FR2872055A1 - Apparatus for transferring active principle molecules into tissue cells using one or two physical forces has two electrode needles and one or more injection needles - Google Patents

Abstract

The apparatus consists of at least two electrode needles (10) connected to an electrical impulse generator (21), and one or more injection needles (11) between the electrode needles, with which they are approximately or precisely aligned, with a container (1) to contain and deliver an active principle in fluid form. The injection needle can also be in the form of an electrode, but with a different polarity to the outer electrode needles, and pressure can be applied to the injected fluid by a motorised piston.

Description

Description

  TECHNICAL FIELD OF THE INVENTION The present invention relates to a device for improving the administration of substances in tissues and in the cells of these tissues, by associating the injection of active ingredient with a physical process.

  In a first embodiment of the invention, the physical process used is the administration of electric fields or electric currents. These fields and / or this current have the effect of improving the penetration of the active ingredient into the tissue. This results from an effect of electrophoresis or iontophoresis on the active principle, by which the molecules of the active principle are driven by electrical convection in the tissue or even within human, animal, plant or bacterial cells.

  Furthermore, in a preferred embodiment of the invention, the electric fields or the electric current also have the effect of permeabilizing the cells transiently. It is then, unlike electrophoresis or iontophoresis, an effect on the biological tissue. By the same token, the penetration of the active ingredient into the cells or within certain compartments of human, animal, plant or bacterial cells is increased, which improves the effectiveness of the active ingredient.

  In a second embodiment of the invention, the physical process used is the hydrostatic pressure generated by the rapid injection of a quantity of liquid simultaneously with the injection of the active ingredient. Such a pressure has the effect of forcing the entry of the molecules into the cells. Such an embodiment is particularly suitable in organs defined by a cell layer or a membrane such as and without limitation, the joints limited by the synovial membrane, the anterior chamber of the eye, or the bladder.

  In a third embodiment of the invention, the association of the two previous embodiments is used.

  State of the prior art. BACKGROUND OF THE INVENTION Antitumor chemotherapy is an example in which the penetration of the active ingredient into tumor cells is necessary for an activity.

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  therapeutic. For example, bleomycin has been used for the treatment of tumors in association with permeabilizing electric fields. This process uses tissue electroporation and is often called electrochemotherapy.

  Gene therapy or DNA or RNA vaccination is a second, non-limiting example, in which it is necessary for the active ingredient (DNA, RNA, interfering RNA or any other molecule having activity on gene expression) to be internalized. within the cell so that the effect on gene expression is observed. This particularly concerns nucleotides or pseudonucleotides.

  Electrotransfer or electroporation of DNA is the use of electric fields to promote the tissue and intracellular penetration of these DNAs, which makes it possible to express them in the form of RNA or protein if these DNAs contain a gene preceded by a promoter and followed by a polyadenylation sequence (a so-called therapeutic expression cassette).

  Electrotransfer has been used for plasmids, i.e., circular DNAs in tissues such as muscle, skin, tumors, brain or liver. This coding DNA may be a plasmid or any other form of genetic material (DNA, RNA or other, commonly referred to herein as the nucleotide) leading to the expression of the product of this gene, this product being an RNA or a protein. . Plasmid electrotransfer using suitable electric fields or currents allowed an increase in the expression of the product of the injected gene of several orders of magnitude in muscle, tumors, skin, and the nervous system or the liver. Electrotransfer consists of injecting the nucleotide into a tissue, and administering simultaneously or after pulses of fields and electric current, which permeabilize the cell wall and thereby promote the entry of the nucleotide into the cell and, in some cases, up to the core. Alternatively, some administered the permeabilizing electric fields prior to nucleotide delivery, or used the electric fields to enhance nucleotide diffusion into the injected tissue.

  In the following description, will be called electrically assisted the above described methods, as well as others not described but using the electric fields to improve the delivery of an active ingredient to biological tissues, as well as its effectiveness. 20

  Many injection systems for electrically assisted delivery have been designed. No. 5,273,525 discloses a syringe having two injection needles which also serve as electrodes for the administration of the electric fields and the current that flows therefrom. A variant of this patent is an electrode needle associated with a second electrode in the form of a knife inserted into the fabric.

  U.S. Patent No. 6,055,453 discloses the use of a plurality of identified electrodes for selecting an electric field application sequence.

  PCT application WO 98/47562 discloses the use of an electrode array of needles having at least three electrodes arranged to form a triangle in a plane of intersection of these three identified individual electrodes, and a means of generating electric fields between these electrodes.

  The PCT application WO 03/0760006 describes an array of at least three odd-numbered electrode needles which, paired together, deliver a maximum resultant electric current in a different region for each pair of electrodes, the injection site of the principle. active being located at the center of gravity of the grid formed by the electrodes.

  The patent application US 2004/0059285 describes the use of two electrodes - injection needles, in which the injection of the active ingredient is enslaved to the insertion of the needles into the tissue, the electrical pulses being delivered during or after the injection. depression of the electrodes. This application also has a pre-deposited guide for inserting the needle-electrode for injecting the active ingredient.

  The set of electrically assisted administration systems described to date can therefore be broken down into two categories.

  In the first group, the electrodes are also used for the injection of the active ingredient. These needle electrodes have the advantage of simplicity. However, one of the main problems related to this design is that the active ingredient is injected in the vicinity of the two electrodes. If part of the active ingredient is well in the most direct field lines between the two electrodes, a part is injected on the other side, and is therefore not in the optimal tissue zone from the point of view electropermeabilization and electrophoresis. This is shown in Figure 1.

  The importance of optimal localization of the active ingredient in the area where the electrical pulses are administered has been illustrated by others, in the case of plasmid electrotransfer in muscle, by the beneficial effect of injection of enzymes digesting extracellular matrices, such as hyaluronidase. Digestion of the extracellular matrix allows a better diffusion of the nucleotide.

