MXPA97008316A - Method of treatment using administration of drugs and genes through electroporac - Google Patents

Abstract

The present invention relates to an apparatus for the application of electric fields to a selected part of a living body, comprising a support means, a configuration of multiple electrodes mounted on said support means, at least a plurality of the electrodes has a needle configuration for penetrating the tissue and an electric pulse generator for applying pulses of high amplitude electrical signals to said electrodes for electroporation of the cells between said electrodes

Description

METHOD OF TREATMENT USING ADMINISTRATION OF DRUGS AND GENES BY MEANS OF ELECTROPORATION TECHNICAL FIELD The present invention relates to the treatment of ailments in humans and other mammals, more specifically, with an improved method and apparatus for the application of controlled electric fields for the in vivo administration of genes and pharmaceutical compounds within living cells of a patient, by means of electroporation.

BACKGROUND IN THE TECHNIQUE In the 70's it was discovered that electric fields can be used to create pores in cells without causing permanent damage. This discovery made it possible to insert large molecules into the cytoplasm of the cell. It is known that genes and other molecules, such as pharmacological compounds, can be incorporated into living cells by means of a process known as eletroporation. The genes or other molecules are mixed with the living cells in a buffer medium and short pulses of intense electric fields are applied to them. The cell membranes become transiently porous and the genes or molecules enter the cells. There they can modify the genome of the cells. Recently, electroporation was suggested as an approach to the treatment of certain diseases such as cancer. For example, in the treatment with chemotherapy of certain types of cancer it is necessary to use a dose of the drug high enough to kill the cancer cells without killing an unacceptably high amount of normal cells. This goal would be achieved if the chemotherapy drug could be inserted directly into the cancer cells. In general, some of the best cancer drugs, for example bleomycin, can not penetrate the membrane of certain cells. However, electroporation makes it possible to insert bleomycin into cells. A therapeutic application of electroporation is for the treatment of cancer. Experiments have been carried out in laboratory mammals, with the following report: Okino, M., E. Kensuke, 1990. The effects of a single high-voltage electrical stimulus with an anticancer drug in growing malignant tumors in vivo . Jap. Journal of Surgery. 20: 197-204. Mir, L.M., S. Orlowski, J. Belehrade Jr., and C. Paoletti. 1991 Potentiation by electrochemotherapy of the anti-tumor effect of Bleomycin by local electrical impulses. Eur. J. Cancer. 27: 68-72. Mir, L.M., M. Belehradek, C. Domenge, S. Orlowski, B. Poddevin, and collaborators carried out and reported clinical trials. 1991. Electrochemotherapy, a new anti-tumor treatment: first clinical trial. C.R. Acad. Sci. Paris. 313: 613-618. This treatment is carried out by the infusion of an anticancer drug directly into the tumor and the application of an electric field to the tumor, between a pair of electrodes. The field strength must be adjusted with reasonable accuracy so that the electroporation of the tumor cells is carried out harmlessly, or at least with minimal damage, to any normal or healthy cell. This can usually be done easily in external tumors, by placing electrodes on opposite sides of the tumor, so that the electric field is between the electrodes. In this way, the distance between the electrodes can be measured to apply the correct voltage to the electrodes according to the formula E = V / d (E = power of the electric field in V / cm, V = voltage in volts, and d = distance In centimetres) . When treating internal tumors, it is not easy to place the electrodes correctly and measure the distance between them. In the aforementioned main application, information is presented of an electrode system for live electroporation, in which the electrodes can be inserted into the body cavities. In the related United States Patent, No. 5,273,525, a syringe for injecting molecules and macromolecules for electroporation uses needles for injection, which also function as electrodes. This structure allows the subsurface placement of electrodes. It would be convenient to have an electrode device having electrodes that can be inserted into the tumors or adjacent to them, in order to be able to generate predetermined electric fields in the tissue for the electroporation of the tumor cells. Studies have also shown that it is possible to introduce large nucleotide sequences within the cells of mammals by means of electroporation (Eanault, et al., Gene (Amsterdam), 144 (2): 205, 1994; Nucleic acid research, 15 (3): 1311, 1987; Knutson et al., Anal Biochemistry. , 164: 4, 1987; Gibson, et al., EMBO J., 6 (8): 2457, 1987; Dower et al., Genetic Engineering, 1_2: 275, 1990; Mozo et al., Molecular Biology of the Plant, 1 ^: 917, 1991), making possible, for example, an efficient method of gene therapy.

EXHIBITING THE INVENTION Accordingly, the main object of the present invention is to provide an improved apparatus that can be conveniently and effectively positioned to generate predetermined electric fields in the previously selected fabric. Another of the main objects of the present invention is to provide an improved apparatus that provides effective and convenient means for placing electrodes within the tissue for the injection of therapeutic compounds into the tissue and the application of electric fields to the tissue.

According to a main aspect of the present invention, an electrode device for the electroporation application to a part of the body of a patient includes a support element, a plurality of electrode needles that are placed in an adjustable manner on said support element for its insertion within the tissue, in the positions and distances, from one another, selected, and means including a signal generator that responds to said distance signal to apply an electrical signal to the electrodes, proportional to the distance between said electrodes, to generate an electric field with the previously determined power. Another aspect of the invention includes needles that function to inject the therapeutic substances into the tissue, and function as electrodes to generate the electric fields for a portion of the tissue cells. In another aspect of the invention there is provided a therapeutic method which uses the apparatus with needles for the treatment of cells, especially tumorigenic cells.

