% and 95% feedback data were statistically different from the 4X4 pulsed group at 200 V/cm for days 7, 10, and 14. Interestingly, the total number of pulses was reduced in the two impedance monitoring groups, but the number of pulses applied varied even within like-treated animals. This indicates that the system and process were working and could compensate for differences in individual animals and treatment sites. These results suggest that impedance-based feedback control can improve gene delivery and optimize treatment efficacy.

[0041] Turning now to FIG. 4, yet another example process 66 for combining tissue heating and impedance feedback during PEF is illustrated. The process 66 of FIG. 4 combines the processes 40, 52 of FIGS. 2 and 3. Thus, as shown in FIG. 4, to start, at step 68, a target tissue is injected with a molecule, electrodes are placed adjacent the target tissue (e.g., on or within the target tissue), heat is applied, and tissue impedance is measured (e.g., via the impedance measurement system 14). At step 70, an electroporation pulse generator 16 is controlled to emit voltage pulses. For example, the processor 22 of the controller 18 controls the pulse generator 16 to emit voltage pulses based on pulse parameters stored in the memory 20 (e.g., by executing computer-readable instructions stored in the memory 20). At step 72, the voltage pulses are applied to the target tissue by the electrodes.

[0042] Furthermore, at step 74, a temperature of the target tissue is monitored and heat is further applied to maintain the elevated tissue temperature (e.g., a predefined target temperature stored in memory, such as 43 degrees Celsius). While step 74 is illustrated as being performed concurrently with step 72, it should be noted that step 74 may be continuously executed during the process 66. Additionally, following step 72 when voltage pulses are applied to the target tissue, tissue impedance is again measured by the impedance measurement system 14 at step 76. The tissue impedance measurement from step 76 is then compared to the pre-pulse impedance measurement from step 68 at step 78. If tissue impedance has not yet been reduced by a set amount (e.g., a predefined amount stored in memory 20), the process 66 returns to step 70 to continue applying pulses to the target tissue. If tissue impedance has been reduced by a set amount, the process 66 proceeds to step 80 to treat another section of tissue, if treatment of another section is required, or the process 66 is completed.

[0043] The combination of moderate heat, impedance feedback control, and PEF was investigated to determine whether it could improve gene delivery. A plasmid encoding luciferase delivered to guinea pig skin was used, with two applied voltages: 45 V (225 V/cm) for standard GET and 35 V (175 V/cm) with either GET with moderate heat (HGET) or GET with moderate heat and impedance monitoring with a drop to 80% (HGET+Imp). The results showed that HGET could achieve the same expression pattern at a reduced voltage, while HGET+Imp achieved a higher expression but with a lower number of applied pulses. These results demonstrate how a system 10 combining moderate heat, impedance monitoring, and GET can be utilized to exert control over tissue variability and pulse delivery to achieve a desired effect.

[0044] One approach for moderating tissue temperature may be performed utilizing an infrared laser as the radiation source 28. According to some embodiments, convective heating can be instead used as the radiation source 28, incorporated into the handle 24, that could blow warm air onto the target tissue, along with a temperature measurement system 30 built into the handle 24, such as a non-contact forward-looking infrared (FLIR) camera or another system (e.g., a temperature sensor such as a thermistor, thermocouple, etc.). For example, FIG. 5 illustrates an example handle 24 according to some embodiments. As shown in FIG. 5, the handle 24 includes an electrode array 26 with electrodes 32, a radiation source 28 in the form of a heating coil (along with a fan or other source of air movement, not shown), and a temperature measurement system 30 in the form of an infrared camera. The electrode array 26 further includes holes 34 between electrodes 32 to allow warm air flow therethrough and to permit tissue imaging by the infrared camera within the handle 24.

