Note: Descriptions are shown in the official language in which they were submitted.
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ULTRASONIC ENHANCEMENT OF DRUG INJECTION
This application is a continuation-in-part of application numbers
09/126,011, filed on July 29, 1998, and 09/255,290, filed on February 22,
1999, the full
disclosures of which are incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to methods and devices for enhancing
cellular absorption of a substance delivered into a target region of a
patient's body.
BACKGROUND OF THE INVENTION
General
A current standard technique for the delivery of drugs or other substances
into the human body is needle injection. A bolus containing the drug is
typically injected
into muscle or fatty tissue and is then absorbed into the interstitial fluid
or directly into
the fatty tissue. Over a period of time, the vascular system of the body takes
over and
flushes the drug out of the interstitial fluid or fat and into the
capillaries. From there, the
cardiovascular system widely distributes the drug into the rest of the
patient's body.
Newly developed drugs often have application only to specific organs or
sections of organs. As such, systemic distribution of the drug throughout the
remainder
of the body can: (1) dilute very expensive drugs, weakening their effects, (2)
generate an
effect systemically instead of locally, and (3) widely distribute a drug which
may be toxic
to other organs in the body. Furthermore, some of the newly developed drugs
include
DNA in various forms, such DNA being degraded very rapidly by natural
mechanisms in
the body if delivered systemically, thus preventing a full dose from reaching
the
designated organ. Accordingly, it would be desirable to provide devices, kits,
and
methods for delivering such site-specific drugs in a manner which enhances
absorption
specifically at the site of their delivery into a target region of a patient's
body.
2. Description of the Background Art
Catheters and methods for intravascular transfections are described in U.S.
Patent No. 5,328,470 and published in PCT applications WO 97/12519; WO
97/11720;
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WO 95/25807; WO 93/00052; and WO 90/11734. See also copending application
no. 09/223,231, the full disclosure of which is incorporated herein by
reference.
Ultrasound-mediated cellular transfection is described or suggested in Kim
et al. (/996) Hum. Gene Ther. 7:1339-1346; Tata et al. (1997) Biochem. Biophy.
Res.
Comm. 234:64-67; and Bao et al. (1997) Ultrasound in Med. & Biol. 23:953-959.
The
effects of ultrasound energy on cell wall permeability and drug delivery are
described in
Harrison et al. (1996) Ultrasound Med. Biol. 22:355-362; Gao et al. (1995)
Gene Ther.
2:710-722; Pohl et al. (1993) Biochem. Biophys. Acta. 1145:279-283; Gambihler
et al.
(1994) J. Membrane Biol. 141:267-275; Bornmannan et al. (1992) Pharma. Res.
9:559-
564; Tata and Dunn (1992) J. Phys. Chem. 96:3548-3555; Levy et al. (1989) J.
Clin.
Invest. 83:2074-2078; Feschheimer et al. (1986) Eur. J. Cell Biol. 40:242-247;
and
Kaufman et al. (1977) Ultrasound Med. Biol. 3:21-25. A device and method for
transfection of endothelial cells suitable for seeding vascular prostheses are
described in
WO 97/13849.
Local gene delivery for the treatment of restenosis following intravascular
intervention is discussed in Bauters and Isner (1998) Progr. Cardiovasc. Dis.
40:107-116
and in Baek and March ( 1998) Cire. Res. 82:295-305.
SUMMARY OF THE INVENTION
The present invention provides methods, devices, and kits for enhancing
cellular absorption of a drug or other substance into a local target region of
a patient's
body, thereby avoiding the undesirable effects of the substance being widely
dispersed
throughout the patient's body by the patient's cardiovascular system. By
"cellular
absorption," it is meant that at least a significant proportion of the total
amount of drug
delivered to the site is absorbed or otherwise taken up by the cells within or
surrounding
the target site. The nature of the cells will vary depending on the target
site. The cells
may be muscle or fat cells receiving transcutaneous, intraoperative, or
percutaneous
injection. In a first preferred aspect of the present invention, these cells
comprise the
patient's myocardial tissue. In a second preferred aspect of the present
invention, the cells
may comprise any solid tissue cell which is a target for gene transfection,
particularly
myocardial and other muscle tissues. The cells may also be endothelial,
epithelial, and/or
other cells which line the interior or exterior of target organs, or brain
cells protected by
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the blood/brain barner, or organ cells in general. Lastly, the cells may also
be specific
organ cells of a target organ.
Specifically, a method is provided for enhancing cellular absorption of a
substance, comprising the steps of (a) delivering the substance to the target
tissue region,
and (b) directing vibrational energy to the target region, wherein the
vibrational energy is
of a type and amount sufficient to enhance absorption of-the substance into
the cells of the
target region. In a preferred aspect of the present invention, the vibrational
energy has a
mechanical index in the range of 0.1 to 20. Devices for emitting ultrasonic
vibrations of a
type and amount sufficient to enhance cellular absorption may comprise a wide
variety of
known transducer systems, such as piezoelectric, magnetostrictive or single
crystal
devices.
The application of such vibrational energy to the target region increases
cellular absorption on the order of 3 to 300 times or more for biological
reporters such as
luciferase and beta-galactosidase genes and for drugs such as heparin,
probucol,
liposome-complexed plasmid DNA, cationic polymer complexed DNA, plus viral
vectors
including adeno-associated viral DNA, vascular endothelial growth factors, and
naked
DNA, relative to their uptake in the absence of the vibrational energy.
The present invention will be useful for delivering a wide variety of drugs,
genes, and other therapeutic and/or diagnostic substances to target tissue
sites. The
substances will usually have a pharmological or biological effect and may
range from
those generally classified as small molecule drugs (usually below 2 kD, more
usually
below 1 kD), such as hormones, peptides, small nucleic acids, carbohydrates,
and the like
to those generally classified as large molecule drugs (usually above 500 kD,
often above
50 kD, and sometimes above 200 kD) such as large proteins, complete regulatory
and
structural genes, large carbohydrates, and the like. The present invention
will be
particularly effective in delivering macromolecules such as biologically
active proteins
and nucleic acids. For delivery to the muscles in general, or the myocardium
in
particular, useful substances, proteins and the genes which encode such
proteins,
e.g., angiogenesis stimulators, such as angiogenic cytokines including
vascular
endothelial growth factor (VEGF) and basic fibroblast growth factor (BFGF).
Other
useful substances and genes include endothelial nitric oxide synthase (eNOS)
for
inhibiting restenosis; brain naturatic peptides; beta-adrenogenic receptors
for preventing
congestive heart failure; erythropoietin (EPO); clotting factors, such as
Factor VIII and
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Factor IX; human growth hormone; insulin; interferons, particularly including
interferon-
A for treating neoplasms; interleukins; and various "secretory proteins" which
are
proteins that are secreted from transfected cells and exert biological effects
on other cells
and tissues. Such secretory proteins may include hepatocyte growth factor,
atrial
S naturiuretic factor, VEGF, a-1 antitrypsin, a- Iduronidase, Iduronate-2-
sulfatase,
glucocerebrosidase, (3-glacuronidase and neurotrophin. For many or most of
these, it will
be preferred to introduce a gene which encodes the desired therapeutic protein
together
with any necessary regulatory nucleic acid sequences to a desired target
tissue. By
transfecting the target tissue with the therapeutic protein gene, the cells
can then produce
the therapeutic proteins in therapeutically effective amounts. Ultrasound in
combination
with DNA-based vaccines would enhance protein expression by improving the
humoral
and cellular immune response.
The delivery of nucleic acids (usually in the form of genes) to target cells
is generally referred to as "transfection." The methods of the present
invention may be
I S advantageously applied to cellular transfection of target tissues since
they are capable of
significantly increasing transfection efficiency, i.e. the amount of nucleic
acid materials
taken up by the muscle cells and cellular nuclei to which they are delivered.