BRIEF DESCRIPTION OF THE DRAWINGS The objects, advantages and features of this invention will be more readily appreciated with the following detailed description, when this description is read in conjunction with the accompanying drawings, in which: Fig. 1 is a side elevational, sectional view of a needle assembly, according to the preferred embodiment of the invention. Fig. 2 is a bottom-up view of the embodiment of Fig. 1. Fig. 3 is an assembly drawing showing a perspective view of an alternative embodiment of the invention.

Fig. 4 is a perspective view of the embodiment of Fig. 3, shown already assembled. Fig. 5 is a perspective view of a uniselect distributor of the electrode assembly of Fig. 4. Figs. 6a and 6b are a schematic illustration of the selected contact positions of the dispenser of Fig. 5. Fig. 7 is a perspective view of another embodiment of the invention. Fig. 8 is a perspective view of yet another embodiment of the invention. Figs. 9a to 9d are a plan view from above illustrating a preferred form of the electrodes and the sequence of use. Figs. 10a and 10b show the volume of the tumor, after 43 days of electroporation with bleomycin, in nude mice with Panc-3 xenon grafting. (D = drug, E = electroporation). Fig. 11 is an illustration of the tumorigenic growth of Panc-3 cells, after ECT with bleomycin to nude mice. Figs. 12a and 12b show the volume of the tumor after 20 and 34 days of ECT with bleomycin, respectively, in nude mice with xenon graft of non-small cell lung carcinoma (NSCLC). (D = drug, E = electroporation) Fig. 13 shows the volume of the tumor after 34 days of ECT with bleomycin, in nude mice with xenon graft of non-small cell lung carcinoma (NSCLC). The arrows indicate the treatment of a mouse on day 27. (D = drug, E = electroporation) Figs. 14a and 14b show the administration of previous pulses with neocarcinostatin to Panc-3 and NSCLC, respectively, in the nude mouse model. Figs. 14c and 14d show the administration of subsequent pulses with neocarcinostatin to Panc-3 and in the nude mouse model.

THE BEST WAY TO CARRY OUT THE INVENTION As used in this document, the term "molecules" includes agents, genes, antibodies or other pharmacological proteins. A therapeutic application of electroporation in humans consists of the infusion of an anticancer drug and the electroporation of the drug into the tumor by applying voltage pulses between electrodes placed on opposite sides of the tumor, called electrochemotherapy (ECT). The present invention was devised mainly to be able to make the ECT, as reported by Okino and Mir and collaborators, can be applied to non-superficial tumors such as those that exist within the body. However, it can be used for other therapeutic applications. Referring to Fig. 1 of the drawings, generally numeral 10 illustrates and designates a set of needles according to the preferred embodiment of the invention. The needle assembly includes an elongated tubular support body 12, which preferably has the shape of a hollow stainless steel shaft. At the lower end of the shaft 12 is mounted a support for the central needle 14, which has a central hole 16 for accepting and guiding a central needle 18. The shaft 12 includes a needle exit groove 20 through which extends the electrode of the needle 18 from the inside thereof to the outside where it is secured by means of a fastener 22 to the outside of the tube 12. The upper end of the electrode 18 can be secured to a screw 24 to connect it to a circuit electric. The lower end of the tubular support 12 includes the threads 26 for threadably accepting a ring 28 for mounting a plurality of needles and a stop collar 30 for stopping or securing the ring 28 in place.

In the slots 34 a plurality of needles 32 are mounted, at equal intervals around the outer surface of the needle ring 28. In this way there is a circular configuration of needles arranged at equal intervals, eight in the illustrated embodiment. The needles are held in place by a band holder 36, the ends of which are held by a screw or nut and bolt 38, and which also serve as an electrical connection for the needles. The band holder attaches and holds the needles directly in place. This set of electrodes is designed to apply electrical energy to living tissue when the needles are inserted into the tissue. The central needle 18 functions as an electrode, as an anode or cathode, and the other needle or the annular needle configuration 32 functions as the opposite electrode. When the fasteners are installed and secured, all of these needles are held in fixed positions. One or more of the needles can be cannulated or tubular in order to inject tissue into the tissue of genes, pharmaceuticals or other substances. In practice it is necessary to adjust the central needle to obtain the desired penetration in the tissue. This is achieved by releasing the pressure from the central needle holder 22 and sliding the central needle 18 outwards or inwards, as seen in Fig. 1, so that it projects from the central needle guide 14. up to the desired penetration distance. The needle is then held in place. After that, the annular needles 32 are adjusted to obtain the desired penetration into the fabric. This is achieved by releasing the pressure of the band holder 36 and sliding the needles 32 to the desired position. It is also possible to make minor adjustments by moving the ring of the needle 28 to bring it closer or further away from the end of the shaft 12. The therapeutic substance can be injected into the tissue by means of one or more of these needles, or by separate means. Once all the needles have been adjusted for the correct penetration, the shaft 12 is taken and the needles are inserted into the tissue at the desired depth. A pulse generator is then connected to the electrode assembly and the correct voltage is applied to the electrodes. Before applying the voltage, the correct quantity of therapeutic substance is injected into the tissue, such as genes or molecules of the chemical or pharmaceutical suitable for the treatment of the tissue. A modification to this electrode assembly could include a solid non-penetrating electrode (not shown) in place of the central needle. The central non-penetrating electrode can be any conductor in a suitable manner, such as a button or a plate attached to the end of the shaft 12 to make contact with the surface tissue. The configuration of annular needles is adjusted to penetrate the tissue to the desired depth when the central electrode is resting on the tissue surface. The electrical energy flows from the needles that penetrate into the tissues and towards the central electrode on the surface. These configurations can be used to treat tumors that are close to the surface, when the circular array of electrodes is designed to enclose the tumor in a circle. The central electrode is placed so that electrical energy flows through the tumor to the central electrode. Another advantage of this electrode assembly is that all the needles 18 and 32 can be adjusted independently to obtain the desired penetration. The ring of the needles 28 can also be adjusted to be placed on the end of the shaft 12, so that the insertion of the central needle and the annular needles can be observed directly. In addition, the needle ring 28 can be of any size or configuration to surround the tissue area to be treated.

