Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/02—Details
  • A61N1/04—Electrodes
  • A61N1/0404—Electrodes for external use
  • A61N1/0408—Use-related aspects
  • A61N1/0412—Specially adapted for transcutaneous electroporation, e.g. including drug reservoirs
  • A61N1/0416—Anode and cathode
  • A61N1/0424—Shape of the electrode
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/02—Details
  • A61N1/04—Electrodes
  • A61N1/0404—Electrodes for external use
  • A61N1/0408—Use-related aspects
  • A61N1/0428—Specially adapted for iontophoresis, e.g. AC, DC or including drug reservoirs
  • A61N1/0432—Anode and cathode
  • A61N1/044—Shape of the electrode
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/02—Details
  • A61N1/04—Electrodes
  • A61N1/0404—Electrodes for external use
  • A61N1/0472—Structure-related aspects
  • A61N1/0476—Array electrodes (including any electrode arrangement with more than one electrode for at least one of the polarities)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/325—Applying electric currents by contact electrodes alternating or intermittent currents for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/326—Applying electric currents by contact electrodes alternating or intermittent currents for promoting growth of cells, e.g. bone cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/327—Applying electric currents by contact electrodes alternating or intermittent currents for enhancing the absorption properties of tissue, e.g. by electroporation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
  • A61N1/30—Apparatus for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body, or cataphoresis
  • A61N1/303—Constructional details
  • A61N1/306—Arrangements where at least part of the apparatus is introduced into the body

Definitions

• the present invention relates to the delivery of electrical waveforms and, more particularly, to the design of electrodes and electrode arrays for efficiently delivering electrical waveforms in vivo.
• Iontophoresis involves the application of electric currents to drive or repel oppositely charged particles through tissue. Iontophoretic devices have been known since the early 1900's. U.S. Pat. Nos. 3,991,755; 4,141,359; 4,398,545 and 4,250,878 disclose examples and some applications of such devices.
• Electro-osmosis occurs when an electric field is applied parallel to a charged surface in contact with a solution. The net ion movement causes a hydrodynamic flow of all molecules which are nearby in the solution (Dimitrov et al. (1990)). During electroporation, the transport of therapeutic agent across the cell membrane is commonly believed to involve diffusion, the movement of molecules from areas of higher concentration to areas of lower concentration. This transmembrane movement into the cytosol of electroporated cells may be enhanced by electro-osmosis (Sowers
• Electroporation refers to the application of electric fields of sufficient intensity and duration as to induce transient increases in cell membrane permeability.
• the cell membrane is a selectively permeable barrier that greatly inhibits the penetration of many therapeutic agents into the cytosol.
• Mir et al. (1992) report a 10, 000-fold increase in the cytotoxic activity of bleomycin, a normally impermeable chemotherapeutic agent, in the electroporated cells.
• Rols et al. (1990) describe electroporation as a threshold dependent phenomenon in that electric field intensity must be higher than a critical threshold to induce cell permeability. They further report that the extent and duration of membrane permeabilization is dependent on pulse duration and number. Provided that the electric field strength was not too high and the pulse duration not too long, electroporation of the cell membrane appears reversible (Zimmerman (1986) pg 177). Thus there exists an opportunity for the use of electroporation of cell membranes to achieve therapeutic benefits. In order to achieve success, the electric fields propagated in tissue by the delivery of specific electrical waveforms must apply sufficient transmembrane voltage and pulse duration to induce cell membrane permeability, yet not exceed inherent upper limits leading to cell lysis (death).
• Nishi, et al. (1996), Ceberg, et al. (1994), Salford, et al. (1993) and Okino, et al. (1987) also describe two electrode systems for tissue electroporation. Where described therein, the electrodes are of rod type (needle) construction, acutely placed in tissue, and spaced approximately 0.5-2cm apart.
• the prior art does not provide clear guidance as to the need for uniformity in the electric field propagation. Additionally, there are not known to be methods for the confinement of threshold level field intensities to the targeted tissue.
• panel A the electric field propagated in tissue by a two electrode system 20a-20b as described in the prior art would be considerably weaker in the region of tissue that is proximal to the midpoint between the electrodes (represented as the dashed box 22).