The methods
of the present invention are useful with a wide variety of nucleic acid types.
For example,
it has been found that significant transfection efficiencies can be obtained
even with
naked DNA, i.e., nucleic acids which are not incorporated into liposomes,
virosomes,
viral vehicles, (eg: adenovirus, retrovirus, lentivirus, and adeno-associated
virus),
plasmids, or other conventional nucleic acid vehicles. The methods are not
limited to
such naked nucleic acids, however, they are also suitable for the delivery of
nucleic acids
incorporated into liposomes and cationic polymer complexes such as virosomes,
vesicles;
viral vectors, including both adenoviral vectors and retroviral vectors;
plasmids, and the
like.
In a preferred method, the substance is delivered to the target cells in the
target tissue of the host. This delivery can be accomplished transcutaneously
or
percutaneously by way of an injection needle or needles, injected in high-
velocity, small-
volume jets of delivery fluid, or delivered interoperatively. The substance
can also be
delivered by a controlled release device such as a microsphere. Substance
delivery could
also be accomplished by positioning the distal end of a delivery device, (such
as a
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catheter or hand held device), proximal to a target region of tissue, wherein
a vibrationaI
energy emitter is positioned at the distal end of the device. For delivery
through the skin
or surgical use, the device may be constructed similarly to a syringe having
an ultrasonic
driver on or near the needle tip. For internal delivery, the device will
typically be formed
as a catheter for intraluminal or endoscopic introduction to a target site.
By "delivery," it is meant that the drug, gene, or other substance is injected
or otherwise physically advanced into a target region of tissue. injection can
be
performed with a needle and a pressurized source of the substance, e.g. a
syringe. A
controlled delivery device or depot containing the drug could also be
implanted within the
target tissue. Substances of interest will typically be delivered through the
internal walls
and membranes of organs (particularly the epicardium and endocardium when
targeting
the myocardium), blood vessels, and the like, as well as through the skin. In
some
instances, the catheter will be percutaneously introduced to a blood vessel or
open body
cavity in order to permit access to the internal organs and gene delivery
sites.
The present invention also provides a device for enhancing cellular
absorption of a substance delivered to a target tissue region of a patient's
body comprising
a substance delivery system and a vibrational energy emitter which is adapted
to emit
vibrational energy of a type and amount sufficient to enhance cellular
absorption in the
tissue. Preferably, the substance delivery system comprises one or more
injection
needles.
In one embodiment, the injection needle and the ultrasound energy emitter
form a small integrated device which is received at the distal end of a
catheter. In one
aspect of this embodiment, the energy emitter may include one or more
vibrational energy
emitting transducers received within the injection needle. In various
embodiments, the
vibrational energy emitter is disposed proximate to the substance delivery
system. In
certain preferred embodiments, the vibrational energy emitter is mounted
directly to the
injection needle. The vibrational energy emitter may also be disposed
concentrically
around the substance delivery system. Specifically, the substance delivery
system may
comprise an injection needle with the vibrational energy emitter mounted
directly on the
injection needle. In various embodiments, the injection needle system
comprises a
plurality of retractable radially extending injection needles which are
positioned at the
distal end of a catheter such that when the catheter is received into an
intraluminal cavity,
the injection needles can be radially extended outward puncturing the wall of
the cavity
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and entering into the underlying tissue. In various preferred embodiments, the
vibrational
energy emitter emits vibrational energy laterally outward in radial directions
away from
the distal end of a catheter such that the catheter can be positioned in
parallel orientation
to the target tissue, such as when the distal end of the catheter is received
in a blood
vessel or other luminal cavity.
Also, specific embodiments of the present invention may include
additional diagnostic, measurement, or monitoring components or capabilities.
For
example, the device for emitting vibrational energy to the target region may
be adapted to
detect the net electromechanical impedance of the target tissue in opposition
with the
vibrational device thus enabling an operator to determine when the distal end
of the
device contacts the target tissue by observing a change in the effective
impedance of the
device. Moreover, an echo ranging transducer positioned on the distal end of
the catheter
or other device can be used to determine the thickness and condition of the
target tissue.
This can be accomplished by operating the ranging transducer in a pulse echo
mode, and
1 ~ characterizing the amplitude, spectral content, and timing of the
returning echoes.
Furthermore, an electrocardiograph monitoring electrode can optionally be
positioned on
the distal end of the catheter adjacent the substance delivery system for
monitoring
potentials in a patients myocardium. This can be useful for providing therapy
to the site
in the myocardium which is responsible for rhythm abnormalities.
Kits according to the present invention may comprise the delivery devices
in combination with instructions for use setting forth any of the above-
described methods.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a microscopic pictorial representation of a substance being
injected into a target tissue by way of an injection needle.
Fig. 2 is a microscopic pictorial representation of cellular absorption of the
substance of Fig. 1 mediated by ultrasound energy.
Fig. 3A is a sectional view of the distal tip of a device for enhancing
cellular absorption of a substance delivered to a region of target tissue.
Fig. 3B is a sectional view of the distal tip of an alternate device for
enhancing cellular absorption of a substance.
Fig. 3C is a sectional view of the distal tip of a third alternative device
for
enhancing cellular absorption of a substance.
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Fig. 4 is a pictorial view of the device of Figs. 3A, 3B or 3C received into
a ventricle of a patient's heart.
Fig. SA is an enlarged pictorial view of the device of Figs. 3A, 3B, 3C, and
4 shown penetrating through a patient's endocardium into the patient's
myocardium.
Fig. SB is an enlarged pictorial view of the device of Figs. 3A, 3B, 3C, and
4 shown penetrating through the wall of a patient's coronary artery and into
the patient's
myocardium.
Fig. SC is an enlarged pictorial view of the device of Figs. 3A, 3B, 3C, and
4 shown penetrating through a patient's epicardium and into the patient's
myocardium, via
an open thoracotomy surgical approach.
Fig. SD is an enlarged pictorial view of needle injection of a drug with
transesophageal ultrasonic enhancement.
Fig. SE is an enlarged pictorial view of an alternate system of needle
injection of a drug with remote, simultaneous ultrasonic enhancement.
1 S Fig. 6A is a side elevation sectional view of a first embodiment of the
vibrational energy emitter of the device of Fig. 3A.
Fig. 6B is a side elevation sectional view of a combined vibrational energy
emitter and injection device of Fig. 3B.
Fig. 7 is a side elevation sectional view of an alternative embodiment of
the vibrational energy emitter of Fig. 6A.
Fig. 8 is a side elevation sectional view of the vibrational emitter of Fig.
6A with an echo ranging transducer and electrophysiology electrode at its
distal end.
Fig. 9 is a representation of the electromechanical impedance magnitude of
a vibrational energy emitter in contact with fluid and with the myocardium.
Fig. 10 is a representation of the electromechanical impedance phase angle
of a vibrational energy emitter in contact with fluid and with the myocardium.
Fig. 11 is a pictorial representation of the range finding transducer emitting
a signal into the myocardium.
Fig. 12 is a representation of the return echo from the range finding
transducer's emitted pulse, shown coming back from the myocardium.
Fig. 13 is a pictorial representation of an alternative embodiment of a
device for enhancing cellular absorption having a vibrational energy emitter
and plurality
of radially outwardly extending retractable injection needles.
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Fig. 14 is a pictorial representation of a patient's leg immersed in a fluidic
environment which is subjected to vibrational energy.
Fig. 15 is an illustration of a kit comprising devices for enhancing cellular
absorption and instructions for its use.
Fig. 16 is an illustration of treatment of soft tissue lesions by combined
needle injection and ultrasonic emission.
Fig. 17 illustrates an ultrasound transducer device that was employed in
the examples described in the Experimental section.
Fig. 18 is a graph referred to in the Experimental section showing
enhanced transfection with ultrasonic treatment in a rabbit model.
