CA2274690A1 - Method and apparatus for administration of substances by ultrasound - Google Patents

Abstract

The invention concerns a method for administration of substances to or through cells, tissues or membranes by exposing the cells, tissues or membranes to two ultrasound stimuli wherein the first stimulus is used to create transient openings in the cells, tissues or membranes and the second stimulus is used in driving the substances through said openings.

Description

WO 98/25655 PCT/8.97/00405 ..
METHOD AND APPARATUS FOR ADMINISTRATION
OF SUBSTANCES BY ULTRASOUND
FIELD OF THE INVENTION
The present invention concerns a method and apparatus for the administration of substances, into and through tissues or membranes using ultrasound.
BACKGROUND OF THE INVENTION
Administration of substances such as drugs, nutrients, vaccines and metabolites into tissues or via membranes, which may be biological or artificial (such as in implants), is often faced with difficulties due to the barrier healthy tissues and biological or artificial membranes present against undesired penetration of foreign components.
Various techniques have been developed for the facilitation of transport of substances across tissues. Examples of such techniques are:
building up concentration gradients of the compounds to be administered;
iontophoresis utilizing an electromagnetic field carried out both to increase the driving force of the administered substance and to cause formation of pores in the tissue or membranes; and utilization of 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.

WO 98/25655 PCT/8.97/00405 -2- _.
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/cm2.
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 (IJ.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.
At times, it is desirable to administer through tissues or biological membranes large 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. Examples of such 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 _T 1 WO 98/25655 PCT/8.97/00405 -3- _.
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, cosmetic or research purposes.
State-of the-art ultrasound-facilitated administration methods are unsuitable to administration of such large complex particles; since applica-tion of ultrasound pulses, sufficient to drive a small-sized molecule through a tissue is insufficient to drive those large complex particles through tissues or biological or artificial membranes. Increase of the duration, frequency or intensity of the ultrasound pulses to levels which are presumably sufFlcient to drive the large complex particles through the tissue or cell membrane, has not been reported probably since it results in irreversible damage to the tissue and in massive cell-death. Similarly, irreversible damage occurs in non-biological membranes of e.g., polyethylene or elastomer (for example those used in implants), when increased intensities or durations of ultrasound irradiation have been used.
It would have been highly desirable to provide a method for administering substances to tissues, cells or membranes (both natural and artificial) utilizing ultrasound, while minimizing the damage to the tissue or cells. It would have further been desirable to provide an ultrasound facilitated method for administration of complex particles having a relatively large size.

WO 98/25655 PCT/IL.97/00405 SUMMARY OF THE INVENTION
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. By utilizing these two stimuli, it was discovered that it was possible to introduce to the tissues or cells large complex particles without causing irreversible damage. The method for the administration of substances to cells, tissues or membranes, in accordance with the invention, comprises the following steps:
(a) exposing the cells, tissues or membranes to a first irntant ultrasound stimulus, being such as to cause transient formation of openings in said cells, said tissues or said membranes without causing any irreversible damage to the bulk of said cells or to most of the cells of said tissues or i 5 without causing irreversible damage to the membranes, the openings being of a size allowing entry therethrough of said substances; and (b) within a time period in which at least a portion of said openings remains open, exposing the cells, tissues or membranes to a second driving ultrasound stimulus, said exposure being carried out in the presence of said substances in a medium which is in contact with said cells, tissues or membranes; said second ultrasound stimulus being effective in driving at least part of said substances through said openings without causing any irreversible damage to the bulk of said cells or most of the cells of said tissues or to the membranes.
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 _T .. T ..__ 5 PCT/a,97/00405 -5) ..
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.
By a preferred embodiment, the substances to be administered are complex particles. The term "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. Examples of 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 impreg-nated 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.
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 WO 98125655 PCT/n.97/00405 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.
The term "openings" when used herein in connection with cells refers to pores formed in the membrane of the cells. The term "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.
The term "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. 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. When the two stimuli are applied simultaneously the substances __ .__._T... T

