U.S. patent application number 11/064572 was filed with the patent office on 2005-08-04 for electrically induced vessel vasodilation.
Invention is credited to Dev, Nagendu B., Dev, Sukhendu B., Hofmann, Gunter A..
Application Number | 20050171575 11/064572 |
Document ID | / |
Family ID | 22187658 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050171575 |
Kind Code |
A1 |
Dev, Nagendu B. ; et
al. |
August 4, 2005 |
Electrically induced vessel vasodilation
Abstract
The invention provides methods for inducing or increasing the
vasodilation of a vessel. The invention further provides methods
for inducing or increasing the flow of fluid through a vessel. An
electrical impulse is applied to the vessel in order to induce or
increase vessel vasodilation or to induce or increase the flow of
fluid through the vessel. In a particular embodiment, a novel
double-balloon catheter system incorporating electroporation
technology has been designed and is used to apply the electrical
impulse endoluminally.
Inventors: |
Dev, Nagendu B.; (San Diego,
CA) ; Dev, Sukhendu B.; (San Diego, CA) ;
Hofmann, Gunter A.; (San Diego, CA) |
Correspondence
Address: |
BIOTECHNOLOGY LAW GROUP
C/O PORTFOLIOIP
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Family ID: |
22187658 |
Appl. No.: |
11/064572 |
Filed: |
February 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11064572 |
Feb 23, 2005 |
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09969367 |
Oct 1, 2001 |
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6865416 |
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09969367 |
Oct 1, 2001 |
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09307216 |
May 7, 1999 |
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6347247 |
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60084857 |
May 8, 1998 |
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Current U.S.
Class: |
607/2 |
Current CPC
Class: |
A61N 1/325 20130101;
Y10S 977/904 20130101; A61N 1/327 20130101; Y10S 977/905 20130101;
Y10S 977/883 20130101 |
Class at
Publication: |
607/002 |
International
Class: |
A61N 001/00 |
Claims
What is claimed is:
1. A method for inducing or increasing vasodilation of a vessel in
a subject comprising applying an electrical impulse to the vessel,
wherein the impulse is of sufficient strength and duration to
induce or increase vasodilation of the vessel, thereby inducing or
increasing vasodilation of the vessel.
2. A method for inducing or increasing the flow of fluid through a
vessel in a subject comprising applying an electrical impulse to
the vessel, wherein the impulse is of sufficient strength and
duration to induce or increase the flow of fluid through the
vessel, thereby inducing or increasing the flow of fluid through
the vessel.
3. The method of claims 1 or 2, further comprising denuding the
endothelial lining of the vessel prior to, simultaneously with or
after applying said electrical impulse.
4. The method of claims 1 or 2, wherein said electrical impulse is
applied with a catheter apparatus.
5. The method of claims 1 or 2, wherein multiple electrical
impulses are applied.
6. The method of claims 1 or 2, wherein said electrical impulse
applied is from about 50 to 90 volts per 1.5 mm.
7. The method of claims 1 or 2, wherein said duration is from about
0.5 ms to 10 ms.
8. The method of claims 1 or 2, wherein said electrical impulse is
applied via electroporation.
9. The method of claim 8, further comprising administering a
composition to the vessel in the subject prior to, simultaneously
with or after the application of said electrical impulse.
10. The method of claim 9, wherein said composition is administered
locally or systemically.
11. The method of claim 9, wherein said composition is delivered
into the tunica media of the vessel.
12. The method of claim 9, wherein said composition is delivered
into the tunica adventitia of the vessel.
13. The method of claim 9, wherein said composition inhibits cell
proliferation.
14. The method of claim 9, wherein said cell proliferation leads to
intimal thickening or hyperplasia.
15. The method of claim 9, wherein said composition is selected
from the group consisting of an angiotensin-converting enzyme
inhibitor, colchicine, somatostatin analog and serotonin
antagonist.
16. The method of claim 9, wherein said composition inhibits
platelet adhesion or aggregation, PDGF action, or matrix
synthesis.
17. The method of claim 9, wherein said composition is selected
from the group consisting of a drug, a polynucleotide, a
polypeptide and a chemotherapeutic agent.
18. The method of claim 9, wherein said composition is selected
from the group consisting of heparin, low molecular weight heparin
and hirudin.
19. A method for inducing or increasing vasodilation of a vessel in
a subject comprising: applying an electrical impulse to the vessel
using an apparatus having: a catheter having at least one
inflatable balloon portion; a first electrode; a second electrode
positioned with respect to the first electrode and the subject such
that an electric field sufficient to induce or increase
vasodilation of the vessel is generated by the electrical impulse,
wherein application of the electrical impulse induces or increases
vasodilation of the vessel.
20. The method of claim 19, wherein said catheter apparatus has at
least one infusion passage for administering a composition into a
vessel of the subject.
21. The method of claim 19, wherein said electrodes are positioned
within the vessel.
Description
RELATED APPLICATIONS
[0001] Under 37 U.S.C. .sctn.119(e)(1), this application claims the
benefit of prior U.S. provisional application 60/084,857, filed May
8, 1998 and U.S. application Ser. No. 09/307,216, filed May 7,
1999.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of vessel
vasodilation and, more particularly, to methods for inducing or
increasing the vasodilation of a vessel and methods for inducing or
increasing the flow of fluid through a vessel by applying an
electrical impulse to the vessel.
BACKGROUND OF THE INVENTION
[0003] Despite procedural success rates greater than 95% achieved
by percutaneous transluminal coronary angioplasty (PTCA), luminal
renarrowing of blood vessels after balloon angioplasty occurs in
30% to 60% of all cases within 3 to 6 months. Smooth muscle cell
proliferation and extracellular matrix remodeling appear to play
pivotal roles in the luminal renarrowing process and negate the
beneficial effect of vascular reconstruction by angioplasty
(Leclercq et al., Arch. Mal. Coeur. Vaiss. 89: 359-365 (1996)). The
use of new technology, such as atherectomy, excimer laser, stent or
rotablator (Hofling et al., Z. Kardiol. 80: 25-34 (1991); Margolis
et al., Clin. Cardiol. 14: 489-493 (1991); Serruys et al., J. Am
Coll. Cardiol. 17: 143B-154B (1991); Warth et al., J. Am. Coll.
Cardiol. 34: 641-648 (1994)) has not been able to reduce the
incidence of restenosis significantly.
[0004] A variety of drugs also have been investigated to prevent
luminal renarrowing in experimental animal and clinical settings,
but without much success. A primary reason for this may be the
failure of systemic administration to achieve effective
concentrations of drugs at the targeted area. To overcome this
deficiency, new endoluminal catheter delivery systems with various
balloon configurations have been employed for localizing drug
delivery. These include: hydrogel balloon, laser-perforated
(Wolinsky balloon), `weeping,` channel and `Dispatch` balloons and
variations thereof (Azrin et al., Circulation 90: 433 (1994);
Consigny et al., J. Vasc. Interv. Radiol 5: 553 (1994); Wolinsky et
al., JACC, 17: 174B (1991); Riessen et al., JACC 23: 1234 (1994);
Schwartz, Restenosis Summit VII, Cleveland, Ohio, 1995, pp
290-294). Delivery capacity with hydrogel balloon is limited and,
during placement, the catheter can lose substantial amount of the
drug or agent that is administered. High pressure jet effect in
Wolinsky balloon can cause vessel injury which can be avoided by
making many holes, <1 .mu.m, (weeping type). The `Dispatch`
catheter has generated a great deal of interest for drug delivery
as it creates circular channels and can be used as a perfusion
device, allowing continuous blood flow. However, each of these
devices have limitations and have not been successful in resolving
the problem of restenosis.
[0005] The cell membrane may be transiently permeabilized by
subjecting cells to a brief, high intensity, electric field. This
pulse-induced permeabilization of cell membranes, termed
electroporation, has been used by investigators to introduce
various compositions such as DNA, RNA, proteins, liposomes, latex
beads, whole virus particles and other macromolecules into living
cells (Hapala, Crit. Rev. Biotechnol. 17: 105-122 (1997)). In
particular, for example, large size nucleotide sequences (up to 630
Kb) can be introduced into mammalian cells via electroporation
(Eanault et al., Gene 144: 205 (1994); Nucl. Acids Res. 15: 1311
(1987); Knutson et al., Anal. Biochem. 164: 44 (1987); Gibson et
al., EMBO J. 6: 2457 (1987); Dower et al., Genetic Engineering 12:
275 (1990); Mozo et al., Plant Molecular Biology 16: 917 (1991)).
These studies show that electroporation affords an efficient means
to deliver therapeutic compositions such as drugs, genes,
polypeptides and the like in vivo by applying an electrical pulse
to particular cells or tissues within a subject.
[0006] Several therapeutic applications of electroporation are now
being explored: treatment of restenosis using angioplasty combined
with electroporation to deliver drugs to a localized portion of
coronary or peripheral arteries (Shapland et al., U.S. Pat. No.
5,498,238); treatment of cancer by electroporation in the presence
of low doses of chemotherapeutic drugs (Mir, U.S. Pat. No.
5,468,223); introduction of functional genes for gene therapy
(Nishi et al., Cancer Research 56: 1050-1055 (1996)),
electroporation of skin for the delivery of drugs into the skin or
for the transdermal delivery of drugs across tissue (Zhang et al.,
Biochem. Biophys. Res. Comm. 220: 633-636 (1996)); Weaver et al.,
U.S. Pat. No. 5,019,034; Prausnitz, Adv. Drug. Deliv. 18: 395-425
(1996)). Hofmann describes a syringe apparatus for electroporating
molecules and macromolecules into tissue regions in vivo in which
the needles of the syringe used to deliver the molecules also
function as electrodes (U.S. Pat. No. 5,273,525). Weaver describes
an apparatus for the delivery of chemical agents into tissues in
vivo via electroporation (U.S. Pat. No. 5,389,069). Hofmann et al.,
describe methods for delivering genes or drugs via electroporation
to treat endothelial and other cells of blood vessels, for example,
and an electroporation catheter device that can be used to practice
the methods (U.S. Pat. No. 5,507,724). Crandell et al. describe the
use of a catheter apparatus for introducing therapeutic
macromolecules via electroporation into endothelial cells of a
patients' blood vessels (U.S. Pat. No. 5,304,120).
