Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
  • A61M37/0092—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin using ultrasonic, sonic or infrasonic vibrations, e.g. phonophoresis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0047—Sonopheresis, i.e. ultrasonically-enhanced transdermal delivery, electroporation of a pharmacologically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
  • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis

Definitions

• the present invention concerns a method and apparatus for the administration of substances, into and through tissues or membranes using ultrasound.
• Ultrasound is defined as a sound having a frequency greater than 20 kHz and is used in a plurality of medical and diagnostic procedures such as imaging of internal organs, sterilization, degassation, superficial eye-lens-epithelium surgery, bile-stone perforation and anti-cancer treatment. Ultrasound is also used for facilitation of transport of various compounds across tissues, typically skin (Mitragotri, M., et al, Science, 269:850-853 (1995)).
• U.S. Patent 5,076,208 discloses a method for the delivery of molecules, the example being gonadotropin-releasing hormone analogue (GnRHa), to aquatic animals in an aquatic medium.
• the molecule to be administered is added to the medium and ultrasound is then applied to enhance the effect of the uptake of the compound by the animal.
• the ultrasound is usually applied at a single frequency for a relatively long period of time, typically 10-15 minutes at an intensity of 1.7 W/cm 2 .
• the ultrasonic delivery was improved by using ultrasound in conjunction with chemical permeation enhancer and/or iontophoresis (U.S. Patent 5,231,975).
• Other methods use ultrasonic waves to exite the local nerves in the way that trauma does, and the nerve excitation opens the epidermal/dermal junction membrane and the capillary endothelial cell joints, which enables the transfer of drugs through the skin and into the blood stream (U.S. Patent No. 5,421,816).
• Prior art administration methods utilizing ultrasound are suitable for the administration of substances such as proteins, nucleic acids and drugs having a small size, which are typically dissolved in a liquid medium, i.e. soluble substances.
• complex particles having a size in the range of 1 nm to tens or hundreds of microns
• these complex particles are essentially inert in the liquid medium in which they are carried.
• complex particles are dead or attenuated virions, bacteria, fungi or parasites or their fragments administered for the purpose of immunization; plasmids administered to tissues or to cultured cells for the purpose of genetic manipulation; nuclei of gametes administered to oocytes for the purpose of fertilization; particles impregnated with medicaments and capable of releasing them at a slow rate to the surrounding tissue administered for the purpose of therapeutic treatment; particles containing compounds that were coated with a protective coating, for example, in order to change the compounds to prevent oxidation, to prevent a hygroscopic effect, to increase resistance to heat or to protect the contents of the particle from biological effects (such as degradation); and administration, topical or systemic administration of particles, for example, magnetic beads or dye particles for local or systemic therapeutic
• the present invention provides a novel method allowing the introduction of substances into or through cells, tissues or membranes.
• This in accordance with the invention, is achieved by utilizing a complex ultrasound stimulus, consisting of a first irritant stimulus and a second driving stimulus.
• a complex ultrasound stimulus consisting of a first irritant stimulus and a second driving stimulus.
• the method of the present invention may be used for therapeutic and cosmetic purposes, as well as for diagnostic and experimental purposes, according to the type of substance to be administered.
• the substances to be administered may be soluble substances such as various medicaments for therapeutic treatment, macromolecules such as DNA molecules for the purpose of genetic manipulation, various dyes for the purpose of diagnosis inside cells, or within a tissue and the like.
• the substances to be administered are complex particles.
• complex particle refers generally to a particle having the size of at least 1 nm ranging to tens or hundreds of microns which is usually composed of several types of molecules, although at times it may be composed of a single type of molecule.
• the complex particles are essentially insoluble in the medium in which they are carried.
• complex particles are attenuated disease-causing agents or parts thereof such as bacteria, virions, fungi, protozoa or parasites administered for the purpose of vaccination; plasmids containing DNA to be inserted into tissues or cultured cells for the purpose of genetic manipulations; nuclei of gametes administered into oocytes for the purpose of fertilization; particles impregnated with medicaments capable of releasing them at a slow rate to the surrounding tissue for the purpose of therapy; particles containing compounds that were coated with a protective coating, for example, in order to form particles having different solubility, to prevent oxidation, to prevent a hygroscopic effect, to increase resistance to heat or to protect the contents of the particle from biological effects (such as degradation); particles comprising a biologically compatible dye for the purpose of tattooing, as for example, in the case of permanent makeup; particles comprising a detectable marker for the purpose of diagnosis, and the like.
• disease-causing agents or parts thereof such as bacteria, virions, fungi, protozoa or parasites
• the cells to which the substances are administered can be any type of eukaryotic or prokaryotic cells, typically cells cultured in a medium.
