U.S. patent application number 12/413357 was filed with the patent office on 2009-10-01 for balloon catheter method for reducing restenosis via irreversible electroporation.
This patent application is currently assigned to AngioDynamics, Inc. The Regents of the University of California. Invention is credited to WILLIAM C. HAMILTON, ANTONI IVORRA, ELAD MAOR, JAMES J. MITCHELL, BORIS RUBINSKY.
Application Number | 20090247933 12/413357 |
Document ID | / |
Family ID | 41114350 |
Filed Date | 2009-10-01 |
United States Patent
Application |
20090247933 |
Kind Code |
A1 |
MAOR; ELAD ; et al. |
October 1, 2009 |
BALLOON CATHETER METHOD FOR REDUCING RESTENOSIS VIA IRREVERSIBLE
ELECTROPORATION
Abstract
Restenosis or neointimal formation may occur following
angioplasty or other trauma to an artery such as by-pass surgery.
This presents a major clinical problem which narrows the artery.
The invention provides a balloon catheter with a particular
electrode configuration. Also provided is a method whereby vascular
cells in the area of the artery subjected to the trauma are
subjected to irreversible electroporation which is a non-thermal,
non-pharmaceutical method of applying electrical pulses to the
cells so that substantially all of the cells in the area are
ablated while leaving the structure of the vessel in place and
substantially unharmed due to the non-thermal nature of the
procedure.
Inventors: |
MAOR; ELAD; (Berkeley,
CA) ; MITCHELL; JAMES J.; (Ballston Spa, NY) ;
IVORRA; ANTONI; (Berkeley, CA) ; HAMILTON; WILLIAM
C.; (Queensbury, NY) ; RUBINSKY; BORIS;
(Givataaim, IL) |
Correspondence
Address: |
UC Berkeley - OTL;Bozicevic, Field & Francis LLP
1900 University Avenue, Suite 200
East Palo Alto
CA
94303
US
|
Assignee: |
The Regents of the University of
California; AngioDynamics, Inc.
|
Family ID: |
41114350 |
Appl. No.: |
12/413357 |
Filed: |
March 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61040110 |
Mar 27, 2008 |
|
|
|
61156368 |
Feb 27, 2009 |
|
|
|
Current U.S.
Class: |
604/20 |
Current CPC
Class: |
A61B 2017/22038
20130101; A61B 2018/0022 20130101; A61B 2018/0041 20130101; A61B
18/14 20130101; A61B 2018/00214 20130101; A61B 2018/1435 20130101;
A61N 1/327 20130101; A61B 2018/00166 20130101; A61M 5/14 20130101;
A61B 18/1492 20130101; A61B 2018/00613 20130101 |
Class at
Publication: |
604/20 |
International
Class: |
A61N 1/30 20060101
A61N001/30 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with government support under
federal grant no. NIH R01 RR018961 awarded by the U.S. National
Institutes of Health (NIH). The United States Government has
certain rights in this invention.
Claims
1. A method of reducing restenosis, comprising: inserting into a
vessel of a human patient a balloon catheter comprised of a
flexible elongated shaft comprising a distal end portion for
insertion into a vessel, wherein the balloon catheter is comprised
of: a balloon positioned at the distal end portion; a first
electrode positioned at the distal end portion of the shaft, the
first electrode being comprised of a conductive material which is
flexible and generally conforms to an outer surface of the balloon
during expansion of the balloon; a second electrode positioned at a
point relative to the first electrode so as to allow electrical
current to flow between the electrodes and through vascular tissue
to the first electrode, the second electrode being comprised of a
conductive material which is flexible and conforms to an outer
surface of the balloon during expansion of the balloon; and
providing electrical pulses to the electrodes for durations and in
amounts sufficient to subject substantially all vascular cells in a
target area of an artery to irreversible electroporation (IRE).
2. The method of claim 1, wherein the first and second electrodes
each encircle the balloon and are positioned on either side of the
target area.
3. The method of claim 2, wherein the first and second electrodes
each have a helical configuration around the balloon and the
electrodes are positioned so that electrical current flows between
the electrodes and through the target area.
4. The device of claim 1, wherein the first and second electrodes
each have an elongated configuration along a proximal end of the
balloon to a distal end of the balloon and the electrodes are
positioned so that electrical current flows between the electrodes
and through the target area.
5. The method of claim 1, further comprising: administering an
anti-restenosis drug; and wherein the IRE is carried out using a
voltage and a current with defined ranges over a defined period of
time and in absence of drug being delivered into the vascular
cells.
6. The method of claim 5, wherein the anti-restenosis drug is
selected from the group consisting of paclitaxel and vasculant and
is administered locally to the target area; and wherein electrical
power source provides electrical pulses having a voltage, current,
and duration so as to avoid thermal damage to a target area and
surrounding tissue while obtaining IRE on the target area.
7. The method of claim 6, wherein the pulses have a duration of
from 50 to 200 microseconds with a current in a range of from 2,000
V/cm to 6,000 V/cm and wherein the electrical power source is
configured to apply between two and twenty-five pulses.
8. A balloon catheter device for insertion into a vessel and
reducing neointima, comprising: a flexible elongated shaft
comprising a distal end portion for insertion into a vessel; a
balloon positioned at the distal end portion; a first electrode
positioned at the distal end portion of the shaft, the first
electrode being comprised of a conductive material which is
flexible and generally conforms to an outer surface of the balloon
during expansion of the balloon; a second electrode positioned at a
point relative to the first electrode so as to allow electrical
current to flow between the electrodes and through vascular tissue
to the first electrode, the second electrode being comprised of a
conductive material which is flexible and conforms to an outer
surface of the balloon during expansion of the balloon; an
electrical power source which provides electrical pulses to the
electrodes for durations and in amounts sufficient to subject
substantially all vascular cells in an area of an artery to
irreversible electroporation (IRE) before neointima occurs.
9. The device of claim 8, wherein the first and second electrodes
each encircle the balloon.
10. The device of claim 9, wherein the first and second electrodes
each have a helical configuration around the balloon.
11. The device of claim 8, wherein the first and second electrodes
each have an elongated configuration along a proximal end of the
balloon to a distal end of the balloon.
12. The device of claim 8, wherein the first and second electrodes
are formed from electrically conductive ink drawn on the
balloon.
13. The device of claim 8, for use wherein the IRE is carried out
using a voltage and a current with defined ranges over a defined
period of time and in absence of drug being delivered into the
vascular cells.
14. The device of claim 13, wherein the electrical power source
provides electrical pulses having a voltage, current, and duration
so as to avoid thermal damage to a target area and surrounding
tissue while obtaining IRE on the target area.
15. The device of claim 14, wherein the electrical power source is
designed to emit pulses wherein the pulses have a duration of from
50 to 200 microseconds.
16. The device of claim 8, wherein electrical power source applies
current in pulses.
17. The device of claim 16, wherein the electrical power source is
configured to apply pulses having a duration of from 50 to 200
microseconds.
18. The device of claim 17, wherein the electrical power source is
configured to apply pulses of a current in a range of from 2,000
V/cm to 6,000 V/cm.
19. The device of claim 18, wherein the electrical power source is
configured to apply between two and twenty-five pulses.