• the field strength in the tissue will weaken geometrically as the distance from either electrode 20a, 20b is increased (panel B), and the field strength in the mid-region of tissue will also weaken geometrically as the distance "L" between the two electrodes is increased.
• electroporation is considered to be a threshold-dependent phenomenon, with inherent upper limits due to the risk of cell lysis, a two electrode system is poorly suited to establish uniform electric field coverage, i.e., uniform electroporation, in the tissue targeted for treatment.
• Plate-type electrodes 24a, 24b aligned in parallel have been proposed to provide a uniform electric field 26 for electrochemotherapy delivered transcutaneously (U.S. Pat. No. 5,468,223 to Mir), and U.S. Pat. No. 5,439,440 to
• Hofmann describes an electrode for in vivo electroporation as spaced apart parallel arrays of needle electrodes 28a, 28b, 28c, 28d mounted on a dielectric support member 30 (see Figure 3).
• This design allows adjustments in needle depth and spacing between the parallel arrays (separated by distance L), but not the spacing of electrodes within each array 32.
• the array design has features that suggest similarity to the plate-type electrodes of Figure 2.
• the spacing 32 between adjacent electrodes 28a, 28b in the same array would need to be in the range of 0.25 L or less.
• the anatomy of the tissue involved may limit the number of electrodes that can be spaced within the targeted region.
• the needle arrays are described as adjustable in depth, there is no disclosure providing a means to confine the field effects for deep tissue applications in the third dimensional axis relative to the electrode arrays.
• the present invention provides electrode and electrode array apparatus which facilitates the efficient delivery of electrical waveforms, and particularly delivery to a predetermined three dimensional region of tissue within a patient. Electrical waveforms of certain parameters, comprising many different therapeutic objectives and techniques, may be efficiently delivered through the present invention, while minimizing the risk of trauma to the patient.
• the invention provides an electrode array having at least three individually addressable electrodes disposed so as to form a triangle in a plane intersecting the electrodes.
• a system wherein the electrode array is located in situ in a patient. When combined with elongate electrodes having defined conductive regions, the system defines a predetermined treatment region within the tissue of the patient.
• a further aspect of the invention provides an elongate electrode apparatus having a means for connecting the electrode to a source of electrical signals, an electrically conductive region located distal to the connecting means, and an electrically nonconductive region located proximal to the connecting means.
• the invention also provides an efficient means of expanding the electrode array "cell,” or any alternative cell design, into a multi-cell grid for controlled electric field propagation in an expanded cross sectional area of the predetermined region.
• individual electrodes may be of varying lengths and proportions, comprising a distal electrically-conductive portion and a proximal electrically- nonconductive portion.
• panel A is a two dimensional schematic view depicting the field lines and approximate electric field intensity for a two electrode model, wherein the intra- electrode distance is L and the dashed box positioned midway between the two electrodes indicates the region of lowest electric field strength, and panel B is a graphic representation of the relationship between the distance from the electrode and the electric field strength;
• Figure 2 is a schematic view depicting the field lines and approximate electric field strength for a plate electrode model, wherein the plates generate approximately uniform fields in the central region and weaker fringe electric fields at the edges;
• Figure 3 depicts an electrode array of the prior art which includes spaced apart parallel arrays of needle electrodes mounted on a dielectric support member;
• Figure 4 depicts an electrode array configuration in accordance with the invention, exemplified as a tricell array mounted on a dielectric support member;
• Figure 5 depicts the electric field generated in the array of Figure 4 in a characteristic trilobe pattern, wherein the shaded area indicates the region where the electric field strength exceeds the electroporation threshold;
• Figure 6 depicts three interlocking trilobe field patterns resulting from the sequentially shifting electric field activation of the array of Figure 4;
• Figure 7 illustrates a box array of needle electrodes with a core electrode mounted on a dielectric support member;
• Figure 8 illustrates a box array of needle electrodes without a core electrode mounted on a dielectric support member
• Figure 9 depicts a hexagonal electrode array of needle electrodes mounted on a dielectric support member
• Figure 10 depicts the expansion of the tricell array of Figure 4 into a trigrid pattern of interlocking tricells
• Figure 11 depicts the use of a four channel control means to activate the tricells in a trigrid, so as to provide the interlocking trilobe field patterns of Figure 6;
• Figure 12 depicts the individual trilobe field patterns resulting from the four channel control means used to activate the tricells in a trigrid, with the arrows indicating the polarity along coincident lines;
• Figure 13 depicts the expansion of the box array of Figure 7 into a multi cell grid pattern of overlapping cells;
• Figure 14 depicts the expansion of the box array of Figure 8 into a multi cell grid pattern of overlapping cells
• Figure 15 depicts the expansion of the hexagonal array of Figure 9 into a multi cell grid pattern of overlapping cells
• Figure 16 depicts the placement of an electrode array of the invention in situ in the brain of a patient
• Figure 17 depicts cascaded electrical signal pulses of increasing electric field intensity
• Figure 18 depicts the cascaded pulses of Figure 17, followed by pulses creating electric fields of decreasing intensity
• Figure 19 is a Kaplan-Meier survival chart for the experimental subjects described in example 2;
• Figure 20 is a Kaplan-Meier survival chart for the experimental subjects described in example 3;
• Figure 21 is a Kaplan-Meier survival chart for the experimental subjects described in example 4.