• the cells may be obtained from a single-cell organism or cells obtained from multicellular organisms.
• the tissue to which the substances are administered are typically epithelial tissues which may be an artificially moistened keratinized epithelial tissues such as skin, or moist-non-keratinized epithelial tissues, for example, the epithelium lining the eyes, digestive, respiratory, or reproductive systems.
• the tissue may also be the moist epithelial tissue covering aquatic animals such as fish, crustaceans or molluscs at different stages of rearing.
• the membranes may be either natural membranes or artificial membranes such as polyethylene or elastomer membranes which form a part of an implant.
• openings when used herein in connection with cells refers to pores formed in the membrane of the cells.
• openings when used herein in connection with tissue refers to pores formed in the membranes of the cells forming the tissue, pores formed in the basal lamina lining the tissue, or opening of the intercellular junctions of the tissue and/or increase of the intercellular space which may be due to elimination of some cells from the tissue, or vibration at or close to the tissue resonance frequency.
• opening used in connection with membranes (both natural and artificial) refers to gaps i.e. incontinuous spaces in the natural or artificial membranes.
• the two stimuli of the ultrasound are applied when the cells, the tissue or the membrane are inside or in contact, as the case may be, with a liquid medium.
• a liquid medium When the tissue is keratinized skin the liquid medium may be a gel adapted for ultrasound at exposure.
• the liquid medium may also be water or water with additives needed for the maintenance of particular cells, tissue or membrane.
• the substance to be administered may be present in the liquid medium a priori, i.e. also during the first irritant stimulus, or alternatively may be present only during the time the second stimulus is applied, either by adding the substance to the liquid medium immediately prior to the application of the second driving stimulus, or by transferring the tissue or cells into a medium containing the substance after the first stimulus has been applied.
• a priori in the medium The substances may be added manually to the medium.
• the specific parameters of the first irritant stimulus, capable of causing a formation of transient openings in the cells or cells in the tissue, and the second driving stimulus capable of driving the administered substances through said openings to the cells or the tissue, should be determined empirically, depending on the exact nature of the cells or tissue and the nature of the administered substance.
• a first set of samples of the tissue, cells or membranes to be administered should be exposed to a variety of ultrasound pulses varying in intensities, frequencies, pulse modes and durations, and concomitantly or immediately after the application of the stimulus, fixated and examined under electron-microscope.
• a corresponding set of stimuli should be given to a second set of samples which are fixated for electron-microscope after several hours from the end of the stimulus in order to determine the level of recovery.
• Parameters which should be chosen are those which are able to cause formation of openings of a desired size, as determined in the first set of samples, preferably without causing irreversible damage to the bulk of the cells or tissue, as determined by the second set of samples.
• the second driving stimulus should be applied while the openings are still open, which is usually within the range of several seconds to several minutes from administration of the first irritant stimulus.
• the exact time window in which the openings caused by the first stimulus are still open may also be determined empirically by monitoring the number and size of openings at various time periods after application of the first stimulus. Alternatively, the two stimuli may be applied simultaneously.
• the exact parameters of the second driving stimulus should also be determined empirically, for example, by using electron dense particles (i.e. tracer) having about the same size as the size of the substance to be administered, and determining which are the exact parameters of the ultrasound required to successfully drive the tracer through the openings formed by the first irritant stimulus, without causing substantial damage to the bulk of cells or tissue.
• electron dense particles i.e. tracer
• the total effect which should be considered as not constituting an irreversible damage is an effect which should never exceed necrosis followed by loss of the superficial pavement cells at the irradiated zone, i.e. a skin peeling of about the one-twelfth of skin layers at ultrasound irradiated zones.
• Acceptable alterations which can be observed one layer deeper are formation of pores in the distal (towards the external part) part of cell membranes. All deeper cells should remain naive, i.e., should show essentially no ultrastructural or physiological alterations. A two-fold enlargement of the intercellular spaces in the 3-4 outermost epithelial layers is also acceptable. Substantial recovery of these phenomena, i.e., that re-epithelization occurred, should be observed within about ten minutes.
• the first irritant stimulus has the following parameters:
• Frequency 20 kHz to 3 MHz, preferably 100 kHz to
• Frequency 20 kHz to 50 MHz, preferably 2 MHz to 15 MHz, most preferably, 3-5 MHz. Duration: 0.01 to 20 mins. preferably 0.1 to 5 mins., most preferably 1-10 sees.
• Intensity 0.1 - 50 W/cm 2 , preferably 0.1 to 10 W/cm 2 most preferably 0.5 to 5 W/cm 2 .
• the intensity is increased, for example, due to use of focusing system such as acoustic lenses, the duration should be decreased.