[0007] However, in view of the limited success in preventing
luminal renarrowing after angioplasty, a need exists for the
development of methods for inducing or increasing vessel
vasodilation in order to treat undesirable vessel narrowing without
therapeutic compositions, many of which elicit adverse side
effects. The present invention satisfies this need and provides
related advantages as well.
SUMMARY OF THE INVENTION
[0008] In accordance with the present invention, there are provided
methods for inducing or increasing vasodilation of a vessel in a
subject by applying an electrical impulse to the vessel, having
sufficient strength and duration to induce or increase vasodilation
of the vessel. Methods for inducing or increasing the flow of fluid
through a vessel in a subject by applying an electrical impulse to
the vessel, having sufficient strength and duration to induce or
increase the flow of fluid through the vessel, also are provided.
For example, a method of the invention employs an electrical
impulse applied via electroporation. A method of the invention
applies an electrical impulse with an electro-catheter apparatus.
Invention methods are useful for treating clinical situations in
which it is desired to increase or induce vessel vasodilation or to
induce or increase the flow of fluid through the vessel.
[0009] Multiple electrical impulses can be applied in a method of
the invention. An electrical impulse can be applied from about 50
to 90 volts per 1.5 mm. An electrical impulse can be applied for
about 0.5 ms to 10 ms. Vessels can be denuded prior to,
simultaneously with or after applying an electrical impulse in a
method of the invention in order to augment the induction of vessel
vasodilation or increase in the flow of fluid through the
vessel.
[0010] Compositions can be administered to the vessel in the
subject prior to, simultaneously with or after the application of
an electrical impulse. Compositions can be administered locally or
systemically. For example, a composition that inhibits cell
proliferation, such as that associated with intimal thickening or
hyperplasia or that inhibits platelet adhesion or aggregation, PDGF
action, or matrix synthesis can be administered. Compositions
useful in a method of the invention include but are not limted to
are heparin, low molecular weight heparin and hirudin as well as
angiotensin-converting enzyme inhibitor, colchicine, somatostatin
analog and serotonin antagonist. Drugs, polynucleotides,
polypeptides and chemotherapeutic agents also are included. A
method of the invention can deliver a composition into the tunica
intima, tunica media or tunica adventitia of the vessel, for
example.
[0011] In another embodiment, the invention includes a method for
inducing or increasing vasodilation of a vessel in a subject by
applying an electrical impulse to the vessel using a catheter
apparatus having at least one inflatable balloon portion, a first
electrode, a second electrode positioned with respect to the first
electrode and the subject where an electric field sufficient to
induce or increase vasodilation of the vessel is generated by the
electrical impulse. For example, a catheter apparatus having at
least one infusion passage for administering a composition into a
vessel of the subject is useful in a method of the invention. In
one aspect, electrodes are positioned within the vessel.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic diagram of an exemplary endoluminal
double-balloon electroporation catheter (EPC) in which the
guidewire for inserting the catheter serves as the return
electrode, and an ECM 600 exponential pulse generator.
[0013] FIG. 2 is a schematic diagram of an exemplary endoluminal
balloon electroporation catheter in which the guidewire for
inserting the catheter serves as the return electrode.
[0014] FIG. 3 is a schematic diagram of a rabbit treated by a
method of the invention, including several of the elements of an
exemplary catheter apparatus in which one electrode is within the
vessel lumen and the other electrode is in contact with the body
surface.
[0015] FIG. 4 is a schematic diagram of an electroporation catheter
having a balloon that is porous.
[0016] FIGS. 5A and 5B show a sample of a tissue section from
either non-pulsed arteries (A) or pulsed arteries (B).
Electroporation of the artery was performed using four exponential
pulses of 66 volts at 9.6 msec over a period of 15 seconds. Tissue
images were taken five hours after applying the pulses. Images were
taken at equal magnification in which the guidewire for inserting
the catheter serves as the return electrode.
[0017] FIG. 6 is a bar graph showing the effect of electrical
pulsing on the luminal area of the artery. The value plotted as the
total number of pixels (Y-axis) is a relative measure of the area
inside the lumen of the vessel. The data was obtained from eight
different experiments; "n" is the number of arterial sections for
each sample in which the guidewire for inserting the catheter
serves as the return electrode.
[0018] FIGS. 7A and 7B show pseudocolor images of the pulsed (A)
and non-pulsed (B) artery. Fluorescence intensity increases from
red (lowest in the color value ladder) to blue, green, yellow,
fire-red and finally to white. The approximate values that
correspond to the indicated colors are as follows: red=2; deep
blue=82; green=176; yellow=215 and white>255. Both pulsed
arteries and non-pulsed arteries received 20 units/Kg F-heparin
locally through a double-balloon catheter in which the guidewire
for inserting the catheter serves as the return electrode.
[0019] FIG. 8 shows the uptake of heparin in the arterial tissue
after applying the electrical pulse. The upper left panel is
without heparin and without electropulsing, the upper right panel
is with heparin and with electropulsing and the bottom panel is
with heparin but without electropulsing. The relative color
intensity increases from top (lightest) to bottom (darkest) in the
color ladder. Arteries receiving heparin received 10 units/Kg
heparin locally, and arteries were excised four hours after
treatment in which the guidewire for inserting the catheter serves
as the return electrode.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention is based on the seminal discovery that
application of an electrical impulse to a vessel can induce the
vasodilation of that vessel. A method of the invention utilizes an
electrical impulse which, when applied to a vessel at a sufficient
strength and duration, induces the vasodilation of the vessel. The
invention therefore provides methods for inducing or increasing
vessel vasodilation in a subject and methods for inducing or
increasing the flow of fluid through a vessel in a subject.
[0021] The methods of the invention are advantageous in several
respects. For example, the methods allow for the induction of
vessel vasodilation or allow for increasing the flow of fluid
through a vessel without the presence of exogenous therapeutic
compositions having potentially toxic side effects. Thus, the
methods of the invention are particularly applicable in treating
clinical situations of vessel narrowing or blockage where the
systemic use of drugs is undesirable, for example, due to toxicity.
The methods of the invention are additionally advantageous when a
composition is administered to the vessel. For example, drugs that
can function to induce or increase vessel vasodilation or that can
function to induce or increase the flow of fluid through a vessel,
when administered to the vessel to which an electrical impulse is
to be applied, can function additively or synergistically with the
electrical impulse to produce a greater induction or increase of
vessel vasodilation or greater induction or increase in the flow of
fluid through a vessel than that produced by electropulsing
alone.
[0022] As used herein, the terms "impulse," "pulse," "electrical
impulse," "electrical pulse," "electropulse" and grammatical
variations thereof are interchangeable and all refer to an
electrical stimulus. Although the various terms are frequently used
herein in the singular, the singular forms of the terms can refer
to multiple pulses. Preferred electrical impulses are those applied
via electroporation.
[0023] As used herein, the term "vessel" means any tube within the
body of a subject or a hollow channel through an organ of a subject
to which an electrical impulse can be applied. Vessels include, for
example, blood vessels, such as arteries, veins and capillaries,
and gastrointestinal vessels such as the esophagus, larynx, small
intestine and large intestine (i.e., colon). Vessels also include
lymphatic vessels, ducts etc. The methods of the invention are
applicable to any vessel of any size in a subject. Preferred
vessels contain smooth muscle cells (e.g., myocytes). Examples of
preferred vessels are the carotid artery, coronary artery, femoral
artery iliac artery and aorta.
[0024] As used herein, the term "lumen" refers to the interior
portion of a vessel. The lumen can be an area substantially devoid
of the material which comprises a vessel, or a "cavity" within a
vessel, in which case the term "luminal cavity" is used, or can
refer to an inner cellular layer of vessel (e.g., vessel
endothelium, extracellular matrix etc.) which surrounds a "cavity,"
in which case the term "luminal vessel layer" or "luminal lining"
is used. Thus, in a blood vessel, a "luminal cavity" would refer to
the portion of the vessel through which blood can flow and a
"luminal vessel layer" would refer to an inner layer or lining of
the vessel that surrounds the luminal cavity.
[0025] The term "vasodilation" or "dilation" or grammatical
variations thereof, when used as a modifier of the term "vessel,"
means that the vessel has expanded in comparison to a
non-electrically pulsed vessel. Vessel expansion generally occurs
in the luminal cavity or in the luminal vessel layer and is
indicated by an increase in the area or diameter of the luminal
cavity, an increase in the area or circumference of the luminal
vessel layer or, by an increase in the circumference or outer
diameter of the vessel. Although not wishing to be bound by any
particular theory, the induction or increase of vessel vasodilation
by an electrical impulse appears to result either from a direct
effect caused by the electrical current applied to the vessel, or
an indirect effect resulting from the release or stimulation of
factors that promote vasodilation, such as the release of
endothelium derived relaxation factors (EDRF) currently identified
as nitric oxide (NO) or other vasodilating substances triggered by
the electrical pulses applied to the cells of the vessel.