[0045] This configuration was tested in excised pig skin using two thermocouples: one placed intradermally and the other in the subdermis. An automated system was developed that can toggle the radiation source on and off based on the temperature reading from the camera. Utilizing this set up, the intradermal temperature was maintained between 42-44 degrees Celsius. The temperature at the level of the subdermis was on average about two degrees lower but took about 15-20 seconds longer to reach temperature. Once both intradermal and subdermal layers were at temperature, temperature could generally be maintained for at least two minutes. This demonstrates that internal tissue temperature can be controlled based on the convective radiation source 28 and temperature measurement system 30 within the handle 24.

[0046] Additionally, the handle design of FIG. 5 was tested using a series of experiments performed using a needle array and evaluating various impedance changes. The first configuration tested was an array of two rows of four needles. One row was electrically connected together and served as four simultaneously positive electrodes. The other row was electrically connected together and served as four simultaneous negative electrodes. This did not yield desired outcomes; a high enough level of disease-free survival could not be achieved regardless of the impedance change algorithm used.

[0047] Next, the electrode array strategy was changed to use a multiple-electrode array (MEA)-type design that improved results dramatically. An example MEA 26 is illustrated in FIGS. 6A and 6B. As shown in FIGS. 6A and 6B, the MEA 26 has nine electrode needles 32 (i.e., three rows of three needles) that are each independently addressable. In use, four needles are active at any one time, creating four separate sectors (or groupings or sections), i.e., sector 1 (SI), sector 2 (S2), sector 3 (S3), and sector 4 (S4), as shown in FIG. 6B. By way of example, sector 1 can be pulsed first in the direction of the red arrows (i.e., toward the right in FIG. 6B) and then blue arrows (i.e., downward in FIG. 6B). Treatment can then be started in sector 2 only after sector 1 treatment is complete. The remaining sectors can be treated in series. Furthermore, FIGS. 7 A and 7B illustrate another example MEA 26 including 16 surface electrodes 32 (i.e., four rows of four) that are each independently addressable. In use, the MEA 26 shown in FIGS. 7A and 7B can include nine total sectors, i.e., S1-S9, as illustrated in FIG.

7B.

[0048] The use of independently addressable electrodes 32 that are closely spaced can compensate for the inhomogeneous nature of tissue because such use provides better control over the therapy. That is, the independently addressable electrodes 32 define subspaces within the target tissue, corresponding to the sectors described above. By defining these subspaces, the impact of the inhomogeneity to the overall treatment site can be reduced as the impedance guidance can compensate in these smaller areas/volumes. The larger the area being treated (i.e., the area between any set of electrodes), the more difficult it is to get a good indication of impedance as there can be pockets of naturally occurring impedance differences.

[0049] Accordingly, smaller spaced sectors between electrodes 32, along with sectorspecific impedance feedback, allow for better control of the electrical treatment to address inhomogeneities within a tissue site. This allows the single placement of an array 26 to be used to treat a large area. Traditionally, a user would have to move the electrode and reinsert to treat a large area. However, using this approach allows the user to treat a large area, but in small, well-controlled sectors, without moving the array 26. By way of example, looking back to the process 40 of FIG. 2, the final step of treating a next sector (i.e., step 50) can simply include reverting back to step 44 with pulse generation for a new sector within the MEA 26.