With reference to Figures 3 and 4, an alternative embodiment of a set of electrode needles with circular arrangement is illustrated therein, which is usually marked with the numeral 40. This needle assembly includes a circular arrangement of needles, from 42 to 52, which are mounted at equal intervals in a cavity 54 mounted on an elongated cylindrical shaft 56. Preferably, the cavity 54 has the diameter correctly selected to obtain the desired diameter of arrangements for placing it around a tumor or other tissue to be treated. One or more of the needles may be hollow to allow the injection of molecules of a therapeutic substance, which will be described in greater detail later in this document. The electrical connector receptacle assembly includes a body part 58 that has a central opening or hole 60 for receiving the shaft 56, and an annular arrangement of a plurality of receptacles, 62 to 72, for receiving the ends of the needles 42. at 52. Receptacles 62 through 72 electrically connect the needles with cables 74 through 84, which are connected to a distribution board, which will be described later.

The receptacles of the electrical connector 58 fit on the shaft 56, with the end of the needles projecting towards the electrical receptacles 62 to 72, to connect the cables 74 to 84. The axis 56 joining the cavity of the needle distribution 54 and the receptacle assembly 58, is mounted on a handle 86 adapted to be held in the hand. The handle 86 has an elongated cylindrical configuration adapted to be able to hold it with the hand and thus be able to handle it. The handle 86 has a front receptacle and includes a tubular shaft 88 that extends forward and has a hole 90 toward which the shaft 56 projects while the shaft 88 projects into an orifice (not shown) within the connecting piece 58. The shaft 56 projects towards the hole 90 and has an annular recess or slot 92 which is joined by means of a retaining clip, which includes a transverse pin 94 in a hole 96 biased to one side and which includes a hole 98 in which the annular groove 92 projects which is supported by the handle. The spring 102 which is in the hole 96 deflects the pin 94 towards the clamping position. The shaft 56 can be released to remove it by pressing the end 100 of the pin 94. The handle, when assembled as shown in Fig. 4, can be held by hand to insert the needles into the selected tissue area. Preferably, the needles 42 through 52 have the spacing and placement to surround the tissue selected for treatment. As explained above, one or more of the needles 42 through 52 may be hollow to allow injection of the desired therapeutic substance. The electrode cables 74 to 84 are subsequently connected with the preferred arrangement to a rotary distributor, as shown in Fig. 5, which allows the opposing pairs of needles to be selected to activate or apply electric current. The dnser, indicated generally with the numeral 104, includes a fixed case 106, which in the illustrated embodiment has generally a cylindrical configuration, and in which a rotor 108 with spaced contacts 110 and 112 connected by a pair of conductors 114 and 116 for driving a power generator 115. The rotor contacts 110 and 112 are placed inside the case 106 to join the annular contacts 118, 120, 122, 124, 126 and 128, to which they are connected the cables 74 to 84. In relation to Figures 6a, b and c, the rotor 108 has an internal part that includes the contacts 110 and 112, each of which acts as a bridge between the contacts 118 to 128, to which they are connected. connected cables 74 to 84, to connect the power supply. The internal contacts 110 and 112 rotate with the rotor 108, and can be selectively placed in conducting relationship with the pairs of internal contacts 118 through 128 to thereby activate opposite pairs of electrode pins. This allows the operator to choose the position of the electrodes surrounding a selected tissue, and selectively apply the direction of the electric field as desired to administer the optimal treatment. The rotor 108 allows the field to be generated selectively around or through the tissue from all directions. Referring to Fig. 7, this illustrates an alternative embodiment of an arrangement for the generation of an electric field with electrodes whose position can be adjusted in parallel, as indicated in the main application. The electrode assembly, generally designated 130, includes a pair of separate sets 132 and 134 of conductive electrode pins 136 and 138 mounted on a dielectric carrier or on a support piece 140. Needle assembly 132 is supported in a fixed fastener 142 that allows the depth of the needles 136 to be adjusted relative to the support 140.