  In the second electrically assisted administration group, the electrodes are dissociated from the injection needle. The insertion of a network greater than three electrodes is preceded or followed by the insertion of an injection needle, possibly using a guide. In a variant of this group, the product is injected into the tissue, followed by the insertion of an array of more than three electrodes. This system has the disadvantage of complexity. Indeed, the insertion of a multiplicity of electrodes can prove difficult when the fabric that one seeks to electrotransfer is small. This is the case, for example, of the muscle of a small pet such as the cat, or that of an infant, or for digital muscles or tumor metastases located near sensitive areas. Moreover, these systems require a long implementation time, since it is necessary to insert in two stages first the electrode array, then the injection needle, or vice versa. At this time is added that necessary for the administration of electrical impulses. This can be prohibitive when a minimum intervention time is necessary, such as the intervention on animal watch, or when the subject treated can not be anesthetized for medical or economic reasons (speed of implementation).

  In all these electrotransfer or electroporation techniques existing, the disadvantages noted require to increase the intensity of the electric field or the electric currents dispensed, which induces a certain toxicity. It is therefore necessary to identify a means of decreasing the intensity of the electrical pulses administered.

  In a second embodiment of the invention, the physical process used is the hydrostatic pressure generated by the rapid injection of a quantity of liquid within a limited volume of an organism. This method will be referred to as hydrostatic pressure assisted delivery. This has been used by others in intra-arterial or intravenous injection.

  In the case of intra-arterial or intravenous injection, a very large volume must be used to obtain a local pressure increase leading to the penetration of macromolecules. For example, one must inject in a few seconds into the vein of a rodent's tail a volume of liquid containing a nucleotide equivalent to the totality of its blood volume. Only under these conditions was good penetration of the nucleotide in liver cells observed. This method has also been used in skeletal muscle members.

  The problem with this approach is that it requires the injection of a very large volume of fluid, which greatly dilutes the active ingredient and is not devoid of toxicity. In addition, only hepatic cells and, to a lesser extent, muscle cells have been permeabilized by this approach.

  PRESENTATION OF THE INVENTION The present invention aims to overcome these disadvantages. Several systems are proposed. They make it possible to inject a solution of active principle in all the tissues and in particular in the muscle, in the tumors, in the joints, in the eye or in the bladder of an animal or human living or not, or in any other volume limited by a membrane.

  By active principle is meant any molecule having a beneficial effect or for analytical purposes. The analysis may consist of imaging, functional or otherwise. In particular, the active principle is understood to mean macromolecules of the peptide or nucleic acid type. Among the nucleic acids, plasmids or strands of synthetically produced linear DNA or RNA are a preferred form. The invention also relates to any nucleic acid, any protein, any sugar or any other biological molecule modified genetically or chemically, or any entirely synthetic molecule, manufactured according to the methods of the person skilled in the art.

  For the electrically assisted administration, the applicant proposes two exemplifications.

  The invention relates to a preferred form of electric field assisted delivery is shown in Figure 2. The system comprises two electrodes (10) and one or more injection needles (11) located between these two electrodes. 10

  The intersection of the pieces (electrodes and injection needles) with a plane roughly forms a line. The injection needle or the plurality of needles and the two electrodes are crimped in a crimping disc (12), and which allows the maintenance in a defined geometry. The set of injection needles, electrodes and crimping disk is called by the plaintiff injection part (2).

  This injection part is characterized, in a preferred form of the invention, by an exact or approximate parallelism between the two electrodes and the injection needle or needles. It is disposable in a preferred form of the invention.

  An additional piece results from the assembly of two half-cylinders (3, 4) forming an arm acting as a casing. This housing allows grip for injection and connection to an electric pulse generator. In a preferred form of the invention, it is reusable.

  As indicated in FIG. 2 by the schematic representation of the diffusion zone of the active principle (36), one of the characteristics of the invention is to position the active principle optimally in the zone of maximum electric fields. This therefore ensures the best conditions for intracellular penetration of the active ingredient, and therefore represents a significant improvement.

  The invention also relates to a second preferred form of electric field assisted administration, which is shown in FIG. 3. The invention proposed by the applicant contains three aligned electrodes, the central electrode (11) of which is used for injection of the active ingredient. One or more series of electrical pulses are given, the central electrode being of opposite sign to the other two This system has the optimum characteristics for administering high electric fields with a low voltage, since the distance between the electrodes is minimal, and that the electric field results from the ratio of the potential difference between the electrodes (voltage), divided by the distance between the electrodes. This represents an improvement over the disadvantage of toxicity observed in the state of the art.

  The intersection of the pieces (electrodes and injection needle) with a plane forms approximately or exactly a straight line. The electrodes are crimped into a crimping disk (12), and which allows the maintenance in a defined geometry.

  The set of needles and electrodes and the crimping disc is called by the plaintiff injection part (2).

  This injection part is characterized, in a preferred form of the invention, by an exact or approximate parallelism between the two electrodes and the injection needle or needles. This injection piece is disposable in a preferred form of the invention.

  An additional piece results from the assembly of two half-cylinders or equivalent forms (3, 4) forming an arm acting as a housing. This housing allows grip for injection and connection to an electric pulse generator. In a preferred form of the invention, it is reusable. Half-cylinders or equivalent forms are interconnected by a hinge constructed according to the rules of the art, for example a metal hinge or obtained by shrinking the thickness of the housing. The two half pieces of the case are then closed using a button, or a ring covering the outside of the case, or a clip, or a screw ring or any other system known to those skilled in the art.

  As indicated in FIG. 3 by the schematic representation of the diffusion zone of the active principle, one of the characteristics of the invention is to position the active principle in a favorable electric field zone. This therefore ensures the best conditions for intracellular penetration of the active ingredient, and therefore represents a significant improvement in the state of the art.