Fig. 19 is a graph referred to in the Experimental section showing
enhanced blood flow with VEGF and ultrasonic treatment in ischemic hind limbs
in
young rabbits.
Fig. 20 is an illustration of the individual test results used to generate
Fig.
19.
Fig. 21 is a graph referred to in the Experimental section showing
enhanced blood flow with VEGF and ultrasonic treatment in ischemic hind limbs
in older
rabbits.
Fig. 22 is a graph referred to in the Experimental section showing
enhanced blood pressure ratio with VEGF and ultrasonic treatment in ischemic
hind limbs
in young rabbits.
Fig. 23 is an illustration of the individual test results used to generate
Fig.
22.
Fig. 24 is a graph referred to in the Experimental section showing
enhanced blood pressure ratio with VEGF and ultrasonic treatment in ischemic
hind limbs
in older rabbits.
Fig. 25 is a graph referred to in the Experimental section showing
enhanced angiographic score with VEGF and ultrasonic treatment in ischemic
hind limbs
in young rabbits.
Fig. 26 is an illustration of the individual test results used to generate
Fig.
25.
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Fig. 27 is a graph referred to in the Experimental section showing
enhanced angiographic score with VEGF and ultrasonic treatment in ischemic
hind limbs
in older rabbits.
Fig. 28 is a graph referred to in the Experimental section showing the
effect of ultrasonic pretreatment, prior to DNA injection.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention provides a method for enhancing cellular absorption
of a drug, gene, or other substance into a local target region of a patient's
body, thereby
avoiding the undesirable effects of the substance being widely dispersed
throughout the
patient's body by the patient's cardiovascular system or having the substance
compromised by the natural cleansing activities of the patients organs, as
follows.
First, the substance is delivered to the target region of a patient's tissues.
Secondly, vibrational energy is directed to the target region, wherein the
vibrational
1 S energy is of a type and amount sufficient to enhance absorption of the
substance into the
cells of the target region, as will be explained.
The methods, systems, and kits of the present invention will be suitable for
delivering virtually any therapeutic, diagnostic, or other substance where it
is desired that
the substances be taken up by individual cells comprising part of a human or
other animal
tissue mass. As a first general example, the substances may be therapeutic
drugs,
proteins, small molecules, or the like, where the drugs are intended to enter
through the
cell walls to have a desired therapeutic or other effect. In a second general
example, the
substances will be nucleic acids intended to transfect the target cells in the
tissue mass.
Such nucleic acids which may delivered by the methods and devices of the
present
invention will comprise nucleic acid molecules in a form suitable for uptake
into target
cells within a host tissue, usually smooth muscle cells lining the blood
vessels, or skeletal
cells or cardial muscle. The nucleic acids will usually be in the form of bare
DNA or
RNA molecules, where the molecules may comprise one or more structural genes,
one or
more regulatory genes, antisense strands, strands capable of triplex
formation, or the like.
Commonly, such nucleic acid constructs will include at least one structural
gene under the
transcriptional and translational control of a suitable regulatory region.
Optionally, but
not necessarily, the nucleic acids may be incorporated in a viral, plasmid, or
liposome
vesicle delivery vehicle to improve transfection efficiency.
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If viral delivery vehicles are employed, they may comprise viral vectors,
such as retroviruses, adenoviruses, and adeno-associated viruses, which have
been
inactivated to prevent self replication but which maintain the native viral
ability to bind a
target host cell, deliver genetic material into the cytoplasm of the target
host cell, and
5 promote expression of structural or other genes which have been incorporated
in the
particle. Suitable retrovirus vectors for mediated gene transfer are described
in Kahn et
al. (1992) CIRC. RES. 71:1508-1517, the disclosure of which is incorporated
herein by
reference. A suitable adenovirus gene delivery is described in Rosenfeld et
al. (1991)
SCIENCE 252:431-434, the disclosure of which is incorporated herein by
reference.
10 Both retroviral and adenovirus delivery systems are described in Friedman
(1989)
SCIENCE 244:1275-1281, the disclosure of which is also incorporated herein by
reference.
The nucleic acids may be present in a lipid delivery vehicle which
enhances delivery of the genes to target smooth muscle cells within the
vascular epithelia
1 S or elsewhere. Transfection with a lipid delivery vehicle is often referred
to as
"lipofection." Such delivery vesicles may be in the form of a liposome where
an outer
lipid bilayer surrounds and encapsulates the nucleic acid materials.
Alternatively, the
nucleic complexes may be in the form of a nucleic acid-lipid dispersion,
nucleic acid-lipid
emulsion, or other combination. In particular, the complexes may comprise
liposomal
transfection vesicles, including both anionic and cationic liposomal
constructs. The use
of anionic liposomes requires that the nucleic acids be entrapped within the
liposome.
Cationic liposomes do not require nucleic acid entrapment and instead may be
formed by
simple mixing of the nucleic acids and liposomes. The cationic liposomes
avidly bind to
the negatively charged nucleic acid molecules, including both DNA and RNA, to
yield
complexes which give reasonable transfection efficiency in many cell types.
See Farhood
et al. (1992) BIOCHEM. BIOPHYS. ACTA. 1111:239-246, the disclosure of which is
incorporated herein by reference. A particularly preferred material for
forming liposomal
vesicles is lipofection which is composed of an equimolar mixture of
dioleylphosphatidyl
ethanolamine (DOPE) and dioleyloxypropyl-triethylammonium (DOTMA), as
described
in Felgner and Ringold (1989) NATURE 337:387-388, the disclosure of which is
incorporated herein by reference.
It is also possible to combine these two types of delivery systems. For
example, Kahn et al. (1992), supra., teaches that a retrovirus vector may be
combined in a
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cationic DEAE-dextran vesicle to further enhance transformation efficiency. It
is also
possible to incorporate nuclear proteins into viral and/or liposomal delivery
vesicles to
even further improve transfection efficiencies. See, Kaneda et al. (1989)
SCIENCE
243:375-378, the disclosure of which is incorporated herein by reference.
The nucleic acids will usually be incorporated into a suitable Garner to
facilitate delivery and release into the blood vessels according to the
present invention.
The carriers will usually be liquids or low viscosity gels, where the nucleic
acids will be
dissolved, suspended, or otherwise combined in the carrier so that the
combination may
be delivered through the catheter and/or carried by the catheter and released
intravascularly at the treatment site. Alternatively, the nucleic acids may be
provided in a
dry or solid form and coated onto or otherwise carried by the catheter or the
vibrational
surface.
Fig. 1 illustrates an injection needle 20 delivering a drug or other
substance 21 into a region of target tissue 22 which is comprised of a
plurality of cells 24.
In the absence of the present invention's application of vibrational energy,
drug 21 will
tend to absorb slowly into cells 24 causing the drug 21 to be distributed
widely in the
patient's body thus either diluting a very expensive drug and thereby
weakening its effect
or generating a systemic effect on the patient instead of the desired local
effect.
However, in accordance with the present invention as shown in Fig. 2, a
vibrational emitter 30, may be used to emit ultrasound waves 32 into the
target tissue 22
in a type and in an amount sufficient such that drug 21 is instead readily
absorbed into
cells 24. As will be explained herein, emitter 30 preferably has a vibrational
energy with
a mechanical index in the range of 0.1 to 20. In addition, this vibrational
energy
preferably has a frequency range of 20 kHz to 3.0 MHz, and more preferably in
the range
of 200 kHz to 1.0 MHz.
The bio-effects of ultrasonic energy are typically mechanical in nature
(cavitational or pressure effects) or thermal in nature (heat due to
absorption of energy or
energy conversion). The American Institute for Ultrasound in Medicine (AIUM)
and the
National Electrical Manufacturers Association (NEMA) in "Standard for Real-
Time
Display of Thermal and Mechanical Indices on Diagnostic Ultrasound Equipment",
1991,
have together defined the term "mechanical index" for medical diagnostic
ultrasound
operating in the frequency range of 1 to 10 MHz, as follows.