The needles 138 are mounted on a movable fastener 146, which is adjustably mounted on a piece of support 140 by means of a fastening screw 148. Each of the needles 136 and 138 has a penetration stop 144. The opening spacer attachment screw 148 secures the fastener 146 at the selected location of the support 140. A sensor of the separation of the opening 150 detects the distance between the needle assemblies 132 and 134 and generates a signal that is sent to the pulse generator by means of the lead wire 152. A pulse generator is connected to the electrode pins by means of the wires 154 and 156. With reference to Fig. 8, this illustrates the details of a needle holder or template for various configurations, useful for establishing a spaced pair of parallel needle configurations. This embodiment includes a base support part 158 having a plurality of parallel adjacent slots 160, in which the selected needles 162 and 164 can be positioned with a selected space ratio. This support can serve to mount a pair of electrode needles with opposite polarization 162 and 164, as illustrated. These can be selectively placed with a selected space ratio to arrange them on opposite sides of a selected fabric. The needles are held within the slots by means of a fastener or a plate 159. Furthermore, the support can be used in combination with an additional support to have multiple multiple arrangements on opposite sides of a selected fabric. The illustrated needles can be connected by means of leads 166 and 168 to the correct pulse generator. With relation to the Figures. 9a to 9d, a further aspect of the invention is illustrated in these. As illustrated more clearly, the combination of electrodes may take the form of separate needles 170 and 172, which may be inserted first into or behind the area of a selected tissue, such as on opposite sides of a tumor 194, as shown in FIG. illustrate The needles can then be connected to a syringe or other source of molecules and used to inject a selected molecular solution into the tissue area. The needles may be non-conductive and a pair of electrodes 176 and 178 may be selectively inserted, as illustrated in Fig. 9b, by the hole or lumen of the respective needles within the tissue, as illustrated, and subsequently removing the needle , as shown in Fig. 9c. Each of the electrodes 176 and 178 has an isolated elongate conductor 180 and 182 with conductive tips 184 and 186.

Subsequently, a pair of conductors 188 and 190 of a suitable power generator can be connected to the tips of the electrode conductors by means of the microsupers 192 and 194, as shown in 9d, and apply an electric potential to the electrodes. This generates a field in the tissue and electroporates the cells of the selected tissue, such as those of a tumor or similar. This electroporation allows the selected molecules to enter the tissue cells and aniquilen or alter the cells efficiently and as desired. This needle and electrode shape can be used with any of the sets described above. The needle electrode assemblies described above allow the live placement of electrodes within or adjacent to subsurface tumors or other tissue. Although the present application is focused on electrochemotherapy, the embodiment of the invention in question can be applied to other treatments, such as gene therapy, to certain organs of the body. The nature of the electric field to be generated is determined by the nature of the tissue, the size of the selected tissue and its location. It is convenient that the tissue be as homogeneous as possible, and that it be of the correct amplitude. A field with excessive power results in the lysis of the cells, while a field with little power reduces the efficiency. The electrodes can be assembled and operated in various ways, including, without this being a limitation, those used in the main application. The electrodes can be manipulated conveniently in internal positions by means of forceps. The waveform of the electrical signal provided by the pulse generator can be an exponential decay pulse, a square pulse, a train of oscillating unipolar pulses or a train of oscillating bipolar pulses. The power of the electric field can be from 0.2kV / cm to lOkV / cm. The pulse duration can be from ten μs to 100 ms. There may be one to a hundred impulses. Of course, the shape of the wave, the power of the electric field and the duration of the impulse also depend on the type of cells and the type of molecules that will be introduced to the cells by means of electroporation. The various parameters, including the power of the electric field, necessary for the electroporation of any known cell, are generally available in the various research documents that exist on the subject, as well as in the Genetronics, Inc. database. , San Diego, California, assignee of the application object of this document. The electric fields required for cellular electroporation in vivo, such as ECT, have an amplitude similar to the fields required for in vitro cells, which are within a range of 100 V / cm at several kV / cm. This has been verified through the experiments of the inventors themselves and others that are reported in scientific publications. The first live application of pulsed electric fields in the field of chemotherapy for the treatment of tumors was reported in 1987 by Okino in Japan. The pulse generators for carrying out the procedures described herein are and have been available in the market for several years. A suitable signal generator is the CELL ELECTROMANIPULATOR Model ECM 600, which has commercially available GENETRONICS, INC. from San Diego, California, E.U.A. The ECM 600 signal generator generates a pulse of the complete discharge of a capacitor that results in a waveform with exponential decay. The electrical signal generated by this signal generator is characterized by the speed with which it rises and by the exponential tail of the impulse. In the signal generator, the power of the electroporation pulse is set by selecting one of ten timing resistors marked Rl to RIO. These are active in both High Voltage Mode (MAV) (fixed capacitance at fifty microfarads) and Low Voltage Mode (MBV) (with capacitance from 25 to 3), 175 microfarads). The ECM 600 signal generator has a control knob that allows to adjust the amplitude of the fixed load voltage applied to the internal capacitors from 50 to 500 volts in MBV and from 0.05 to 2.5kV in the MAV. The amplitude of the electrical signal appears on the screen included in the ECM 600 signal generator. This device also includes a plurality of push button switches to control the pulse length, in the Low Voltage mode, by means of of a simultaneous combination of parallel resistors to the output and to a bank of seven additional capacitors that can be selected. The ECM 600 signal generator also includes a single push button for automatic charging and impulse. This button can be pressed either to start charging the internal capacitors to set the voltage or to trigger a pulse to the outer electrodes in an automatic cycle that lasts less than five seconds. The manual button can be pressed in sequence to repeatedly apply the previously determined electric field. Preferably, the therapeutic method of the invention utilizes a square wave pulse electroporation system. For example, you can use the ElectroSquarePorator (T820), which also has available GENETRONICS, INC. Square-wave electroporation systems emit controlled electrical impulses that rapidly rise to a fixed voltage, remain at that level for a fixed time (pulse length), and rapidly drop to zero. This type of systems has a transformation efficiency efficiency for the electroporation of the plant protoplast and mammalian cell lines that is better than that of the exponential decay system. The ElectroSquarePorator (T820) is the first square wave electroporation system available on the market, which is capable of generating up to 3000 volts. The pulse length can be adjusted from 5μsec to 99msec. The square wave electroporation pulses have a smoother effect on the cells, which results in greater cell viability.