• Figure 22 is a Kaplan-Meier survival chart for the experimental subjects described in example 5.
• the present invention provides apparatus and systems which facilitate the efficient delivery of electrical waveforms, and particularly such waveform delivery to a predetermined three dimensional region of tissue within a patient. Electrical waveforms of certain parameters, comprising many different therapeutic objectives and techniques, may be efficiently delivered through the present invention, while minimizing the risk of surgical trauma to the patient.
• the invention provides an electrode array having at least three individually addressable electrodes disposed so as to form a triangle in a plane intersecting the electrodes.
• the electrodes can be provided in any form capable of conducting an electrical signal and establishing an electric field between oppositely- charged electrodes. Desirably, such electrodes will be of surgical quality, bio- compatible, and capable of withstanding the demands of implantation and use in a patient. Materials commonly employed in the construction of such electrodes include nickel titanium, gold, silver, stainless steel, platinum, platinum iridium alloys, graphite, ceramic, and the like.
• the electrodes will be elongate, for example rod electrodes 34a, 34b, 34c (see Figure 4), so as to simplify the surgical implantation of the electrode in the patient.
• rod electrodes such as acupuncture needles, which are usually of surgical quality, strength and (presumptively) bio-compatible stainless steel construction.
• needles such as acupuncture needles, which are usually of surgical quality, strength and (presumptively) bio-compatible stainless steel construction.
• the relatively sharp point of such a needle electrode may provoke arcing of the electric field, particularly at the electrode delivering the electrons in the propagation of the electric field (i.e. the "pulse electrode").
• the point of the electrode distal from the source of electrical signals includes a radius of curvature sufficient to substantially eliminate arcing at the electric field strengths utilized in the practice of the present invention, for example in the range of 0.1 to 1mm. It has also been found that the diameter of the rod electrode has a similar effect on arcing potential, due in part to the relationship of the electrode diameter to the radius of curvature of the point.
• the rod electrode itself will desirably also be of sufficient diameter to satisfy the arcing-reduction conditions as well.
• Such elongate electrodes will desirably include a means for connecting to a source of electrical signals 36 and a non-conductive region 38 located proximal to the signal source.
• non-conductive is taken to mean that the electrode does not permit an electric field to propagate radially from the electrode in the non-conductive region, while allowing the electrical signal or waveform to proceed axially or longitudinally through the non-conductive region.
• Such electrodes will also include a conductive region 40 located distal from the signal source.
• these electrode regions 38, 40 can be provided by shielding portions of the electrode 34 with a dielectric material, for example, with plastic, Teflon ® , ceramic, graphite, polyimide, and the like.
• the electrode may be of composite construction, for example where the cross sectional diameter of the electrode is maintained relatively constant in order to facilitate insertion of the electrode 34 into the tissue of the patient.
• such an electrode 34 with a nonconductive region 38 will permit the establishment of a predetermined region of tissue for treatment, where two of the dimensions are established by the geometry of the electrode array, and the third dimension is established by the length of the conductive region 40 of the electrode 34, i.e. the "effective electrode length.”
• one aspect of the invention is a three electrode array (termed a "tricell") comprising three elongate electrodes of predetermined length, disposed approximately parallel to each other, and spatially oriented in the "X" and "Y" dimensions of a three dimensional predetermined region, desirably in the approximate geometrical shape of an equilateral triangle.