WO 98/25655 PCT/8.97/00405 should be present, a priori in the medium. The substances may be added manually to the medium. Alternatively, it is possible to construct a venturi tube and use the pumping effect created by the ultrasound wave itself, to deliver the substances present in specially constructed reservoirs to the medium, at rates according to the specific requirements.
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.
In order to determine the parameters of the first irritant stimulus, 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. Preferably 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. With respect to the fish skin, 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.
Generally speaking, the first irritant stimulus has the following parameters:
Frequency: 20 kHz to 3 MHz, preferably 100 kHz to 1.5 MHz, most preferably, 1 MHz or less.
Duration: 0.01 sec. to 20 mins. preferably 0.1 sec. to secs. most preferably less than but close to 1 sec.
Intensity: 0.1 - 500 W/cm2, preferably 0.1 - 100 W/cm2, most preferably 3-50 W/cm2.
. . _ _ r.~__T.-_.~_... _.

WO 98/25655 PCT/a,97/00405 The parameters of the second driving stimuli are:
Frequency: 20 kHz to 50 MHz, preferably 2 MHz to 15 MHz, most preferably, 3-S MHz.
Duration: 0.01 to 20 mins. preferably 0.1 to 5 mins., most preferably 1-10 secs.
Intensity: 0.1 - 50 W/cm2, preferably 0.1 to 10 W/cm2 most preferably 0.5 to 5 W/cm2.
It should be appreciated that there exists a reversal proportion between the intensity and the duration. Where the intensity is increased, for example, due to use of focusing system such as acoustic lenses, the duration should be decreased. Furthermore, cells present in vitro should be treated with lower intensities and shorter durations than cells present in vivo.
Occasionally, cells under in vitYO conditions might have to be adhered, prior to ultrasound application, to a particular sheet to enable controlled irradiation 1 S in order to reduce streaming effects on particular cells.
Preferably, 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.
In accordance with a second aspect of the invention, there is provided a method of destroying cells or tissues by irradiation of irradia-tion-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, 73:93 7-944, ( 1996)).
There have been reports (Miyoshi et al., Radiat. Res., 143:194-202 (1995)) of cancer treatment based on the combined effect of a photosensitizer and ultrasound which apparently is capable of activating some compounds previously reported as being activated by light.
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.
By a second mode, the activating irradiation, which again may be light, ultrasound or another source of energy, may be applied simultaneously with the second driving stimulus. This caused the irradiation-activated substances to cause their tissue-destroying affect already during their penetration route so that essentially all the region from the site of administration (the outer layer of the skin) to the region where the substances reached is essentially destroyed.
Examples of irradiation-activated substances which are activated by light are photofrin, pheophorbide, porphyrin, boronated porphyrin, phtalocyanine, hematoporphyrin and chlorin.
Examples of 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 mufti-frequency signal generator, a signal amplifier, a matching unit and at least one transducer ~.~_._...._.._.._ _~_..~T_._ WO 98/25655 PCT/a,97/00405 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.
In the following the invention will be further illustrated with reference to some non-limiting drawings and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows electron microscopy of the epidermis of fish irradiated with a continuous wave of ultrasound at 1 MHz, 1.5 w/cm2 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/cm2 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/cm2 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/cm2 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/cm2 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/cm2 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 ~~:AIZJS 17 DEC 199g the tracer lanthanum (arrow) 1.7 w/cm2 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/cm2 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/cm2 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;
Fig. 22 shows a transdermal delivery system for professional purposes;
Fig. 23 shows a transdermal delivery system: 23A in an expanded form and 23B in an assembled form;
Fig. 24A, 24B and 24C show schematic drawings of parts of the treated system intended for personal transdermal delivery. Fig. 24D describes a device for creating suctions;
Fig. 25 shows a personal transdermal delivery system - 25A in the position used in the first irradiation phase and 25B in the position used in the second irradiation phase;
r~

12a ~~~~~ I 7 DEC 199 Fig. 26 shows another personal transdermal delivery system - 26A in the position used in the first irradiation phase and 26B in the position used in the second irradiation phase; and Fig. 27 shows yet another personal delivery system - 27A in the position used in the first irradiation phase and 27B in ,the position used in the second irradiation phase.
DETAILED DESCRIPTION OF THE INVENTION
I. EXPERIMENTAL PROCEDURES
A. Ultrasound instruments The therapeutic ultrasound used was a Sonicator 720 (Mettler Electronics, California, USA), with a probe surface area of 5 cm2 and 10 cm2 for two possible frequencies of 1.0 and 3.0 MHz (~ 5%), respectively. Power output up to 2.2 w/cm2, pulse mode 20% duty cycle or continuous wave.