The T820 ElectroSquarePorator is active in both High Voltage (MAV) Mode (100 to 3000 volts) and Low Voltage Mode (MBV) (50 to 500 volts). The pulse length in MBV is approximately 0.3 to 99 msec and in MAV it is 5 to 99 μsec. The T820 has the capacity to generate approximately 1 to 99 pulses. Mir and others have used square-wave pulses for electrochemotherapy, which allow the insertion of chemotherapeutic agents into cancerous tumors. Mice were injected with a low dose of bleomycin. Next, electroporation was applied to the cancerous tumors, with the result of a reduction or total elimination of the tumors (Mir, L.M. Eur. J. Cancer, 27 (1); 68, 1991). Saunders compared the square wave with the pulses with exponential decrease, in the electroporation of the plant protoplast. Electroporation by square wave produced a higher transformation efficiency than pulses with exponential decay. It was also reported that it is much easier to improve the parameters of electroporation with square wave pulses, since sufficient transformation efficiency can be produced in a larger range of voltages (Saunders, Guide for Electroporation and Electrofusion, pp .227-247, 1991). The therapeutic method of the invention includes electrotherapy, which is also referred to herein as electroporation therapy, using the apparatus of the invention to carry the macromolecules to the cell or tissue. As previously described, the term "macromolecule" or "molecule" is used herein to refer to drugs (eg, chemotherapeutic agents), nucleic acids (eg, polynucleotides), peptides and polypeptides, including antibodies. The term polynucleotides includes DNA, cDNA and RNA sequences. The drugs whose use is contemplated in the method of the invention are typically chemotherapeutic agents that have an antitumor or cytotoxic effect. Such drugs or agents include bleomycin, neocarcinostatin, suramin, and cisplatin. Those skilled in the art will know other chemotherapeutic agents (for example, see the Merck index). The chemical composition of the agent will dictate the most convenient time for the administration of the agent, in relation to the administration of the electrical impulse. For example, without wanting to be bound by a particular theory, it is thought that a drug that has a low isoelectric point (for example, neocarcinostatin, isoelectric point = 3.78), may be more effective if electroporation is administered afterwards, to avoid electrostatic interaction of the drug with a high charge within the field. In addition, drugs such as bleomycin have a very negative P log (where P is the partition coefficient between octanol and water), their size is very large (MW = 1400), and they are hydrophilic, therefore they are intimately related to the lipid membrane, they diffuse very slowly inside the tumorigenic cell and are usually administered before the electrical impulse, or simultaneously. Electroporation facilitates the entry of bleomycin and other similar drugs into the tumorigenic cell by creating pores in the cell membrane. It may be advisable to modulate the expression of a gene in a cell by introducing a molecule using the method of the invention. The term "modular" supposes suppressing the expression of a gene when it is overexpressed, or increasing the expression when it is under-expressed. It is possible to use the nucleic acid sequences that interfere with the expression of the gene at the translation level, when the alteration of cell proliferation is related to the expression of a gene. This approach uses, for example, the nucleic acid without direction, riboenzymes, or triplex agents to block the transcription or translation of a specific mRNA, either by covering that mRNA with a nucleic acid without direction or with a triplex agent, or by unfolding it with a riboenzyme.