20. The device of claim 19, wherein electrical power source is
configured to apply 10 pulses for 100 microseconds each at a
current of 3,800 V/cm.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 61/040,110, filed Mar. 28, 2008 and 61/156,368,
filed Feb. 27, 2009, which applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0003] The present invention relates to a medical device and method
for the prevention of vascular re-stenosis using electroporation.
More particularly, the present invention relates to a balloon
catheter device with electrodes for electroporating the inner wall
of a vascular structure to prevent re-stenosis.
BACKGROUND OF THE INVENTION
[0004] Catheters, and more particularly, balloon catheters have
been used to treat stenosis of a vascular or other anatomical
tubular structure. In one such procedure, called percutaneous
transluminal angioplasty or PTA, a balloon catheter is inserted
into a vessel and advanced to the site of the stenosis or lesion
where the balloon is inflated against the lesion. Pressure applied
to the stenosis by the surface of inflated balloon compresses the
lesion, pushing it radially outward and widening or restoring the
luminal diameter of the vessel. Various forms of PTA have been used
to treat peripheral arterial stenosis, coronary lesions and other
non-vascular tubular structures such as biliary ducts.
[0005] Notwithstanding the importance of PTA procedures in
restoring normal blood flow to an anatomical region, one problem
associated with PTA procedures is the undesired re-growth of the
lesion, commonly known as re-stenosis. Re-stenosis, a re-narrowing
of the vessel lumen, usually occurs within three to six months
after the angioplasty procedure. Studies have demonstrated a
re-stenosis rate after angioplasty in up to 50% of patients
treated. Although the use of stents has reduced the re-stenosis
rate to approximately 30% of the procedures, re-stenosis remains a
significant clinical problem, particularly for those patients whose
general health is not conducive to repeat interventional
procedures.
[0006] The main cause of re-stenosis following angioplasty
procedures is due to vessel wall trauma created during the
procedure. Evidence has shown that scar tissue forms as endothelial
cells that line the inner wall of the blood vessel re-generate in
response to the vessel wall injury created during angioplasty. An
overgrowth of endothelial cells triggered by the trauma leads to a
re-narrowing of the vessel and eventual re-stenosis of the treated
area. Cutting wire balloon catheters, also known in the art, have
been used to "score" a stenotic lesion in a more controlled,
precise manner. Although it is contemplated that scoring a lesion
will lead to less procedural vessel trauma, endothelial cell
re-growth and re-stenosis, to date there are no studies that
effectively demonstrate this.
[0007] Recently, advances in stent technology have included
drug-eluting stents which are intended to reduce the occurrence of
re-stenosis even further. These types of stents are coated with a
drug designed to suppress growth of scar tissue along the inner
vessel wall over an extended period of time. The drug is slowly
released or eluted, thus reducing the occurrence and extent of
re-stenosis when compared with bare stents. Although shown to be
effective in further reducing re-stenosis, there are several known
problems with drug-eluting stents including an increased risk in
some patient populations of localized blood clots after the drug
has been completely eluted, usually after six or more months. Clot
formation in the coronary system can lead to heart attack and
death. Other problems include stent fracture and other known risks
associated with long-term implants.
[0008] Therefore, it is desirable to provide a device and method
for the prevention of re-stenosis associated with primary
angioplasty and/or stenting procedures that is safe, easy and does
not require placement of a stent.
SUMMARY OF THE INVENTION
[0009] A catheter device for insertion into a vessel which device
is used for reducing neotima or reducing the occurrence of
restenosis is disclosed. The present invention can utilize basic
structural configurations of a balloon catheter device modified to
incorporate electrodes which can be electrically connected to a
power source for the administration of electrical pulses which can
provide for irreversible electroporation. Thus, a device of the
invention includes a basic balloon catheter configuration having a
first electrode positioned at a distal end of the catheter. A
second electrode is positioned at a point relative to the first
electrode so as to allow electrical current to flow between the
first and second electrodes and through vascular tissue. The device
includes a power source and electrical connections from the power
source to the electrodes. The power source provides electrical
pulses to the electrodes for durations, voltages, current amounts
and combinations thereof so as to provide sufficient electrical
flow to substantially all of the vascular cells in the area of an
artery (which has been subjected to trauma) to irreversible
electroporation (IRE) which is preferably done before neointima
occurs.
[0010] In an aspect of the invention the catheter is a balloon
catheter and the electrodes may encircle the catheter in a spiral
configuration.
[0011] In one embodiment the first and second electrodes are
designed for use following a by-pass surgery or alternatively are
designed for use following angioplasty with a balloon angioplasty
device which may be the same device to which the electrodes are
connected.
[0012] The system of the invention may be comprised of two separate
catheter devices wherein a first catheter device is a balloon
catheter which is used for carrying out balloon angioplasty and a
second catheter which is specific for use in the IRE.
[0013] The system of the invention is designed for use wherein the
IRE is carried out using a voltage, and a current within defined
ranges over a defined period of time and in the absence of drug
being delivered into the vascular cells.
[0014] In another embodiment of the invention the device comprises
an electrical power source which provides electrical pulses which
provide voltage, current and are provided for a duration so as to
avoid thermal damage to a target area and surrounding tissues while
obtaining the IRE on the target area.
[0015] In another embodiment of the device the power source is
designed to emit pulses wherein the pulses have a duration from 50
to 200 microseconds and the device may be designed for carrying out
the IRE immediately following balloon angioplasty or alternatively
the IRE may be carried out immediately prior to balloon
angioplastly.
[0016] The system of the invention includes an electrical power
source which is specifically designed for carrying out the IRE so
as to reduce restenosis or neointimal and avoid thermal damages.
The power source may be designed to deliver a range of different
voltages, currents and duration of pulses as well as number of
pulses. The system may be designed to provide for pulse durations
from about 50 to 200 microseconds and may administer a current in a
range of from about 2,000 V/cm to about 6,000 V/cm. The power
source may provide between 2 and 25 pulses upon activation and may
be designed to provide a specific number of pulses which are at a
specific known duration and with a specific amount of current. For
example, the power source may be designed upon activation to
provide 10 pulses for 100 microseconds each providing a current of
3,800 V/cm.+-.50%, .+-.25%, .+-.10%, .+-.5%.
[0017] A method of reducing, attenuating or eliminating the intimal
formation on a patient that has undergone a surgical procedure in a
target area of an artery is disclosed. The method first involves
diagnosing a subject which may be a human subject suffering from
coronary artery disease and specifically identifying a target area
of an artery in the subject which is partially blocked by plaque. A
procedure is performed whereby blockage in the target area is moved
or removed from the artery so as to increase blood flow through the
target area of the artery. This procedure can be balloon
angioplasty whereby the plaque is forced away from the area of flow
or can involve by-pass surgery whereby the blocked area of the
artery is completely removed.
[0018] After the procedure is carried out vascular cells in the
area subjected to trauma by the angioplasty or surgery are
subjected to irreversible electroporation (IRE). The IRE may be
carried out (1) before, (2) at substantially the same time, or (3)
just after the procedure (e.g. angioplasty) is carried out, but is
carried out before restenosis occurs to obtain the best results.
The IRE may be carried out by the use of electrodes which are
present on or near the balloon portion of the balloon catheter used
in the angioplasty. The IRE is carried out using a voltage and
current within defined ranges over a defined period of time.