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Mechanical index, (hereafter "MI"), is defined as the peak rarefactional
pressure (in MPa) at the point of effectivity (corrected for attenuation along
the beam
path) in the tissue divided by the square root of the frequency (in MHz), or
MI=P (MPa) / sqrt (f [MHz])
Typical ultrasound devices specify operating conditions based on
frequency (kHz or MHz) and intensity (W/cm2). As defined above, MI effects
embody
frequency and intensity, and therefore for the purpose of this invention,
ultrasound
conditions will be specified solely in terms of MI.
The tolerated range for diagnostic imaging equipment is up to an MI of
1.9. MI values over 1 to 2 represent acoustic levels which can cause
mechanical bio-
effects including excessive membrane damage and cell necrosis due to inertial
cavitation,
microstreaming, or radiation pressure. In addition, MI's above specific
diagnostic limits,
(such as MI's in excess of 20), are typically regarded as potentially damaging
to tissue,
1 S and are instead exploited by various therapeutic devices for tissue
destruction, ablation,
and deep heating.
Moreover, as the MI increases, the temperature elevation in the tissue will
also tend to increase. Unfortunately, biological dangers also increase
concurrent with
excessive temperature elevation in the tissue. Specifically, a temperature
increase in
normal vascularized muscle tissue of more than 5 degrees Centigrade may cause
unwanted formation and accumulation of clot. Moreover, a temperature elevation
in the
tissue of greater than 5 degrees can cause significant heating of the tissue
resulting in
denaturation and necrosis. Moreover, increased temperatures of tissue may
cause
inflammation in the area of treatment. Accordingly, temperature elevations
within the
tissue are typically kept 5 degrees or less to avoid such clotting,
inflammation or other
tissue damage.
Accordingly, in a preferred aspect of the present invention, ultrasound
energy is employed within a "therapeutic window" wherein the range of
ultrasound
energy is generally above the level used for diagnostic purposes, yet below
the level
where profound tissue damage occurs.
In particular, in an aspect of the present invention, ultrasound conditions
which favor a high mechanical index yet preferably produce only a low
temperature
elevation in the tissue are used to induce a preferred cellular response which
promotes
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increased porosity and subsequent uptake of therapeutic agents. In accordance
with the
present invention, a preferred range for enhanced drug delivery is an MI of
0.1 to 20, and
more preferably, a MI of 0.3 to 15, still more preferably a MI of 0.5 to 10,
and most
preferably a MI of 0.5 to 5. Preferably, the duty cycle of the transducer of
the present
invention is set such that the temperature elevation in the tissue remains
less than about S
degrees Celsius.
By virtue of controlled mechanical action on the tissue interfaces and
cellular membranes, temporary disruption of membranes occurs, thereby
increasing
porosity and perfusion of adjacent liquids into cells. Ultrasound induced
membrane
disruption has moderate durability, with most cells returning to normal. In
accordance
with an aspect of the present invention, controlled disruption of membranes
allows
therapeutic agents to more readily pass into the cells and cell organelles
including the
nucleus. An advantage of this system is its improvement in the efficiency of
gene
transfection and subsequent expression of genes.
I S In contrast to the present invention, existing ultrasound transducers used
for diagnostic purposes are typically highly damped, have low sensitivity, and
have a
broad bandwidth response. Such transducers are designed to generate very short
ultrasound pulses and to receive highly complex and irregular return echoes
which are
used to generate images or other diagnostic information. Moreover, these
transducers
tend to generate a high temperature rise in the tissue when operated at a high
duty cycle
(i.e., the fraction of time during which the ultrasound field is energized)
such as greater
than SO%, but are incapable of generating a high MI, primarily because of
their high
operating frequency (3 MHz and above) and heavy damping. As the above equation
for
MI indicates, at constant pressure, MI decreases by the square root of
frequency.
Accordingly, these diagnostic device transducers cannot generate enough
pressure
(amplitude) to overcome the frequency related loss. These restrictions apply
to
transcutaneous as well as intravascular diagnostic ultrasound devices.
Ultrasound transducers used for therapeutic purposes generally fall into
two categories: thermal devices having high frequency for thermal effects,
i.e., deep
heating, and mechanical devices having low frequency for mechanical effects,
i.e., lithotripsy and clot lysis.
Thermal devices are used transcutaneously for deep heating and tissue
destruction, invasively for destroying pathological tissue, and percutaneously
for ablation.
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In contrast to the present invention, these devices operate at a high duty
cycle (greater
than 50%) and typically raise the tissue temperature on the order of at least
4 degrees
Celsius. Similar to the existing ultrasonic diagnostic transducers, ultrasonic
therapeutic
transducers are generally incapable of operating with a high MI due to their
high
operating frequency and heavy damping.
Mechanical devices for lithotripsy, or disintegration of concretions within
the body, are exclusively transcutaneous and not invasive. They operate at low
frequency
(20-500 kHz} and have very large acoustic apertures which allow the ultrasound
energy to
be focussed within the body. By virtue of their frequency and application,
these devices
operate with a high MI. Clot lysis using low frequency ultrasound energy is
achieved by
positioning a transducer external to the body and percutaneously transferring
vibrational
energy into veins and arteries through a translating wire coupled to the
transducer. These
devices suffer from high frictional power loss when used in curved arteries
and lumens.
Transfection of mammalian cells in vitro was reported in Kim et al.
(199G), supra. In that publication, the most efficient transfection of
fibroblasts and
chondrocytes was achieved with continuous exposure of 1 MHz ultrasonic energy
of peak
pressures up to 400 kPa. (ie: having an MI of 0.4). Employing an MI of greater
than 0.4,
such devices were found to fragment plasma DNA and therefore were not used in
their
transfection studies..
In contrast, the present invention can achieve gene transfection employing
much higher MI's with higher peak pressures by setting its duty cycle
sufficiently low
enough to prevent DNA fragmentation induced by ultrasound. For example, as
will be
shown in Experiment Number one, employing a duty cycle of 6% with an MI'of
1.8, a
24.5 times increase in beta-galactosidase transfection was achieved. In this
way, higher
transfection efficiencies can be achieved without significantly damaging the
nucleic acids
being delivered.
In the present invention, vibrational energy emitters capable of high MI yet
maintaining low temperature increases in the tissue were coupled to a
substance delivery
device which can be used transcutaneously or intraoperatively in the form of a
hand held
probe or injection device, and percutaneously in the form of a catheter are
provided.
In the present invention, the application of vibrational energy to the target
region increases cellular absorption on the order of 3 to 300 times or more
for drugs such
as hormones, peptides, proteins, nucleic acids, genes, carbohydrates, DNA
vaccines, and
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angiogenesis stimulators relative to their uptake in the absence of such
vibrational energy.
For example, cellular transfections (DNA transfer into the nucleus of the
cell, as
manifested by altered expression of the cell) of a reporter gene Beta
galactosidase into
muscle tissue has been shown to increase by over a factor of 25 at an MI of
0.05 to 5Ø
5 For cellular transfection in target muscles and other tissues, there is
concern not only with damage to the tissue but also to the DNA/RNA structures
being
delivered. It has been observed (as described above) that high peak pressures,
e.g., over
400 kPa at 1 MHz (ie: 0.4 MI), can have a deleterious effect on the DNA/RNA
structures
being delivered. In ir: vitro transfection the use of ultrasonic energy having
lower peak
10 pressures, however, is disadvantageous because of a potentially significant
reduction in
transfection efficiency. The present invention, in contrast, has recognized
that higher
mechanical indices (and higher peak pressures) can be utilized by employing a
limited
duty cycle, usually below 50%, more usually below 25%, typically in the range
from
0.1% to 10%, more typically in the range from 1% to 10%. The duty cycle is
defined as
15 the percentage of time during which the transducer is active or energized.