• cells present in vitro should be treated with lower intensities and shorter durations than cells present in vivo.
• the duration and frequency of the second driving stimulus should be longer and higher, respectively, than those of the first irritant stimulus, while the intensity should be lower.
• irradiation-activated compounds which may be activated by light, by ultrasound or by other energy sources. These compounds are administered according to the administration method of the invention.
• the irradiation-activated compounds are several substances known to be activated by irradiation and to release free radicals. They are therefore used in medicine to cause damage to the surrounding tissues.
• Several substances are activated by light, in photodynamic therapy, in order to selectively destroy target tissue, typically neoplasmic tissue (Orenstein et al, Br. J. Cancer, 73:937-944, (1996)).
• target tissue typically neoplasmic tissue
• Prior art methods do not teach, however, the manner in which the irradiation-activated compounds, which may be soluble or particulate reach their target zone by topical application. This can be achieved by the method of the present invention. Once they reach their desired zone, for example, a certain epithelial-layer several layers deep, the activating radiation, which may be light, ultrasound or another source of energy is applied. By this mode, only cells or tissue in the region which the administered compound reached are selectively destroyed, while cells and tissue present outward of this region can remain intact.
• the activating irradiation which again may be light, ultrasound or another source of energy, may be applied simultaneously with the second driving stimulus.
• irradiation-activated substances which are activated by light are photofrin, pheophorbide, po hyrin, boronated porphyrin, phtalocyanine, hematoporphyrin and chlorin.
• irradiation-activated substances which are activated by ultrasound are dimethylformamide, N-methylformamide, dimethylsulfoxide and gallium porphyrin.
• the present invention also concerns a system for use in the above method.
• the system generally comprises a multi-frequency signal generator, a signal amplifier, a matching unit and at least one transducer which may be attached to a focusing system (for example focusing lens) in order to increase the intensity at a desired site.
• the system may be also attached to any ultrasonic pumping unit attached to the first or second transducer.
• Fig. 1 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz, 1.5 w/cm 2 for 5 min. (x 3,900);
• Fig. 2 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz, 1.5 w/cm 2 for 50 sec. (x 7,500);
• Fig. 3 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz, 3 w/cm 2 for 1 sec. (x 12,450);
• Figs. 4,5 show electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MHz, 1.7 w/cm for 5 min. (Fig. 4 - x 4,800; Fig. 5 - x 3,900);
• Fig. 6 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MHz, 1.7 w/cm 2 for 50 sec. (x 8,700);
• Figs. 7,8 show electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MHz, 1.7 w/cm for 10 sec. (Fig. 7 - x 18,000; Fig. 8 - x 19,400);
• Figs. 9,10 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 3 MHz in the presence of the tracer lanthanum (arrow) 1.7 w/cm 2 for 5 mins. (Fig. 9 - x 19,500, Fig. 10 - x 9,900);
• Figs. 11,12 and 13 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz and the presence of the tracer lanthanum (arrow) in the cells and intercellular spaces 1.7 w/cm 2 for 5 mins. (Fig. 11 - x 12,000;, Fig. 12 - x 15,000; Fig. 13 - x 3,000);
• Fig. 14 shows a higher magnification of part of Fig. 13 (x 19,000);
• Fig. 15 shows electron microscopy of the epidermis after irradiation of 1 MHz at 2 w/cm for 10 sec, followed by irradiation at 3 MHz for 50 sec. in the presence of the tracer (arrows) in the tissue and sampled immediately after irradiation (x 9,000);
• Figs. 16-18 show the ultrastructure of fish skin treated as indicated in Fig. 15 above, 10 min. after irradiation. Tracer is present in naive cells (Fig. 16 - x 24,000; Fig. 17 - x 24,000; Fig. 18 - x 27,000); Fig. 19 shows a schematic representation of the ultrasound system of the invention;
• Fig. 20 shows a schematic drawing of acoustic lens used in the system of the invention.
• Fig. 21 shows a schematic representation of an ultrasonic pump.
• the therapeutic ultrasound used was a Sonicator 720 (Mettler Electronics, California, USA), with a probe surface area of 5 cm 2 and 10 cm 2 for two possible frequencies of 1.0 and 3.0 MHz ( ⁇ 5%), respectively. Power output up to 2.2 w/cm 2 , pulse mode 20% duty cycle or continuous wave.
• Skin biopsies (3x3 mm and thickness of 0.5 mm) were taken at different times after the ultrasound irradiation.
• the biopsies were taken from the dorsal part of the head of fish that were lightly anesthetized (MS222) and the biopsies were taken without killing the fish.