-' 3 - _.
B. Application of ultrasound to fish Over I20 specimens of fish (tilapia and carp species) weighing 4-100 gr. were treated with ultrasound in sea water. Ultrasonic irradiation was performed on 4-6 fish concomitantly, when the fish were held in the same container (of about 3,000 mI) and each of the fish was stabilized to his location using a special holder. Ultrasound irradiation was performed from above as demonstrated in the schematic drawing of Fig. 17. Except for one exposure, in which the fish were transferred to another container comprising the tracer, in all other experiments the fish remained in the same container during the two stimuli. The lanthanum tracer was added either immediately before the second irradiation or was present in the medium from the beginning of the first irradiation. The time of introducing the tracer did not significantly affect the results. The experiment was later carried on again.
This time the lanthanum tracer was replaced by bacterins (streptococcus) that was introduced to the water during the second irradiation.
C. Histological preparation for electron microscopy analysis 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 ( I %) 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. Samples of fish treated in the presence of the tracer were not contrasted so that visualization of the tracer was enhanced. All samples were examined in a -14- w Jeol 100 CX transmission electron microscope (Iger Y. and Wendelaar Bonga, S.E., Cell Tissue Res., 275:481-492 (1994)).
D. Ultrasound system 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 carned 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.
In practice, an irritant stimulus is applied and later, or concomi-tartly, 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.
E. Acoustic lenses 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 _._ _..~___. ~ ~__ __ _._ . T. T._..~... __ . __._._ _ _ _.._ .

WO 98/25655 PCT/8.97/00405 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 aid analog signal processing, Prehtice Hall ~nc., Englewood Cliffs, New 3ersey, p. 652.
The calculation of F is as follows:
F= r 1 (1._ 1 ) n wherein r = curvature of lens;
n=Cp2 Cw Cp = the speed of sound in matrix (in plexiglass 2.7/km/hr);
Cw = the speed of sound in water at 20°C ( 1.48 km/hr).
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.
F. Ultrasound pump 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 WO 98/25655 PCT/II,97/00405 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.
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 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.
Example 1 Morphological effects of ultrasound application Over 120 specimens of fish (tilapia and carp species), weighing 4-100 gr, were treated with ultrasound. All fish survived the irradiation and the periods after that for all the time they were still monitored (30 to 50 days).
Macroscopically, fish (either naive or anesthetized) exposed to 1 MHz ultrasound were covered with cavitation bubbles, whereas fish (if under 10 gr) exposed to 3 MHz, were slightly forced downwards by the acoustic pressure.
Using different ultrasound parameters, the rupture of cellular junctions was initiated in the epidermis, which was (macroscopically) ,, _ _ _ _ _ __ __.~ ~__ T __ T__ _.~