[0026] As used herein, the term "fluid" refers to a mobile
composition that passes or transits through a vessel. A fluid
therefore includes, for example, blood, lymphatic fluid, urine, or
enzyme containing liquids such as bile as well as any material
which passes through the gastrointestinal tract, such as food or
liquid that passes through the esophagus, and the digested material
that passes through the various stages of the gastrointestinal
tract (i.e., small intestine and colon). The phrase "flow of fluid"
refers to the velocity or volume of a fluid that passes through a
vessel. Thus, the phrase "inducing the flow of fluid" means that
the fluid now proceeds through the vessel in which, prior to
applying an electrical impulse, essentially no fluid flow occurred
(e.g., a blocked vessel). The phrase "increasing the flow of fluid"
means that the amount of fluid that passes through the vessel is
greater than that which occurs in the vessel, for example, prior to
applying an electrical impulse (e.g., a partially obstructed or
narrowed vessel).
[0027] As used herein, the term "subject" refers to any animal that
has a vessel. It is envisioned that the methods for inducing or
increasing vasodilation of a vessel and the methods for inducing or
increasing the flow of fluid through a vessel described herein can
be performed on any animal. Preferably, the subject is a human.
[0028] The invention provides a method for inducing or increasing
the vasodilation of a vessel in a subject by applying an electrical
impulse to the vessel at a sufficient strength and duration to
induce or increase vasodilation of the vessel. The invention
further provides a method for inducing or increasing the flow of
fluid through a vessel in a subject by applying an electrical
impulse to the vessel at a sufficient strength and duration to
induce or increase the flow of fluid through the vessel.
[0029] Any electrical impulse capable of inducing or increasing
vessel vasodilation or inducing or increasing the flow of fluid
through a vessel can be used to practice the methods of the
invention. Exemplary means with which to apply an electrical
impulse to a vessel is via electroporation. Exemplary
electropulsing parameters for inducing the vasodilation of an
artery via electroporation were as follows: one pulse having a
pulse width of about 9.0 msec at approximately 63 volts applied to
the vessel endoluminally (Example II). The effect of an electrical
impulse applied via electroporation to the lumen layer of an
artery, as observed histologically, is shown in FIG. 5. The results
showing an increase in the area of the luminal cavity of the artery
after electropulsing are shown in FIG. 6.
[0030] Thus, in one embodiment, the methods of the invention are
practiced by applying an electrical impulse to a vessel via
electroporation.
[0031] The electropulsing parameters exemplified for arteries are
applicable to blood vessels in general as blood vessels have
similar cell structure, electrical resistance, matrix structure
etc. Additionally, other vessels which have a comparable smooth
muscle cell content and electrical resistance, etc., to blood
vessels also can be electropulsed using electropulsing parameters
similar to those exemplified for blood vessels. For example,
although the gastrointestinal tract (e.g., small, large intestine)
is functionally distinct from blood vessels, it is contemplated
that the electropulsing parameters will be comparable to blood
vessels due, in part, to the similar smooth muscle cell content and
electrical resistance.
[0032] Differences between vessel cell type, the presence or
absence of extracellular matrix, wall thickness, the presence of a
stenotic lesion (soft plaque vs. hard), and electrical resistance
are likely to require manipulation of the electropulsing parameters
for inducing or increasing vessel vasodilation or for inducing or
increasing the flow of fluid for particular vessels. The
electropulsing parameters that may be manipulated include, for
example, applying the pulse to the vessel exo- or endo-luminally,
the position of the electrodes relative to the vessel (closer or
further), the type and number of electrodes, the length and
diameter of the vessel region pulsed and the administration of a
composition prior to, substantially contemporaneously with or after
electropulsing. For example, for an electrically resistive vessel
(e.g., one containing a hard stenosis) pulse duration or the number
of pulses can be increased. Ultrafast CT scan can be used to
determine the presence or extent of stenotic calcification. By
placing the electrode in direct contact with the vessel, reduced
pulse length can be used to induce a similar degree of vessel
vasodilation.
[0033] Electropulsing parameters may include denuding a vessel
before, during or after an electrical impulse is applied. As used
herein, the term "denude" or "denuding" refers to the removal of
all, a substantial portion, or any part of the cells that comprise
a vessel. Denuding the endothelial lining of a vessel prior to
applying an electrical impulse may "potentiate" or "augment" the
induction of vessel vasodilation or an increase in the flow of
fluid through the vessel. As used herein, the term "potentiate" or
"augment" means any action (e.g., mechanical, physical) or
composition that can enhance, stimulate, or promote the induction
of vessel vasodilation or the increase in flow of fluid through a
vessel produced by an electropulse. "Potentiating" compositions
include compositions that either induce vessel vasodilation or the
flow of fluid through a vessel independent of an electrical impulse
(e.g., heparin) or compositions whose function is associated with
an electrical impulse (i.e., generally do not function to induce
vessel vasodilation or to increase flow of fluid through a vessel
independent of an electrical pulse).
[0034] Electropulsing parameters also include electrical
parameters. Appropriate electrical parameters may vary and will
depend on the vessel chosen, whether or not the vessel is blocked
and, if blocked, to what extent. For example, for a rabbit blood
vessel, about six pulses having a voltage ranging from about 50 to
90 volts with a duration of about 10 to 15 msec is applied to the
vessel when one of the electrodes is positioned between two
balloons of an endoluminally inserted catheter is preferred.
Electrical parameters that can be manipulated therefore include
pulse strength, pulse wave form, duration, the number of pulses
applied and the time between pulses, for example. The particular
electropulsing and electrical parameters for inducing or increasing
vessel vasodilation or for inducing or increasing the flow of fluid
through any vessel can be determined using the teachings herein and
the general knowledge of those having skill in the art.
[0035] Suitable electric pulses for practicing the invention
methods include, for example, square wave pulses, exponential
waves, unipolar oscillating wave forms, bipolar oscillating wave
forms, other wave forms generating electric fields, or a
combination of any of these forms. Each pulse wave form has
particular advantages. For example, square wave form pulses are
advantageous due to increased cell transformation efficiencies in
comparison to exponential decay wave form pulses, and the ease of
optimization over a broad range of voltages (Saunders, "Guide to
Electroporation and Electrofusion," 1991, pp. 227-47). Preferably,
the waveform used for a method of the invention is an exponential
or a square wave pulse. In the methods of the invention where
compositions are administered in order to deliver the composition
into a vessel, square wave electrical pulses are preferred.
[0036] It is desired that the electric field produced by a pulse be
as homogeneous as possible and of the correct amplitude so as to
prevent excessive cell lysing. Generally, the strength of the
electric field will range from about 50 volts/cm to about several
KV/cm. The field strength is calculated by dividing the voltage by
the distance (calculated for 1 cm separation; expressed in cm)
between the electrodes. Thus, if the voltage is 500 volts between
two electrode faces which are 0.5 cm apart, then the field strength
is 500/(0.5) or 1000 volts/cm or 1 KV/cm. Preferably, the amount of
voltage applied between the electrodes is in the range of about 10
volts to 200 volts, and more preferably from about 50 to 90
volts.
[0037] The pulse length can be from about 100 microseconds (.mu.s)
to 100 milliseconds (ms), preferably from about 500 .mu.s to 100 ms
and more preferably from about 1 ms to 10 ms. There can be from
about 1 to 100 pulses applied to a vessel. Preferably, the number
of pulses is from about 1 to 50 pulses and more preferably from
about 1 to 10 pulses. The time between pulses can be one second or
longer. The electric field strength, waveform type, pulse duration
and number of pulses depend upon the construction of the device
used to apply the electrical pulse and can be adjusted as
appropriate according to the particular vessel to which the
electric pulse is applied, and whether compositions are to be
administered before or substantially contemporaneously with the
electrical pulse.
[0038] The various electrical parameters, including electric field
strengths, are similar to the electric fields needed for in vivo
cell electroporation, which are similar in amplitude to the
electric fields required for in vitro cell electroporation. Each
cell has its own critical field strength for optimum
electroporation due to cell size, membrane make-up and individual
characteristics of the cell membrane itself. For example, the field
strength required generally varies inversely with the size of the
cell. Mammalian cells typically require about 0.5 to 5.0 KV/cm
before cell death or electroporation occur. A database containing
the various electrical parameters for in vivo and in vitro cell
electroporation is maintained by Genetronics, Inc. (San Diego,
Calif.). The electrical parameters used for in vitro and in vivo
electroporation of various cell types also are known in the art and
can be found in research papers and in various electroporation
protocols provided by commercial vendors (e.g., Bio-Rad Catalogue,
1996, pp. 293-302, and the references cited at page 302).
[0039] Therefore, appropriate electrical parameters for inducing
the vasodilation of a vessel or for increasing the flow of fluid
through a vessel can be based upon the electrical parameters used
for the in vitro electroporation of the predominant cell type that
comprises the vessel. Alternatively, electrical parameters can be
empirically determined by applying an impulse and detecting an
induction or increase of vessel vasodilation or an induction or
increase in the flow of fluid through the vessel. For example, an
electrical impulse can be applied to a vessel and induction of
vessel vasodilation or an increase in the flow of fluid through a
vessel can be detected using the histological methods exemplified
herein or other methods known in the art. If the induction of
vessel vasodilation or increase in the flow of fluid through the
vessel is less than desired, the strength or duration of the pulse
can be increased from the initial setting, or multiple pulses can
be applied. Multiple electrical impulses also can be applied in
order to regulate the rate at which vessel vasodilation is induced
(rapid vs. slow) or to increase the degree to which a vessel is
vasodilated. Similarly, multiple electrical impulses can be applied
in order to increase the rate at which the flow of fluid through a
vessel is increased.
[0040] As the induction or increase of vessel vasodilation is
detected by an expansion of the luminal cavity, the luminal vessel
layer or of the outer diameter of a vessel, a variety of methods
can be employed in order to detect vessel vasodilation. In
particular, for example, the histological methods set forth in
Example II can be used to detect expansion of the luminal vessel
layer and the vessel cavity of an artery in response to the
electrical impulse. Specific (e.g., tissue, cell, etc.) or
non-specific marker stains or labeling agents, which can visualize
smaller vessels or increase the detail of the visualized vessel can
be used alone or in combination with these histological methods. If
the vessel is physically large enough or the induction of vessel
vasodilation is great enough, visual inspection of the vessel can
detect induction or an increase of vessel vasodilation. Other
methods useful for the detection of vessel vasodilation are known
in the art including, for example, intravascular ultrasound (IVUS)
and vascular angioscopy which measures luminal cross sectional area
of a vessel (Nissen et al., Circulation 88: 1087-99 (1993); Mallery
et al., Am. Heart J. 119: 1392-1400 (1990); McPherson, Sci.