  In a preferred form of the invention, a device allows the injection of the active ingredient by the central needle electrode at an intermediate depth in the tissue. This can be controlled by a removable button, or a wedge that is removed, or by any other technique familiar to those skilled in the art. The three electrodes are then pushed deeper into the tissue. The electric fields are then delivered by means of a generator connected to the apparatus by any technique familiar to those skilled in the art. This improvement makes it possible to inject the active ingredient into an area that will be positioned optimally vis-à-vis the electric field generated by the electrodes. Indeed, the active ingredient is approximately in the zone where the field lines are linear between the two electrodes and are of maximum intensity.

  The systems set out in FIGS. 2 and 3 have the following advantages: Injection of the active ingredient into the optimum zone of the maximum electric fields delivered by the electrodes. This allows, for an efficiency identical to that of other systems, to use electrical pulses of less intensity or shorter duration, which reduces the toxicity and the duration of the intervention.

  - Minimum implementation time by co-crimping a pair of electrodes and an injection needle. This allows a quick surgical procedure, with only three steps: insertion, injection, pulse administration. On the other hand, a servo system can be designed, combining the injection of the active ingredient to a simultaneous administration of electrical pulses.

  - A system compatible with the rules of medical safety, since the electrode assembly and injection needle, collected in the injection part, are disposable, for a relatively low construction cost.

  - A reduced size related to the use of at most three electrodes or injection needles, in a preferred form of the invention.

  The invention also relates to a third preferred form of electric field assisted delivery, which is shown in FIG. 4. This invention is adapted for gene transfer into the joints or into an organ circumscribed by a membrane, such as the bladder.

  In the example of FIG. 4, an electrical pulse conducting shell, which may, for example, take the form of a curved plate, covers the articulation or the outside of the organ. It constitutes the first electrode connected to one of the terminals of a generator. This shell is in contact with the tissue via the use of a conductive gel, such as, for example, the gels used for electrocardiograms.

  The other electrodes consist of one or a plurality of needles, all connected to the second terminal of the generator. These needles pass through the conductive shell while being isolated by insulating sheaths surrounding these needles or crimped on the shell. The electric fields are generated by potential difference between the electrode needles (53) and (54), and the conductive curve plate. The number of electrode needles is variable (54). In a preferred form, these electrode needles can also be hollow and be used for the injection of the active ingredient. The device presented makes it possible to increase the volume traversed by electric fields in the vicinity of the membrane of the articulation or of the membrane defining the member, as represented in FIG. 4 by the field lines.

  In the example of the apparatuses of FIGS. 2, 3 and 4, the electrodes are electrically connected to an electric pulse generator using the techniques of those skilled in the art, such as clamp, chisel, etc. In a preferred embodiment of the invention of Figures 2 and 3, the electrical contact between the electrodes and a cable connected to the generator is obtained by a system of one or two metal blades which are pressed against the conductive part of the electrodes at the time closing the case.

  The invention also relates to a preferred form of hydrostatic or hydrodynamic pressure assisted delivery, characterized by the insertion of one or more needles into a volume limited by a membrane. The biological volume concerned is for example, non-limiting way, a joint, the eye or the bladder.

  This needle or set of several needles are associated with a system for injecting rapidly, in a controlled time ranging from a fraction of a second to several minutes, a large volume of liquid. This makes it possible to create a hydrodynamic and hydrostatic pressure in the zone circumscribed by the membrane wall. This wall is, in the case of the joint, the synovial membrane. In the vitreous of the eye, it is the retina, choroid and lens. In the case of the bladder, it is the bladder epithelium.

  An example of the joints is given in Figure 5. A system comprising a needle array is pressed against the joint, so that the needles penetrate the synovial area without damaging the cartilaginous or bone tissue. This needle network, which can contain only one needle, is connected to an injection reservoir, syringe type or any other reservoir known to those skilled in the art, such as a compressible compartment.

  In a preferred form of pressure-assisted administration, several syringes are used, so as to inject simultaneously and under pressure a large volume of active ingredient in multiple areas of the joint. In this case, an intermediate reservoir makes it possible to connect the reservoir initially containing the active ingredient to the multiplicity of needles. In a preferred form, the injection needles are crimped into a part allowing maintenance in a defined geometry. An additional piece, a body, allows the grip for the connection to the tank containing the active ingredient and for the procedure on the patient.

  As indicated in FIG. 5, by the schematic representation of the diffusion zone of the active principle, one of the characteristics of the invention is to position the active ingredient in a favorable and diffuse zone within the zone to be treated. This therefore ensures the best conditions for intracellular penetration of the active ingredient after implementation of the hydrostatic or hydrodynamic pressure force, and therefore represents a significant improvement in the state of the art.

  Another preferred form of the invention is characterized by the joint use of pressure forces and electric fields. For this, an apparatus such as that shown in Figures 2 and 3 is provided with a piston actuated electrically or mechanically. This piston injects the active ingredient under pressure, during or before the administration of electric fields. An example of an apparatus is illustrated in FIGS. 17 and 18.

  Another preferred form of the invention relates to tissue volumes delimited by a membrane, such as the joint or bladder. The invention is characterized by the use of the apparatus shown in FIG. 5, representing the setting in play of pressure forces, and, simultaneously or consecutively, the electroporation technique shown in FIG. In this case, the needle electrodes used are also used for the injection of the active ingredient under pressure. They are connected to a single tank allowing the simultaneous injection of a large volume of active ingredient in the tissue area concerned. These electrodes are, moreover, connected to the same terminal of a pulse generator. The second terminal of the generator is connected to the conductive shell in contact with the tissue volume. This device has the advantage of combining two physical techniques of administration of active ingredients, and thereby to achieve a synergistic effect leading to maximum efficiency.