[0050] Additionally, in some applications, tissue heating can be accomplished on a persector basis. Thus, the multi el ectrode array 26 also allows for better heating control. For example, a radiation source 28 can include multiple heating coils. A voltage or current to a particular heating coil adjacent a sector to be treated can be modulated based upon images taken through the holes 34 with the IR camera (i.e., the temperature measurement system 30), as such images can provide temperature measurements adjacent all holes 34 in the multi el ectrode array 26. In another example, looking to the MEA 26 of FIG. 5, certain holes 34 can be blocked to direct heated air only toward certain sectors. Doing this allows for targeting the effect of heat application to specific sectors. More specifically, as noted above, one advantage of heat application is the ability to use much lower voltages to achieve the same therapeutic effect. In some applications, voltages can be lowered so much that they are only therapeutically effective where heat is applied. As such, the therapeutic effect of PEF would only be seen where heat is applied and would not have an effect on areas that are not heated. [0051] Furthermore, in some embodiments, concurrent, individualized pulse applications can be executed across multiple sectors. That is, while the processes are described above as treating a subsequent sector only after treatment of a first sector is completed, in some applications, the processes can treat multiple sectors concurrently. By way of example, looking back to the process 66 of FIG. 4, multiple sets of steps 72-80 can be performed concurrently. Such a process may be beneficial when two different types of DNA or therapeutic molecules are delivered during the same treatment. For example, part of a tissue area covered by an MEA 26 can be injected with a chemotherapeutic, while another part is injected with DNA encoding a therapeutic agent. The independently addressable electrodes could be configured to administer different pulse parameters, heating, and/or impedance control algorithms in accordance with the processes described herein to deliver each type of molecule, as different parameters be required for optimal delivery of the two different molecules. As another example, different treatment types can be applied to different sectors, e.g., part of a tissue area covered by an MEA 26 can receive an IRE treatment, while another part receives a GET treatment or an ECT treatment.

[0052] Accordingly, moderate heat, impedance feedback control, and/or independently addressable electrode arrays can be used to improve PEF therapies by allowing for a reduction in the applied voltage to a target tissue or an increase in treatment areas and more targeted treatments over specified treatment areas. Additionally, further enhancements to one or more of these principles can provide additional improvements.

[0053] For example, FIG. 8 illustrates another process 82, according to some embodiments, for using impedance feedback during electroporation. To start, at step 84, a target tissue is injected with a molecule, electrodes 32 are placed adjacent the target tissue (e.g., on or within the target tissue), heat is applied, and, optionally, tissue impedance is measured (e.g., via the impedance measurement system 14). At step 86, an electroporation pulse generator 16 is controlled to emit voltage pulses. For example, the processor 22 of the controller 18 controls the pulse generator 16 to emit voltage pulses based on pulse parameters stored in the memory 20 (e.g., by executing computer-readable instructions stored in the memory 20). At step 88, the voltage pulses are applied to the target tissue by the electrodes 32. Furthermore, at step 90, a temperature of the target tissue is monitored and heat is further applied to maintain the elevated tissue temperature. While step 90 is illustrated as being performed concurrently with step 88, it should be noted that step 90 may be continuously executed during the process 82. Additionally, in some applications, the process 82 may eliminate heat application and monitoring altogether (i.e., may only be directed toward impedance feedback).

[0054] Referring still to FIG. 8, following step 88 when voltage pulses are applied to the target tissue, tissue impedance is measured by the impedance measurement system 14 at step 92. The tissue impedance measurement from step 92 is then analyzed to determine whether it has reached steady state at step 94. If tissue impedance is still changing (“NO” at step 94), the process 82 returns to step 86 to continue applying pulses to the target tissue. If tissue impedance is no longer changing, e.g., has reached steady state (“YES” at step 94), an additional X number of pulses can be applied at step 96, and then the process 82 proceeds to step 98 to treat another section of tissue, if treatment of another section is required, or the process 82 is completed.

[0055] Looking back to the process 52 of FIG. 3 described above, the feedback loop compares a post-pulse impedance measurement (from step 60) to a baseline pre-pulse impedance measurement (from step 54) to determine if enough pulses have been applied for the treatment to be effective. That is, a user inputs a percentage change in impedance that signals stopping the process. The process 82 of FIG. 8, on the other hand, does not rely on prepulse measurements but, rather, looks for when impedance stops changing. Thus, the process 82 of FIG. 8 compares an impedance measurement to an immediately previous impedance measurement after each pulse application. In some applications, “stops changing” or “steady state” may mean that the impedance measurement and the immediately previous impedance measurement are equal. In other applications, “stops changing” or “steady state” may mean that the impedance measurement and the immediately previous impedance measurement are not significantly different within a specified percentage of error (i.e., impedance values have changed by no more than Z percent, wherein Z is a predefined value stored in memory 20).