American March/April, pp. 22-31 (1996); and Baptista et al., Eur.
Heart J. 16: 1603-12 (1995), which are incorporated herein by
reference).
[0041] An induction or increase in the flow of fluid through a
vessel can similarly be detected using the exemplified histological
methods as well as the above-described methods known in the art for
detecting vessel vasodilation. Additionally, other assays known in
the art can be useful for detecting an induction or increase in the
flow of fluid through a vessel. For example, illumination of a
vessel by barium can be used to detect the induction or an increase
in the flow of fluid through the gastrointestinal tract.
[0042] In another embodiment, an electrical impulse is applied to a
vessel with a catheter apparatus. In one aspect, an electroporation
catheter (EPC) or similar apparatus inserted endo-luminally into a
vessel is used to apply an electrical impulse. In this aspect, the
electrodes are positioned within the vessel near the region where
the electrical impulse is to be applied. After one or more pulses
are applied, the EPC is withdrawn from the vessel.
[0043] An endoluminal electroporation catheter useful in practicing
the methods invention of the invention needs to satisfy several
requirements: A pulsed field with a field strength of about 100
volts/cm needs to be created at the vessel wall; the pulse length
is typically in the order of milliseconds. The pulsed field should
generally be confined to the area of the vessel to be dilated, with
the main vector of the electric field pointing into the vessel
wall. Other design aspects concern the electrode geometry and the
electric field for a given potential difference (voltage) between
electrodes; it is desirable to maximize the electric field for a
given potential difference (electrical efficiency) (Hofmann, Cells
in electric fields: physical and practical electronic aspects of
Electro cell fusion and electroporation. In Neumann E, Sowers A and
Jordan C (eds): "Electroporation and Electrofusion in Cell
Biology," New York: Plenum Press, 1989, pp 389-407). Electrostatic
field plots of catheter configurations can be useful in determining
this electrical efficiency.
[0044] Catheters (e.g., balloon type) having various balloon sizes
suitable for endoluminal use in a variety of vessel types and sizes
(e.g., blood vessels, large and small intestine, urethra, etc.) are
commercially available. Such commercially available catheters can
be modified to include electrodes suitable for applying an
electrical impulse. The catheter may be, for example, a modified
Berman catheter (Arrow International, Inc., Reading, Pa.). One of
skill in the art will know of other catheter devices that can be
modified for endoluminal electropulsing based on the teachings
herein. Exemplary endoluminal catheter devices useful in the
methods of the invention are modified as described herein, and are
shown in FIGS. 1 to 3. FIG. 4 shows a porous balloon catheter
having multiple holes on the balloon surface and a guidewire
positioned within the balloon shaft which serves as the electrode.
The holes can be used to optionally administer compositions into
the vessel.
[0045] A catheter 100 (FIG. 1) includes at least one inflatable
balloon 102 near the distal end of the catheter 100, and at least
one inflation port 104 for inflating the at least one inflatable
balloon 102, in a conventional manner. The catheter 100 also
includes a first electrode 110 and a second electrode 112 that are
coupled by wires to a voltage source generator 114, which may be,
for example, an ECM 600 exponential pulse generator (BTX, a
division of Genetronics, Inc., San Diego, Calif.).
[0046] In one embodiment, the catheter 100 has at least one
infusion port for introducing a composition into a vessel of the
subject. As used herein, the term "infusion port" refers to a part
of an apparatus that is capable of introducing a composition, such
as a drug (e.g., heparin), via infusion. Infusion openings 120
capable of delivering the introduced composition endoluminally can
be made during or after manufacture of the catheter 100, and can be
placed on one or both sides of the first electrode 110, or within
the bounds of the first electrode 110. The first electrode 110 is
preferably placed close to the at least one infusion opening 120.
In one embodiment, the infusion openings 120 may be coincident with
the first electrode 110, such that the first electrode 110
completely surrounds the at least one infusion opening 120.
[0047] The first electrode 110 is preferably made of an
electrically conductive material that is biologically compatible,
e.g., biologically inert, with a subject. Examples of such material
include silver or platinum wire wrapped around or laid on or near
the surface of the catheter 100; a plated or painted coating of
conductive material, such as silver paint, on some portion of the
catheter 100; or a region of the catheter 100 that has been made
conductive by implantation (during or after manufacture, such as by
ion implantation) of electrically conductive materials, such as
powdered metal or conductive fibers. The conductor need not be
limited to metal, but can be a conductive plastic or ceramic. For
ease of manufacture, the embodiments shown in FIGS. 1 and 3 use
conductive silver paint for the first electrode 110 as a coating on
approximately 2.5 cm of the length of the catheter 100 near the
infusion openings 120.
[0048] The second electrode 112 (FIG. 1) similarly comprises an
electrically conductive material, and can be of the same or
different type of conductive material as the first electrode 110.
The second electrode 112 may be formed in a manner similar to the
first electrode 110 and positioned between the first electrode 110
and the infusion openings 120, or positioned with the infusion
openings 120 between the first electrode 110 and the second
electrode 112 (FIG. 1). Other configurations of the first electrode
110 and the second electrode 112 can be utilized, such as
interdigitated electrodes with infusion openings 120 nearby or
between the interdigitated "fingers" of the electrodes, or as
concentric rings with the infusion openings within the centermost
ring, between the centermost and outermost ring, or outside of the
outermost ring. Additional catheter configurations are within the
scope of the present invention so long as they provide a structure
that, when supplied by voltage from the voltage source 114,
generates an electric field sufficient to induce the vasodilation
of a vessel or to increase the flow of fluid through a vessel.
[0049] The first electrode 110 and the second electrode 112 are
coupled to the voltage source 114 by conductors, which may be, for
example, silver or platinum wires, but can be any conductive
structure, such as flexible conductive ink within the catheter 100
for connecting the first electrode 110.
[0050] In an alternative embodiment shown in FIG. 3, the second
electrode comprises a silver plate 112a configured to be applied to
a portion of the body of a subject such that an electric field
sufficient to induce vessel vasodilation or to increase the flow of
fluid through a vessel is generated when voltage from the voltage
source 114 is applied to the first electrode 110 and the second
electrode 112a. The second electrode, when placed externally, is
preferably placed on bare skin (e.g., shaved abdominal muscle of
the subject), preferably using a conductive gel for better
contact.
[0051] In operation, the catheter 100 is inserted into a vessel
(e.g., an artery or vein) endoluminally and is positioned so that a
balloon 102 traverses or crosses the vessel region to be pulsed.
The balloon 102 is then inflated to expand the vessel, if desired,
and a electrical pulse from the voltage source 114 is applied to
the first electrode 110 and second electrode 112 so as to induce
the vasodilation of the vessel or increase the flow of fluid
through the vessel. If desired, a composition is administered into
the vessel via the infusion openings 120, at some point in time
before, during, or after applying an electrical pulse or between
electrical pulses.
[0052] Thus, in another embodiment, the invention provides a method
for inducing the vasodilation of a vessel in a subject by applying
an electrical impulse to the vessel using a catheter apparatus
having at least one inflatable balloon portion, a first electrode,
a second electrode positioned with respect to the first electrode
and the subject such that an electric field sufficient to induce
vasodilation of the vessel is generated by the electrical impulse,
wherein application of the electrical impulse induces vasodilation
of the vessel.
[0053] In one aspect of this embodiment, the methods of the
invention employ a modified electroporation catheter apparatus
having a double balloon configuration, as illustrated in FIG. 2
(Danforth Biomedical, Calif.). The double balloon catheter contains
two 2.5 mm PET balloons of about 10 mm in length each with
radio-opaque markers mounted on a catheter shaft of about 1 mm in
diameter and about 60 cm in length. The balloons are separated by a
space of about 20 mm. Both balloons can be infusion lumen of the
catheter. An active electrode of about 7 mm in length is tightly
wrapped around the infusion outlet holes, which connects via a
lumen to an external power supply. In this aspect, a clinical 0.014
inch guidewire through the guidewire lumen of the catheter serves
as a return electrode. In operation, both electrodes of the EPC are
within the vessel lumen, the electrodes are positioned near where
the electrical pulse is to be applied, and the voltage applied
between the first and second electrode is of sufficient strength
and duration to induce the vasodilation of the vessel or to
increase the flow of fluid through the vessel.
[0054] Pulse generators useful for delivering the electrical pulses
are commercially available. For example, a pulse generator such as
the ECM Model 600 (BTX, a division of Genetronics, Inc., San Diego,
Calif.) can be used to practice the methods of the invention (FIG.
1). The ECM 600 generates an electric pulse from the complete
discharge of a capacitor is characterized by a fast rise time and
an exponentially decaying waveform tail. In the ECM 600, the pulse
length is set by selecting one often timing resistors marked R1
through R10. They are active in both High Voltage Mode (HVM)
(capacitance fixed at fifty microfarads) and Low Voltage Mode (LVM)
(with a capacitance range from 25 to 3,175 microfarads).
[0055] The ECM 600 pulse generator has a control knob that permits
the adjustment of the amplitude of the set charging voltage applied
to the internal capacitors from 50 to 500 volts in LVM and from
0.05 to 2.5 kV in the HVM; the maximum amplitude of the electrical
pulse is shown on a display. This device further includes a
plurality of push button switches for controlling pulse length, in
the LVM mode, by a simultaneous combination of resistors parallel
to the output and a bank of seven selectable additive
capacitors.