Brief description of the figures

  FIG. 1 schematizes the location of the active principle in the electroporation or electrotransfer processes known to those skilled in the art. The active principle is not optimally located in the areas of maximum electric field generated by the electrodes. FIG. 2 shows the open housing of the device of the invention, for a device with two electrodes and one or more central injection needles, as well as the injection part.

  Figure 3 shows the open housing of a variant of the device of the invention, for a device with three electrodes.

  Figure 4 depicts a device for electrotransfer or electroporation in an area delimited by a membrane, such as a joint or bladder. The device here using a curved shell connected to the + terminal of a generator and three needle electrodes connected to the generator terminal, needles also used for the injection of the active ingredient.

  FIG. 5 discloses a device for injecting simultaneously and under pressure a large volume of active ingredient into multiple areas of a joint or organ delimited by a membrane, so as to generate a pressure leading to better tissue diffusion. and cellular internalisation of the active ingredient.

  FIG. 6 represents the open housing of the device of FIGS. 3 and 4, in particular for a variant of the device of the invention composed of a device with three electrodes, with the injection system placed next to the housing.

  FIG. 7 represents a section of the closed casing of the device of FIGS. 3 and 4 of the invention, in particular for a variant of the device of the invention composed of a device with three electrodes.

  Figure 8 shows the bottom of the open housing of the device of the invention with the injection part placed in its compartment.

  FIG. 9 shows a section of the injection part of the device of FIG. 2 of the invention. FIG. 10 represents a section of the injection double needle injection device of the device of FIG. 2 of the invention. .

  FIG. 11 shows a cross-section of the double-electrode injection part, in particular for a variant of the device of the invention composed of a device containing a sliding injection needle.

  FIG. 12 describes the positioning movement of the injection part in the injection box, in particular for a variant of the device of the invention composed of a device with three electrodes.

  FIG. 13 represents the act of closing the housing, in particular for a variant of the device of the invention composed of a device with three electrodes. FIG. 14 represents the installation act of the locking system of the housing of a variant. of the device of the invention, for a device with three electrodes.

  FIG. 15 represents the act of locking the casing and placing the syringe in the locked casing, in particular for a variant of the device of the invention composed of a device with three electrodes.

  FIG. 16 represents the act of injecting the active principle into the tissue and triggering electrotransfer, in particular for a variant of the device of the invention composed of a device with three electrodes. . FIG. 17 represents the injection of active ingredient at constant speed and under pressure into a tissue with a simple syringe, for a variant of the device of the invention with an injection needle.

  FIG. 18 shows the use of a device for injecting active ingredient at a constant speed and under pressure into a fabric, for a variant of the device of the invention of FIG. 3 composed of a device with three electrodes.

  Figure 19 shows the bottom of the open housing, with the injection part placed in its compartment for a variant of the device of the invention consists of a device with three electrodes.

  Figure 20 shows the location of the electrodes of the device of the invention of Figure 3, having four external electrodes and an inner needle-electrode.

  DETAILED DESCRIPTION OF AN EMBODIMENT OF THE INVENTION The applicant now describes examples of preferred forms of the invention. Other analogous examples can be obtained by all the techniques of the person skilled in the art leading to operation and identical implementation in the principle presented above.

  The injection system, described in Figure 2, is composed of two parts: The first part, called housing ", is composed of two half-cylinders (3) and (4) connected by a hinge.The second part (2) , called "injection part" is composed of one or two crimping disc crossed by an injection needle possibly also serving as an electrode, and several electrode needles.

  The housing serves as a passenger compartment for the injection part. It also serves as an electrical connection with the electrical terminals of the injection part (2) and the electrical wires (7) connected to the electric generator (21) for electrotransfer. The housing also serves as a cabin for the syringe (1) containing the active ingredient (36) (biological or chemical solution) to be injected between the two external electrodes of the injection part (2). Figure 2 shows the assembled device, Figure 6 shows the two main parts (3 and 4) making up the device with the injection part (2) outside of its cabin.

  The housing is composed of 2 half-cylinders or equivalent forms leading to the same result (3 and 4), connected to each other by a hinge. In a preferred form of the invention, the thickness of each half-cylinder (3 and 4) must be sufficient to ensure the rigidity of the assembly, and is by way of non-limiting example between 0.1 and 20 mm. In a preferred form of the invention, the diameter of the semi-cylinders is by way of non-limiting example between 3 and 100 mm. These two half-cylinders pivot around the hinge to form, when pressed against each other, a cylindrical or equivalent shape, the essential feature being that this form is easily manipulated and ensures the isolation of the manipulator vis-à-vis -vis electric fields, as shown in Figure 7.

  8 shows the lower part of each half-cylinder (3 and 4) having geometric shapes for fixing the injection part (2), and thus preventing it from moving in translation in its axis. This device may for example be formed by a circular notch (9) hollowed out in the half cylinders (3 and 4). The housing provides electrical contact between each electrode needle and the electrical contact wires (7) with the electrotransport pulse generator (21). One of the two half cylinders (3) has shapes (8) on which is fixed the electrical contact device with the needle-electrodes (10). One or each shape (8) of this half-cylinder will support a metal electrical contact piece (42) with the electrode needles (10) of the injection piece, which may be flat or curved to fit the shape of the needle and can be rigid or flexible.

  In a preferred form of the invention, each contact piece consists of a curved metal strip. Thus, when closing the housing (1), each electrode (10) is compressed between the metal strip (42) and the opposite shape (8) located in the corresponding half-cylinder (4), in order to improve the electric contact. Any other system to improve the contact (for example use a spring, fix on the curved blade a metal half-cylinder conforming to the shape of the tip) can be used. These lamellae (42) are fixed to their respective shape (40) and connected to the electrical wires (7), thus allowing the connection with the electrical pulse generator for electrotransfer (21). In a variant of the device, the switch triggering the electrical pulses can be fixed on the housing, to facilitate the act of the operator and reduce its duration.