A 100% duty
cycle represents a substantially continuous energy emission from the
transducer. The
duty cycle will generally be controlled by energizing and de-energizing the
ultrasonic
transducer at a fairly rapid rate, typically having a relatively short "burst"
length, i.e., the
length time for a single burst of vibrational energy. The on/off frequency
will generally
be referred to as the pulse repetition frequency (PRF), and the vibrational
energy will
usually be applied in short bursts of relatively high intensity (power)
interspersed in
relatively long periods of no excitation (or much lower excitation). Thus, for
a 1 % duty
cycle, the energy will emanate 1 % of the time, but frequently at a relatively
rapid on/off
rate, with exemplary PRF being in the range from 10 to 10,000, often from 100
to 5,000,
and more often from 300 to 3,000 {usually expressed as Hz).
Broad, preferred, and exemplary values for each of the ultrasonic energy
parameters are set forth below.
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BROAD, PREFERRED, AND EXEMPLARY
TREATMENT CONDITIONS
Broad Preferred Exemplary
Mechanical Index (MI) 0.1 to 20 0.3 to 1 0.5 to S
S
Frequency (kHz) 100 to 5,000300 to 3,000S00 to 1,500
Duty Cycle (%) 0.1 to 50 0.5 to 20 1 to 10
Pulse Repetition Frequency10 to 10,000100 to 5,000300 to 3,000
(Hz) ~ [
In preferred embodiments, emitter 30 has a generally planar vibrational
surface 31 which is positioned proximate to, or engages, a surface of target
tissue 22, such
as the external surface of the patient's skin or an external surface of an
organ comprising
target tissue 22.
The step of delivering the substance 21 by way of injection needle 20 or
otherwise can be accomplished either prior to, concurrently with, or after the
step of
directing vibrational energy emitted by emitter 30 to cells 24 of target
region 22 as is
shown in Fig. 2. In an alternative method, the step of delivering substance 21
into target
region 22 can be accomplished by forming an incision in the patient's skin and
depositing
the substance 21 (e.g. in the form of an implantable release depot) into the
incision.
In preferred embodiments, target region 22 can be the myocardium of the
patient and substance 21 can be any substance which promotes angiogenesis, for
example
VEGF, BFBF, and the like, and their corresponding genes. It is to be
understood,
~ however, that target region 22 is illustrative of any target tissue region
in the patient's
body and substance 21 is illustrative of any of a variety of drugs or other
substances
which have a therapeutic effect upon a local region of the patient's tissues.
For example,
substance 21 can include any drug useful in the treatment of vascular
diseases, including
proteins, such as growth factors, clotting factors, clotting factor
inhibitors; nucleic acids,
such as the genes which encode the listed proteins, antisense genes, and other
secretory
proteins. It may also include chemotherapeutic agents for the treatment of
cancer and
other hyperproliferative diseases; and may also include vaccines and any other
type of
therapeutic substance or agent.
As is shown in Fig. 3A, the injection needle 20 of Fig. 1 and the
vibrational energy emitter 30 of Fig. 2 can preferably be combined into an
integrated
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device 40 which is preferably positioned at a distal end of a catheter 42. In
a preferred
embodiment, vibrational emitter 30 completely surrounds injection needle 20.
An
advantage of integrated device 40 is that a drug can be delivered to a target
tissue region
by way of injection needle 20 concurrently with the application of vibrational
energy to
S the target region by emitter 30. As is seen in Fig. 3A, vibrational emitter
30 preferably
comprises an inertial mass 3I and a head mass 33 with a piezostack 35
positioned
therebetween. Inertial mass 31 and head mass 33 are preferably linked together
by way
of an internal rod 34 and the mass of radiating head 33 and the dimensions of
piezostack 35 would be adjusted to achieve the final desired frequency and
output
displacement. Piezostack 35 would typically comprise on the order,of twenty
layers of
ceramic material. Emitter 30 may altemaiively include a piezoelectric tube, a
magnetostrictive device, or transducer bars. Emission may be in either the
forward or
lateral directions.
Preferably, injection needle 20 is slidably received in lumen 44 of
1 ~ catheter 42 and in Lumen 41 of emitter 30. As such, injection needle 20
can be easily
advanced or withdrawn to project out of the distal end of emitter 30, as
desired. For
example, the distal end 42 of catheter 40 can first be safely introduced into
a patient's
body and positioned proximal a target tissue region. Subsequently, injection
needle 20
can then be advanced to project out of the emitter 30 and into the target
tissue, thereby
delivering a drug or other substance to the target tissue.
Fig. 3B shows an alternative embodiment corresponding to Fig. 3A, but
with the injection needle 20a being connected directly to energy emitter 30a.
Energy
emitter 30a may comprise a piezostack 35a. Vibration of piezostack 35a causes
needle 20a to vibrate. Needle 20a, being short and stiff in nature, will
oscillate axially at
the same frequency and at the same amplitude of the piezostack 35a. The tip of
the
needle 20a will thus act as an ultrasound emitter. An advantage of this device
is that, as
energy emitter 30a is attached to the substance delivery injection needle 20a,
very
effective application of ultrasound energy at the exact point of drug delivery
is achieved.
The present needle 20a is preferably fabricated from stainless steel, while
the emitter is
fabricated from piezoelectric material. This system is an improvement over
prior
developments because the needle 20a will thus oscillate at the site of
injection causing
formation of microbubbles as the liquid agent is injected. The presence of
microbubbles
enhances cavitation which improves the efficiency of transfections. Needle 20a
will thus
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act as an extension of transducer 30a with the tip of the needle vibrating at
the same
frequency and amplitude as that of the transducer. Similar to the device of
Fig. 3a, the
device of Fig. 3B is preferably received in the system of catheter 40, as
previously
described.
S Fig. 3C shows a third alternative embodiment of a device for enhancing
cellular absorption of a substance comprising an integrated injection needle
and
ultrasound energy emitter system 30b. System 30b comprises an injection needle
20b
having a penetration tip 20c and one or more portals 20d. Penetration tip 20c
preferably
has a 14-30 gauge diameter for easy skin penetration. One or more internally
mounted
transducers 20e are provided. Preferably, transducers 20e are located proximal
and distal
or just proximal or just distal the locations of portal or portals 20d.
Accordingly, drug
injection can be provided such that ultrasonic energy emitted by transducers
20e is
adjacent to the point of drug delivery through portals 20d, thus ensuring that
ultrasound
energy is applied directly to the target tissue, thereby increasing drug
delivery
effectiveness into the target cells.
Fig. 4 is a pictorial view of a preferred embodiment of the device of
Fig. 3A, 3B, or 3C as inserted into the left ventricle LV of a patient's heart
with
catheter 42 positioned proximate a diseased region 25 of a patient's
myocardium 26.
Catheter 42 is preferably provided with a guidewire 45, a distal tip
deflection actuator 46
for controlling the position of distal end 43 of the catheter 42. A flush luer
48 is adapted
to provide plumbing for contrast dye or for the irrigation of the guidewire
lumen.
Catheter 42 would preferably be rigid enough to allow for pushability and
torqueability as
required for typical intracardiac procedures. Electrical connectors 49 are
also typically
provided for powering emitter 30 and other transducers which will be described
herein.
The internal guidewire 45 used to direct the distal tip 43 of catheter 42
would preferably be received in a separate lumen from that of injection needle
20/20a
lie: injection needle 20 or 20a). However, it is to be understood that
guidewire 45 may
itself comprise injection needle 20 or 20a. Conceivably, however, guidewire 45
and
injection needle 20/20a can both be received in the central lumen 44 with
guidewire 45
first positioning distal head 43 of catheter 42. Subsequently, guidewire 45
would then be
removed such that injection needle 20/20a can be slidably received in central
lumen 44
such that injection needle 20/20a passes out of distal end 43 of catheter 42
to a location
past vibration energy emitter 30 and into the target tissue.