[0056] The ECM 600 also includes a single automatic charge and
pulse push button. This button may be depressed to initiate both
charging of the internal capacitors to the set voltage and to
deliver a pulse to the outside electrodes in an automatic cycle
that takes less than five seconds. If desired, the manual button
may be pressed repeatedly to apply multiple electric pulses to the
vessel.
[0057] The ECM 600 provides the voltage (in Volts) that travels
across the gap (in cm) between the electrodes. This potential
difference defines what is called the electric field strength where
E equals Volts/cm. Thus, the distance between the electrodes can be
measured and a suitable voltage according to the formula E=V/d can
then be applied to the electrodes (E=electric field strength in
V/cm; V=voltage in volts; and d=distance in cm). The waveforms of
the pulse provided by the ECM600 in the power pack deliver an
exponentially decaying pulse.
[0058] It is understood that other pulse generating systems can be
utilized in the methods of the invention. For example, the
ElectroSquarePorator (T820) can be used to apply a square wave
pulse to a vessel, if desired (Genetronics, Inc., San Diego,
Calif.). Square wave electrical pulses rise quickly to the set
voltage and stay at that level for a set length of time (pulse
length) and then rapidly decay to zero. This type of electrical
pulse is preferred where a composition is to be delivered into
cells via electroporation, as discussed below.
[0059] The T820 is capable of generating up to 3000 volts. The
pulse length is adjustable from 5 .mu.s to 99 msec. The T820 is
active in both High Voltage Mode (100 to 3000 volts) and Low
Voltage Mode (10 to 500 volts) and has multiple pulsing capability
(1 to 99 pulses).
[0060] The methods of the invention for inducing or increasing
vasodilation of a vessel in a subject are useful in treating
clinical situations in which a subject exhibits a blockage,
undesirable narrowing or a situation in which it is desired to
dilate a vessel. Thus, a method of the invention can be used to
treat vessel blockages or narrowing caused by events that occur
within the vessel, for example, the luminal renarrowing that occurs
within an artery during restenosis or that occurs as a result of a
blood clot.
[0061] A method of the invention also can be used to treat vessel
blockage or narrowing caused by events that occur external to the
vessel, for example, where an external mass, such as a tumor,
causes vessel constriction or blockage as a result of pressure.
Tumor masses often cause the obstruction or blockage of the
gastrointestinal system or blood vessels that supply blood to one
or more vital organs and therefore, obstruction or blockage can be
treated by using a method of the invention. Prostate hyperplasia,
which often causes urethra constriction or obstruction, is another
example of a clinical situation that can be treated with a method
of the invention. Such constrictions or blockages can lead to
severe discomfort, organ failure, or an inability to consume food.
Accordingly, the methods of the invention also can be useful for
relieving the symptoms associated with vessel obstruction or
blockage caused by an external mass or other form of external
pressure (e.g., pain, inability to consume food, difficult or
frequent urination, etc.).
[0062] The methods of the invention for inducing or increasing the
flow of fluid through a vessel in a subject are useful in treating
clinical situations for which a blockage or an undesirable decrease
in the flow of fluid through a vessel occurs, or for situations in
which it is desired to induce or increase the flow of fluid through
a vessel. For example, the above-described obstructions or
blockages can be treated. The invention methods for inducing or
increasing the flow of fluid through a vessel can be used to treat
various clinical situations, for example, an artery which supplies
blood to the heart in which a blockage, narrowing or occlusion is
present in the artery, such as that caused by restenosis or a blood
clot. Acute or chronic angina, which can be caused by a lack of
oxygen to the heart, can be treated. Additionally, a vessel
supplying blood to a transplanted organ in which it is desired to
induce or increase the delivery of nutrients or oxygen to the
transplanted organ can be treated. Similarly, a blood vessel that
supplies blood to a tumorous organ can be treated in order to
induce or increase delivery of a chemotherapeutic agent more
efficiently to the afflicted organ.
[0063] In another embodiment, a composition is administered to the
vessel in the subject prior to, substantially contemporaneously
with or after the application of the electrical impulse. In one
aspect, an electrical impulse is of the appropriate strength and
duration to electroporate at least one cell thereby allowing the
composition to be delivered into the at least one electroporated
cell.
[0064] As used herein, the term "substantially contemporaneously"
means that the composition is administered and the electrical pulse
is applied reasonably close together in time. Preferably, the
composition is administered concurrently with electropulsing, or at
some point in time before electropulsing. Thus, in another
embodiment the composition is administered either prior to or
substantially contemporaneously with the application of the
electropulse. When applying multiple electrical impulses, the
composition can be administered before or after each of the pulses,
or at any time between the electrical pulses.
[0065] In another embodiment, the composition is delivered locally.
As used herein, the term "local" means in a confined or in a
particular region. Thus, the term "local" when used in reference to
the delivery of a composition means that the composition remains
near the delivery site. The skilled artisan will recognize that a
composition delivered locally can, over time, be distributed
throughout a subject depending on various factors, for example, the
concentration of the composition, the half-life of the composition,
the site of delivery, the efficiency of cell electroporation and
the degree of leakage from the delivery site that occurs, for
example, as a result of the porosity of the vessel into which the
composition is delivered. The localized delivery of compositions to
cells of a vessel in a subject via electroporation is described in
U.S. application Ser. No. 08/668,725, which is herein incorporated
by reference.
[0066] Vasoconstrictor agents or mechanical devices can be used to
keep the therapeutic composition localized prior to, during or
after pulsing. For example, a catheter apparatus having a double
balloon configuration can hold the composition in place between the
two balloons for localized delivery (see for example, FIG. 1).
[0067] In another embodiment, the composition is delivered
systemically. As used herein, the term "systemic" means throughout
the body of the subject. Thus, a composition that is delivered
systemically is generally present throughout the body. A
composition delivered systemically can be followed by pulsing at a
vessel blockage or narrowing in order to deliver the compositions
into the site, for example.
[0068] The chemical structure of the composition, its intended
function and the preparation administered will dictate the most
appropriate time to administer the composition in relation to
applying the electrical pulse. Such factors include, for example,
the particular clinical situation, the condition of the patient,
the size of the composition, the presence of a carrier and the
half-life of the composition. One skilled in the art, depending on
the desired effect of the electrical pulsing and an administered
composition can readily determine when the composition should be
administered in relationship to applying the electrical
impulse.
[0069] Administration of the composition prior to or substantially
contemporaneously with an electrical impulse applied via
electroporation allows the composition to enter at least one
electroporated cell. The at least one electroporated cell will
generally be near the region at which the electrical impulse is
applied, i.e., in the vessel cavity (e.g., blood) or in the vessel
wall. For example, in a blood vessel, the composition can be
delivered into the tunica intima, tunica media or tunica adventitia
of the vessel. One skilled in the art will readily recognize that
although the terms "tunica intima," "tunica media" and "tunica
adventitia" are used in the art of cardiology to refer to the
increasing depth of a blood vessel wall as measured from the lumen,
that compositions can be delivered into the vessel wall of other
vessels (e.g., lymph vessel, G.I. vessel etc.) at depths
corresponding to the tunica intima, tunica media and tunica
adventitia.
[0070] The compositions delivered into the at least one
electroporated cell can be retained by the electroporated cell
thereby resulting in sustained delivery of the composition. As used
herein, the term "sustained" refers to the presence of the
composition over a relatively prolonged period of time during which
there is no appreciable washout. In general, "sustained" delivery
of a composition means that the composition is present for about 12
hours, but can be longer, for example, steadily over 24 to 72
hours. Sustained delivery generally means that the compositions are
present for a longer period of time than if the composition were
delivered by conventional methods (e.g., by injection into the
bloodstream). In the case of genes, for example, gene expression
can last for several weeks.
[0071] As disclosed herein (e.g., Example III), a double-balloon
catheter system incorporating electroporation technology was used
to deliver heparin into a vessel in an overstretch balloon injury
animal model. Following arterial injury, the double-balloon
catheter was inserted endoluminally, fluoresceinated heparin was
administered into the space between the two inflated balloons, and
the artery was subjected to an electrical pulse. Catheter
deployment and endoluminal electrical pulsing were well tolerated
in all animals (N=21) without adverse hemodynamic and histological
changes. Periodic arterial blood samples revealed no abnormalities
in the clotting profile or any gross morphological changes in the
blood cells up to 8 hours after treatment. Histochemical staining
of the tissue showed intracellular localization of heparin (FIGS. 7
and 8). Furthermore, heparin fluorescence was detected throughout
the vessel layers in the pulsed arteries for at least 12 hours in
comparison to the unpulsed control.
[0072] It is contemplated that the electropulsing parameters can be
manipulated in order to modulate the delivery of a composition into
particular areas of a vessel. It is further contemplated that the
electropulsing parameters can be manipulated as appropriate in
order to deliver a composition beyond the exterior vessel wall,
i.e., into a tissue or space that surrounds a vessel.
[0073] As used herein, the term "modulate," when used in reference
to the delivery of a composition, means to control or to regulate
the area of the vessel into which the composition is delivered. For
example, where it is desired to deliver a composition into cells at
the luminal surface of a vessel (i.e., the tunica intima), an
impulse having a particular strength and duration can be applied to
the vessel. Where delivery of a composition into the tunica media
or into the tunica adventitia is desired, an impulse having a
greater strength, duration or multiple pulses will be applied to
the vessel. Delivery of a composition into a stenotic lesion can
similarly be achieved by increasing pulse number, strength or
duration. Denuding the vessel prior to electropulsing can also be
used to modulate delivery of a composition. For example, denuding
blood vessel epithelium prior to applying the electrical pulse
facilitated the delivery of heparin into deeper areas of the blood
vessel, such as into the tunica media and into the tunica
adventitia (FIGS. 7 and 8).