• the "Z" or third dimension of the predetermined region is dependent on the effective electrode length established by the conductive region(s).
• a system is provided wherein the present electrode array is located in situ in a patient. When combined with elongate electrodes having defined conductive regions establishing effective electrode lengths, the system defines a predetermined treatment region within the tissue of the patient.
• This predetermined treatment region can then be subject to the electropermeabilization effects of the electric fields, promoting the introduction of therapeutic agents into cells within the region.
• the present invention can be readily applied in any environment in which the delivery of electrical waveforms is considered desirable, the invention is particularly suited for the delivery of electrical waveforms in vivo to patients expected to benefit from such treatment.
• Such therapeutic uses presently include bone repair, dermal wound healing, nerve regeneration, transport and delivery of therapeutic agents, and the like.
• the invention will find use in aiding in the administration of therapeutic agents in situations in which such administration has been considered problematic. Among these situations are included chemotherapy, and particularly chemotherapeutic treatment of brain tumors.
• the term "therapeutic agent” includes pharmacological agents, chemotherapeutic agents, nucleic acids, genes, antibodies and all other compositions which provide therapeutic benefit to a patient in need of treatment.
• One therapeutic application of electroporation consists of infusion of an anti-cancer drug into the patient, and electroporation of the drug into the tumor cells by applying electric fields between the electrodes.
• the present invention provides an array of individual electrodes organized into a geometry which efficiently delivers electrical waveforms to a predetermined region of tissue within a patient.
• One aspect of this efficiency is to maximize the electrical waveform intensity and uniformity for a given intra-electrode distance while minimizing the actual number of electrodes which must be placed in order to insure complete coverage of the predetermined treatment region
• the present invention thus provides an apparatus comprising an electrode array of a pre-determined geometry which can be implanted as a system in situ in a patient.
• the electrodes are each connected through conductive cables to a high voltage switching device, for example a Cyto PulseTM PA- 101 sequencer (available from Cyto Pulse Sciences, Inc., Columbia, Maryland) with software modifications to permit simultaneous activation of multiple electrodes.
• the switching device will in turn be connected to a suitable power supply or pulse generator, for example, a Cyto PulseTM PA 2000 or PA 4000 power supply, or a BTXTM T820 power supply (available from Genetronics, San Diego, California).
• the switching of electrical signals between the individual electrodes can be accomplished by numerous means, e.g. manually, mechanically, electrically, or by means of a circuit controlled by a programmed digital computer.
• the precise mechanism used in switching the electrical signals between individual electrodes will largely be chosen as a matter of convenience.
• the amplitude of the waveform applied to the array is based on the intra-electrode distance and should be selected so as to propagate electric field strengths sufficient to obtain the intended therapeutic benefits.
• the present array will allow for the efficient delivery of the electrical waveforms to the predetermined tissue region within the patient.
• an array as described above is disposed within the tissue of a host patient so as to adequately define the intended region for therapeutic treatment. Once the array is established and connected to the pulse generator, electric signals are directed to the electrodes in order to propagate electric fields of predetermined amplitude and duration through the tissue between the electrodes.
• the parameters of the signal are generally selected so that the tissue between the electrodes is subjected to electric fields strengths of high intensity and short duration.
• the voltage will be adjusted so that the generated field has the desired intensity.
• These fields will make the membranes of preselected cells in the tissue transiently permeable in order to permit the therapeutic agents to enter the cells in the predetermined region. This transient increase in permeability is believed to result from the temporary formation of pores within the cell membranes large enough to permit migration of the agent through the membrane and into the cytosol.
• the present array can be desirably established as conductive rod electrodes 34a, 34b, 34c, optionally mounted on an insulated or dielectric carrier or support member 45 and held in a predetermined geometry while allowing the electrodes to be inserted to a desired depth in the tissue.
• the electrodes can be implanted individually, and then optionally affixed to a support or directly to the patient (as depicted schematically in Figure 16).
• the electrodes can be provided with a penetration stop so that the predetermined depth of insertion can be achieved in minimal time.
• the geometry of the array established on the carrier can be subject to adjustment for intra-electrode distance, and a distance spacing sensor may be used to generate a signal either to the operator or directly to the pulse generator so that voltage adjustments can be made in order to establish the desired electric field.