Further, the IRE is carried out in the absence of a drug being
delivered to the vascular cells in a manner which would effect the
growth of the cells.
[0019] The IRE is not carried out in order to provide for
reversible electroporation of substantially all of the cells.
Reversible electroporation is carried out when the pores of the
cells are temporarily opened and after the procedure go back to
normal size and the cells survive. Others carry out electroporation
in a manner so as to prevent excessive cell lysing (see U.S. Pat.
Nos. 6,865,416 and 6,342,247). With irreversible electroporation
the pores of the cells are opened and are opened to a degree that
they do not return to normal size and the cells die, so excessive
lysing of cells is desired. Thus, irreversible electroporation
requires more voltage, current or time in order to obtain the
desired result as compared to reversible electroporation. The
amount of current used and the time it is applied must be
controlled in accordance with the invention in order to avoid
thermal damage. The result sought per the present invention is to
have substantially all of the vascular cells of the targeted area
of the artery ablated or killed but to not raise the temperature of
that area sufficiently to cause thermal damage and denature
proteins. By avoiding thermal damage the structure of the artery
and surrounding tissue remains in place. However, due to the
irreversible electroporation the vascular cells are killed and as
such do not form scar tissue (neointimal) in the area thereby
reducing or avoiding restenosis.
[0020] The methodology of the invention may involve carrying out
the IRE at substantially the same time the balloon angioplasty or
by-pass surgery is carried out. It would be possible to carry out
the IRE prior to angioplasty or by-pass surgery or other trauma
event or carry out the IRE at substantially immediately after the
balloon angioplasty or by-pass surgery or other trauma event is
carried out. The timing of carrying out the IRE relative to the
timing of the trauma event is important in order to avoid the
occurrence of restenosis and avoid as much as possible the artery
being blocked with respect to blood flow.
[0021] In addition to the timing of the IRE the parameters of the
IRE in terms of voltage/current/pulse duration are important. These
parameters are important so as to go beyond reversible
electroporation and obtaining irreversible electroporation.
Further, the parameters are important so as to avoid thermal
damage. It is undesirable to heat the area in that too much heat
can cause denaturation of the proteins. Denaturation of the
proteins results in breakdown of those proteins which thereafter
can result in structural breakdown of the vessel which is
undesirable. Thus, the method of the invention is intended to go
beyond reversible electroporation to obtain irreversible
electroporation but not obtain thermal damage.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1A is a plan view of an embodiment of an
electroporation balloon catheter of the present invention with a
balloon and electrode assembly at the distal portion of the
catheter.
[0023] FIG. 1B illustrates an enlarged partial plan view and a
cross-sectional view of the catheter shaft of FIG. 1A.
[0024] FIG. 2 depicts a plan view of the balloon and electrode
assembly of the electroporation balloon catheter of the present
invention.
[0025] FIG. 3A is a plan view of a first longitudinal electrode of
the electrode assembly.
[0026] FIG. 3B is a plan view of a second longitudinal electrode of
the electrode assembly.
[0027] FIG. 4A illustrates a plan view of the electrode assembly
comprising the first and second longitudinal electrodes.
[0028] FIG. 4B depicts end views of the electrode assembly taken
from lines A-A and B-B of FIG. 4A.
[0029] FIGS. 5A and 5B depict partial cross-sectional views of the
proximal section of the balloon and electrode assembly taken along
lines A-A of FIG. 2.
[0030] FIGS. 6A and 6B depict partial cross-sectional views of the
distal section of the balloon and electrode assembly taken along
lines B-B of FIG. 2.
[0031] FIGS. 7A and 7B illustrate end views of the balloon and
electrode assembly taken along lines C-C and D-D of FIG. 1A.
[0032] FIG. 8A illustrates a plan view of the combined
balloon/electrode assembly in an expanded position.
[0033] FIG. 8B illustrates a plan view of the combined
balloon/electrode assembly shown in a collapsed position.
[0034] FIG. 9 shows an end view of the balloon electrode assembly
taken along lines D-D of FIG. 1 illustrating the electrical current
generated between the electrodes.
[0035] FIG. 10 is a plan view of another embodiment of an
electroporation balloon catheter of the present invention with a
balloon and electrode assembly at the distal portion of the
catheter.
[0036] FIG. 11 illustrates an enlarged cross-sectional view of the
catheter shaft of FIG. 10 taken along lines A-A.
[0037] FIG. 12 is a plan view of the balloon and electrode assembly
of the electroporation balloon catheter of FIG. 10.
[0038] FIG. 13A is a plan view of a first spiral electrode of the
electrode assembly of FIG. 10.
[0039] FIG. 13B is a plan view of the second spiral electrode of
the electrode assembly of FIG. 10.
[0040] FIG. 14 illustrates a plan view of a spiral electrode
assembly comprising the first and second spiral electrodes.
[0041] FIG. 15A is a partial longitudinal cross-sectional view of
the distal portion of the electroporation catheter taken along
lines A-A of FIG. 12 illustrating the electrodes, shaft and
balloon.
[0042] FIG. 15B is a partial longitudinal cross-sectional view of
the distal portion of the electroporation catheter taken along
lines A-A of FIG. 12 after 90 degree rotation from FIG. 15A.
[0043] FIG. 16 is a flowchart representing the steps of one method
of the invention.
[0044] FIG. 17A-E depicts the steps in the method of treatment of
restenosis within a vessel.
[0045] FIG. 18A-D illustrate other embodiments of the electrode
configurations of the electrode balloon catheter of the current
invention.
[0046] FIGS. 19-23 relate to Example 1.
[0047] FIG. 19 includes FIG. 19A which is a schematic drawing and
FIG. 19B which is a photograph of a custom made electrode clamp
employed to induce irreversible electroporation of the carotid
artery. FIG. 19A shows the clamp is comprised of two printed
circuit boards (1.5 mm thickness) with disk electrodes (diameter=5
mm) made of copper (70 microns thickness) plated with gold
(manufacturing process by Sierra Proto Express, Sunnyvale, Calif.,
USA). FIG. 19B shows a photograph where the clamp is used for
clamping the carotid artery, where the distance between electrodes
was approximately 0.3 mm
[0048] FIG. 20 shows a graph of examples of conductance of the
arterial wall during repetitive direct current pulses. Conductance
is measured only during the 100 microseconds pulses and here it is
displayed without the 100 ms intervals between pulses. Two cases
are shown: a trial in which successful irreversible electroporation
was achieved and a case in which the voltage pulses apparently were
not able to cause electroporation
[0049] FIG. 21A is an actual photograph of a right common carotid
artery. This slide is an example of the appearance of a normal
right carotid artery. (L--Intra-arterial lumen; TM--Tunica
media).
[0050] FIG. 21B is an actual photograph of a left common carotid
artery 28 days after intimal damage, showing high neointima to
media ratio. (L--Intra-arterial lumen; NI--Neointimal
Formation).
[0051] FIG. 21C is an actual photograph of a left common carotid
artery 28 days after intimal damage in an IRE treated rat, showing
the scarcity of neointimal formation compared with FIG. 21B
(L--Intra-arterial lumen; Arrow--minimal neointimal formation).
[0052] FIG. 22A is an actual photograph of a right common carotid
artery. This slide is an example of the appearance of a normal
control endothelial layer (L--Intra-arterial lumen; TM--Tunica
media; Arrow--Endothelial layer).