• the tissues were fixed in 3% glutaraldehyde in sodiumcacodylate buffer (0.09M, pH 7.3), washed in the same buffer and post fixed in osmium-tetroxide (1%) in the same buffer. Ethanol dehydrated tissues were embedded in Agar 100 resin. Thin sections were collected in 300 mesh copper grids. Samples of fish treated without tracer were further contrasted with uranyl acetate and lead citrate.
• the system 1 shown in Fig. 19, is a conceptual model which was used to perform the following experiments is composed of multi frequency signal generator and signal amplifier and matching unit all designated as 2 and a transducer 3. Irradiation is always carried out via aquatic medium 4, i.e. gel, or water as shown in the figure inside container 5.
• the active side of the transducers 6 is placed in aquatic medium 4.
• the transducer for the irritant stimuli may be attached to focusing lens (not shown; to increase its intensity) via regular inertic sonic-coupling gel.
• the lens is made of condensed material, preferably transparent plexiglass.
• an irritant stimulus is applied and later, or concomi- tantly, the tissue or cells are exposed to the second driving stimuli.
• the substances to be delivered are then introduced to the medium 4 either, a priori, before or simultaneously with the exposure to the second stimulus.
• the second transducer (not shown) might be also attached to lens in order to further increase the intensity.
• the fish 7 are held in place by a particular stand 8 acting as a clasp to immobilize them during the irradiation.
• a rubber lamina 9 is placed in the far part of the irradiated tissue, to absorb the energy and prevent formulation of standing wave resulting in unneeded elevation of the ultrasonic intensity.
• Plexiglass lenses were made from plexiglass cylinder.
• the curvature of the etched lens was 28 mm, i.e. the physical focus was at r- 28 mm.
• the F, (acoustic focus) was F-40 mm.
• a schematic drawing of the lens is shown in Fig. 20, wherein F represents the acoustic focus and r the curvature of lens.
• Region 10 is made of plexiglass and region 11 is made of water.
• Calculation of lens' parameter was carried out in accordance with Gordon S.K., Acoustic waves, devices, imaging and analog signal processing, Prentice Hall Inc., Englewood Cliffs, New Jersey, p. 652. The calculation of F is as follows:
• F can be determined also experimentally. Since the intensity is the highest and the affected area is the smallest at the exact focal point, it should cause a smallest mark to appear in the least amount of time on exposed materials. For calibration, a moveable thin disc of plastic was used. The distance where ultrasound caused the desired smallest "scar" on the plastic was then chosen.
• the ultrasonic force may be used in order to cause flow of the medium and thus bring the substances to be delivered to the site of administration.
• the medium flow causes suction activity at its surroundings, based on the effect known as "venturi tube”, and this suction activity can be used to introduce the desired elements into the medium. This will occur if the wave is enclosed in a hollowed shape, for example, a cylinder.
• Fig. 21 shows an administration system 20.
• the system contains a hollow cylinder 21 closed at one end by a flat disc 22 and closed at the other end by acoustic lens 23 attached to an ultrasound transducer 24.
• Cylinder 21 has about in its middle zone three openings 25 (only one shown in the figure) allowing passage of medium 29 from the tank 30 in which the cylinder is held into the cylinder 21 in the direction of the curved arrows.
• Disk 22 has a hole 26 which allows passage of liquid from the cylinder outward in the direction of the straight arrow.
• On top of the disc 22 are present two open-ended tubes 27 each attached at one end to reservoir 28 containing the substance to be administered and having the other open-ended tube present adjacent to opening 26 or disc 22.
• the medium 29 present in tank 30 Upon ultrasound application the medium 29 present in tank 30 will flow into cylinder 21 through openings 25 and then out of the cylinder through hole 26 in the direction of the arrows. While passing through hole 26, the suction activity initiated will pump out substances present in reservoir 28 through tubes 27. The substances will then be released into medium 29.
• the rate of substance release, from the reservoir via the tube, is proportional, among other parameters, to the relation between areas of openings 25 and the diameter of the cylinder 21. The intensity of suction and release can be justified according to the needs.
• Fig. 1 Application of 1 MHz at an intensity of 1.5 w/cm 2 for 5 min. (Fig. 1) resulted in a local degeneration effect. Outmost cells shown at the top of the figure featured severe damage and numerous membrane holes. Cells positioned in deeper levels were less affected and inner cell layers (bottom of Fig.) appeared normal. Application of 1.5 w/cm for 50 sec. (Fig. 2) resulted in necrotic superficial cells. Remnants of outer cells are visible while cells positioned at a deeper level appeared normal. Application of 30 w/cm 2 , carried out by applying an acoustic lens for a period of 1 sec. (Fig. 3) caused degeneration of cells which feature agglutination of nuclear hetrochronation and rupture of membranes.

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