-' 7 _ _.
followed by skin sloughing. Vasodilation and skin hemorrhages was also initiated when high intensities were used for prolonged periods.
Example 2 Ultrastructural effects of ultrasound application Effect of ultrasound phases having various parameters and the results are shown in Figs. 1 to 8.
Application of 1 MHz at an intensity of 1.5 w/cm2 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/cmz 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/cm2, 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.
Application of 3 MHz at 1.7 w/cm2 for 5 min. (Figs. 4 and 5) caused rupture and fusion of cells located at the 4-5 epidermal layers from the surface while outer cells remain in tact. Five to fifteen minutes after application of the ultrasound skin peeling of outer layers occurred.
Application of 3 MHz at 1.7 w/cm2 for 50 sec. (Fig. 6) caused detachment of cells at the site of ultrasound administration. Celts located outer to the place of detachment were sloughed from the epidermis as compact layers. Remnants of ruptured membranes can be observed.
Application of 3 MHz 1.7 w/cm2 for 10 sec. (Figs. 7 and 8) caused formation of vesicles unbound by membranes, which are probably cavitation bubbles in superficial cells.
It was found that application of 1 MHz of ultrasound could break and cause sloughing of the previous unstirred microclimate of mucus, surrounding the epithelium. Formation of holes in cell membranes and membranes rupturing, up to necrosis, of the 2-3 outermost layers of the epidermis (the outermost 20-30 performed for longer than 2 minutes, sloughing of the affected epithelial tissues followed the application. Application of such an intensity for periods below 2 minutes allowed many of the cells to recover and remain an integral part of the epidermal tissue. Deeper cells were, at least apparently, unaffected. The intercellular spaces of the external epithelial layers were slightly increased.
Using special focusing lenses described in Experimental Procedure D, time of exposure of the fish was dropped to 1-2 seconds, yet having effects similar to those mentioned above.
Ezample 3 Sonophoresis Lanthanum hydroxide {LH) having a size of about 60-90 nm was used as a colloidal tracer. Fish were immersed in a bath containing 0.1 % w/w of LH. LH was added to the water immediately before the second ultrasonic stimuli. In biopsies of control fish exposed to LH without ultrasound, the tracer was only rarely observed on the skin surface. In fish irradiated with 3 MHz in the presence of LH, and sampled immediately afterwards, LH had adhered heavily to the epidermal surface, (Figs. 9-10), but the tracer did not penetrate the cells. In fish irradiated with 1 MHz and sampled immediately afterwards, the tracer penetrated well into the cells with ruptured membranes and also into the intercellular spaces of deeper and naive cells located one or two levels deeper (Figs. 11-14).
Fish were irradiated with 1 MHz followed by irradiation with 3 MHz, in the presence of a tracer. In fish sampled immediately afterwards (Fig. 15) the tracer penetrated deeper, up to 5-b layers from the surface and at a higher quantity into the epidermal intercellular spaces than was possible rl _ _... __._._~.. ~

..
~~i~l~lS 17 DEC 1999 only by a single application of 1 MHz. In fish sampled 10 minutes after the irradiation (F igs. 16-18), particles - of tracer were foi,uld not only in the intercellular spaces, but also in the cytoplasm of naive. cells, located at deeper epidermal zones- close to the basal lamina. The uptake of such particles into the cells was probably carried out by phagocytosis since they were mostly engulfed into phagosomes. After 10 minutes particles were found also in the dermal zone.
Similar results were obtained when LH was replaced by bacterins (which are formalin inactivated streptococcus) u'~at was introduced to the water just before the second irradiation to create concentrations of Sx 10' bacterins/ml. Bacterin particles were not observed in tissue immersed in bacteria-containing water, without ultrasound irradiation. After irradiation with 1 MHz followed by 3 MHz, bacteria particles penetrated the skin. They were found in the intercellular spaces of the epidermis and in the cytoplasm of superFcial cells, as well as in the cytoplasm of deeper and naive cells.
These results demonstrate that it is possible to bypass the nonspecific barriers at the fish skin surface, at the cellular and intercellular levels, to force delivery of particles into epithelial cells and deeper dermal zones, when a two phase delivery of the invention is used.
Reference is now made to Fig. 22, which shows a transdermal delivery system 30 intended for professional purposes for use in clinics, hospitals, etc.
The system 30 comprises a treatment cylinder 34 which is effective in delivery the substance to the skin 31, and a transducer 36, movable within transducer housing 35. The treatment cylinder is secured to skin 31 through suction forces.
The system also comprises reservoir 64 holding the agent to be administered transdermally, and balloon 55 which holds a gas to facilitate entry of the agent as will be explained hereinbelow.