• an array as above described is selected and optionally mounted on a suitable carrier which can then be stereotaxically positioned in relationship to the patient so as to facilitate the accurate and rapid establishment of the electrode array in situ in the patient.
• a suitable carrier which can then be stereotaxically positioned in relationship to the patient so as to facilitate the accurate and rapid establishment of the electrode array in situ in the patient.
• acute placement of the electrodes will be made into the peripheral regions of the cavity remaining after surgical resection of a tumor mass.
• Electrode array geometry can be maintained by a support, while electrode placement and depth would be determined by the surgeon.
• the therapeutic agent will then be infused into the patient either systemically or by direct injection into the vasculature of the predetermined tissue region or directly into the tissue itself, or by methods as described in Application Serial No. 08/476,714.
• the function of the pulse or signal generator is to generate a predetermined electrical waveform which, when applied to the electrodes, results in the propagation of electric fields of the predetermined amplitude and duration through the tissue that is positioned between the electrodes.
• these waveforms are applied repeatedly so as to optimize the electropermeability effect.
• two elements are ordinarily controlled to provide the intended benefits: i) the electrical waveform parameters delivered by the signal generator, and ii) the electrical state of each individual electrode within the array during the delivery of the waveform.
• the electrical waveform parameters which can be controlled include the type of waveform, the signal amplitude, and the pulse duration. These individual parameters determine the characteristics of a single pulse. In certain embodiments it is desirable to vary these characteristics between pulses. Pulse-to-pulse variability dictates that one or more individual parameters, such as amplitude, be manipulated from one pulse to the next in order to increase safety or therapeutic efficacy.
• Figure 18 depicts a pulse train wherein a high amplitude pulse 46 is followed by a series of lower amplitude pulses 48.
• Pulse sequences determine the manner in which individual pulses are integrated. In this regard, the number of, and time interval between, individual pulses in a sequence is regulated, as well as the number of pulse sequences in a course of therapeutic treatment (e.g. 46, 46', 46" in Figure 18).
• Control of the electrical state of the individual electrodes involves a system capable of individually addressing electrodes within an array.
• One embodiment of such control would incorporate a high voltage switching device wherein the electrical state of each electrode is controlled by an individual switch.
• Each switch would have three settings: i) pulse positive (+), ii) pulse negative (-), and iii) open (i.e. off). Together these switches could be electrically activated in any of numerous possible combinations.
• an array of "tricells" can be pulsed in a "trigrid” pattern with only four channels (as depicted in Figure 11), system flexibility in establishing alternative array patterns suggests the use of individual switches for each electrode in the array.
• the integrated control of these and other elements of the invention can be accomplished by utilizing microprocessor or digital computer technologies.
• data pertaining to realtime waveform delivery can be digitized and acquired for evaluation and verification of proper system function. Desirably, such data could be stored within the local system until uploaded to a central computer for further use.
• Electric fields will be established within the target tissue by delivery of electrical waveforms to preselected electrodes.
• the parameters of the signal will define the electric field strength E (in kV/cm) within the tissue.
• E the electric field strength
• Each protocol and tissue will have its own characteristics which determine the critical field strength for optimum therapeutic effect. This tissue variation is due to cell size, membrane composition and the individual characteristics of the cell membrane itself. Generally, the required field strength varies inversely with the size of the cells contained within the tissue. Mammalian tissue generally requires field strengths of between approximately 0.2 kV/cm to 3 kV/cm for electroporation.
• the electrical waveform provided by the generator can be an exponentially decaying pulse, a square pulse, a unipolar oscillating pulse train or a bipolar oscillating pulse train. Square pulses are generally sufficient to implement the majority of techniques.
• the electric field strength can desirably be between 0.2 kV/cm to 20 kV/cm, more commonly 0.5 kV/cm to 3 kV/cm.
• the pulse duration can be from 100 nanoseconds to 100 milliseconds and there can be from 1 to 10,000 pulses per second.
• the waveform, strength and pulse duration will be dependent on the tissue in the predetermined region and the type of therapeutic agent that is to permeate the cells via electroporation.
• the present invention provides an apparatus and system for the transport and delivery of therapeutic agents in vivo to a predetermined region of tissue within the host.