[0056] Additionally, the process 82 of FIG. 8 includes the step 96 of applying X pulses after impedance steady state is reached. In some applications, the number of pulses (X) can be treatment-specific and can be input by a user and/or can be a predefined value stored in memory 20. Furthermore, in some applications, this additional step 96 can also be applied to the process 52 of FIG. 3. For example, after impedance has been reduced by a set percentage (step 62), an additional X pulses are applied.

[0057] Furthermore, in some applications, rather than looking for steady state to be reached, as in the process 82 of FIG. 8, or comparing to a pre-pulse impedance measurement, as in the process 52 of FIG. 3, either process can incorporate applying pulses until a predefined absolute value of impedance is achieved. In this manner, impedance measurements are not compared to prior values or pre-pulse values but, rather, to a value stored in memory 20. Additionally, in some applications, combinations of processes may be used on a sectionspecific basis of an MEA 26, e.g., where treatment in one section of the MEA 26 is performed based on the process 52 of FIG. 3, and treatment in another section of the MEA 26 is performed based on the process 82 of FIG. 8, or different predefined values may be used for different sections.

[0058] Testing was performed to demonstrating the effects of the process 82 of FIG. 8 in a C57B1/6 mouse model with B16.F10 melanoma cells. More specifically, a variety of impedance algorithms were tested to determine which would give the best results. Impedance reductions of 40% and 60% gave reasonable results, but it was observed that, on occasion, the prescribed impedance change could not be achieved and would instead stabilize to a level steady-state. The next approach was to monitor until steady state was achieved and then an additional one, two, or three pulses were applied. It was found that best results were achieved particularly at lower applied voltages when utilizing steady-state plus three additional pulses. This process still allows for a reduced total voltage delivery to the tissues and, therefore, minimal side effects. Another observation that resulted from this set of experiments was that it was clear that utilizing the MEA type approach (e.g., applying treatment on a per-segment basis using an electrode array) was beneficial as different number of pulses were administered to each of the four segments.

[0059] Using the heating and impedance process 82 of FIG. 8, additional efficacy experiments were initiated. For example, standard GET for interleukin- 12 plasmid (pIL-12) delivery was performed utilizing 100 microsecond (ps) pulses at a field strength of 1300 V/cm. These conditions were utilized as a comparison group. Then, GET using a pulse width of 100 ps was applied with varied the field strength, testing 1300 (standard GET only), 1000, 800, 600 and 400 V/cm. The best results for long-term, disease-free survival for standard GET was at a field strength of 1300 and 1000 V/cm and achieved long-term survival in 75% of mice. Groups treated with the heat and impedance feedback process achieved 100% disease-free survival at 1000, 800 and 600 V/cm. Response could also be increased at a lower field strength with this strategy. In addition, disease-free surviving mice were reinjected with B16.F10 cells on the opposite flank and all remained disease free. Replicate experiments have confirmed these results.

[0060] Additionally, in some applications, one or more of the above processes, such as the process 82 of FIG. 8, can be used to deliver plasmids encoding peptides or proteins designed to block PD1-PDL1 (programmed cell death protein 1 and programmed deadline-ligand 1) binding, e.g., for cancer treatment. Further, this plasmid could be delivered in combination with a plasmid encoding a cytokine that could modify the tumor microenvironment from cold to hot. The two together would potentially elevate the immune response. To test the feasibility of this concept, B16.F10 tumors were established in the left flank of mice and delivery was performed when tumors were approximately 50 mm^A3. 100 micrograms (pg) of each plasmid was injected and followed by application of GET with or without heat. GET was performed as follows: field strength of 600 V/cm at 5 millisecond (ms) pulse width and 10 pulses; 600 V/cm at 5 ms pulse width and 10 pulses with heat; or 150 V/cm at 150 ms pulse width and 10 pulses with heat. Two days after delivery, mice were humanely euthanized and tumors removed. Half of the tumors were evaluated by flow cytometry and half were evaluated by immunohistochemistry (IHC). The indicator was detecting PD1 peptide on CD45- cells, suggesting that a plasmid encoding PD1 extracellular domain (pPDlex) is expressed and secreted and in turn is binding to PDL1 on the surface of these cells. Flow cytometry revealed that the use of GET plus heat further increased production and subsequent binding of PD1 to CD45- cells. Using IHC, tumors were stained for the presence of MelanA (a marker on B16 cells) and PD1. Following delivery of the two plasmids there were clearly dual stained cells. While levels of binding were not high, this was evaluated after a single treatment and a single dose which may not have been the correct dose. It does, however, show that binding can be achieved.