a ~~I~$17 D E C 1998 Transducer 36 is operated by driving unit 51 which is composed of a signal generator, amplifier and a matching unit (not shown).
Driving unit 51, is controlled by control unit 52, which can give the driving unit pre-set instructions which preferably comprises a computerized component. Control unit 52, also receives inputs from various components of the system in order to give feedback inputs as will be explained hereinbelow.
For professional purposes the system is attached to the skin 31, by base housing 32 which contacts the skin 31 by gently applying a vacuum force in the suction groove 33 until the base is secure. Attachment to skin may be made in a plurality of manners well known in the art. The treatment cylinder 34 is secured to base housing 32 by suitable grooves.
Transducer housing 35 together with the transducer 36 are attached to the treatment cylinder 34. After the initial irradiating phase is completed, the 1 S transducer housing 35 is lowered to a second, lower position either by the motorized linear driver 59 or else by manual movement downwards. The transducer housing's new position is secured by inserting the locking pin 60 via hole 61 from lock hole 62 into lock hole 63 or 64. Preferably housing 35 is lowered to the level of hole 63 during the second irradiation phase and secured to that wall.
The liquid llow from reservoir 37 to the treatment cylinder 34 operates as follows: When water reservoir valve 36 is opened and water is pumped from the reservoir 37 via the pump 38, and pipe 39, into the treatment cylinder 34 through entry holes 40 and 41. Air trapped in the system is released from openings 42, 43 and 44. Once the treatment cylinder 34 is filled and free of air bubbles, the ultrasonic treatment can initiate with waves transmitted from the transducer irradiating face 45 towards the skin 31.

~~A~IS 17 DEC 199 Water return line valve 47 can be shut during application of ultrasonic forces, or can circulate the water via pipe 48 and filter 49 back to reservoir 37, and again into treatment cylinder 34. The constant circulation of water, especially through opening 40, clears excess bubble formation from the transducer irradiating face. Excess air will drain through suitable drain openings.
Excess water is drained through the drain holes 42, 43 and 44.
Once the treatment apparatus is ready for the second irradiation phase which drives transdermally the desired substance, the reservoir valve 65 is opened and the compound to be delivered is pumped from container 64 via tube 67. The compound is pumped into the space of container 34, which space remained after the lowering of housing 35 and transducer 36 to the new, low position. The compound enters the space of cylinder 34, via entry hole 60, displacing any excess water drained through hole 44. Additional suction can be created over the treated skin area by closing valve 65 and allowing drain valve 47 to remain open for a period sufficient to create aforementioned suction. The suction attachment is a preferred embodiment.
It creates a partial vacuum, enabling better attachment of the treatment cylinder to the skin. In addition during the first irradiation phase the partial vacuum can be utilized since it reduces the threshold level for cavitation, enabling better irritant effect. During the second irradiation phase, the partial vacuum improves delivery since the vacuum force causes enlargement of blood vessels, enabling better diffusive of the ultrasonic delivered compounds. After the treatment is complete, excess solutions are drained, the suction released and the apparatus removed.
The control unit 52 can monitor gas concentration in the water reservoir via the reservoir sensor 53 and, if needed, compensates for gas loss, (for example due to cavitation during the first irradiation phase).
Compensation is carried out by opening the gas balloon valve 54 and 22 i~~A~IS 1 ?' DEC 1998 allowing gas to escape from the balloon 55 via pipe 57 into reservoir 37. By this mechanism the dissolved gas content of cylinder 34 remains constant.
Gas pressure in the balloon is also monitored by the computer by use of a pressure sensor (not shown). The computer maintains a desired water pressure in the cylinder 34 by adjusting the pump speed.
Fig. 23 shows a schematic drawing of treatment system 100 in Fig. 23A in an expanded form and in Fig. 23B in an assembled form. The system is intended for personal use, for example, for a patient who needs daily transdermal administration of a certain medicament. Preferably the system is in a shape of cylinder. It is composed of transducer unit 101, treatment cylinder 102 and suction cup 103. The transducer unit 101 is composed of housing 104 and transducer unit 105. The transducer unit 105 can be an independent irradiation device, composed of signal card, amplifier card and energy battery source which are elements coupled together to create desired sequences at desired frequencies and intensities at the irradiating part of the transducer itself. Alternatively, the transducer unit 105 can be composed of transducer itself, which is electrically connected to another external device (not shown) comprising signal generator, amplifier and energy source. Treatment device 102, is composed of device wall 107 and a protruding lip 108. Suction cup 103 is preferably from a flexible material, e.g., rubber. It has special shape, with groove 110 at one side and suction tunnel 109 at the other side. When connected together (Fig. 23B), Transducer unit 101' is attached to treatment device 102'. Device 102' is further attached to the suction cup 103' by placing the protrusing lip into the groove.
Fig. 24A, B, C and D show schematic drawing of parts of the treatment device intended for personal transdermal delivery.
Fig. 24A shows schematically parts of the transdermal device and the treatment cylinder 201 that has a protruding lip intended for 23 ~S 1 ? DEC 199 sealing 202 that fits into the seal groove of the suction base (not shown).
The compound to be transdermally delivered is placed in the holding unit 203 and the cylinder is fastened to the suction cup (not shown) and attached to the skin 208. Water is filled through the top of the device and the transducer unit (not shown) is attached from above, near valve 209 opposite to skin 211 forcing excess water out through the valve 209. During the initial irradiation phase, shield 210, which can be reflector or absorbent, is located between the irradiation source and the said compound, protecting compound 203 and preventing destruction effects of the cavitation bubbles on the compound. Skin irradiating zone at this position is 211.
. Fig. 24B describes the position of the system during the second irradiation phase. At this time the shield 210 is flipped to the other side of the cylinder so compound 203 is exposed to the second driving stimulus. During the second irradiation phase area 203 is placed over the area 211', which correspond to the same area irradiated during the first irradiation phase (area 211 in Fig. 24A). At this position, irradiation affect the compound and cause its delivery into the initially irradiated zone. The system can have also two irradiating transducers located one over water and skin 211 and one over compound 203. The two transducers will work in synchronization and thus no shield will be required.
Fig. 24C schematically describes device in which the compound to be transdermally administered is retained in reservoir 220 attached to side of cylinder. After attachment of the device to skin via suction base (not shown), the cylinder is filled with water and the transducer is placed at level E1. After pre-treatment, water is removed through syringe 221 and transducer is lowered to level E2. Said compound to be administered is dispensed from the reservoir 220 into the gap between the lower treatment line E2 and the skin. A suction force can be created over the skin with the synnge.