[0074] Compositions contemplated for use in the methods of the
invention include those that elicit a biological effect or response
such as drugs (e.g., vessel vasodilators, cell proliferation
inhibitors, anticancer agents, angioproliferative inhibitors,
antibiotics, antiviral agents, fungicides and antimycobacterial
agents), polynucleotides (e.g., genes used in gene therapy,
antisense nucleotides, ribozymes), polypeptides (e.g., proteins,
antibodies, fragments thereof, functional derivatives thereof
including, for example, protease resistant analogues). Additional
useful compositions include dyes, stains, radionuclides (e.g.,
barium), luminescent and fluorescent agents (e.g., fluorescein),
and other compositions (e.g., gold particles) for the visualization
of a vessel cavity or vessel lumen, or for the visualization of a
tissue or space that surrounds a vessel.
[0075] Administering a composition that induces or increases vessel
vasodilation or that induces or increases the flow of fluid through
a vessel in combination with electropulsing can provide an additive
or synergistic effect. Compositions known to induce or increase
vessel vasodilation or induce or increase the flow of fluid through
a vessel are therefore particularly useful in treating various
clinical situations characterized by undesirable vessel narrowing,
such as restenosis, as described further below. Specific examples
of such compositions include, for example, high and low molecular
weight heparin and fragments thereof, which may control post-PTCA
vascular renarrowing caused by intimal thickening and hyperplasia.
Heparin also facilitates blood flow, modulates some growth factors
and provides cytoprotective action (Black et al., Cardiovas. Res.
29: 629-636 (1995)) and has an inhibitory effect on smooth muscle,
Schwann and epithelial cell proliferation in vitro (Clowes et al.,
Circ. Res. 58: 839-845 (1986)), U-937 leukemia cell proliferation
(Volpi et al., Exp. Cell. Res. 215: 119-130 (1994)) and on
thrombogenicity (Araki et al., Circ. Res. 71: 577-584 (1992)).
[0076] Additional examples of preferred compositions include
antithrombotic, antirestenotic, antiplatelet, and antiproliferative
compositions, for example, platelet receptor and mediator
inhibitors, smooth muscle cell proliferation inhibitors, growth
factor inhibitors, GpIIb/IIIa antagonists, cell adhesion and
aggregation inhibitors (e.g., platelet adhesion and aggregation),
PDGF inhibitors, matrix synthesis inhibitors, thromboxane receptor
inhibitors, fibrinogen receptor inhibitors, serotonin inhibitors,
fibrosis inhibitors and the like.
[0077] Thus, in another embodiment, the composition that is
administered to the vessel inhibits cell proliferation. In one
aspect, the cell proliferation inhibited is associated with vessel
intimal thickening or hyperplasia.
[0078] Particular useful compositions therefore, include
angiotensin converting enzyme (ACE) inhibitors, colchicine,
somatostatin analogues, hirudin, hirulog, tissue plasminogen
activator (tPA), urokinase, streptokinase, warfarin,
PDGF-antibodies, proteases such as elastase and collagenase,
serotonin, prostaglandins, vasoconstrictors, vasodilators,
anti-angiogenesis factors, Factor VIII or Factor IX, TNF, tissue
factor, VLA-4, gax, L-arginine, GR32191, sulotroban, ketanserin,
fish oil, enoxaprin, cilazapril, forinopril, lovastatin,
angiopeptin, cyclosporin A, steroids, trapidil, colchicine, DMSO,
retinoids, thrombin inhibitors, antibodies to von Willebrand
factor, antibodies to glycoprotein IIb/IIIa and calcium chelating
agents, for example.
[0079] Anticancer or chemotherapeutic drugs having an antitumor or
cyotoxic effect also are useful compositions. Anticancer drugs
include, for example, cell proliferation, angiogenesis and
metastases inhibitors (e.g., cell attachment inhibitors), as well
as inducers or promoters of apoptosis, growth arrest and the like.
Chemotherapeutic drugs include, for example, bleomycin, cisplatin,
5-fluorouracil, doxorubicin, taxol, suramin, neocarcinostatin.
Other compositions known in the art are applicable in the methods
of the invention (e.g., Remington's Pharmaceutical Sciences, 18th
ed., Mack Publishing Co., Easton, Pa., 1990; The Merck Index, 12th
ed., Merck Publishing Group, Whitehouse, N.J., 1996, which are
herein incorporated by reference).
[0080] Polynucleotides useful in the methods of the invention
include DNA, cDNA, RNA, and oligonucleotides thereof, either
unmodified or modified (e.g., nuclease resistant forms, conjugated
forms, e.g., to beads or to biotin and the like). Such
polynucleotides can encode proteins or polypeptides that inhibit or
induce cell proliferation (e.g., growth-arrest homeobox gene), or
angioproliferation, or have antithrombotic, antirestenotic,
antiplatelet, antifibrotic or anticancer activity. Preferred
polynucleotides function additively or synergistically for inducing
vessel vasodilation or for increasing the flow of fluid through a
vessel.
[0081] Antisense polynucleotides complementary to RNA molecules
(e.g., mRNA) that promote or inhibit the above-described activities
(cell proliferation, adhesion, clotting, angioproliferation,
fibrosis etc.) also are contemplated for use. Although not wishing
to be bound by a particular theory, it is believed that antisense
molecules hybridize to a complementary mRNA in the cell thereby
inhibiting translation of the mRNA. Preferred antisense
oligonucleotides are about 15 to 30 base pairs to minimize the
possibility of the antisense forming an internal secondary
structure. Oligonucleotides that form triplexes with double-helical
DNA can be used where it is desired to inhibit transcription of a
particular DNA sequence in cells. Such triplex oligonucleotides can
be designed to recognize a unique site on a chosen gene, for
example (Maher et al., Antisense Res. and Dev. 1: 227 (1991);
Helene, Anticancer Drug Design 6: 569 (1991)).
[0082] Ribozymes are RNA molecules that have the ability to cleave
other single-stranded RNA molecules at particular nucleotide sites.
Thus, it is possible to engineer ribozymes for the purpose of
cleaving specific RNA molecules which have the nucleotide sequences
cleaved (Czech, J. Amer. Med. Assn. 260: 3030 (1988)). Only those
RNA molecules having the particular nucleotide sites will be
cleaved and subsequently inactivated. As the hammerhead-type of
ribozymes have longer recognition motifs (11 to 18 base pairs) than
tetrahymena-type ribozymes (four base pairs), hammerhead-types are
preferably used for inactivating particular RNA molecules.
[0083] The polynucleotides of the invention can, if desired, be
naked or be contained in a vector (e.g., retroviral vector,
adenoviral vectors and the like), in a carrier (e.g., DNA-liposome
complex), or conjugated to inert beads or other functional moieties
(e.g., biotin, streptavidin, lectins, etc.), or appropriate
compositions disclosed herein. Such polynucleotides can be
modified, for example, to be resistant to nucleases. Thus, both
viral and non-viral means of polynucleotide delivery can be
achieved and are contemplated using the methods of the
invention.
[0084] Modified compositions that are biologically functional
analogues of the compositions described herein also are useful in
the methods of the invention. Such modified compositions can have
sulfate groups, phosphate groups, or hydrophobic groups such as
aliphatic or aromatic aglycones added or removed. For example,
modified heparin can include the addition of non-heparin saccharide
residues such as sialic acid, galactose, fucose, glucose, and
xylose. When heparin is used as the composition, it may include a
fragment of naturally occurring heparin or heparin-like molecule
such as heparin sulfate or other glycosaminoglycans, or may be
synthetic fragments. The synthetic fragments could be modified in
saccharide linkage in order to produce more effective blockers of
selecting binding. Methods for producing such saccharides will be
known by those of skill in the art (see for example, M. Petitou,
Chemical Synthesis of Heparin, in Heparin, Chemical and Biological
Properties, Clinical Applications, CRC Press, Boca Raton, Fla., D.
A. Lane and V. Lindahl, eds., 1989, pp. 65-79).
[0085] The compositions administered by a method of the invention
can be administered parenterally by injection or by gradual
perfusion over time. The composition can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, or transdermally, and preferably is administered
intravascularly at or near the site of electroporation.
Compositions also can be administered with a catheter apparatus
having at least one infusion port for introducing the composition
into the vessel.
[0086] The compositions administered will be in a "pharmaceutically
acceptable" or "physiologically acceptable" preparation. As used
herein, the terms "pharmaceutically acceptable" and
"physiologically acceptable" refer to carriers, diluents,
excipients and the like that can be administered to a subject,
preferably without excessive adverse side effects (e.g., nausea,
headaches, etc.).
[0087] Such preparations for administration include sterile aqueous
or non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Vehicles include sodium chloride
solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's, or fixed oils. Intravenous vehicles include fluid and
nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobial,
anti-oxidants, chelating agents, and inert gases and the like.
[0088] Controlling the duration of action or controlled delivery of
an administered composition can be achieved by incorporating the
composition into particles or a polymeric substance such as
polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone,
ethylene-vinylacetate, methylcellulose, carboxymethylcellulose,
protamine sulfate, or lactide/glycolide copolymers,
polylactide/glycolide copolymers, or ethylenevinylacetate
copolymers. The rate of release of the composition may be
controlled by altering the concentration or composition of such
macromolecules.
[0089] For example, it is possible to entrap a composition in
micro-capsules prepared by coacervation techniques or by
interfacial polymerization, for example, by the use of
hydroxymethylcellulose or gelatin-microcapsules or
poly(methylmethacrolate) microcapsules, respectively, or in a
colloid drug delivery system. Colloidal dispersion systems include
macromolecule complexes, nano-capsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes.
[0090] The above-described compositions and others not specifically
described herein are useful in various clinical situations and can
be administered alone, or in a combination with other compositions
by a method of the invention. The compositions administered alone,
or in combination include combinations of tPA, urokinase,
prourokinase, heparin, and streptokinase, for example.