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In the preferred method of enhancing cellular absorption of a substance
delivered into a patient's myocardial tissue, the first step is the
positioning of distal end 43
of catheter 42 proximal diseased tissue region 25. The present invention
comprises a
variety of different preferred approaches to positioning distal end 43 of
catheter 42 into
the patient's myocardium. Specifically, Fig. 5A illustrates an approach
through the
endocardium, Fig. 5B illustrates an approach through a coronary artery, Fig.
5C illustrates
an open chest procedure approach through the epicardium, Fig. 5D illustrates
an approach
wherein the needle and ultrasonic source are separated, wherein the needle
approaches
through the endocardium with the ultrasonic emission originating from a
transesophageal
device or a transthoracic device, and Fig. 5E illustrates an approach wherein
the needle
and ultrasonic source are separated, wherein the needle approaches through the
endocardium with the ultrasonic emission originating from an external location
on the
surface of the patient's skin.
As is shown in Fig. 5A, needle 20/20a is extended through the patient's
endocardium 27 to penetrate into diseased region 25 and a fluid suspension
containing the
substance to be delivered is then passed into target region 25 by injection
needle 20/20a.
The energizing of transducer 30/30a generates ultrasound waves 32 which cause
the
tissue in target region 25 to vibrate by an amount sufficient to enhance
absorption of the
injected substance into this tissue. Preferably, planar vibrational surface 31
will be
positioned in flush contact to endocardium 27, thereby providing optimal
vibration energy
transfer and controlling the penetration depth of injection needle 20/20a.
As is shown in Fig. 5B, access to myocardium 26 can also be achieved
with catheter 42 -positioned intravascularly in coronary artery or vein 28
with injection
needle 20/20a passing through the lumen wall and into myocardium 26.
As is shown in Fig. 5C, access to myocardium 26 can also be achieved
with device 42 positioned in or over the patient's pericardium 29 with
injection
needle 20/20a passing through epicardium 19. This approach may be accomplished
in
surgical procedures in which access to the patient's heart is achieved either
through the
sternum or between the ribs 23.
As is shown in Fig. 5D, access to the myocardium is achieved by the
system depicted in Fig. 5A. It is to be understood, however, that access to
the
myocardium could also be achieved by the systems depicted in Figs. 5B or 5C.
As
depicted in Fig. 5D, the source of ultrasonic emission, however, is separated
from the
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substance delivery system such that the ultrasonic energy may be emitted in
the
esophagus 300 from a large aperture focused transducer 301 or transducer array
on a
transesophagal probe. Acoustic waves 32 may be divergent or focussed on a
small spot
consistent with the resolution of the ultrasonic emitter device.
As is shown in Fig. SE, access to the myocardium is achieved by the
system depicted in Fig. SA. It is to be understood, however, that access to
the
myocardium could also be achieved by the systems depicted in Figs. SB or SC.
As
depicted in Fig. SE, the source of ultrasonic emission, however, is separated
from the
substance delivery system such that the ultrasonic energy may be emitted from
a location
10 on the patient's skin by a transducer 303. A transducer 304 may be
positioned at the
distal end of the catheter adjacent the injection needle 20/20a, with
transducer 304
operating as a receiving transducer, measuring the dose of ultrasound energy
received
adjacent needle 20/20a.
In the specific case of a transducer or transducer array located in the
1 S esophageus, a higher frequency device may preferably be employed, such
that a level of
beam focussing may be achieved. As such, it may not be necessary to achieve
the
typically 1-2 millimeter beam profile of diagnostic imaging system; instead,
beam
profiles on the order of 0.5 to 1.0 cm may be preferred. Operating frequencies
in the
range of 0.5 to 1.5 MHz from plastic piezoelectric ceramics may also be
preferred.
20 Fig. 6A shows a sectional view of vibration energy emitter 350 similar to
emitter 30 shown in Fig. 3A. Fig. 7 shows an embodiment of a vibration emitter
351 in
which head mass 33a and inertial mass 31a are held together by an outer
external casing
or tensioning skin 36. An internal insulator 37 is provided as a conduit for
an injection
needle. The systems of Figs. GA and 7 are ideally suited for catheter based
applications.
In an alternative embodiment, vibration energy emitter 352, as shown in
Fig. 8, further includes an echo ranging transducer 39 which is used to detect
contact with
the target tissue. Using this embodiment of the present invention in a
preferred method,
contact with the target tissue is confirmed by observing a change in the
impedance of the
ranging transducer 39. When ranging transducer 39 contacts the myocardial
wall, the
additional rigidity of the tissue typically pulls down the resonant frequency
of the
transducer by as much as 5%. As is illustrated in the electrical impedance
plat of Fig. 9,
this effect can easily be measured, and it can be used to affirm direct
contact between
ranging transducer 39 and the myocardial wall. Fig. 10 shows the corresponding
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impedance phase shift as ranging transducer 39 contacts the myocardial wall.
As can be
seen, this effect can also be easily measured.
An electrical lead 38, positioned over then distal face of the transducer as
shown in Fig. 8, can be used for electrocardiograph monitoring which permits
the
electrocardiograph function to be traced and mapped onto commercially
available
electrophysiological equipment such that the location of a specific lesion in
the
myocardium can be precisely determined, thereby allowing the drug delivery
system at
the distal end of the catheter to be guided to an optimal location for drug
delivery.
Alternatively, electrical lead 38 can be in the form of band wrapped around
the
circumference of the distal end of the catheter. Electrical lead 38 can be
used for
electrophysiology.
As shown in Fig. 11, ranging transducer 39 can also be used to measure
the thickness of the myocardium, as follows. As the tip of catheter 40
approaches the
endocardium, ranging transducer 39 is repetitively pulsed in a pulse echo, or
A-scan
1 S mode. Ultrasound waves 50 will reflect off of the tissue, creating echoes
which return to
ranging transducer 39. The amplitude and duration of the returning echoes are
determined by fluctuations in the acoustic impedance of the tissue and its
thickness.
As shown in Fig. 12, which represents the amplitude and duration of the
ultrasound echo, the distance from the ranging transducer 39 to the myocardial
surface is
represented by a low amplitude blood field echo. The myocardium presence is
represented by a high amplitude echo, the duration of which is proportional to
its
thickness, and the pericardial fluid is represented by a low amplitude echo.
Accordingly,
measurements can be easily made to determine the thickness of the myocardium.
The
ranging transducer and therapy transducers may be separate piezoelectric
ceramic
devices, although electrode patterning may allow the use of a single
piezoelectric
component.
As the operator moves the present device from site to site making multiple
injections and applying vibrational energy, the echo ranging transducer 39
would first
ascertain whether direct contact has been made with the myocardial wall.
Thereafter,
transducer 39 could be used to determine the wall thickness such that the
proper depth
setting far the injection needle plunge could be determined. Doppler signal
processing of
the A-mode traces 50 might further help delineate the margin. Software may
then
compute the thickness of the myocardium.
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Fig. 6B shows a sectional view of vibration energy emitter 30a as was
shown in Fig. 3B, having a ranging transducer 39a and electrical lead 38a
positioned
thereon, operating similar to ranging transducer 39 and electrical lead 38,
described
herein.
In yet another embodiment of the present device, as illustrated in Fig. 13,
catheter 42a is received into a intraluminal cavity 60. Intraluminal cavity 60
can either be
a naturally occurnng cavity in a patient's body or a cavity formed by
injection of a needle
into the patient's body. A drug or other substance is delivered into a target
region of
tissue by puncturing cavity wall 62 of intraluminal cavity 60 by injection
needles 20,.
Preferably, injection needles 201 are disposed to extend radially outward from
catheter 42a as shown. In addition, injection needles 20a are preferably
retractable into
catheter 42a such that in a preferred method, catheter 42a can first be
conveniently
inserted into lumen 60, and subsequently, injection needles 201 can then be
radially
extended such that they puncture wall 62 at a variety of radial locations.