[0061] These initial experiments demonstrate the advantage of using the heating and impedance based feedback of some embodiments for delivering gene-based therapeutics for immunotherapy of cancer and that it is clearly superior to older PEF technology. In addition, while there is clear evidence of the efficacy of delivering pIL-12 via GET for both local and distant response in preclinical and clinical studies, it has also been shown that combining this approach with checkpoint inhibitors elevates the response compared to administering either as a single agent. [0062] Another evaluation was performed to determine if combining pIL-12 with a plasmid encoding PD1 extracellular domain (pPDlex) could also induce a robust anti -tumor immune response. For this experiment, a C57B1/6 mouse model was utilized with B16.F10 melanoma cells. Tumors were established on the left flank as described above. On the day of the first treatment, 5X104 B16.F10 RedLuc cells (stably expressing luciferase) were injected via intraperitoneal route into each mouse. Treatments were performed on five groups of mice on days 1, 5 and 8 and consisted of injection of plasmid(s) directly into tumor followed by treatment with pulse protocol. The subcutaneous tumor response was similar to what was observed in the experiments described above. One interesting result from this experiment was related to tumor growth in the peritoneal cavity, which was assessed via imaging with an in vivo imaging system (IVIS). Combining the two plasmids and delivering with the system 10 according to processes described herein blocked growth within the peritoneum and resulted in 100% tumor free survival of the mice through the entire monitoring period (100 days). Using a single plasmid or both plasmids with standard GET resulted in increased growth and, thus, could not achieve the same survival levels. The results from this preliminary experiment are illustrated in FIG. 9 and demonstrate the feasibility and the potential of the system 10 and processes to induce a robust anti-tumor immune response.

[0063] While the above-described processes are directed toward electroporation, that is, PEF for delivery of molecules (e.g., ECT and GET), it should be noted that any of the processes can be adapted for direct ablation (e.g., IRE), that is, PEF for causing irreversible cell membrane damage to induce cell death. For example, in such processes with respect to IRE, the steps may be similar as that described above, though the step of injecting tissue with a molecule may be eliminated. Also, in such processes, the electroporation pulses have a combination of pulse parameters adapted for IRE (e.g., higher voltages).

[0064] For example, the system 10 and processes were tested for administering IRE as a means to ablate pancreatic cancers in a mouse model. Tumors were induced by injecting 1x106 KPC cells into B6129SFl/j mice. Tumors were allowed to grow to 5-6 mm in diameter prior to initiation of treatment. A series of experiments were performed using the system 10 described above. It was tested using different configurations of a needle array 26 and evaluating various impedance changes. Initially, an array of two rows of four needles was used. This did not yield desired outcomes as disease-free survival was not achieved. The strategy was changed to using a nine-needle MEA 26, which improved results dramatically. The results were long- term disease-free survival in up to 60% of the mice. Interestingly, but not surprising due to the inhomogeneity of the tumor, each sector required a different number of pulsing sequences to achieve the desired change. Each pulsing sequence included 15 pulses in each of two directions and pulses were administered at 1800 V/cm. The best results for long-term disease-free survival were in a group that received IRE with both heat and impedance (60% disease free survival). IRE with heat resulted in 40% disease free survival, and standard IRE resulted in 20%. The results from this experiment are illustrated in FIG. 10.