[0053] FIG. 22B is an actual photograph of a left common carotid
artery, 28 days after intimal damage and IRE. This slide shows the
overall preserved appearance of the endothelial layer.
(A--Intra-arterial lumen artifact; TM--Tunica media;
Arrow--Endothelial layer).
[0054] FIG. 22C is an actual photograph of a left common carotid
artery, 28 days after intimal damage. This slide shows the damaged
and irregular endothelial layer in the control group. (L
Intra-arterial lumen; NI--Neointimal Formation; Arrow--irregular
endothelial layer).
[0055] FIG. 23 is a Table showing results obtained on 8 animal
models.
[0056] FIGS. 24-32 relate to Example 2.
[0057] FIG. 24 is a Table showing the eight different
electroporation parameters used in this study. Groups differ in the
magnitude of the applied electric field, the number of the pulses
and their frequency. All pulses were square pulses, 100 .mu.s in
length. Frequency of 10 Hz was used for the 10-pulse protocols, and
was reduced to 4 Hz for 45 or 90 pulse protocols to prevent
significant heating.
[0058] FIG. 25 is a Table showing data of the four different
10-pulse protocols. All data are shown as average with standard
deviation, and include the percentage of IRE values compared with
control. Cell number is the average number of VSMC nuclei
identified in the Tunica Media. Concentration is the ratio between
the number of cells and the area of the Tunica Media (10.sup.-3
mm.sup.2). Area is the total area of the Tunica Media (10.sup.-1
mm.sup.2), and the thickness is the thickness of the Tunica Media
based on five different measurements in each section in
micrometers.
[0059] FIG. 26 is a Table showing data for the four different
protocols with more than 10 pulses. All data are shown in the same
manner as in FIG. 25.
[0060] FIG. 27 is a bar graph showing the ablation effect due to
different NTIRE protocols. The reduction in five of the groups was
statistically significant (P<0.001, bars marked with an
asterisk). Ablation effect is shown as the percentage of VSMC cells
in the treated artery compared with the right carotid artery of the
same animal.
[0061] FIG. 28 is an actual photograph showing complete ablation of
VSMC population one week following NTIRE with 90 pulses of 1,750
V/cm (right picture) compared with right carotid artery of the same
animal that was used as a control (left picture). Note the complete
absence of VSMC cells compared with notable repopulation of the
endothelial layer with endothelial cells.
[0062] FIG. 29 is a bar graph showing the effect on the sub-layer
of the Tunica Media. Inner most, middle and outer sub-layers are in
the first, second and third positions, left to right, respectively.
Ablation effect is shown as the percentage of VSMC cells in the
sub-layer compared with the same sub-layer in the right carotid
artery of the same animal. Note the relative sparing of the inner
most VSMC cells in all five groups, compared with the complete
ablation of VSMC in the outer layers with 1750 V/cm (second and
third groups in FIG. 29).
[0063] FIG. 30 shows three actual photographs taken at
magnification (.times.40) of the effect of NTIRE on blood vessels
after one week. Top picture shows a control artery, middle picture
shows a partial effect due to 45 pulses of 875 V/cm (Group 6),
lower picture shows a complete ablation of the arterial VSMC
population. In the case of the partial effect--all surviving VSMC
are located in the innermost layer of the Tunica Media. Also, note
in the lower picture the repopulation of the endothelial layer with
endothelial cells, compared with total absence of VSMC.
[0064] FIG. 31 is a graph showing the conductance change during
NTIRE application. X-axis shows the eight study groups. Y-axis
shows the change as the ratio between the conductance value
measured at the last electroporation pulse and the value at the
first pulse. Groups 3 and 4 (875.times.10 and 437.5.times.10,
respectively) show no change in conductivity, which correlates well
with the no ablation effect (see FIG. 28). Group 2 (1,750.times.10)
shows partial reduction in conductivity, correlating well with
minor ablation effect.
[0065] FIG. 32 shows six actual photographs of tissues subjected to
histology staining. Left column shows control arteries and right
column shows IRE-treated arteries. Top row--EVG stain showing
undamaged elastic fibers in IRE-treated arteries (elastic Van
Gieson, X40). Middle row--Masson Trichrome stain showing mild
fibrosis in the perivascular area with dominance of collagen fibers
in the Tunica Media of the IRE-treated Arteries (Masson Trichrome,
X40). Lower row--Negative staining of both arteries with CD34
antibodies at higher magnification (.times.60). Note the similar
morphology and distribution of the endothelial cells.
DETAILED DESCRIPTION OF THE INVENTION
[0066] Before the present method of treating restenosis and device
and system used for same are described, it is to be understood that
this invention is not limited to particular devices or method steps
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting, since the scope of the present invention will be limited
only by the appended claims.
[0067] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0068] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0069] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a target area" includes a plurality of such
target area and reference to "restenosis" includes reference to one
or more areas of restenosis and equivalents thereof known to those
skilled in the art, and so forth.
[0070] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Electroporation
[0071] Electroporation is defined as a phenomenon that makes cell
membranes permeable by exposing them to certain electric pulses. As
a function of the electrical parameters, electroporation pulses can
have two different effects on the permeability of the cell
membrane. The permeabilization of the cell membrane can be
reversible or irreversible as a function of the electrical
parameters used. Reversible electroporation is the process by which
the cellular membranes are made temporarily permeable. The cell
membrane will reseal a certain time after the pulses cease, and the
cell will survive. Reversible electroporation is most commonly used
for the introduction of therapeutic or genetic material into the
cell. Irreversible electroporation, also creates pores in the cell
membrane but these pores do not reseal, resulting in cell
death.
[0072] Irreversible electroporation has recently been discovered as
a viable alternative for the ablation of undesired tissue. See, in
particular, PCT Application No. PCT/US04/43477, filed Dec. 21,
2004. An important advantage of irreversible electroporation, as
described in the above reference application, is that the undesired
tissue can be destroyed without creating a thermal effect. When
tissue is ablated with thermal effects, not only are the cells
destroyed, but the elastin, collagen and other extra-cellular
matrix components (tissue scaffolding) of blood vessels are also
destroyed. This thermal mode of damage detrimentally affects the
tissue, that is, it destroys the vasculature structure and bile
ducts, and produces collateral damage.
[0073] Irreversible and reversible electroporation without thermal
effect to ablate tissue offers many advantages. One advantage is
that it does not result in thermal damage to target tissue or other
tissue surrounding the target tissue. Another advantage is that it
only ablates cells and does not damage blood vessel structure
itself. Accordingly, irreversible electroporation may be used to
treat the inner wall of a blood vessel during or immediately
following balloon angioplasty to prevent the re-growth of
endothelial cells.
[0074] Human arteries and veins are comprised of three layers; the
intima which is the thinnest and innermost layer; the media which
is the thickest and middle layer; and an outer adventitia layer
comprised of connective tissue. The medial layer is comprised
mainly of smooth muscle cells which play a prominent role in
re-stenosis of previously treated vessels. It is believed that in
reaction to the vessel wall trauma associated with balloon
angioplasty, the smooth muscle cells within the medial layer
proliferate causing a thickening of the overall vessel wall and
consequently, a reduction in the luminal diameter of the vessel.
This is also known as hyperplasia of the smooth muscle cells.