Administration of heparin with tissue plasminogen activator would
reduce the dose of tissue plasminogen activator that would be
required, thereby reducing the risk of clot formation which is
often associated with the conclusion of tissue plasminogen
activator and other thrombolytic or fibrinolytic therapies.
Further, compositions containing heparin may include a mixture of
molecules containing from about 2 to about 50 saccharide units or
may be homogeneous fragments as long as the number of saccharide
units is 2 or more, but not greater than about 50.
[0091] Preferred clinical situations which can be treated using a
method of the invention include but are not limited to: 1) acute
arterial thrombotic occlusion including coronary, cerebral or
peripheral arteries; 2) acute thrombotic occlusion or restenosis
after angioplasty; 3) reocclusion or restenosis after thrombolytic
therapy (e.g., in an ishemic tissue); 4) vascular graft occlusion;
5) hemodialysis; 6) cardiopulmonary bypass surgery; 7) left
ventricular cardiac assist device; 8) total artificial heart and
left ventricular assist devices; 9) septic shock; 10) other
arterial thromboses (e.g., thrombolism where current therapeutic
measures are either contraindicated or ineffective).
[0092] Alternatively, various nucleic acid sequences encoding a
protein of interest can be used for treatment of cardiovascular
disorders. In particular, the expression of the growth factors
PDGF-B, FGF-1 and TGF.beta.1 has been associated with intimal
hyperplasia. Thus, either increasing (deliver sense constructs) or
decreasing (deliver antisense constructs) such gene expression can
be useful in treating such disorders. For example, whereas PDGF-B
is associated with smooth muscle cell (SMC) proliferation and
migration, FGF-1 stimulates angiogenesis and TGF .beta.1
accelerates procollagen synthesis. Thus, a nucleic acid that
encodes an inhibitor of SMC proliferation, migration, platelet
aggregation, extracellular remodeling or matrix formation also is
desirable for use in the methods of the invention. Such
compositions further include interferon-.gamma., which inhibits
proliferation and expression of .alpha.-smooth muscle actin in
arterial SMCs and non-protein mediators such as prostaglandin of
the E series.
[0093] Examples of other genes to be delivered by a method of the
invention include vascular endothelial growth factor (VEGF) and
endothelial specific mitogen, which can stimulate angiogenesis and
regulate both physiologic and pathologic angiogenesis.
[0094] Administration of the composition by a method of the
invention can be used for ameliorating post-reperfusion injury, for
example. When treating arterial thrombosis, induction of
reperfusion by clot lysing agents such as tPA is often associated
with tissue damage.
[0095] The methods of the invention also are useful for delivering
compositions that inhibit microbial infection which can be useful
in the treatment of microbial infections. Many microbes, such as
bacteria, rickettsia, various parasites, and viruses, bind to
vascular endothelium and leukocytes. Thus, the methods of the
invention may be used to deliver a composition to a patient to
prevent binding of a microbe which uses a particular receptor
(e.g., selectin) as its binding target molecule, thereby inhibiting
or reducing the microbial infection.
[0096] The methods of the invention can be used to treat vasculitis
by administering a composition described above to a patient. Tissue
damage associated with focal adhesion of leukocytes to the
endothelial lining of blood vessels is inhibited by blocking the
P-selectin and L-selectin receptors, for example.
[0097] The doses needed for clinical efficacy of the administered
compositions are those large enough to produce a desired effect in
which the signs or symptoms of the clinical situation are
ameliorated. The dose should not be so large as to cause excessive
adverse side effects. Generally, the dose will vary with the age,
condition, sex and extent of the disease in the patient and can be
determined by one of skill in the art. The dose can be adjusted by
the individual physician in the event of any complication. When
used for the treatment of inflammation, post-reperfusion injury,
microbial/viral infection, or vasculitis, or inhibition of the
metastatic spread of tumor cells, for example, the composition may
be administered at a dose which can vary from about 1 mg/Kg to
about 1000 mg/Kg, preferably about 1 mg/Kg to about 50 mg/Kg, in
one or more dose administrations.
[0098] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures and applications of the invention methods
known to those skilled in the art may alternatively be used.
EXAMPLE I
[0099] This example describes applying an electrical impulse to a
vessel in a subject.
[0100] Animal Preparation and Surgical Approach:
[0101] This study conformed to the care and use of laboratory
animals and standard euthanasia procedures policies by the NIH
guide set forth in the Institutional Animal Care and Use Committee,
and to the position of the American Heart Association on Research
Animal Use. New Zealand White rabbits weighing 3.2-4.2 kg of both
sexes (N=21) were utilized in the studies. After withdrawal of food
for 12-18 hrs, but not water, the animals were sedated using 2
mg/Kg intramuscular xylazine (Miles Inc., KS) and 50 mg/Kg ketamine
(Fort Dodge Lab, IA). Sedated animals were anesthetized with 30
mg/Kg .alpha.-chloralose (Fisher Scientific, NJ) throughout the ear
vein and endotracheally intubated. Once anesthetized, the animal
was strapped supine on the surgical table and ventilated with a
volume controlled Harvard 665 ventilator (Harvard Apparatus,
Natick, Mass.). Corneal and toe-pinching reflexes monitored the
state of anesthesia. A supplemental dose of .alpha.-chloralose (10
mg/Kg every hour) was injected intravenously for adequate
anesthesia. Throughout the experimental procedure, PO.sub.2 and
pCO.sub.2 were maintained at physiological levels and the body
temperature was maintained at 37.degree. C. using an infrared
heating lamp or thermostatic blanket. A polyethylene cannula
connected to a pressure transducer (Statham P23 Db) was introduced
into a femoral artery for continuous monitoring (DC-300 Hz) of
systemic blood pressure. EKG monitoring (0.05 Hz-1 KHz) was
undertaken using Lead I or Lead II connected to a differential
amplifier. Expiratory CO.sub.2 was monitored with a respiratory
CO.sub.2 analyzer (223 Puritan-Bennett). Blood pressure, EKG and
end-tidal CO.sub.2 signals were suitably amplified and output
signals fed in parallel to a storage oscilloscope (Tektronix,
Oreg.) and to an analog chart recorder (Gould, Ohio).
[0102] The common carotid arteries (CCA) of the animals were
accessed either by making a midline incision in the cervical region
and isolating them from the surrounding vago-sympathetic trunk
(N=16) or through the femoral artery under fluoroscopic guidance
(N=5).
[0103] Catheterization Procedure and Experimental Protocol:
[0104] I) Retrograde approach: A small vascular clip was applied
onto the caudal end of one common carotid artery (CCA), and the
catheter was pushed up to the junction of the CCA and the
innominate artery through a small incision at the rostral end of
the CCA. The balloons were next inflated to 2-3 atm inside the
carotid lumen, rubbed and pulled against the length of the CCA for
45 to 60 seconds to denude the endothelium of the artery. Following
this, the balloons were inflated at 4 atm to keep the lumen
occluded during the intervention period. Fluoresceinated heparin
(F-heparin; Molecular Probes, OR) at a dose of 8 to 30 units/Kg was
delivered over a period of 30 seconds through the infusion lumen of
the catheter. Immediately after the introduction of F-heparin the
catheter electrodes were connected to an ECM 600 exponential pulse
generator (BTX, San Diego, Calif.) and four pulses varying from 63
to 90 volts having a pulse width of 7.0 to 9.65 msec in different
experiments were applied endoluminally over a period of
approximately 60 to 90 seconds. Following electropulsing, the
balloons were deflated, the catheter was withdrawn and the incised
rostral end of the artery was ligated or an arterial repair was
made and the vascular clip was taken off to restore cranial blood
flow. The contralateral artery received the same treatment, but no
electropulsing.
[0105] II) Antgrade approach: Under fluoroscopy the EPC was
inserted into the femoral artery through a 5 F arterial sheath
(Cook, Inc., IN). It was first passed into one CCA and then
partially withdrawn and passed into the contralateral CCA. Drug
delivery and electropulsing were performed as described above for
the retrograde approach.
[0106] Tissue Processing:
[0107] Between 1 to 12 hours after pulsing, the animal was
sacrificed. The carotid arteries were quickly excised, embedded in
optimum cutting temperature compound (OCT), and frozen in
isopentane dipped in liquid nitrogen. Serial sections (10 to 15
microns in thickness) of the tissue were prepared using a
Reichert-Jung microtome. Frozen tissue sections mounted in glycerol
were viewed with a confocal laser scanning or epifluorescence
microscope (Zeiss Axiovert) connected to a Hamamatsu CCD camera and
Argus image processor. The image signal was fed into a video
monitor and stored in an optical disk for further processing and
analysis using NIH image analysis software. Some tissues were
dipped in formalin for 24 to 36 hours and paraffin embedded. Serial
sections (10 to 15 microns in thickness) stained with
hematoxylin-eosin (H-E) or van Giesson were observed by light
microscopy for any evidence of gross tissue damage.
[0108] Safety Data:
[0109] Blood Pressure
[0110] Intravascular pulsing of CCA had no influence on systemic
arterial pressure monitored throughout the studies, except that
each pulse in the stimulation sequence caused a very transient
reduction of the mean arterial pressure in the range of 5 to 20
torr, which immediately returned to baseline levels on cessation of
pulsing stimuli.
[0111] Electrocardiogram
[0112] Randomly induced impulse given at different phases of the
cardiac did not change the prepulsing lead H EKG pattern. There was
no appreciable change in heart rate nor did the pulsing elicit any
arrhythmia or atrial/ventricular fibrillation. No deviation from
the normal P-R interval (0.05 to 0.08 sec) on pulsed stimuli was
noticed, suggesting that intraluminal pulsing did not evoke any
atrioventricular conduction defect.