[0065] Now referring back to the multiple-electrode arrays 26 of the system 10 of FIG. 1, as discussed above, the use of MEAs 26 can improve PEF therapies. MEAs 26 utilize independently addressable electrodes that can be designed in a variety of different configurations to best cover the area to be treated, such as the 3x3 needle electrode MEA 26 shown in FIGS. 6 A and 6B, or the 4x4 surface electrode MEA 26 shown in FIGS. 7 A and 7B. The MEAs 26 of some embodiments further permit tissue heating and impedance monitoring during pulsing (e.g., via holes in the circuit board between the electrodes 32).

[0066] While the distance between electrodes 32 can generally remain fixed, the size of a treatment area can be increased by adding rows or columns of additional electrodes 32 to an MEA 26. Additionally, in some applications, the fixed distance can also be modulated to accommodate the size of the treatment area. Furthermore, in some embodiments, the pulsing sequence (as controlled by the controller 18) can be designed to cover specific areas within a treatment site.

[0067] To better accommodate such variations in a clinical system, FIG. 11 illustrates another handle 24A according to some embodiments. The handle 24A can be used with the system 10 of FIG. 1 and can be used to execute any of the processes described herein. Generally, the handle 24A can include any of the features and/or functions of the handle 24 described above and, thus, any description above related to the handle 24 may be equally applicable to the handle 24 A. Furthermore, as shown in FIG. 11, the handle 24 A can include a replaceable MEA cartridge 100 comprising an MEA 26 of independently addressable electrodes 32 that can be used for pulse application and impedance monitoring.

[0068] As shown in FIGS. 11, 12A, and 12B, according to some embodiments, each removable electrode cartridge 100 can include N independently addressable electrodes that are used for electropulsation and impedance measurement, where N may be two or more. The electrodes 32 can be needles, as shown in FIG. 11, or can be nonpenetrating, surface electrodes 32. Furthermore, each electrode cartridge 100 can have holes 34 between electrodes 32 to accommodate heating a target tissue and for an IR camera to measure temperature and provide a means be used as a feedback control of the heating process so that an appropriate temperature is achieved and maintained.

[0069] For example, an MEA cartridge 100 can include an MxM array of equispaced electrodes 32 or unequally spaced electrodes 32. The electrodes 32 can be in any geometry such as square, rectangular, triangular, circular, elliptical, or any geometry. Example arrays include the square arrays illustrated in FIGS. 6A-7B. These square arrays illustrate rectangular or square sectors comprising four electrodes each. However, in some embodiments, additional geometry sectors with more than four electrodes can be used. For example, further example arrays are illustrated in FIGS. 13 and 14. That is, FIG. 13 illustrates an array 26 comprising four circular sectors or groupings with 28 total electrodes (8 electrodes per sector), and FIG. 14 illustrates an array comprising four circular sectors with 18 total electrodes (6 electrodes per sector). Accordingly, a sector within the array of electrodes 32 in an MEA cartridge 100 can comprise two electrodes, three electrodes, four electrodes, or more than four electrodes, in any geometry (e.g., triangle, square, circle, etc.).

[0070] Referring back to FIGS. 11 and 12A, generally, an MEA cartridge 100 can include a mechanical interface and an electrical interface to mechanically and electrically couple the MEA cartridge 100 to mating mechanical and electrical interfaces of the handle 24 A. For example, as shown in FIG. 11, the handle 24A can include a recess 102. The MEA cartridge 100 can having a matching shape as the handle recess 102, allowing the MEA cartridge 100 to be received within the recess 102. The recess 102 can also include plugs (e.g., guide pins) 104 serving as a mechanical interface, to allow the MEA cartridge 100 (with mating plug features 106, shown in FIG. 12A) to be plugged into the recess 102. While the recess 102 and/or plugs 104/106 can serve as a mechanical interface, it should be noted that other mechanical interfaces can be used in some applications. For example, snap-fit connections, press-fit connections, friction-fit connections, mechanical connectors such as latches, etc., or other mechanical interfaces are contemplated. The recess 102 can further include pogo pins 108 or other electrical contacts, serving as an electrical interface, to allow the MEA cartridge (with corresponding electrical contacts) to be plugged into the recess 102. Furthermore, within the recess 102, the handle 24 A can include an orifice 110 that permits airflow therethrough to direct heat through the holes 34 in the cartridge 100.