[0075] In another aspect of the invention, smooth muscle cells of
the vessel are selectively destroyed without damage to the
non-cellular tissue of the vessel. By selectively destroying smooth
muscle cells through irreversible electroporation, the
proliferation response of the vessel is suppressed. As irreversible
electroporation is a non-thermal treatment modality, adjacent
structures are not damaged by the electrical field. As an example,
the connective non-cellular tissue of the vessel (collagen, elastin
and other extra-cellular components) is not impacted by the
non-thermal electrical current. Instead, the treated vessel wall is
gradually repopulated with endothelial cells that regenerate over a
period of time but do proliferate or thicken into a stenotic
lesion.
[0076] In another aspect of the invention, the electroporation
catheter of the current invention may be used to treat native
stenotic lesions as well as stenoses or strictures of other bodily
organs. Target treatment areas may include claudication of
peripheral arteries, stenotic buildup in dialysis fistulas and
grafts, carotid artery stenosis and renal artery strictures as well
as venous lesions. Also within the scope of this invention are
non-vessel lumens including but not limited to biliary tract
blockages, bowel obstructions, gastric outflow strictures as well
as any other bodily lumen narrowing or occlusion.
[0077] Thus in one aspect of the invention, a method of treating
stenotic lesions is presented wherein an electrical field ablates
vessel wall cells to prevent re-growth of the lesion after
angioplasty or other treatment. By suppressing re-proliferation of
vessel wall cells, re-stenosis after angioplasty or stenting may be
prevented. In addition, the method described herein may be used in
lieu of drug-eluting stents which have demonstrated only limited
success in preventing stent re-stenosis. In yet another aspect of
the invention, the electrical parameters may be set to create an
electrical field that temporarily or reversibly electroporate
cellular structures. The smooth muscle cells comprising the target
lesion will temporarily permiablize, allowing the transport of a
drug into the intracellular structure. Drugs may include
anti-stenotic agents that may further prevent smooth cell
proliferation or cytotoxic drugs, such as a chemotherapy agent if
the stricture is caused by a cancerous growth.
Specific Embodiments
[0078] There are a range of different catheter device type
configurations which can be used in connection with the present
invention. Some examples of devices which could be modified to
obtain the basic objects of the invention include the balloon
catheter device of U.S. Pat. No. 7,150,723 teaching a medical
device including guidewire and balloon catheter for curing a
coronary artery. Another catheter device which might be modified to
utilize the aspects of the invention is the device of U.S. Pat. No.
7,273,487 disclosing a balloon catheter having a multi-layered
shaft with variable flexibility. Still another balloon catheter
device is taught within U.S. Pat. No. 7,351,214 disclosing a
steerable balloon catheter. Yet another device is taught within
U.S. Pat. No. 7,481,800 disclosing a triple lumen stone balloon
catheter and method. The present invention is not specific to any
of these embodiments and other embodiments can be used to provide
various catheter configurations which include first and second
electrodes connected to a power source which provides to the
electrodes a sufficient amount of electrical energy to carry out
irreversible electroporation on substantially all of the cells in
the vessel target area without subjecting the target area or
surrounding area to thermal damage.
[0079] Others have endeavored to develop devices and methods for
preventing restenosis. The present invention can be used by itself.
However, it is also contemplated to utilize the device and methods
of the present invention in combination with other methods for
reducing restenosis. A possible example includes the device and
method disclosed within U.S. Pat. No. 5,947,889 which discloses a
balloon catheter to prevent restenosis after angioplasty and
process for producing a balloon catheter.
[0080] Those skilled in the art will understand that these specific
examples provided here are carried out in order to demonstrate the
utility of the present invention and that modification of the
devices and methodology may be carried out in order to obtain
specific preferred embodiments which are intended to be within the
scope of the present invention. An example of a specific embodiment
is provided below.
[0081] Restenosis following coronary angioplasty represents a major
clinical problem. Irreversible Electroporation (IRE) is a
non-thermal, non-pharmacological cell ablation method. IRE utilizes
a sequence of electrical pulses that produce permanent damage to
tissue within a few seconds. Examples provided here show that the
left carotid arteries of 8 rats underwent in vivo intimal damage
using 2 Fogarty angioplasty catheters. The procedure was
immediately followed by IRE ablation in 4 rats, while the remaining
4 were used as the control group. The IRE ablation was performed
using a sequence of 10 direct current pulses of 3800 V/cm, 100
.mu.s each, at a frequency of 10 pulses per second, applied across
the blood vessel between two parallel electrodes. The electrical
conductance of the treated tissue was measured during the
electroporation to provide real time feedback of the process. Left
carotid arteries were excised and fixated after a 28-day follow-up
period. Neointimal formation was evaluated histologically. The use
of IRE was successful in 3 out of 4 animals in a way that is
consistent with the measurements of blood vessel electrical
properties. The integrity of the endothelial layer was recovered in
the IRE-treated animals, compared with control. Successful IRE
reduced neointima to media ratio (0.57.+-.0.4 vs. 1.88.+-.1.0,
P=0.02). The present invention shows that the in vivo results of
attenuation of neointimal formation using IRE. The invention
provides a method which uses IRE to attenuate neointimal formation
after angioplasty damage in a mammal such as a human and provides a
method of treating coronary artery restenosis after balloon
angioplasty.
Balloon Catheter Embodiments
[0082] FIG. 1A illustrates a plan view of an embodiment of the
electroporation balloon catheter 10 of the current invention.
Catheter 10 is comprised of a hub 13, a flexible catheter shaft 15
extending distally from hub 13 to an expandable member such as a
balloon 19 and terminating at catheter distal tip 17. Hub 13
includes a port opening 21 in communication with a shaft lumen (not
shown) for the injection and aspiration of fluid to inflate and
deflate the balloon 19 during use. Shaft 15 extends from hub 13
distally through the interior of balloon 19 before terminating in
distal tip 17. Balloon 19 is coaxially arranged around catheter
shaft 15 near the distal end and is shown in an expanded state.
Although not shown in FIG. 1A, catheter 10 may also include a
side-arm extension on hub 13 with an opening to allow the insertion
of a guidewire to facilitate tracking through the vessel. Extending
from hub 13 are electrical cable wires 9 which terminate in
connectors 11. Connectors 11 are connected to an electrical
generator or electrical power source (not shown) to provide an
electrical current to a first and second longitudinal electrodes 25
and 27, which are positioned in a longitudinal arrangement around
the outer surface of balloon 19.
[0083] Catheter shaft 15 construction is illustrated in more detail
in FIG. 1B, which includes the exploded partial plan view from "A"
of FIG. 1A and an enlarged cross-sectional view of the shaft 15
taken along lines B-B of FIG. 1A. As shown in Detail "A" and
Section B-B, shaft 15 is comprised of an electrically conductive
tubing 110 with a through lumen 41, an insulating layer 112
coaxially surrounding tubing 110 and an outer insulative layer 116
coaxially surrounding the inner insulating layer 112. Wedged
between the inner and outer insulating layers 112 and 116, is
positioned an electrically conducting wire 114. Inner electrically
conductive tubing is preferably comprised of a flexible nitinol
shaft or other electrically conductive material to provide a
pathway for the electrical current from the electrical generator to
the distal end portion of the catheter when in use. It may be
dimensioned with appropriate inner diameter and outer diameter.