[0113] Blood Chemistry and Morphology
[0114] Prothrombin time (PT), activated partial thromboplastin time
(APTT) and fibrinogen were measured at various intervals throughout
the studies. Plasma was collected from whole blood by
centrifugation at 3250 RPM for 15 minutes. Erythrocyte
sedimentation rate (ESR) and differential blood count pre- and
post-pulsing were evaluated to determine potential alterations
associated with electroporation.
[0115] ESR of the post-pulse samples were obtained over a four hour
interval and tested by the Westergren method. These values remained
in the normal range (0 to 20 mm/hr). Routine clotting assays
performed included pre- and post-pulsing measurement of PT,
fibrinogen and APTT at various time intervals throughout the
experiment, and were normal as well. The PT ranged from 6.2 to 8.0
seconds before pulsing to 6.4 to 9.3 seconds after pulsing. The
APTT changed from 14.0 to 19.0 seconds pre- and post-pulse states,
ranging from 177 to 202 mg/dL.
[0116] Post-pulsing blood cell count were within the normal
physiological range. Blood films stained with Wright or Giemsa
procedures showed no cellular deformation.
[0117] Light Microspcopy of Arterial Tissue
[0118] Electropulsing had no effect upon tissue morphology. Damage
caused by balloon injury to the endothelial layer and, less
frequently, to the internal elastic lamina was observed in the
paraffin-embedded tissue sections stained with hematoxylin-eosin
and observed under a light microscope at low and high power
magnification.
EXAMPLE II
[0119] This example shows that an electrical impulse applied to a
vessel of an animal increases the vessel luminal area.
[0120] Luminal area measurements of the arterial tissue sections
were made by NIH image analysis software. Measurement of areas
enclosed by the lumen in pulsed artery samples and non-pulsed
(control) samples were performed in multiple sections in a given
experiment. Statistical testing was performed with "Instat"
software (Graphpad, San Diego, Calif.). Results were considered
statistically significant when the probability of error was
p.sub.--0.05.
[0121] A total of 120 serial tissue sections obtained from eight
animals were examined. Fifty-seven sections were from electrically
pulsed arteries and sixty-three sections were from non-electrically
pulsed arteries. All experiments but one showed expansion of the
vessel lumen after electropulsing at approximately 65 volts and a
pulse length of 9 ms. The luminal vessel layer of the pulsed artery
showed an average increase of 76% in area over the non-pulsed. The
Wilcoxon signed rank test for paired non-parametric data showed a
significant increase (2p.sub.--0.0156) in lumenal vessel area of
the electropulsed artery. The effect of applying an electrical
impulse via electroporation on the luminal area of an artery, as
observed histologically and graphically are shown in FIGS. 5 and 6,
respectively.
[0122] The observed expansion of the vessel lumen does not appear
to be due to artifacts resulting from the histological preparation
of the arteries because Kakuta et al. (Circulation 89: 2809-2815
(1994)) has reported a linear correlation of the lumen area
obtained by histology and angiography and the atherosclerotic
rabbit model.
[0123] Expansion of the arterial luminal area as a result of
electropulsing can provide an avenue to achieve maximum dilatation
during PTCA. The expansion of vessel lumen observed in pulsed
arteries may be due to the activation of endothelium derived
relaxation factors (EDRF), now believed to be the same as nitric
oxide (NO), or the release of other vasodilating substances
triggered by electrical pulses. If this vasodilation mechanism
involves a nitrous oxide medicated event, it would probably entail
inhibition of platelet aggregation, cell adhesion and also
vasospasm, which can occur during PTCA.
EXAMPLE III
[0124] This example shows that the application of an electrical
impulse to a vessel of an animal can deliver a composition into the
vessel.
[0125] Fluoresceinated heparin (F-heparin; 167 units/mg of
activity) was used as a probe. To ensure that the covalent bonded
fluorescein had not been dissociated by electroporation, in vitro
experiments were performed in an electroporation cuvette containing
F-heparin. The solution was pulsed using the same electrical
parameters as used in the in vivo studies. The sample was then
loaded in a LKB ultropak column (TSK G4000sw) connected to an
Anspec HPLC pump with a Shimadzu (SPD-6AV) UV-VIS
spectrophotometric detector for analysis. The hard copy of the HPLC
profile was obtained using Hitachi D-2500 chromato-integrator.
Individual samples were also analyzed in a luminescence
spectrometer (Perkin-Elmer LB-5, CA) at 490 nm excitation and
emission at 520 nm.
[0126] Luminescence spectrometry and HPLC of electroporated
F-heparin showed no differences from the non-electroporated sample.
We also obtained an HPLC analysis of FITC in the non-electroporated
condition. Analysis of F-heparin electroplated in vitro at voltages
and pulse lengths used in in vivo rabbit experiments did not show
presence of free FITC. Thus, pulsing does not appear to change the
structure of F-heparin, and therefore, the fluorescence observed in
control and treated tissue samples is predominantly that of
FITC-conjugated heparin.
[0127] F-heparin penetration into the arterial wall was tested by
both direct and indirect visualization. For direct visualization of
heparin fluorescence, tissues were excited at 488 nm and emission
observed at 520 nm in a fluorescent microscope with fluorescent
isothiocyanate (FITC) filter. Pseudocolor images coding for
fluorescence emission were obtained from a linear look-up table
(`LUT`) using NIH image analysis software. Photographs were
obtained by dye-sublimation photocopy with a Tektronix phaser
440-color printer.
[0128] For indirect visualization, tissue sections were hydrated in
Tris-buffered saline (TBS, pH 7.6) and treated with 3%
H.sub.2O.sub.2 for 30 minutes to inhibit an endogenous peroxidase
activity. After incubation with 10% normal goat serum to block
non-specific binding sites, the monoclonal antibody to FITC (1:250,
clone FL-D6; Sigma, Mo.) was applied for 16 hours at 4.degree. C.
After rinsing with 1% normal goat serum in TBS and incubation for
10 minutes in 10% of the same serum, goat antisera to mouse IgG
(1:50) was applied for 30 minutes, followed by incubation with the
mouse peroxidase complex (1:250; Sternberger monoclonals). Sections
were developed using 3, 3, M-{circumflex over (
)}S-diaminobenzidine in 0.3% H.sub.2O.sub.2, 50 mM Tris-HC I (pH
7.6) for identical periods, so direct comparison could be made.
[0129] As shown in FIG. 7B, pseudocolor image manipulation by NIH
image analysis software of the fluorescence emission by
FITC-conjugated heparin showed intense fluorescence of the pulsed
artery in both the tunica media and tunica adventitia of the
arterial cross section. Heparin was found to be inhomogeneously
distributed around the arterial circumference, which could be due
to non-uniformity of the electrical field strength in different
parts of the artery caused by variations in local tissue
resistance. It is clear, however, that pulsing increases heparin
penetration to the deeper part of the tissue several folds with
respect to the control.
[0130] A qualitative measurement of heparin in the tissue with
monoclonal antibody to FITC, followed by peroxidase staining, also
shows that pulsing facilitates heparin binding in the tissue (FIG.
8). At very high magnification of the image, one could clearly see
the presence of the antibody in the cytosol.
[0131] These data show the feasibility of using an electroporation
catheter for the endovascular delivery of molecules, such as
heparin. The observation of increased F-heparin fluorescence by
direct visualization under a fluorescence microscope and increased
binding of antibody to fluorescein by indirect visualization in
electroporated arteries confirms the ability of electroporation to
facilitate entry and retention of heparin into the arterial wall.
Thus, this data show that endovascular electropulsing may provide
better delivery, retention and greater therapeutic efficacy than
that achieved by conventional delivery of heparin. Electropulsing
is therefore applicable for clinical situations in which catheters
have traditionally been used, including those other than
endovascular diseases.
[0132] In earlier studies we demonstrated the penetration of a
marker gene (lacZ driven by a CMV promoter) into the arterial wall
by pulsing with caliper electrodes placed across the extraluminal
adventitial surface. The punctate location of gene expression
inside the arterial wall of rabbit carotid arteries remained for
three weeks (Giordano et al., Abstract #7804, Amer. Coll. Cardiol.
Meeting, Orlando, Fla., March 1996).
[0133] Subsequently, antisense RNA directed towards PCNA and CDC-2
kinase sequences has been successfully delivered in porcine
arteries using an electroporation catheter. Pulsed arteries showed
higher uptake of the antisense oligonucleotide compared to the
control. These results show that delivery of heparin by
electroporation is possible (Wolinsky et al., J. Am. Coll. Cardiol.
15: 475-481 (1990); Gimple et al., Circulation 86: 1536-1546
(1992); Femandez-Ortiz et al., Circulation 89: 1518-1522 (1994))
and suggest the usefulness of electrical pulse-assisted local
delivery of other molecules. For example, pulse-assisted delivery
can be useful for the local or systemic delivery of other molecules
which are of low effectiveness when delivered conventionally
(Fareed et al., Sem. Thromb. Hemos. 17: 455470 (1991)).
[0134] The data show that vasodilation of a vessel can be induced
by applying an electrical impulse. The data also show that the flow
of fluid through a vessel can be increased by applying an
electrical impulse. Based on the pulse-enhanced induction of vessel
vasodilation and the increase of flow of fluid through the vessel,
it is contemplated that the methods of the invention when applied
to clinical situations, would avoid complications due to the side
effects associated with various drugs. Additionally, the data show
that compositions, such as heparin, can be effectively delivered
into vessel tissue using the methods of the invention. This also
would avoid clinical complications that arise due to excess drug in
the systemic circulation. The methods additionally allow the
delivery of compositions into different depths of the artery simply
by changing the electrical parameters. Endovascular electroporation
can therefore provide better retention and higher therapeutic
efficacy than that achieved by conventional systemic delivery of
heparin at clinically safe concentrations.
* * * * *