Nucleic acids without direction are DNA or RNA molecules complementary to at least part of a specific mRNA molecule (Weintraub, Scientific American, 262: 40, 1990). In the cell, nucleic acids without direction hybridize the corresponding mRNA, forming a double-stranded molecule. Nucleic acids without direction interfere with the translation of mRNA, since the cell will not translate a mRNA that is double-stranded. Older oligomers without a direction of approximately 15 nucleotides are preferred, since they are easily synthesized and the possibility of causing problems is less than with larger molecules when they are introduced into the target cell. The use of non-directed methods to inhibit in vitro translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem., 172: 289, 1988). The use of an oligonucleotide to stop transcription is known as the triple strategy since the oligo is rolled into a double helix DNA forming a three-strand helix. Therefore, these triple compounds can be designed to recognize a single site in the chosen gene (Maher et al., Research and Development Without Address, 1 (3): 227, 1991; Helene, C, Design of Anticancer Drugs, 6 ( 6): 569, 1991). The riboenzymes are RNA molecules that have the ability to unfold other single filament RNA, analogously to the endonucleases that restrict DNA. By modifying the nucleotide sequences encoding these RNAs, it is possible to create molecules that recognize specific nucleotide sequences in an RNA molecule and unfold it (Cech, J. Amer. Med. Assn., 260: 3030, 1988) . One of the main advantages of this approach is that because they have a specific sequence, only RNAs that have a particular sequence are inactivated. There are two basic types of riboenzymes, ie, tetrahymena type (Hasselhoff, Nature, 334: 585, 1988) and type "hammerhead". Tetrahymena-type riboenzymes recognize sequences that are four-base long, whereas "hammer-head" riboenzymes recognize base sequences with lengths of 11 to 18 bases. The longer the recognition sequence, the greater the possibility that the sequence occurs exclusively in the target mRNA species. Furthermore, to inactivate a specific species of mRNA, hammerhead riboenzymes are preferred to tetrahymena-type riboenzymes, and 18-base recognition sequences are preferred to short recognition sequences. The present invention also provides a therapy by gene for the treatment of immunological alterations or cell proliferation mediated by a particular gene or by the absence thereof. This therapy can reach its therapeutic effect by introducing a polynucleotide with or without direction inside the cells that undergo the alteration. The administration of the polynucleotides can be carried out using a recombinant expression vector as a chimeric virus, or the polynucleotide can be administered as an "isolated" DNA, for example. The various viral vectors that can be used in gene therapy, as indicated herein, include adenoviruses, herpes virus, vaccinia, or, preferably, an RNA virus as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or bird retrovirus. Some examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), murine sarcoma virus Harvey (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (SV). When the subject is a human, a vector such as the gibbon monkey leukemia virus can be used (GaLV). There are several other retroviral vectors that can incorporate several genes. All these vectors can transfer or incorporate a gene as a marker, so that the transduced cells can be identified and generated. Therapeutic peptides or polypeptides may also be included in the therapeutic method of the invention. For example, immunomodulatory agents or other biological reaction modifiers may be administered to be incorporated by a cell. The term "biological reaction modifiers" tries to cover the substances that are involved in the modification of the immune reaction. Examples of the immune reaction modifiers include compounds such as lymphokines. Lymphokines include tumor necrosis factor, interleukins 1, 2, and 3, lymphotoxins, macrophage activation factor, migration inhibition factor, colony stimulating factor, and interferon alpha, interferon beta, interferon gamma and its subtypes. Also included are polynucleotides that encode enzymes and metabolic proteins, including antiangiogenesis compounds, for example Factor VIII or Factor IX. The macromolecule of the invention also includes antibody molecules. The term "antibody", as used herein, includes intact molecules as well as fragments thereof, such as Fab and F (ab ') 2. With the method of the invention, the administration of a drug, polynucleotide or polypeptide, may for example be parenteral by injection, rapid infusion, nasopharyngeal absorption, absorption by dermis, and oral. In the case of a tumor, for example, a chemotherapeutic or other agent can be administered locally, systemically or injected directly into the tumor. When, for example, a drug is administered directly into the tumor, it is convenient to inject the drug in a "fanned" manner. The term "fanning" refers to administering the drug by changing the direction of the needle as the drug is being injected or by the application of several injections in several directions, similar to the action of opening a fan, instead of applying it to the patient. one place, so that there is a greater distribution of the drug throughout the tumor. In comparison with the volume normally used in the art, it is convenient to increase the volume of the solution containing the drug, when it is administered intratumorally (for example by injection), in order to ensure the proper distribution of the drug in all cases. the tumor For example, in the EXAMPLES included in this document, a person with knowledge in the art normally injects 50 μl of the solution containing the drug, however, the results improve greatly by increasing the volume to 150 μl. Preferably, the injection should be applied very slowly and at the periphery, instead of at the center of the tumor, where the interstitial pressure is very high. Preferably, the molecule is administered substantially contemporaneously with the electroporation treatment. The term "substantially contemporaneous" means that the molecule and the electroporation treatment are reasonably administered together with respect to time. The administration of the molecule or therapeutic agent can be carried out at any interval, depending on factors such as the nature of the tumor, the condition of the patient, the size and chemical characteristics of the molecule and the half-life of the molecule. Preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions and emulsions. Some examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters such as ethyl oleate. It is possible to use a carrier in the occlusion dressings in order to increase the permeability of the skin and improve the absorption of the antigen. Liquid dosage forms for oral administration may, generally, include a liposome solution containing the liquid dose. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups and elixirs containing the inert diluents commonly used in the art, such as purified water. In addition to the inert diluents, such compounds may also include adjuvants, wetting agents, and emulsifying and suspending agents. Moreover, vasoconstrictive agents can be used to keep the therapeutic agent localized before the impulses are applied. By means of the method any cell can be treated. The illustrative examples provided herein demonstrate the use of the method of the invention for the treatment of cancer cells, for example of the pancreas and lung. Other alterations of cell proliferation are also susceptible to treatment by the electroporation method of the invention. The term "alteration of cell proliferation" denotes populations of malignant as well as non-malignant cells, which often differ both morphologically and genotypically from the tissue surrounding them. Malignant cells (ie tumors or cancer) develop as a result of a process that includes several steps. The method of the invention is useful for treating malignant cells or other alterations of various organ systems, especially for example, the cells of the pancreas and lung, including also the cells of the heart, kidney, muscle, breast, colon, prostate, thymus, testes and ovaries. Preferably the subject is human. The following examples are intended to illustrate but not to limit the invention. Although they are typical of those that could be used, it is also possible to use other methods known to those having knowledge of the art.

EXAMPLES The following examples illustrate the use of electrochemotherapy (ECT) in a pancreatic tumor poorly differentiated from human (Panc-3), with a subcutaneous xenon graft on the left flank of a naked mouse. The single treatment procedure involved the intratumoral injection of bleomycin (0.5 units in 0.15 ml of saline), using the fan method as described in this document, followed by the application of six square-wave electric impulses, ten minutes then, using the needle configuration of the mark, placing the electrodes along the circumference of a circle with a diameter of 1 cm. Needle configurations with other diameters (for example 0.5 cm, 0.75 cm and 1.5 cm) can also be used to adjust to tumors of other sizes. In the center of the configuration, stops of different heights can be inserted to make the penetration depth of the needles within the tumor variable. An interconstructed mechanism allowed the electrodes to be changed so that the field to which the impulses were applied had maximum coverage of the tumor. The electrical parameters were: 1300 V / cm and pulses of 6 x 99 μs spaced at intervals of 1 second.