• Such transport and delivery consists of, in part: i) the localized circulatory extravasation of systemically administered therapeutic agents; (ii) the local extracellular infusion of therapeutic agents; (iii) the distribution and concentration of therapeutic agent within the predetermined region of tissue; (iv) the electroporation of cell membranes within the predetermined region of tissue; and (v) the transport of the therapeutic agents into the cytosol of the electroporated cells within the predetermined region of tissue.
• one embodiment of the electrode array representing a single electrode cell (a tricell as depicted in Figure 4), is comprised of three individually addressable penetrating electrodes ordinarily of approximately uniform predetermined lengths, desirably of biologically compatible construction and with electrically nonconductive proximal regions and electrically conductive distal regions; such electrically conductive distal regions defining the effective electrode length.
• the effective electrode length of each individual electrode may be different and will be of a predetermined value in order to confine the electric field effects to the predetermined region for tissue treatment.
• Such predetermination of electrode length shall be of sufficient value as to assure appropriate consideration for the weakened electric field effects encountered at either end of the electrode, as depicted in Figure 2 (i.e. the field-fringe effect 33).
• the present invention also provides an efficient means of expanding the electrode array tricell, or any alternative cell design, into a multi-cell grid for controlled electric field propagation in an expanded cross sectional area of the predetermined region.
• a tricell as depicted in Figure 4 can be expanded in a multi-cell pattern (i.e., a "trigrid") to provide therapeutic coverage of the "X" and "Y" dimension of a predetermined treatment region which exceeds the linear dimension L (see Figure 10).
• the trigrid When compared to other electrode designs configured with the same L (see Figures 13, 14 and 15), the trigrid offers considerable advantages in uniformity of electric field coverage and efficiency when directly comparing the number of electrodes required to treat a given predetermined treatment region. This advantage is an important consideration in any clinical/surgical application.
• One benefit of this expansion into a trigrid is that once the basic tricell is established, each additional electrode placed in the array creates at least one and possibly two additional cells in the array grid.
• the manner in which the trigrid can be pulsed in a tricell-interlocking manner is the manner in which the trigrid can be pulsed in a tricell-interlocking manner, as depicted in Figure 12.
• This pattern provides complete therapeutic coverage of the predetermined treatment region with only three pulse combinations (see Figure 6), and requires only four electrode channels (see Figure 11) regardless of the number of electrodes in the complete array. Overall tissue trauma during surgery is thus minimized, a unique characteristic of the trigrid array.
• the optimal trigrid pattern and effective electrode length can be determined, thus defining the predetermined region targeted for treatment.
• these electrodes may be placed acutely for single treatment or chronically for a series of treatments to the same predetermined region in the patient.
• a localized hyperconductance of the extracellular fluids may be desirable in order to partially shunt or shift field intensities away from the electrode milieu and toward the more central portions of L in the predetermined region. This may be accomplished by direct injection of hyperconductive solutions by local injection, or by infusion through a hollow core in the electrode. Alternatively, in certain embodiments, such as electroporation, electrical pulse parameters determined to be of therapeutic benefit may be adapted in such a way as to initially induce localized increases in the conductance of the electrode milieu.
• One method of inducing such effect is a cascaded pulse sequence (see Figure 17) where applied voltages are cascaded from lower values initially 50, being progressively increased in each subsequent round 52, 54, and reaching optimal therapeutic pulse parameters in a final round of pulsing.
• electrically charged therapeutic agents are desirably distributed through and concentrated within the predetermined region by means of electrical waveforms delivered through the individually addressable electrodes of the present invention.
• the transmembrane delivery of therapeutic agent into the cytosol of the permeabilized cells is commonly believed to occur by diffusion.
• Electroosmosis may enhance this transmembrane delivery.
• electrical waveforms of a lesser intensity 48, 48' , 48" in Figure 18
• the electroporation waveforms after the electroporation waveforms have been delivered and before cell membrane resealing, in order to enhance the transport of the extracellular therapeutic agents into the cytosol of the electroporated cells.
• the following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
• Example 1 Tumor model used in survival studies.
• the experimental brain tumor model employed in the following examples was the 9L gliosarcoma in the female Fischer rat.