  A clasp (35), for example a hook or a clip, keeps the housing closed.

  The injection part (2) can have a protective cover (5) as for conventional syringes. The injection part (2) is sterile and can be disposable.

  Figure 9 shows an enlargement of the injection part for a device with 2 electrodes. The two external electrode tips (10) and the injection needle (11) are placed exactly at equal distances between the two electrodes. An isolation disk (41) protects the housing (1) from direct contact with the skin and blood of the subject. The electrode tips may be solid or hollow, and their shape in their upper part may be adapted to achieve the best electrical contact with the metal contacts (shown in Figure 8, (42)) corresponding to the housing. The injection needle (11) is shallower than the two external electrode needles (10), so that the active ingredient injected is well distributed between the two electrodes. 15 20

  In a preferred form of the invention, an electrical insulating film (15), for example but not limited to a Teflon film, may be applied over a few mm under the crimping disc (12) securing the needles. , on the electrode needles, or even the central hand, to prevent the electric fields from spreading just under the skin of the subject.

  In a variant of the invention shown in FIG. 3 and FIG. 19, the injection needle (11) serves as an electrode, the two external electrode tips (10) being traversed during electrotransfer with a reverse polarity with respect to the injection needle (11). This will have the particular advantage of reducing by half the distance between two electrodes of inverse polarity, and therefore of requiring a lower voltage to obtain the same ratio of volt / cm during the electrotransfer. This has the effect of making, for a given value of the electric field in volt / cm, necessary the use of a lower voltage and therefore an act less painful for the patient and less destructive for the cells crossed by the electric fields. In this variant, the central electrode needle has the same depth as the external needles, in order to obtain an optimum electrotransfer effect. In a variant of the invention, the number of external electrode needles (10) may be greater than two, and without limitation. They must surround the central hand in a circle. FIG. 20 shows, viewed from above, a device with 4 external electrode needles (10) with the central injection electrode needle (11). The generator electrical connection system for the injection needle (11) may be the same as that used for the external electrode needles (10). A slot is embedded in the housing to receive an electrically insulating crimping disk (41) forming part of the injection part (2) to isolate the lower electric poles in case of accidental dispersion of liquid during the act. .

  In order to improve the distribution of the active principle between the two needles, a double needle system (shown in FIG. 10), or even a triple needle system, terminating at different depths can be implemented. For the same purpose, an injection needle with several orifices on its side walls could be used. For the same purpose, presented in FIG. 11, an injection system with sliding central needle makes it possible to inject the active ingredient at two distinct depths. The central rod (16), coupled with a stop system in the housing (3 and 4), makes it possible to move the central hand in the direction of its axis (19).

  Injection needles and injection needle-needles (11) and external electrode needles (10) are made of medical-grade stainless steel materials. The external electrode needles (10) can be solid or hollow. Thus, common syringe needles from which the upper part has been removed to the required height can be used. The electrical contacts (42) of the housing are made of stainless metal materials. There is no special specification for electrical cables and connection terminals to the injector. All other components are made of plastic, resin or any non-electrically conductive material, and can withstand the temperatures of sterilization devices. The film responsible for electrically insulating a portion of the electrodes and electrode needles is sufficiently thin so as not to slow the penetration of the needles, and may be composed for example of Teflon.

  An example of use of the device of the invention, in a variant with three electrode needles is given here. This example is illustrated in FIGS. 12 to 15. The operator places the crimping disk in the space reserved for this purpose of the open housing, then closes the housing. The operator locks the housing by means of the locking cylinder (22) and inserts the stop (20) or any other system to prevent movement of the cylinder along the axis of the device. The parts 20 and 22 make it possible to accurately perform the injection of the active ingredient (36) between the two external electrodes (10). The operator then inserts the syringe (1) containing the active ingredient into the housing, connects the electrical terminals (7) to the electrical pulse generator (21), removes the protective cap from the needles (5), and plants the device in the patient's tissue (50), according to medical practices and gestures usually in practice for planting a needle with a syringe.

  So that the central electrode needle (11) is located within the injected active principle (36), the active principle can be injected when the central needle is partially embedded in the tissue, but this is not necessary in the case hydrostatic pressure assisted administration. The locking cylinder (22) and its abutment system (20) block the insertion of the needles into the tissue. Once the injection is complete, the abutment (20) is removed, the device can be depressed to the guard in the patient's tissue, and then the electrical pulses are delivered.

  For the same purpose of distributing the active ingredient (36) around the central electrode needle (11), an injection needle plugged at its end in the tissue, and perforated in its side walls with small holes would allow to spread the active principle (36) around the needle. This has the advantage of avoiding performing the act of penetration of the injection part in the tissues of the patient in two stages.

  After the electrotransport, the operator removes the housing (3 and 4) and the attached injection part (2) and discards the consumables (syringe (1), injection part (2) and its protective cap (5) locking cylinder (20, 22)).

  FIG. 17 illustrates an exemplary embodiment of a variant of the device for injecting active ingredient at a constant speed and under pressure into a fabric, in a preferred form of the invention. The system is composed of a shell (23) containing an electric motor (27), which in a preferred form of the invention can be found at its top. This shell (23) will also serve as a receptacle for the syringe (1) and the injection needle of the active ingredient (29). The electric motor rotates a threaded rod (25) which causes the movement (28) of a cylindrical block (24) which can not pivot (for example by being locked by a groove). This cylindrical block (24) pushes the piston of the syringe (26) by supporting the pressure related to the injection of the active ingredient. Figure 18 illustrates the same principle, but coupled to the electrotransport device of the invention in its three-electrode variant in this example. During or after the injection, the electrotransport is triggered. Other analogous examples can be obtained by all the techniques of the person skilled in the art leading to identical operation and implementation in the principle presented above.