This radial
puncturing of the wall of the interluminal cavity would operate to center
catheter 42a
within the intraluminal cavity 60. In this embodiment, vibrational energy
emitter 30a
would emit vibrational energy radially outward as shown by ultrasonic waves
SOa.
Fluoroscopic imaging may be used to define a luminal diameter and allow the
preferred
setting of vibrational energy per the observed distance between the catheter
42a to
wall 62.
The present invention also includes a kit 90, as seen in Fig. 15, which
includes any of the preferred systems for enhancing cellular absorption of a
substance as
described herein, for example, a catheter 42 having an ultrasonic emitter
30/30a and an
injection needle 20/20a, as has been described. Also included in kit 90 are
instructions
for use 92 which may be in the form of literature accompanying the system,
writing on
packaging material, information stored on video or audio discs,
electromagnetic data
storage formats, or other data storage and presentation media. Instructions
for use 92 set
forth any of the preferred methods described herein.
In another aspect of the present invention, as seen in Fig. 14, a patient's
leg 70 is received into a fluidic bath 72. A plurality of ultrasonic
vibrational energy
emitters 74 are provided to subject the fluidic bath to ultrasonic vibrational
energy. The
apparatus shown in Fig. 14 is particularly useful for patients requiring
treatment for
ischemia and other vascular problems in the leg, such as may result from
cardiovascular
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disease or diabetes. In a preferred method, which would improve vasculature
and reduce
pain, a series of multiple injections are typically made in the patients leg
from just below
the knee to the ankles. The apparatus of Fig. 14 then permits the entire leg
of the patient
to be subjected to an ultrasonic environment, with the ultrasound vibrations
enhancing the
cellular absorption of a drug or other substance into the leg. Alternatively,
such a
treatment method and apparatus may be employed on the patient's arm, hand or
foot. An
advantage of this apparatus is that a large area of the patient's body can be
subjected to
ultrasound without the problems of acoustic beam spreading and unwanted
amplification,
as follows.
Sharply focusing an acoustic beam at a target tissue region substantially
amplifies the acoustic power at any point, but then the beam will need to be
swept back
and forth over the entire surface area to achieve therapeutic levels over a
large volume of
tissue. This sweeping may require an unacceptable amount of time. To eliminate
the
need for such sweeping, the acoustic beam might be defocussed to provide
acoustic
energy over a large volume, at a lower power level.
A fluidic environment will transmit ultrasonic energy more readily than a
gaseous environment. Accordingly, with the present invention, the use of
fluidic bath 72
will overcome the problem of acoustic beam spreading which would have required
the
beam to be focused and amplified at any particular location in the leg. As
such, the
problem of topical administration of ultrasound is overcome.
In another aspect of the present invention, needle injection and sonication
can be applied in man made lumens within the body, such as those depicted in
Fig. 16 for
treating soft tissue lesions 400. A semi-rigid tube 401 similar to the
catheter
configuration previously described is inserted into the subject's body, and
directly into the
lesion side, by conventional clinical techniques. Semi-rigid tube 401 contains
ultrasonic
emission surfaces 402 at it's distal tip and an injection needle 403 also
protruding from it's
distal tip. This technique can be useful for treating typically cancerous
lesions of the
brain, breast or liver.
The following examples are offered by way of illustration, not by way of
limitation.
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EXPERIMENTAL
Experiment Number One:
Materials and Methods:
Samples of plasmid DNA (0.5 ml) were injected at a depth of about 4 mm
into the thigh muscle of New Zealand white rabbits. The DNA, pCMV-beta-
galactosidase, was formulated at 200 mg/ml in saline. The injected sample was
found to
widely disperse, covering a length of about 4 cm parallel to the muscle fibers
and at
depths within the tissue which varied from injection to injection. Immediately
after the
sample injection, an ultrasonic (US) transducer was contacted to the muscle
surface and
ultrasound energy was applied. There were five overlapping US treatments, each
for
1 minute, covering a length of about 3 cm parallel to the muscle fibers. The
multiple
treatments intended to cover sufficient area assuring the tissue injected with
DNA was
subjected to the ultrasound. Two different transducer designs were tested. One
operated
at 1 MHz and produced ultrasound with a beam diameter of about 1 cm (Fig. 17).
The
other operated at 193 kHz with similar beam characteristics.
The wide beam transducer illustrated in Fig. 17 provides a wide beam
ultrasound delivery system which has the advantage of delivering therapeutic
ultrasound
energy over a large tissue volume such that, in preferred aspects, ultrasound
energy can
be uniformly distributed over the region in which a therapeutic substance has
been
injected intramuscularly. An advantage of the present invention is that by
distributing a
uniform field of ultrasound energy over a large tissue volume, cellular uptake
of injected
substances such as therapeutic DNA can be substantially enhanced over the
entire region
in which the injected DNA spreads.
The wide beam ultrasound delivery system of Fig. 17 comprises a housing
having an opening at its distal end with an ultrasound transducer suspended
within the
housing. The ultrasound transducer is positioned in contact with an acoustic
couplant
material which substantially fills the housing. In the present experiment, the
acoustic
couplant material was water.
A flexible skin-contact window is disposed across the opening at the distal
end of the housing. The skin-contact window was positioned adjacent to the
patient's skin
such that therapeutic ultrasound energy was conducted from the ultrasound
transducer
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along through the fluid-filled housing and then through the skin-contact
window and into
the patient.
The housing of the ultrasound delivery system was generally cylindrical
and tapers to a narrow distal end which assists in focusing the ultrasound
energy emitted
by the transducer. Accordingly, the ultrasound energy beam was focused through
a
narrow region which may be disposed within the housing, or alternatively, the
ultrasound
energy can be focussed at a transdermal depth.
The experimental ultrasound transducer was generally planar and circular
in shape. The transducer of the present invention preferably have a large
surface area
10 which may be constructed to range from 1 in3, to 1000 in3.
The fluid which substantially fills the housing of the ultrasound delivery
system operates as an acoustic couplant material which transmits the
ultrasound energy
generated by the transducer therethrough to the skin-contact window and into
the patient.
Specifically, Fig. 17 is a sectional side elevation view of the wide aperture
15 beam delivery system. Ultrasound delivery system 520 comprises a housing
521 having a
proximal end 522 and a distal end 524. An ultrasound transducer 525 is
disposed at the
proximal end 522 of housing 521 as shown. Housing 521 was generally
cylindrical in
shape and was tapered to a narrow distal end 524, as shown. Transducer 525 was
made
of a ceramic material. Distal end 524 of housing 521 was covered by a flexible
skin-
20 contact window 527 which was supported against the skin of patient P. A
standard
acoustic coupling gel was applied between window 527 and the skin of patient
P, to
facilitate the transmission of therapeutic ultrasound energy to the patient.
Housing 521 is filled with an acoustically couplant material, which
comprised a fluid 523, in this case water. Fluid 523 operated to conduct a
beam of
25 ultrasound energy therethrough from transducer 525 to skin-contact window
527. An
advantage of substantially filling housing 521 with fluid 523 was that a beam
B of
ultrasound energy (shown as a dotted line) was passed therethrough as a wide
beam of
ultrasound energy which can be selectively focussed to pass through a
particular
therapeutic target focal region 529 at a preferred transdermal depth in the
patient.
It was observed that the present wide aperture beam delivery system
produced a generally uniform ultrasound field at a transdermal depth of about
2 to 5 cm,
and especially at 3 to 4 cm.
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An air pocket 528 was provided on one side of transducer 525 such that
substantially all of the ultrasound energy emitted by transducer 525 was then
directed
distally through fluid 523 towards skin-contact window 527 at distal end 524
of housing
521, due to air pocket 528 being an extremely poor conductor of ultrasound
energy.