• Tumor implants consisted of 200,000 9L gliosarcoma cells suspended in 2.3 ⁇ L of Dulbecco's Phosphate Buffered Saline with 2% rat serum. After anesthetization by intra peritoneal injection of a ketamine/xylazine mixture, the scalp was shaved and swabbed with Betadine ® solution. After placement in a stereotaxic apparatus, a 1cm incision was made in the scalp above bregma. A burr hole was drilled in the skull 1mm anterior and 3mm lateral of bregma.
• Example 2 Single Treatment - Triangle Electrode Array (Tricell).
• the rats were again anesthetized with an i.p. injection of a ketamine/xylazine mixture.
• the scalp was again disinfected with Betadine ® .
• Another incision was made in the scalp and the burr hole was located.
• An electrode array comprising three elongate rod electrodes oriented in an approximate equilateral triangle was then placed in the stereotactic arm. The array was then placed around the burr hole so that each electrode was approximately equidistant from the implant hole. New burr holes were drilled to accommodate the electrodes. The array was then lowered with the stereotaxic arm to a depth of 5mm.
• the x-axis represents days of survival and the y- axis represents the percentage of subject alive on the selected day.
• Figure 19 demonstrates, 120 days after implant 43% of the treated animals survived, and showed no outward signs of the presence of a tumor mass.
• the dashed line represents a control group which received no treatment.
• Example 3 Multiple Treatments - Triangle Electrode Array (Tricell)
• Example 2 The tumor model from Example 1 was again employed for these experiments. Animal subjects were treated on day eight post-implantation with the protocol outlined in Example 2 for the triangle electrode arrays. On day eleven, the animals were re-anesthetized and, after reopening the scalp and removing the bone wax plugs, the electrodes were lowered into the existing burr holes. The identical treatment procedure was then performed on the animal. Results: In Figure 20, a Kaplan-Meier survival chart illustrates the survival rates for the multiple treatment groups. Fifteen animals received the two treatment procedure as described. As Figure 20 demonstrates, 120 days after implant 87% of the treated animals survived, and showed no outward signs of the presence of a tumor mass. The dashed line represents a control group which received no treatment.
• the brains of six of the 13 surviving subjects were then prepared according to the histological procedure of Example 2. Pathological examination of the tissue sections revealed no residual tumor.
• Example 1 Four electrodes were arranged into two approximately equilateral triangles with sides of 3mm. The preparation procedure from example 3 was followed for each subject. The electrode array was lowered to a depth of 5 mm. The pulsing regimen was based on the trigrid pulsing pattern outlined in Figure 12.
• Tri-grid pulsing Thirty minutes after the injection of the bleomycin agent, the array was pulsed four times in each of three combinations with a magnitude of 300 volts (1 kV/cm based on electrode spacing) and a pulse duration of lOO ⁇ sec. The electrodes were pulsed so that each "cell” was configured with a single primary and two secondary electrodes. All four of the electrodes were active in each configuration, creating an interlocking triangle array.
• a Kaplan-Meier survival chart illustrates the survival rates for the animals treated as described. As Figure 21 demonstrates, 120 days after implant 75 % of the treated animals survived, and showed no outward signs of the presence of a tumor mass. The dashed line represents a control group which received no treatment.
• the brains of the six surviving subjects were then prepared according to the histological procedure of Example 2. Pathological examination of the tissue sections revealed no residual tumor.
• Example 2 Three control protocols were performed to compare with the treatment groups. All subjects were implanted according to the procedure outlined in Example 1. The first group of 35 rats received no treatment (Drug (D) -, Electrical Pulsing (EP) -). The second group of eight rats were treated on day eight post implant in accordance with the procedure of Example 2, except they did not receive the therapeutic agent (D-, EP+). The third group of eight rats were treated on day eight post implant in accordance with the procedure of Example 2, except they did not receive the electrical pulses (D + , EP-). Results: In Figure 22, a Kaplan-Meier survival chart illustrates the survival rates for the animals in the control protocols. As Figure 22 demonstrates, no animals from any of the control protocols survived beyond 32 days after implant. There was no significant difference in survival between any of the groups (D- EP-, D- EP+ , D + , EP-), demonstrating that both the therapeutic agent and the electrical pulse regimen are necessary for significant increase in survival.

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• 1997
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