  FIG. 4 describes a variant of the device of the invention adapted to a method of electrotransfer in a volume defined by a membrane, such as for example a hinge. Electrotransfer can be delivered following an injection of active principle causing a hydrostatic pressure effect, the main objective being to cause a strong penetration of the active ingredient into the cell membrane (52) surrounding the tissue (55) in which the active ingredient is injected. The active ingredient is introduced under pressure through the injection electrode needles (56). Due to the hydrostatic or hydrodynamic pressure, the active ingredient enters the membrane (52), and can also penetrate the cells. If the concentration concentration obtained from the active principle in the cells of the membrane is not sufficient, this rate can be increased by electrotransfer, the device presented making it possible to increase the area traversed by electric fields, the latter being circular around the electrodes needles (three in this example). The electric fields are generated by potential difference between the electrode needles (54 and 56) and the conductive curve plate (51). This plate is in contact with the tissue via the use of a conductive gel (57). The number -1s- of electrode needles (54 and 56) is variable. These electrode needles can also be injection needles. An electrically insulating film (50) covers the outside of the conductive plate (51). An electrically insulating film (53) isolates a portion of the electrode needles (54 and 56) from the conductive plate (51) and the traversed membrane (52).

  INDUSTRIAL APPLICATION OF THE INVENTION The industrial applications of the administration of nucleotides assisted by electric fields or by hydrostatic pressure are numerous. The device according to the invention is particularly intended for the administration of medicaments, in particular drugs based on DNA, in human and veterinary medicine.

  The therapeutic applications in humans or animals concern, in particular but obviously not exhaustive, the treatment of tumors and the production of blood proteins.

  The production of proteins in the blood concerns the treatment of hemophilia, growth disorders, myopathies, lysosomal and metabolic diseases in general, chronic renal failure and beta-thalassemia by the endogenous production of erythropoietin. Other fields of application concern neoangiogenesis, atherosclerosis, using the protective effect of cytokines such as IL-10, vaccination, the use of antisense oligonucleotides, or the prevention of peripheral neuropathy. induced by cisplatin by electrotransfer of a plasmid encoding a neurotrophin. A particular focus is currently on use in rheumatoid arthritis or in inflammatory conditions of the eye, using the protective effect of IL-10, anti-TNF or other cytokines. The use of growth factors is also rich in potential in neurodegenerative or degenerative diseases of the joint (osteoarthritis) or the eye (blindness).

  In addition, are also concerned all pathologies that can benefit from the local expression of a secreted protein, such as an anti-inflammatory protein secreted in the joint for the treatment of arthritis, or an anti-angiogenic protein for the treatment of cancer or disorders of the macula. Similarly, production in a growth factor joint may be considered for the treatment of osteoarthritis. The use of angiogenic proteins secreted locally for the treatment of peripheral arteritis is also conceivable.

  It is also necessary to mention the therapeutic applications for which the intracellular expression of a protein is necessary, such as numerous neuromuscular diseases (myopathy), tumors, etc.

  Device delivering a pressure for administering an active ingredient in a biological tissue or a cavity circumscribed by a membrane in humans or animals, characterized in that it comprises from 1 to 100 needles in contact with a common reservoir so as to inject simultaneously and under pressure a large volume of active ingredient in multiple areas of the fabric by acting by pressure on the reservoir.

Annex

LIST OF APPLICABLE PATENTS

  Günter A. Hofmann. Injection and electroporation apparatus for drug and gene delivery. US Patent No. 5,273,525 (Dec. 28, 1993).

  Günter A. Hofmann. Applicator for the electroporation of drugs and genes into surface cells. US Pat. No. 5,318,514 (Jun. 7, 1994).

  Günter A. Hofmann; Sukhendu B. DEV; Steven C. Dimmer; Jeffrey I. Levatter; Gurvinder S. NANDA. For electroporation therapy. US Patent, Patent Number 6,055,453 (Apr. 25, 2000).

  Jacob Mathiesen. Method for genetic immunization. U.S. Patent No. 4,610,044 B2 (Aug. 26, 2003).

  Robert Bernard. Electrodes and electrode arrays. PCT / US98 / 08183 (Apr. 28, 1998); International Publication number WO 98/47562 (Oct. 29, 1998).

  Allan Wesyersten; Ruxandra Draghia-Akli; Robert H. Carpenter; Douglas R. Kern; William R. Wilkinson. Electrode assembly for constant-current electroporation and use. PCT / US03 / 06833 (March 6, 2003); International Publication number WO 03/076006 (Sept. 18, 2003).

  Relevant references Bachy, M., Boudet, F., Bureau, M., Girerd-Chambaz, Y., Wils, P., Scherman, D., Meric, C.- Electric pulses increase the immunogenecity of an influenza DNA vaccine injected intramuscularly in the mouse. Vacinnology 2000. Vaccine 2001 Feb 8 19: 13-14 10 15 35 1688-93 Bettan M., Ivanov M., Mir L., Boissiere F., Delaere P. and Scherman D. Efficient DNA electrotransfer into tumors. Bioelectrochemistry, 52, 8390, 2000.

  Bettan M, Emmanuel F, Darteil R, Caillaud JM, Soubrier F, Delaere P, Branelec D, Mahfoudi A, Duverger N, Scherman D - High-Ievel protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle - Mol Ther, 2: 204-10, 2001.

  Bureau M.F., J. Gehl, V. Deleuze, L.M. Mir, And D. Scherman. - Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochim. and Biophys. Acta General Subjects, 2000, 1474, 353-359.

  Celiker MY, Wang M, Atsidaftos E, Liu X, Jiang Y, Valderrama E, Goldberg ID, Shi YE - Inhibition of Wilms' tumor growth by intramuscular administration of tissue inhibitor of metalloproreiases-4 plasmid DNA. Oncogene, 20: 4337-4343, 2001.