2 4 ~~~151 ?' DEC 1998 Fig. 24D schematically describes another device for creating suction. Here, the suction is created by depressing and releasing a rubber seal 230 that comprises part of the cylinder wall. Pressing the seal before attachment and leaving the seal after attachment, creates a vacuum for which the desired suction shows. The rubber seal is further demonstrated in Fig. 24E as 230 in the B-B section.
Fig. 258 shows schematically personal transdermal delivery system 300, when used for first irradiation phase, with the reflector-shield-door 301 attached to hinge 302 and is located in the open position. The transducer 303 is attached to the treatment device 304, so that the transducer irradiating zone 305 is capable of irradiating ultrasonic energy via cylinder wall 319. The treatment device is attached to the suction unit in such a way that protruding lip 307 is placed in the appropriate groove. The device is pressed to the skin 308 in a way that it covers the desired treatment area 309 and filled with treatment liquid holding the agent to be delivered until its level passes the transducer face 305. The reservoir cover 318 is closed. Cavitation bubbles 310 are created during the initial radiation (i.e.
the initial pulse). Further air bubbles created during treatment are collected in the air trap 312 or in the compound holder 313. During this irradiation phase, the compound to be delivered 314 remained non-irradiated and lip 315 remained unattached.
Fig. 25B shows the same device shown in 258, during the second irradiation phase. At this configuration protruding lip 315' is located in the suction groove. The compound 314', (present for example in gel or patch), is placed and attached to area 309' of skin 308', the same area that was irradiated during the first stimulus. Reflector-shield-door 301', connected to hinge 302' is closed manually, or due to the gravitation, and is located in its new position. During the second irradiation, ultrasonic waves formed in irradiating zone 305' of the transducer unit are reflected from 2 5 ~ ~~~I~IS 17 D E C 19 98 reflector 301' via compound 314' into the skin zone. Loss of liquid can be replenished by adding through the reservoir cover 318. The change of position forces the ultrasonic waves to pass via the compound attached to the skin and thus cause its delivery of the compound to the area where openings were formed in the skin due to the irradiation of the first pulse.
Fig. 26A describes another personal treatment system 400 in which the transducer 401 is attached to the conduction unit 402 facing the reflection wall. The unit is filled with water and placed over the treatment chamber 403. The pretreatment chamber 404, used for the irradiant exposure, is filled with water and the compound to be delivered is placed in at least part of holding unit 405. During pretreatment, the pretreatment chamber will cover the skin 406 over the area 407 and the unaffected compound holder will cover area 408. During sonication, the ultrasonic wave will progress from the transducer irradiating zone 410, via wall 411, being parallel to the skin, reflect off the reflection wall 418 to a perpendicular angle towards the skin, pass through wa11406 into the treatment chamber 404, creating cavitation bubbles 412 and finally reaches the skin. Bubbles created in container 402 accumulate in zone 413, and balance can be achieved by opening of closure 414 allowing excess air to leave the system or adding water into the system. Air removal or water adding to device 403 is carried out via opening 415.
In Fig. 26B, position of system components during the second phase is described. The treatment chamber 402' slides from zone 404' via track (not shown) over the compound holding unit 405' . This time, before the second irradiation, the whole device is moved in such a way that compound container 405' is located over skin zone 407', which was irradiated during the first irradiation phase. Sonication is carried out at this phase via the compound to be delivered, into the skin.