Both the inner and outer insulating layers 112 and 116 are thin
layers of appropriate thickness and are made of a non-conductive
material such as nylon, polyamide, PET or other plastic
material.
[0084] The electrically conductive wire 114 is comprised of an
electrically conductive material such as nitinol or copper and may
be dimensioned at 0.004'' thick by 0.015'' wide. The inner and
outer insulative layers which block electrical current ensure that
the electrical current path of the inner electrically conductive
tubing 110 remains isolated from the current path of the
electrically conductive wire 114. Although not shown in FIG. 1B, in
one embodiment, the catheter 10 may include a separate lumen for
insertion of a guidewire to assist in advancing the catheter to the
target site.
[0085] FIG. 2 depicts an enlarged plan view of the distal portion
of the preferred embodiment of the electroporation balloon catheter
of FIG. 1A in an inflated state. Balloon 19 is coaxially arranged
around catheter shaft 15 near the distal end of the shaft. Expanded
balloon 19 includes a balloon body 35 portion of constant
cross-sectional diameter, proximal 32 and distal 34 cone portions
which taper inwardly away from the balloon body 35, and proximal 37
and distal 39 neck portions of reduced diameter relative to the
balloon body 35. Neck portions 37 and 39 are bonded to the outer
surface of the catheter shaft 15 using adhesive or other bonding
methods known in the art. Also illustrated is electrode assembly
26, which is comprised of first and second longitudinal electrodes
25 and 27. The two longitudinal electrodes overlap to form a cage
with a series of legs arranged to be in contact with the surface of
the balloon when inflated.
[0086] Referring to FIGS. 3A and 3B, first and second longitudinal
electrodes 25 and 27 are shown prior to assembly with the balloon.
The electrodes are comprised of suitable electrically conductive
material including but not limited stainless steel, gold, silver
and other metals including shape-memory materials such as nitinol.
Nitinol is an alloy with super-elastic characteristics which
enables it to return to a pre-determined expanded shape upon
release from a constrained position.
[0087] FIG. 3A which depicts the unassembled first longitudinal
electrode 25 which includes a proximal collar 40 with lumen 49
through which is located catheter shaft 15 when assembled. Flexible
first and second electrode legs 44 and 42 extend from collar 40 in
a distal direction to leg end portions 48 and 46 respectively. The
legs 44 and 42 take on the general profile of the expanded balloon
shape as can be seen by tapered portions 51, 52, 53 and 54 which
correspond with the proximal and distal balloon cone sections 32
and 34. A plurality of electrically insulative elements 105 and 107
covers portions of the first longitudinal electrode 25. The
electrically insulating elements 105 and 107 may be of a PET,
polyamide or other similar material in the form a tubular structure
or heat-shrinkable material. As shown in FIG. 3A, insulative
element 105 covers the proximal collar 40 and proximal tapering
portions 51 and 52 of electrode legs 44 and 42. The insulating
element 105 terminates at a point on the electrode legs 44 and 42
that correspond with the junction of the proximal balloon cone and
body (reference FIGS. 2A and 2B). In a similar manner, insulative
element 107 covers the distal portions of legs 44 and 42 and extend
proximally from distal leg ends 48 and 46, taper portions 53 and 54
to a point on the legs 44 and 42 that correspond with the junction
of the distal balloon cone and balloon body. The arrangement of the
insulative elements over the legs 44 and 42 create an active
electrode portion 45 and 47 through which electrical current will
freely pass.
[0088] FIG. 3B illustrates the unassembled second longitudinal
electrode 27, which is comprised of a distal collar 55 including
lumen 65 through which the distal portion of catheter shaft 15 is
positioned when assembled. Extending proximally from collar 55 are
a first electrode leg 57 and second electrode leg 59 which
terminate in proximal leg ends 61 and 63 respectively. Electrode
legs 57 and 59 take on the general profile of the expanded balloon
shape as can be seen by proximal tapered portions 69 and 70 which
correspond with the proximal balloon cone section 32. Distal
tapered portions 67 and 68 correspond with the distal balloon cone
section 34. Proximal and distal outer insulative layers 101 and 103
cover portions of longitudinal electrode 27. As shown in FIG. 3B,
distal insulative layer 103 covers the distal collar 55 and tapered
portions 67 and 68. Distal insulative layer 103 terminates at a
point on the legs 57 and 59 which correspond with the junction of
the distal balloon cone and balloon body (reference FIGS. 2A and
2B). In a similar manner, proximal outer insulative layer 101
covers the proximal portions of legs 57 and 59 and extends from
proximal leg ends 61 and 63, tapered portions 69 and 70 to a point
on the legs 57 and 59 that correspond with the junction of the
proximal balloon cone and balloon body. The straight, un-insulated
portion of legs 57 and 59 are the active electrode portions 77 and
79. While the insulated portions of electrode 27 will not conduct
electrical current, the active electrode portions 77 and 79 of
longitudinal electrode 27 will generate a therapeutic electrical
field when electrical energy from the electrical generator is
applied to the assembly.
[0089] When assembled, first and second longitudinal electrodes 25
and 27 are in an overlapping arrangement relative to each other as
shown in FIG. 4A, which illustrates the electrode assembly 26 with
second longitudinal electrode 27 rotated 90 degrees clockwise from
the view in FIG. 3B. Second longitudinal electrode 27 is positioned
in an overlapping relationship with first longitudinal electrode 25
such that collar 55 of second electrode 27 extends distally beyond
the leg ends 48 and 46 of first electrode 25. In a similar manner,
collar 40 of first electrode 25 extends proximally of leg ends 61
and 63 of second longitudinal electrode 27. When assembled
together, the active electrode portions 45, 47, 77 and 79 of both
electrodes 25 and 27 are in alignment with each other relative to
the longitudinal axis of the electrode assembly 26 and together
form an active electrode region 115.
[0090] The electrode assembly 26 of FIG. 4A is held together as a
single unit at the proximal 40 and distal 55 collars. Specifically,
distal portions of legs 44 and 42 of electrode 25 are immovably
attached to the outer surface of collar 55 of second longitudinal
electrode 27. Conversely, proximal sections of legs 57 and 59 of
electrode 27 are immovably attached to the outer surface of collar
40 of first longitudinal electrode 25. Various methods known in the
art such as welding, bonding, or application of adhesive may be
used to form the attachment between the collars and legs.
[0091] FIG. 4B illustrate end views of the electrode assembly 26
taken from line A-A and B-B of FIG. 4A. Referring first to Detail
A-A, proximal collar 40 is shown with through lumen 49 and
surrounding outer insulative layer 105. Outwardly tapering sections
51 and 52 with insulative layer 105 of first longitudinal electrode
25 are shown extending distally from collar 40 to reach a larger
constant diameter of legs 44 and 42. The proximal most ends 61 and
63 of second electrode 27 are shown bonded to the outer surface of
collar 40/insulative layer 105 at weld joints 73 and 75
respectively. Outwardly tapering sections 69 and 70 extend
horizontally and distally from collar 40 to reach a larger constant
diameter of legs 57 and 59 respectively. A partial view of distal
collar 55 of second longitudinal electrode 27 with insulative layer
103 is also shown.