  Draghia Akli R, Fiorotto ML, Hill LA, Malone PB, Deaver DR, Schwartz RJ 20 Myogenic expression of an injectable protease-resistant growth hormone-releasing hormone augments long-term growth in pigs - Nat Biotechnol 17: 1179-83, 1999.

  Faria M., Spiller D.G., Dubertret C., Nelson J.S., White M.R.H., Scherman D., Helene C., Giovannangeli C. - Phosphoramidate oligonucleotides as antisense potent molecules in cells and in vivo. Nature Biotech, 2001, 19, 40-44.

  Heller, L., Pottinger, C., Jaroszeski, MJ, Gilbert, R., Heller, R. In vivo elctroporation of plasmids encoding GM-CSF or interleukin-2 into existing B 16 melanomas combined with electrochemotherapy induces longterm antitumour immunity - Melanoma Research, 10, 577-583, 2000.

  Heller R., Jaroszeski M., Atkin A., Moradpour D., Gilbert R., Wands J. and Nicolau C. In vivo gene electroinjection and expression in rat liver. FEBS Letters, 389, 225-228, 1996.

  Kishida T, Asada H, Satoh E, Anaka S, Shinya M, Hirai H, Iwai M, Tahara H, Imanishi J, Mazda 0 - In vivo electroporation-mediated transfer of interleukin-12 and - 20 - 2872055 - 21 - interleukin- 18 significant genes antitumor effects against melanoma in mice - Gene Ther, 8: 1234-1240, 2001.

  Kreiss P, Bettan M, Crouzet J, Scherman D - Erythropoietin secretion and physiological effect in mouse after intramuscular DNA plasmid electrotransfer - J Gene Med, 1: 245-50, 1999.

  Leroy P., M. Office, And Scherman D. - Gene Therapy and Electrotransfer of DNA. Biofutur's technoscope, issue 132, Biofutur n 210, April 2001.

  Li, Zhang, X., Xia, X., Zhou, L., Breau, R., Suen, J., Hanna, E. Intramuscular delivery of IFN-alpha gene therapy for tumor growth at a remote site . Gene Ther, 8, 400407, 2001.

  Li S, Xia X, Zhang X, Suen J - Regression of tumors by IFN-gamma electroporation gene therapy and analysis of the responsible genes by cDNA array. Gene Ther, 9: 390-397, 2002.

  Mallat Z., Besnard S., Duriez M., Deleuze V., Emmanuel F., Bureau MF, Soubrier F., Esposito B., Duez H., Fievet C., Staels B., Duverger N., Scherman D. , Tedgui A. - Protective role of interleukin-10 in atherosclerosis. Circulation Research, 1999, 85 October 15, 1-8.

  Martin J.B., Young, J.L., Benoit, J.N., Dean, D.A. (2000). "Gene transfer to intact mesenteric arteries by electroporation." J. Vasc. Res 37: 372-380 Mathiesen, I. - Electropermeabilization of skeletal muscle enhancing gene transfer in vivo. - Gene Ther. 6, 508-514, 1999.

  Mir L., Office M. F., Rangara R., Schwartz B. and Scherman D. - Long-term, high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle. - Proceedings of the Academy of Sciences, Life Sciences I Life Sciences, 321, 893-899, 1998.

  Mir L., M. Office, Gehl J., Rangara R., Rouy D., Caillaud JM., Delaere P., Branellec D., Schwartz B. and Scherman D. - High efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc. Nat / Acad. Sci. USA, 1999, 96, 4262-4267.

  2872055 - 22 - Neumann E., Schaefer-Ridder M., Wand Y., and Hofschneider P. - Gene transfert dans una lyoma cells by electroporation in high electric fields. - The EMBO Journal, 1, 841-845, 1982.

  Poiler, H. Electroporation in biology: Methods, applications, and instrumentation. Anal Biochem 174, 361-373, 1988.

  Oshima, Y., Sakamoto, T., Yamanaka, I., Nishi, T., Ishibashi, T., Inomata, H. - Targeted gene transfer to corneal endothelium in vivo by electric pulse. - Gene. Ther. 5: 1347-1354, 1998.

  Saito T, Nakatsuji N: Efficient gene transfer in the embryonic mouse brain using in vivo electroporation. Dev Biot, 240: 237-246, 2001.

  Payen E., Bettan M., Rouyer-Fessard P., Beuzard Y. and Scherman D. Improvement of mouse beta-thalassemia by electrotransfer of erythropoietin cDNA. Exp. Hematology, 2001, 29, 295-300.

  Pradat P.F., Finiels F., Kennel P., Naimi S., Orsini C., Delaere P., Revah F., Mallet J. - Partial prevention of cisplatin-induced neuropathy by electroporation-mediated nonviral gene transfer. Human Gene Therapy, 2001 March, 12, 367-375.

  Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, Costa P, Mathiesen I, Cortese R, Ciliberto G, Laufer R, La Monica N, Fattori E Efficient and Regulated Erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci U A 96: 6417-22, 1999.

  M. K, Delteil C., Golzio M., Dumond P., Cros S. and Tessie J. In vivo electrically mediated protein and gene transfer in murine melanoma. Nature Biotechnology, 16, 168-171, 1998.

  Saidenberg-Kermanach, N., Bessis, N., Deleuze, V., Bloquel, C., Bureau, MF, Scherman, D., Boissier, MC. "Efficacy of interleukin-10 gene electrotransfer into skeletal muscle of mice with collagen -induced arthritis - Journal of Gene Medicine, 2002 in press.

  Shibata MA, Morimoto J, Otsuki Y - Suppression of murine mammary carcinoma growth and metastasis by HSVtk / GCV gene therapy using in vivo electroporation - Cancer Gene Ther, 9: 16-27, 2002.