26 ~~rus ~ 7 oEC ~9sg Fig. 27 schematically describes another delivery system for personal treatment. In Fig. 27A system 500 is attached to the skin 501 by suction groove 502 situated in the seal ring 503. The system has another attachment rubber 504, used to stabilize the system. The transducer 505 is attached to the treatment chamber 506, so the transducer irradiation zone 507 coupled to wall 508 facing the reflection wall 509. Ultrasonic waves leave the transducer face 507, pass through the chamber and reach the skin after reflecting off the reflection wall 509. Compound 510 to be delivered is placed in the holder 511 and the holder is pushed into its holding tunnel all the way until stopped by stopper 512. The chamber is filled with water, after which the valve 513 is replaced. The valve can be used also for suction activity of the whole content of the device and attached skin. During the initial irradiation phase, slope 517 of the handle is at a certain distance from stopper 512.
Either this first irradiation, or second irradiation described below can be carried out under further suction activity, performed via opening 513. The suction activity can be used either to increase cavitation, to enlarge blood vessels or both as described above.
Fig. 27B shows the apparatus in the second drug stimulus wherein transdermal delivery takes place. The holder 511 is pushed into the tunnel until step stopper 512. The slope 517 reaches stopper 512, forcing the whole handle 511 and compound 510 towards the skin 501. At this position the second drug irradiation is carried out.
The pulses, both the first (causing openings in the skin) and the second (causing delivery of the compound through said opening), used for each of the embodiments may be composed of several pulses, each of these many pulses being in the range relevant to that pulse (i.e. the first or second).
For example, the first pulse can have frequencies of 0.5, 0.7, 0.9 and 1 mHz simultaneously using wide-band transducers. The second pulse may be composed similarly.

Claims (32)

CLAIMS:

1. A method for the administration of substances to and/or through cells, tissues or membranes, comprising the following steps:
(a) exposing the cells, tissues or membranes to a first irritant ultrasound stimulus, being such as to cause transient formation of openings in said cells, said tissues or said membranes without causing any irreversible damage to the bulk of said cells or to most of the cells of said tissues or without causing irreversible damage to the membranes, the openings being of a size allowing entry therethrough of said substances; and (b) within a time period in which at least a portion of said openings remains open, exposing the cells, tissues or membranes to a second driving ultrasound stimulus, said exposure being carried out in the presence of said substances in a medium which is in contact with said cells, tissues or membranes; said second ultrasound stimulus being effective in driving at least part of said substances through said openings without causing any irreversible damage to the bulk of said cells or most of the cells of said tissues or to the membranes.