[0071] Accordingly, various MEA cartridges 100 having different MEAs 26 can be plugged into the handle 24 A so long as they have the same footprint matching the recess 102 and mating mechanical interface, enabling a “plug and play” feature of the system 10. As such, certain MEA cartridges 100 can be used to treat specific target tissues. For example, different geometric array configurations can be used to better match a tumor or other target tissue. And further, as the MEA cartridges 100 contain independently configurable electrodes 32, an MEA cartridge 100, having a specific electrode array geometry, can be further adapted to a target tissue by independently controlling which sectors are pulsed.

[0072] Although the use of PEF has been explored for multiple applications, there are still issues that need to be overcome to gain wider acceptance of the technology and advance its capabilities to a point where it can be a significant tool in treating solid tumors. In light of the above, systems and methods of some embodiments can be used to better control how the PEF therapy is delivered to overcome these issues. Features of such systems and methods include the ability to reduce the applied voltage as well as a means to monitor the treatment site to obtain a signal indicating when the therapy has been accomplished. The applied voltage can be reduced by moderately raising the tissue temperature. By utilizing this feature and reducing the voltage opens two distinct possibilities: either a reduction in the applied voltage or being able to treat a larger area with the same voltage. Additionally, tissue impedance can be used as the measured parameter to monitor the reaction of the tissue to the applied voltage. Changes in the tissue impedance can be utilized as an indicator for when the therapy has been successfully completed, as opposed to using empirically established protocols, which are often established from treating other tissue types or applications. Furthermore, a next-generation electrode array can be used to administer electric fields. The electrode array brings all the components together to make a user-friendly and effective approach for applying PEF therapies. The array contains, e.g., between 4-16 independently addressable electrodes. By making the electrodes independently addressable, treatment, including the application of moderate heat and impedance monitoring, can be applied to specific sections of the target tissue. This reduces or removes the issue of tissue inhomogeneity, which can reduce treatment effectiveness for electrically based therapies. [0073] It is to be understood that the invention is not limited in its application to the details of construction and the arrangement of components set forth in the above description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Unless specified or limited otherwise, the terms “mounted,” “connected,” “supported,” and “coupled” and variations thereof are used broadly and encompass both direct and indirect mountings, connections, supports, and couplings. Further, “connected” and “coupled” are not restricted to physical or mechanical connections or couplings.

[0074] The above discussion is presented to enable a person skilled in the art to make and use embodiments of the invention. Various modifications to the illustrated embodiments will be readily apparent to those skilled in the art, and the generic principles herein can be applied to other embodiments and applications without departing from embodiments of the invention. Thus, embodiments of the invention are not intended to be limited to embodiments shown, but are to be accorded the widest scope consistent with the principles and features disclosed herein. The above detailed description is to be read with reference to the figures, in which like elements in different figures have like reference numerals. The figures, which are not necessarily to scale, depict selected embodiments and are not intended to limit the scope of embodiments of the invention. Skilled artisans will recognize the examples provided herein have many useful alternatives and fall within the scope of embodiments of the invention.

[0075] It will be appreciated by those skilled in the art that while the invention has been described above in connection with particular embodiments and examples, the invention is not necessarily so limited, and that numerous other embodiments, examples, uses, modifications and departures from the embodiments, examples and uses are intended to be encompassed by the claims attached hereto. The entire disclosure of each patent and publication cited herein is incorporated by reference, as if each such patent or publication were individually incorporated by reference herein. Various features and advantages of the invention are set forth in the following claims.