S Transducer 525 yielding a generally uniform ultrasound beam having a width
of at least
0.1 cm, but generally over 0.5 cm, and even over 1.Ocm.
After five days the animals were sacrificed. Each thigh had 9 samples
collected in a 3 by 3 array in the area exposed to ultrasound. The muscle
samples had
dimensions of about 1 x 1 x0.5 cm (WxLxH). Protein was then extracted from the
tissue
and measured for beta-galactosidase enzyme activity and total protein. Beta-
galactosidase activity was normalized to the protein content and expressed as
activity per
protein mass. For each rabbit thigh, an average beta-galactosidase activity
was then
calculated from the 9 samples. Tables 1 and 2, and Fig. 18 present the
results.
Results:
The results are summarized in Table 1 where "No US" and three US
conditions are compared. Expression levels are presented for each treatment
comprising
the mean beta- galactosidase activity from 9 to 11 rabbits for each group. The
ultrasound
condition, 1 MHz, 1.8 MI (mechanical index), 6% duty cycle, yielded the best
results
showing about a 25 fold enhancement of transfection versus the "No US"
exposure
conditions as set forth below.
TABLE 1
INTRAMUSCULAR GENE
DELIVERY:
RESULTS
Treatment N B-gaUmg Bkgrnd Crrct.US/no US
No US 10 49.8 +/-30. S.5 ---
1 MHz, 2 MI, 1.5% 11 102.3 +/-103*58.0 I 10.5
DC
1 MHz, 0.5 MI, 25% 9 124.0 +/-81.2**79.7 14.5
DC
1 MHz, 1.8 MI, 6% 9 179.1 +/-77.7**134.8 I 24.5
DC
tfacKgTOUna: 44.3
* p = 0.0153
* * p = 0.0001
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From the same experiment, the distribution of beta=galactosidase
expression levels for the individual rabbits was plotted as a histogram in
Fig. 18 showing
the "No US" and the 1 MHz, 1.8 MI, 6% DC conditions. All 9 of the rabbits
treated with
this ultrasound condition showed elevations in beta-galactosidase expression.
In the low frequency exposures at 193 kHz with similar transfection
conditions, the effect of the ultrasound was studied and results are presented
in Table 2.
With 193 KHz, 1.09 MI, 1.3% duty cycle about a nine fold increase in beta-
galactosidase
expression was observed compared to the "No US" conditions.
TABLE 2
INTRAA~IUSCULAR GENE
DELIVERY:
RESULTS
Treatment N B-gaUmg Bkgrnd Crrct.US/no US
No US 3 I 14.2 +/-123.947.G ---
194 kHz, 1.09 Mi, 1.3%3 526.0 +/- 459.4 9.7
DC 43.2
t3acKground: 66.6
In a second part of this experiment, an ultrasound pre-treatment was
applied. Specifically, the above experiment was repeated as set out above with
the 5 US
exposures carried out at 1 MHz, 1.8 MI, 6% DC conditions, however, the US was
applied
prior to the VEGF DNA injection. As illustrated in the histogram of Fig. 28,
the US
pretreatment achieved a 10.5 fold 08/5.5) increase in VEGF transfection, (as
compared
to the 24.5 fold (135/5.5)increase in VEGF transfection achieved by applying
the US after
the VEGF injection, as illustrated in Table 1 above and in Fig. 28.
Experiment Number Two:
Materials and Methods:
An ischemic condition was created in one of the hind limbs of each of a
group of New Zealand white rabbits by excising their femoral arteries. Ten
days
thereafter, VEGF DNA was injected into the ischemic muscle and therapeutic
ultrasound
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at 1 MHz, 1.8 MI, and 6% duty cycle was applied for 1 minute in 9 ultrasound
exposures
along the length of the thigh. At 40 days after the creation of the ischemic
condition, a
variety of angiogenesis parameters, including blood flow, blood pressure
ratio, and
angiographic score, were tested.
In a first part of this experiment, (illustrated in Figs. 19, 20, 22, 23, 25,
and
26), the VEGF DNA was prepared at a dosage of 100 ug/rabbit and was given to
younger
rabbits being about 6 months in age.
In a second part of the experiment, (illustrated in Figs. 21, 24 and 27), the
VEGF DNA was prepared at a dosage of 500 ug/rabbit, and was given over S
injections
to older rabbits being about 5 years in age.
Older rabbits were selected for the higher dose DNA since age is known to
impair the angiogenic effect of VEGF, presenting an additional barrier for
ultrasound
gene delivery. Therefore, older rabbits were used for the high DNA dose
because there
was a concern that with young rabbits the higher dose may have produced the
maximal
angiographic response in the rabbit ischemic hind limbs model making it
impossible to
detect further angiogenesis when ultrasound was employed. Since older rabbits
are
angiogenically impaired, they would produce a lower biological response with
the high
DNA dose alone.
In both the first and second parts of the experiment, ultrasound in the range
of 1 MHz, 1.8 MI and 6% duty cycle was applied with the wide beam delivery
system
illustrated in Fig. 17. Comparisons were made to a rabbit control group and
between
rabbit groups to which ultrasound was, and was not, applied concurrent with
VEGF
injection.
Results:
Fig. 19 shows the increased blood flow as measured by a Cardiometrics
Doppler wire, from a young rabbit 100 ug VEGF DNA dose control average of 22.2
mL/min to 36.3 mL/min when VEGF DNA was injected concurrent with the
application
of ultrasound energy. Fig. 20 shows the resulting measurement data for
individual rabbits
which is presented as an average in Fig. 19.
Fig. 22 shows the increased blood pressure ratio comparing ischemic thigh
and normal untreated thigh from a young rabbit control group average of 0.512
to 0.832
when VEGF DNA was injected concurrent with the application of ultrasound
energy.
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Fig. 23 shows the resulting measurement data for individual rabbits which is
presented as
an average in Fig. 22. Lower limb calf blood pressure was measured using a
Doppler
flowmeter to detect the pulse of the posterior tibial artery and a 2.5 cm wide
inflatable
cuff was applied over the upper calf to detect the systolic pressure.
Fig. 25 shows the increased angiographic score from a young rabbit
control average of 48.2 to 67.6 when VEGF DNA was injected concurrent with the
application of ultrasound energy. Fig. 26 shows the resulting measurement data
for
individual rabbits which is presented as an average in Fig. 25.
Angiograms were performed with a Medrad angiographic injector
delivering contrast media to the internal iliac artery. Angiographic score was
determined
by overlaying a grid of 2.5 mm circles spaced 5 mm on the angiographic film
and
counting the number of opacified arteries crossing the circles then dividing
by the total
number of circles.
Fig. 21 shows the increased blood flow from an older rabbit control
average of 23 mL/min to 41 mL/min when VEGF DNA was injected concurrent with
the
application of ultrasound energy.
Fig. 24 shows the increased blood pressure ratio from an older rabbit
control average of 0.49 to 0.89 when VEGF DNA was injected concurrent with the
application of ultrasound energy.
Fig. 27 shows the increased angiographic score from an older rabbit
control average of 48 to 80 when VEGF DNA was injected concurrent with the
application of ultrasound energy.
As can be seen in Figs. 19, 22 and 25, blood flow, blood pressure ratio and
angiographic score all increase for the younger rabbits when ultrasound energy
is applied
concurrently with VEGF DNA injection
As can be seen in Figs. 21, 24, and 27, blood flow, blood pressure ratio
and angiographic score all increase for the older rabbits with VEGF DNA
injection alone,
but all increase to a greater degree when ultrasound energy is applied
concurrently with
VEGF DNA injection.
While the above is a complete description of the preferred embodiments of
the invention, various alternatives, modifications, and equivalents may be
used.
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Therefore, the above description should not be taken as limiting the scope of
the
invention which is defined by the appended claims.