2. A method according to Claim 1, wherein the frequency and duration of said second stimulus is higher and longer, respectively, than those of the first stimulus, while the intensity of the first stimulus is higher than that of the second stimulus.

3. The method of Claim 1, wherein the compounds to be administered are complex particles.

4. A method according to Claim 3, wherein the complex particles are selected from the group consisting of:
i. bacteria;
ii. viruses or virions;
iii. fungi;

iv. protozoa;
v. parasites;
vi. fragments of (i-v);
vii. plasmids;
viii. nuclei;
ix. particles impregnated with medicaments;
x. particles comprising biologically compatible dye; and xi. particles coated with a coat capable of changing their properties.

5. A method according to Claims 1 to 4 , wherein the first and second stimulus are applied simultaneously.

6. A method according to Claims 1 to 4, wherein the second driving stimulus is applied subsequent to application of the first stimulus.

7. A method according to Claim 6, wherein the interval between the first and the second stimulus is up to 15 mins.

8. The method of Claim 1, wherein the tissue is a moist epithelial tissue, or artificially moistured keratinized epithelial tissue.

9. The method of Claim 8, wherein the epithelial tissue is of an aquatic animal.

10. A method according to Claim 1, wherein the frequency of the first stimulus is 20 kHz to 3 MHz.

11. A method according to Claim 10, wherein the frequency is about 1 MHz.

12. A method according to Claim 1, wherein the frequency of the second stimulus is 20 kHz to 50 MHz.

13. A method according to Claim 12, wherein the frequency of the second stimulus is 3 to 5 MHz.

14. A method according to Claims 1 to 13, wherein the duration of the first stimulus is 0.01 secs. to 20 mins.

15. A method according to Claim 14, wherein the duration of the first stimulus is about 1 sec.

16. A method according to Claims 1 to 15, wherein the duration of the second stimulus is 0.01 secs. to 20 mins.

17. A method according to Claim 16, wherein the duration of the second stimulus is 1-10 secs.

18. A method of Claims 1-17, wherein the first ultrasound stimulus is applied at intensities of 0.1-500 w/cm2.

19. A method according to Claim 17, wherein the intensity is 3-5 w/cm2 at the near-zone ultrasonic field, 30-100 w/cm2 close to or at focal point where a focusing system is used.

20. A method according to Claims 1-19, wherein the second ultrasound stimulus is applied at intensities of 0.1-50 w/cm2.

21. A method according to Claim 20, wherein the intensity is 0.5-5 w/cm2 at the near zone ultrasonic field or 5-50 w/cm2 where a focusing system is used.

22. A method according to Claims 1-21, wherein the ultrasound of the first and second stimulus is applied at duty cycles between 10% to a continuous wave.

23. A method for destroying cells or tissue by a substance capable of being activated by irradiation comprising:
(a) topical administering said substance to and/or through the cells or tissues by the method of Claim 1; and (b) applying irradiation to the cells or tissue having an intensity frequency and duration capable of activating said substance.

24. A method according to Claim 23, wherein the substance is activated by irradiation of light, and wherein the irradiation of step (b) is light.

25. A method according to Claim 23, wherein the substance is activated by irradiation ultrasound and wherein the irradiation of step (b) is ultrasound.

26. A method according to Claim 24, wherein the substance is selected from the group consisting of photofrin, pheophorbide, porphyrin, boronated porphyrin, phtalocyanine, hematoporphyrin and chlorin.

27. A method according to Claim 25, wherein the substance is selected from the group consisting of: dimethylformamide, N-methyl-formamide, dimethylsulfoxide and gallium porphyrin.

28. A method according to Claim 23, wherein step (a) and step (b) are carried out simultaneously.

29. A method according to Claim 23, wherein step (b) is carried out subsequently to step (a).

30. A system for use in the method of any one of the preceding claims.

31. A method according to Claim 1, substantially as hereinbefore described.

32. A system according to Claim 30, substantially as hereinbefore described.