[0092] Detail B-B of FIG. 4A illustrates an end view of electrode
assembly 26 taken along line B-B in FIG. 4A. Distal collar 55 is
shown with through lumen 65 and outer insulative layer 103. A
partial view of proximal collar 40, which is of a smaller diameter
than distal collar 55, is shown within lumen 65. Outwardly tapering
sections 67 and 68 of conducting element 27 are shown extending
from collar 55 to reach a larger constant diameter of legs 57 and
59. The distal ends 48 and 46 of first longitudinal electrode 25
are shown bonded to the collar 55 of weld joints 62 and 64
respectively. Other attachment methods may be used to create joints
62 and 64, as is known in the art. Outwardly tapering sections 53
and 54 of electrode 25 are shown extending vertically and
proximally from collar 55 to reach a larger constant diameter of
legs 44 and 42 respectively.
[0093] In operation, first and second longitudinal electrodes 25
and 27 each may carry an opposite polarity electrical charge. For
example, first electrode 25 may carry a negative electrical charge
and second electrode 27 may carry a positive electrical charge. As
a result of this arrangement, an electrical field is created
between active electrode zones 115 of the first and second
electrodes 25 and 27 which are of opposite polarity. For example an
electrical current may be created between positively charged leg 57
of second electrode 27 and negatively charged leg 44 of first
electrode 25. In the same manner, an electrical current may be
created between legs 44 and 59, between legs 59 and 42, and between
legs 42 and 57.
[0094] As will be explained in more detail below, the resulting
electrical field created by the application of electrical energy of
opposite polarities to the legs of the first and second creates a
substantially 360 degree electrical field zone surrounding the
balloon, which when inflated is in contact with the inner wall of
the vessel. Consequentially, the entire circumference of the inner
wall of the target vessel is subject to a therapeutic electrical
field. The electrical field is restricted to the active electrode
portions 45, 47, 77, and 79 of the longitudinal electrodes 25 and
27 because these portions are not insulated. As previously
mentioned, the un-insulated portions of electrodes 25 and 27
correspond to the constant diameter body portion of the balloon.
Those portions of electrodes 25 and 27 that correspond to the
proximal and distal balloon cones 32, 34 and necks 37, 39 are
insulated and accordingly will not generate and electrical field.
Only the vessel wall is treated; any blood present within the
vessel lumen is not impacted by the treatment as no electrical
field is generated from the insulated portions of the device.
[0095] FIGS. 5A and 5B illustrate details of the two electrical
pathways of electrical current to the active electrode zone 115
(reference FIG. 4A). FIG. 5A illustrates an enlarged
cross-sectional partial view of proximal section electrode assembly
26 showing the electrical connection between the wire 114 and the
second electrode 27, which in one embodiment may be a positive
electrical pathway. Catheter shaft lumen 41 is formed by
electrically conductive shaft tubing 110, which includes an outer
insulative layer 112, to which collar 40 and proximal balloon neck
37 are attached. Positioned on the surface of insulated shaft 110
is electrically conductive wire 114, which is positioned between
insulative layer 112 of the shaft and outer insulative layer 116 to
ensure isolation of the positive and negative electrical pathways.
At the junction of the shaft 110 and collar 40, electrically
conductive wire 114 extends outwardly and distally over collar 40
and over electrode leg 57. The wire is positioned between
electrically insulative layer 101 of electrode leg 57 and an
outermost insulating layer 116. Layer 116, which extend from hub 13
of catheter 10 (reference FIG. 1A) and over the distal portion of
electrode assembly 26, not only ensures that wire 114 is
electrically insulated from conductive shaft tubing 110, but also
serves to encompasses the various individual component pieces
comprising the distal portion of assembly 26.
[0096] As shown in FIG. 5A, insulation layer 101 has been removed
from the distal portion of leg 57 so as to allow for a direct
electrical connection between wire 114 and leg 57 of electrode 27.
Wire 114 is attached directly to leg 57 by bonding, welding,
soldering or other known means at attachment zone 141. Thus, in one
embodiment, the positive polarity electrical current is passed from
the generator to the electroporation device 10, electrical energy
is transmitted through the shaft by way of the wire 114, to the leg
57 at the un-insulated attachment zone 141 and to the remaining
portions of second electrode 27 (reference FIG. 3B). In this
manner, the active electrode portions 77 and 79 of legs 57 and 59
(reference FIG. 4A) of longitudinal electrode 27 is energized with
positive polarity.
[0097] FIG. 5B depicts the proximal section details of the
electroporation catheter 10 rotated longitudinally 90 degrees from
the FIG. 5A orientation. This cross-sectional view illustrates the
second electrical pathway, which in one embodiment is a negative
polarity current. Negative electrical current originating from an
electrical generator is transmitted through the electrically
conductive shaft tubing 110 to collar 40 of electrically conductive
element 25. Although shaft 110 includes outer insulative layer 112
for the majority of the shaft length, the layer 112 is removed
allowing for direct attachment between shaft 110 and collar 40 at
region 111. Region 111 may include a weld bond. The resulting
contact region 111 creates an electrical current pathway between
the generator and longitudinal electrode 25. Electrical energy will
be transmitted through shaft tubing 110 to the collar 40 at contact
region 111 and to active electrode regions 45 and 47 of electrode
legs 42 and 44. Thus, with this overlapping arrangement of
electrodes 25 and 27, electrical energy of opposite polarities is
transmitted to the electrically active portion of each pairs of
longitudinal legs.
[0098] The distal portion of electrode assembly 26 is illustrated
in two enlarged, cross-sectional partial views of FIG. 6. The
second enlarged view is shown at a 90 degree longitudinal rotation
from the first view. Electrically conductive shaft 110 is shown
with outer insulative layer 112 which extends through the electrode
assembly 26 to a distal tip end 17. Coaxially arranged around the
insulated shaft 110/112 is the distal neck 39 of balloon 19. Collar
55 of longitudinal electrode 27 also coaxially surrounds shaft 110.
Collar 55 is proximate to but not in contact with insulated shaft
110/112. With a catheter shaft of appropriate outer diameter and
the collar 40 lumen 65 of appropriate diameter, an annular gap 66
can be made to exist between the two components. Annular gap 66
extends from collar 55 proximal edge 104 to collar distal edge 108.
The purpose of gap 66 is to allow electrode assembly 26 to slide
freely over shaft 110 as it foreshortens and lengthens during
balloon expansion and deflation, as will be explained in more
detail with reference to FIG. 8. Also shown in the first view of
FIG. 6 are inwardly bowing leg portions 53 and 54 of legs 44 and
42. Insulated legs 44 and 42 are attached to insulated collar 55 at
attachment regions 81 and 83 respectively. Adhesive or other known
attachment mechanisms may be used to form the connection. Due to
the insulative layers 103 of collar 55 and insulative layer 103 of
legs 44 and 42, attachment regions 81 and 83 are not electrically
conductive and no direct electrical current pathway exists between
collar 55 and electrode assembly 25.
[0099] FIG. 7 illustrates end views of the balloon 19/electrode
assembly 26 taken along lines C-C and D-D of FIG. 1A. Referring
first to Section C-C, proximal balloon cone 32 of balloon 19 is
shown in an inflated position. Electrode legs 42 extend in a
vertical over the surface of balloon 19. Electrically conductive
shaft tubing 110 surrounds shaft lumen 41. Collar 40 is in
electrical connection with shaft 110 by weld region 111. Collar 40
is coaxially surrounded by electrode assembly insulative layer 101
which electrically isolates collar 40 from wire 114. Outer
insulative layer 116 coaxially surrounds the shaft assembly and
electrically conductive wire 114. Also shown is an end view of
insulative layer 116 as it extends over leg ends 61 and 63.