  - 23 - Scherman D., Bessodes M., Cameron B., Herscovici J., Hofland H., Pitard B., Soubrier F., Wils P., Crouzet J. - Application of Lipids and Plasmids for Delivery to Mammalian cells. Curr. Opin. Biotechnol., 1998, 9 (5), 480-485.

  Slack A, Bovenzi V, Bigey P, Ivanov MA, Ramchandani S, Bhattacharya S, TenOever B, Lamrihi B, Scherman D, Szyf M - Antisense MBD2 Gene Therapy Inhibitors Tumorigenesis - J Gene Med, prepublished online May 17, DOI 10. 1002 /jgm.288, 2002.

  Tamura T, Nishi T, Goto T, Takeshima H, Dev SB, Ushio Y, Sakata T Intratumoral delivery of interleukin 12 expression plasmids with in vivo electroporation is effective for colon and renal cancer. Human Gene Ther, 12: 1265-1276, 2001 Titomirov A., Sukharev S. and Kristanova E. In vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNA. Biochimica and Biophysica Acta, 1088, 131-134, 1991.

  Tupin, E., Poirier, B., Bureau, MF, Khallou-Lashet, J., Vranckx, R, Caligiuri, G., Gaston, Anh-Thu., Duong Van Huyen, JP, Scherman, D., Bariety, J., Michel, JB, Nicoletti, A. Non viral gene transfer of murine spleen cells performed by in vivo electroporation. Gen Ther in press, 2002.

  Vicat, JM, Boisseau, S., Jourde, P., Wool, M., Wion, D., BoualiBenazzouz, R. Benabib, AL, and Berger, F. - Muscle transfection by electroporation with high voltage and short-pulse currents provide highlevel and long lasting gene expression. - Hum. Gene. Ther. 11, 909-916, 2000.

  Vilquin J.T., P. Kennel, M. Paturneau-Jouas, P. Chapdelaine, N. Boissel, P. Delaere, J. Tremblay, D. Scherman, M. Fiszman And K. Schwartz. Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies. Gene Therapy, 2001, 8, 1097-1107.

  Watanabe K, Nakazawa M, Fuse K, Hanawa H, Kodama M, Aizawa Y, Ohnuki T, Gejyo F, Maruyama H, Miyazaki JI - Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation Circulation, 104: 1098-1100 , 2001.

  Widera, G., Austin, M., Rabussay, D., Goldbeck, C., Barnett, SW, Chen, M., Leung, L., Otten, GR, Thudium, K., Selby, MJ and Ulmer, JB Increased DNA delivery and immunogenicity by in vivo electroporation. J. Immunol 164: 4635-4640, 2000.

  Xue F, Takahara T, Yata Y, Minemura M, Morioka CY, Takahara S, Yamato E, Dono K, Watanabe A - Attenuated acute liver injury in mice by naked hapatocyte growth factor gene transfer into skeletal muscle with electroporation - Gut, 50: 558-562, 2002.

  Yamashita Y, Shimada M, Tanaka S, Okamamoto M, Miyazaki J, Sugimachi K 10 Electroporation-mediated tumor necrosis factor-related apoptosisinducing ligand (TRAIL) / Apo2L gene therapy for hepatocellular carcinoma.Human Gene Ther, 13: 275-286, 2002 .

20 25 30

Claims (7)

- 25 - Claims   1 - Device for improving the penetration of molecules of active principle into the cells of a human or animal tissue, especially in the context of chemotherapy or gene therapy, characterized in that it comprises: - at least two electrodes - needles (10) connected to an electrical pulse generator generating an electric field or current and - one or more injection needles of the active ingredient (11) placed between these electrodes (10), and being approximately or exactly aligned with these and a means (1) for containing and delivering an active ingredient in fluid form.   2 Device according to claim 1, characterized in that it comprises at least three electrodes (10, 11), the central electrode (11) being of different polarity of the adjacent electrodes (10) and being used as injection needle of the active ingredient.   3 Device according to one of claims 1 and 2, characterized in that it comprises a piston (1) driven by a motor (27) for generating a hydrostatic or hydrodynamic pressure at or after the injection of the active principle, this pressure providing an additional means for improving the penetration of the active ingredient into human or animal tissue and cells of this tissue.   4 - Device according to any one of the preceding claims, characterized in that it comprises a plurality of injection needles in contact with a common reservoir, so as to simultaneously inject a large volume of active ingredient in multiple areas of the fabric by acting by pressing on the tank.     Device according to any one of the preceding claims, characterized in that one or more of the needle electrodes are hollow and are also used to deliver the active ingredient.   6 - Device according to any one of the preceding claims, characterized in that it comprises a stop system (20) which allows the injection of the active ingredient by the injection needles to an intermediate depth of tissue that can be predefined the electrodes are then pushed deeper into the tissue, at an intermediate depth of the tissue that can be predefined.   - 26 - 7 - Device according to any one of the preceding claims, characterized in that one or more electrodes are partially covered with an electrical insulator.   8 - Device according to any one of the preceding claims, characterized in that one or more injection needles are partially covered with an electrical insulator (15).   9 - Device according to any one of the preceding claims, characterized in that the injection needles and the electrodes are made integral in a single injection piece.   - Device according to any one of the preceding claims, characterized in that the injection needles and the electrodes are secured by crimping in the single injection part (2).   11 - Device according to any one of the preceding claims, characterized in that the injection member (2) is inserted into a housing (3, 4) providing the electrical connection with a pulse generator and facilitating the use of the device.   12 - Device according to any one of the preceding claims, characterized in that the electrodes are brought into contact by means of metal blades (8) with a cable (7) connected to a generator of current or electrical potential.   13 Apparatus according to any one of the preceding claims, characterized in that the electrodes are electrically isolated from the container containing the active ingredient.