The results showed severe necrosis and edema in almost the entire treatment site of the mice. A substantial reduction in tumor volume (after a slight initial increase due to edema) of the treatment group mice (D + E +, D = drug, E = electric field) was observed, whereas in the animals of the group of control it increased drastically (D + E-). Twenty-eight days later an almost total regression of the tumor was observed in 90% of the mice treated with ECT. No reaction was observed in 10% of the mice. In 60% of the cases a total regression without palpable tumor was observed at 77 days after the initial treatment. However, there was a recurrence of tumor growth in 20% of the mice at 35 days after treatment, but with a slower growth rate compared to the control animals. This observation has been related to an incomplete treatment of large primary tumors, in which the depth of the needle was less than the Z dimension of the tumor. The histological analysis of the tumor samples showed ghosts of necrotic tumorigenic cells in the D + E + group, in comparison with a mixture of viable and necrotic cells in the D + E group. Preliminary studies with human non-small cell lung cancer (NSCLC) tumors, engrafted by xenon to nude mice, also showed very encouraging results with the treatment of ECT with bleomycin.

Example 1: AntiCancer, Inc., San Diego, provided the Panc-3 tumor cell line, a line of poorly differentiated pancreatic adenocarcinoma cells. For the ECT experiments, the tissues taken from the stock of mice in which the tumor line was maintained were thawed and cut into very small pieces, approximately 1 mm each, to be surgically applied by xenon grafting from 8 to 10 pieces in a subcutaneous sac practiced on the left flank of nude mice, which was closed with surgical suture 6.0. Once the average tumor size reached approximately 5mm, the mice were randomly divided with palpable tumors, 10 mice for the control group (D + E-, D = drug, E = electric field) and 10 mice for the control group. treatment with ECT, in this case with bleomycin injection followed by a pulse application (D + E +) with a BTX Square Wave Generator T820. The dimensions of the tumor were measured and the tumor volume was calculated using the formula: (II / 6 xaxbxc where a, g, and e are respectively the iargp, incho and coarse tumor.) 0.5 units of Bleomycin were dissolved (Sigma Chemicals) in 0.15 ml of 0.9% NaCl ^, and this was injected intratumously, using the fan technique, to each of the mice of the control group (D + E-) and the group of treated * i ento (D + E +). Ten: minutes after injection to each of the mice of group D + E +, pulses were applied with the BTX T820 square-wave electraporadar, using a set of electrodes with the needle configuration described in the present invention. The electrical parameters used were the following: field strength 13QQ V / cm, 6 pulses of 99 μs each, at intervals of 1 second. His daily monitored the mortality of the mice and any signs of illness were noted. The dimensions of the tumor were measured at regular intervals and the regression / progression of tumor growth was monitored. Another group of nude mice with non-small cell line xenon graft of lung cancer was also treated with the i but the procedure used for Panc-3 tumors. Figures 10a and 10b show the analysis of tumor volume, determined during a period of 43 days after ECT, using bleomycin for Panc-3 tumors. In terms of tumor volume, a drastic difference was observed between the untreated and the treated mice. Basically, there was no detectable tumor after approximately 24 days of treatment. A summary of the results in Figure 10 is presented in Table 1 below. An illustration of the actual regression of the tumor is shown in Figure 11.

TABLE 1 Cell line: human pancreatic tumor (panc3), poorly differentiated Mouse model: nude mouse Transplant: subcutaneous graft by xenon Control mice: Cl and C2 Treated mice: TI and T2 The Panc-3 experiment was repeated using a non-small cell lung cancer cell line. { NSCLC), 177 (AntiCancer, San Diego, CA). The results were similar to those found with bleomycin and Panc-3, as shown in Figures 12a and 12b. In one experiment, day 27 (Figure 13) and 7 days later a tumor that had relapsed was treated. No evidence of tumor was observed. The Panc-3 and NSCLC models were used with the drug neocarcinostatin (NCS), following the same procedures indicated above. As shown in Figure 14a and 14b, application of the NCS dose prior to the NCS pulses, similar to that used in the bleomycin studies, was not effective in reducing tumor size. It is thought that due to the low isoelectric point of the NCS, the electrostatic interaction prevented the drug from entering the tumorigenic cell. Therefore, this experiment was repeated by first applying the impulses and the injection of NCS after the impulses (pp). Figure 14c shows the initial tumor volume (I) compared to the final volume thereof (F) on day 13 of seven treated mice (Mice No. 1 to 7). In several of the mice (No. 1, 2, 4, and 7) an increase in the 4Q tumor volume, but this was due to edema. However, as shown in Figure 14d, when examining a group of 5 mice on day 23, all of them showed a marked reduction in tumor volume. A comparison of Figures 14a and 14b with 14c and 14d indicates that the application of the impulses before administering the NCS was more effective than administering the NCS before the impulses.

Summary These Examples illustrate that poorly differentiated pancreatic cancer (Panc-3) and non-small cell lung cancer (NSCLC) applied by xenon grafting to nude mice can be treated effectively by means of the electrochemotherapy protocol, using bleomycin. or NCS and an electrode needle configuration. There are other similar therapeutic agents that can also be used effectively with the method of the invention. The results show that a total regression of Panc-3 tumors was obtained in 60% of the treated group, with no palpable tumors observed even 77 days after a single treatment. A partial regression was observed (reduction of 80% of the volume of the tumor) in 30% of the cases, while only 10% did not react (Table 2). Histological studies clearly demonstrated severe necrosis in the tumor region of the group subject to ECT, although no apparent necrosis was observed in the control group. It was found that the intratumoral injection of the drug with a greater volume of bleomycin, in combination with the application in a fan to maximize the uniform distribution of the drug throughout the tumor volume, is more effective compared to the conventional way of injecting the drug. drug before the application of the impulses.