Claims

1. A pulsed electric field method comprising: applying an electroporation pulse to a target tissue; measuring an impedance of the target tissue; continuing to apply the electroporation pulse and measure the impedance until a steady state impedance is reached; and further applying an additional electroporation pulse to the target tissue after the steady state impedance is reached.

2. The method of claim 1, wherein the steady state impedance is reached when a previous impedance measurement is equal to an immediately subsequent impedance measurement.

3. The method of claim 1, wherein the steady state impedance is reached when a subsequent impedance measurement has changed compared to an immediately previous impedance measurement by no more than a predefined percentage.

4. The method of claim 1, further comprising heating the target tissue to a preset temperature.

5. The method of claim 4, further comprising monitoring a temperature of the target tissue and controlling a radiation source to maintain the preset temperature.

6. The method of claim 1, wherein the steps of applying the electroporation pulse, measuring the impedance, continuing to apply the electroporation pulse and measure the impedance until the steady state impedance is reached, and further applying the additional electroporation pulse are performed in a first section of a multi el ectrode array; and further comprising repeating the steps of applying the electroporation pulse, measuring the impedance, continuing to apply the electroporation pulse and measure the impedance until the steady state impedance is reached, and further applying the additional electroporation pulse in a second section of a multi el ectrode array.

7. The method of claim 1, further comprising injecting the target tissue with a molecule.

8. The method of claim 1, wherein applying the electroporation pulse to the target tissue including applying the electroporation pulse with a multi el ectrode array comprising multiple sectors; and further comprising applying the electroporation pulse with different pulse parameters to different sectors of the multiple sectors to achieve different therapeutic effects in the target tissue adjacent the different sectors or to deliver different molecules to the target tissue adjacent the different sectors.

9. A pulsed electric field method comprising: injecting a target tissue with a first molecule and a second molecule; and treating the target tissue with a multi el ectrode array comprising a first sector and a second sector, including for each of the first sector and the second sector: applying an electroporation pulse to the target tissue adapted for a respective molecule of the first molecule or the second molecule, measuring an impedance of the target tissue adjacent a respective sector, and continuing to apply the electroporation pulse and measure the impedance until a desired impedance is reached.

10. The method of claim 9, further comprising, for each of the first sector and the second sector: further applying an additional electroporation pulse to the target tissue after the desired impedance is reached.

11. The method of claim 9, wherein the desired impedance is a steady state impedance.

12. The method of claim 9, wherein the desired impedance is a predefined percentage of a pre-pulse impedance measurement.

13. The method of claim 9, wherein the desired impedance is a predefined impedance value.

14. The method of claim 9, wherein the desired impedance is different for the first sector and the second sector.

15. The method of claim 9, wherein a pulse parameter of the electroporation pulse is different for the first sector and the second sector.

16. The method of claim 9, further comprising heating the target tissue to a preset temperature.

17. The method of claim 9, wherein the first molecule and the second molecule are each one of a vaccine, a therapeutic drug, a gene, a protein, a nucleic acid sequence, or a plasmid DNA.

18. A system for applying a pulsed electric field therapy, the system comprising: a control unit comprising a controller, an impedance measurement system in communication with the controller, and a pulse generator controlled by the controller; a handle coupled to the control unit, the handle comprising a radiation source controlled by the controller and a temperature measurement system in communication with the controller; and an electrode array cartridge removably coupled to the handle, the electrode array cartridge comprising an array of independently addressable electrodes in communication with the impedance measurement system and the pulse generator, and a series of holes between the electrodes to enable heat from the radiation source in the handle to reach a target tissue and temperature measurements of the target tissue to be obtained by the temperature measurement system in the handle.

19. The system of claim 18, wherein the electrode array cartridge is configured to be plugged into the handle.

20. The system of claim 18, wherein the controller comprising a memory and a processor, wherein the memory comprises instructions stored thereon that, when executed by the processor, cause the processor to control the pulse generator to apply an electroporation pulse via individual groupings of electrodes within the array of independently addressable electrodes.