TABLE 2 Electrochemotherapy of Panc-3 with Bleomycin Number of treated mice: 10 CR: Total regression PR: Partial regression NR: No reaction * 1 mouse died after the second treatment 1 mouse died after surviving 64 days Although the invention has been described with reference to the presently preferred embodiment, it should be understood that it is possible to make several modifications without departing from the spirit of the invention. Similarly, the invention is limited only by the following claims.

Claims (24)

1. An electrode device for applying electric fields to a selected part of a living body, which includes: support means; a configuration of electrodes mounted on said support means with a certain space ratio between them, at least one of said electrodes having a needle configuration for penetrating the tissue and performing an in vivo electroporation of the tissue cells; and an electrical pulse generator for applying pulses of high amplitude electrical signals to the electrodes, proportional to the distance between said electrodes, for the electroporation of the cells located between said electrodes.

2. An apparatus according to Claim 1, wherein said electrode needle has a cannula for introducing molecules into said tissue.

3. An apparatus according to Claim 2, wherein said electrode configuration includes a central electrode of a first polarity and a plurality of electrodes of a second polarity enclosing said central electrode in a circle.

4. An apparatus according to Claim 3, wherein said support means includes a central tubular shaft, and said central electrode is adjustably mounted on said shaft and can be adjustably extended from said shaft to selectable lengths for depths. of penetration selectable within the tissue.

5. An apparatus according to Claim 4, wherein said support means includes a ring mounted on said shaft, and said electrodes have no needles, in a circular configuration supported on said ring.

6. An apparatus according to Claim 5, wherein said apparatus includes a rotatable distributor selectively positionable to connect to said pulse generator alternating opposite pairs of electrodes.

7. An apparatus according to Claim 1, wherein said support means includes a ring mounted on said shaft and said electrodes have a circular configuration of needles supported on said ring.

8. An apparatus according to Claim 1, wherein said electrode configuration includes a circular configuration of electrode needles, and a distributor for selectively changing the polarity of the electrodes opposite said electrodes.

9. An apparatus according to Claim 7, wherein at least one of said electrode needles has a cannula for injecting molecules into said tissue.

10. An apparatus according to Claim 1, wherein said electrode configuration includes a linear electrode needle configuration, and said electrodes can be adjusted relative to said support means to adjust the depth of penetration.

11. An apparatus according to Claim 1, wherein said electrode configuration includes a pair of spaced and linearly configured electrode needles, and wherein the ratio of said configurations can be adjusted on a support part, and includes to detect the distance between these configurations.

12. An apparatus according to Claim 1, wherein said support means includes a pair of tubular needles for inserting them into the selected tissue, and said electrodes are conductors that can be inserted by said needles into said tissue.

13. An apparatus according to Claim 12, wherein said needles can be removed from said electrodes.

14. An apparatus according to Claim 7 »wherein the field generator generates an electric field whose power is approximately between 0.2 kV / cm and 20 kV / cm, and between approximately one pulse and one hundred pulses for its application to a tissue.

15. The use of electroporation to introduce molecules into cells, including: providing an electrode configuration, having at least one of said electrodes a needle configuration for penetrating tissue; inserting said electrode needle into the selected tissue to introduce molecules into the tissue; placing a second electrode of said electrode configuration in conductive relationship with said selected tissue, such that said tissue is between said first and second electrodes; provide an electric pulse generator; connecting said electric pulse generator to said electrodes; and operating said electric pulse generator to apply pulses of electrical signals of high amplitude to the electrodes, proportionally to the distance between said electrodes, to make the electroporation of the cells of the tissue.

16. The use of Claim 15, wherein said step of providing said electrode configuration includes providing a first electrode needle and a second electrode needle, including a pair of tubular needles for inserting them into the selected tissue, and said electrodes are separable conductors that they can be inserted by said needles into said tissue.

17. The use of Claim 16, wherein said needles can be removed from said electrodes

18. The use of Claim 15, wherein said step of providing said electrode configuration includes providing a central electrode of a first polarity and a plurality of electrodes of a second polarity, said central electrode enclosing a circle.

19. The use of Claim 18, including the step of providing a dispenser including a rotary distributor that can be selectively positioned to connect to said pulse generator alternating opposite pairs of electrodes.

20. The use of Claim 15, wherein the molecule is selected from a group consisting of chemotherapeutic agents, a palinucleotide and a polypeptide.

21. The use of Claim 20, wherein the chemotherapeutic agent is bleomycin.

22. The use of Claim 15, wherein the tissue is selected from a group consisting of pancreas, lung, heart, kidney, muscle, breast, colon, prostate, thymus, testes and ovaries.

23. The use of Claim 15, wherein said step of providing said electrode configuration includes providing a multiple electrode needle configuration, including a plurality of opposing pairs of electrode needles to insert them into the selected tissue, and said step of applying pulses to said electrodes including the selective application of pulses to opposite pairs of electrodes.

24. The use of Claim 23, wherein said step of applying i pulses to said electrode configuration includes applying said pulses sequentially to pairs of electrodes in said configuration.