[0100] Detail D-D of FIG. 7 depicts an end view of the catheter
assembly taken along lines D-D of FIG. 1A. The distal tip 17 of
shaft 19 is positioned within lumen 65 of collar 55 which is also
shown as annular gap 66. Electrode legs 57 and 59 extend from
collar 55 in a horizontal direction over the surface of balloon 19.
Ends 48 and 46 of electrode legs 44 respectively are attached to
collar 55 at weld joints 64 and 62.
[0101] FIG. 8A-B illustrates the longitudinal movement of electrode
assembly 26 relative to the catheter shaft 19 during use. FIG. 8A
depicts catheter 10 with balloon 19 inflated. Assembly 26 is
positioned in a surrounding relationship over inflated balloon 35.
Legs 44 and 47 are shown with proximal taper portions 51 and 52
bowing radially outward and away from shaft 15. Distal taper
portions 53 and 54 are also in an expanded state. In one embodiment
the electrode assembly 26 is comprised of shape memory material
such as nitinol which returns to a pre-determined shape upon
release. As such, assembly 26 may retain its expanded profile as
shown in 8A even if the balloon is not inflated. When in its
natural, unconstrained state, the distal edge 108 of collar 55 is
positioned a distance L1 from the catheter distal tip 17.
[0102] In a constrained state, as shown in FIG. 8B, electrode
assembly 26 is in a collapsed position around shaft 15 and balloon
19. Legs 44, 47, 57 and 59 (not visible) become linear in profile
with all tapering portions 51, 52, 53 and 54 flattening out so that
they are positioned parallel with the shaft. When the assembly is
collapsed, collar 55 slides in a distal direction. Movement of the
electrode assembly 26 relative to the shaft 19 and balloon 25
occurs because the distal portion assembly 26 is not attached to
shaft 15 at collar 55. When electrode assembly 26 is completely
collapsed, the leading edge 108 of collar 55 is positioned a
distance L2 away from catheter distal tip 17. Total distal movement
of the assembly collar 55 is for a length of L1-L2. More
specifically, catheter 10 is typically inserted into a target
vessel through a sheath or other introducer device.
[0103] FIG. 9 illustrates the electrical current flow pattern from
an end view of the electroporation catheter device 10. Electrical
energy will be transmitted from an electrical generator through
shaft tubing 110 to electrode legs 42 and 44 of electrode assembly
25 as previously described. In one embodiment, this electrical
pathway is of a positive polarity as indicated by the "+" signs in
FIG. 9. Electrical energy of a negative polarity may be transmitted
through wire 114 to longitudinal electrode assembly 27 through wire
114 to leg 59 connection 111. In one embodiment, this second
electrical pathway is of a negative polarity as indicated by the
"-" signs in FIG. 9. The electrodes can be electrically energized
one pair at a time and selectively switched to cover all four
pairs. In the embodiment shown, all electrodes are simultaneously
energized, causing electrical current to flow from positive
polarity legs to negative polarity legs. As an example, electrical
current will flow from leg 44 with a positive polarity to leg 59
with a negative polarity, creating an electrical field zone 168.
Electrical current from leg 59 will also flow to negative polarity
leg 57, creating an electrical field zone 174. In a similar manner,
positive polarity leg 42 will transmit electrical current to both
negative polarity legs 57 and 59, creating electrical fields 172
and 170. Although not shown in FIG. 9, the flow of electrical
current will be restricted to the un-insulated portions of the
electrode legs, which correspond with maximum diameter of the
inflated balloon 19. The resulting combined electrical fields
created by the application of electrical energy of opposite
polarities to the electrode legs 44, 59, 42 and 57 create a
substantially 360 degree electrical field zone surrounding the
balloon body 35. When the catheter is in position in a target
vessel, this combined electrical field zone extends radially
outward and into the inner wall of the vessel. In this manner, the
entire circumference of the inner wall of the target vessel is
subject to a therapeutic electrical field.
[0104] An alternative embodiment of the electroporation balloon
catheter of the present invention with a balloon and electrode
assembly is shown in a plan view in FIG. 10. Catheter 121 is
comprised of a hub 13, a catheter shaft 119 extending distally to a
balloon 19 and terminating at a catheter open distal end 139, which
is sized to pass a guidewire. Balloon 19 is coaxially arranged
around catheter shaft 119 near the distal end and is shown in an
expanded state. Hub 3 may include port opening 13 and side-arm
extension opening 12 to allow the insertion of a guidewire and
injection of fluid to inflate the balloon 19. Extending from hub 3
are electrical cable wires 9 which terminate in connectors 11.
Connectors 11 are connected to an electrical generator (not shown)
to provide an electrical current to the spiral electrode assembly
123 which is coaxially arrangement over balloon 19.
[0105] FIG. 11 represents a cross-sectional view of the catheter
shaft 119 taken along lines A-A of FIG. 10. Shaft 119, which in one
embodiment has a sufficient outer diameter so that it may include
four lumens. Guidewire lumen 125 may be approximately 0.039'' in
diameter and in communication with side arm extension opening 1 to
accept a standard guidewire which is used for tracking device 121
to the target location within a vessel. Inflation/deflation lumen
131 is in communication with hub opening 21 for injection and
withdrawal of fluid to and from the balloon 35 interior. First and
second electrode wire lumens 127 and 129 are configured to contain
electrode wires 133 and 135 respectively. Wires 133 and 135 are
electrically insulated from each other by inner shaft septum 137,
which is comprised of a plastic or other non-conductive
material.
[0106] FIG. 12 illustrates an enlarged plan view of the distal end
section of the electroporation balloon catheter 121 which is shown
inflated. Catheter shaft 119 extends into and through balloon 35,
terminating at open distal tip 139. Helically surrounding the
balloon surface is spiral electrode assembly 123, which is
comprised of a first and second spiral electrode 85 and 87. In one
embodiment, first spiral electrode element 85 is of a positive
polarity and second electrode 87 is of a negative polarity. Spiral
electrode 85 is connected to first electrode wire 133 and spiral
electrode 87 is connected to second electrode wire 135.
[0107] Details of the spiral electrodes are shown in FIG. 13. First
spiral electrode 85, shown in FIG. 13A, is comprised of a distal
collar 89 containing a lumen 144, a spiral shaped body 91 of an
expanded diameter relative to the collar 89 and a proximal tail 93.
A second spiral electrode 87 is shown in FIG. 13B. Second spiral
electrode 87 is comprised of a proximal collar 95 with through
lumen 146, a spiral shaped body 97 of an expanded diameter relative
to collar 95, and a distal tail 99. The spiral shaped bodies 91 and
97 are formed of a series of helical turns 148. The two spiral
electrodes 85 and 87 may be assembled together in an overlapping
relationship on the same longitudinal axis to form a double helix
was shown in FIG. 13C. The plurality of helical turns 148 of the
first spiral electrode 85 are positioned adjacent to corresponding
helical turns 148 of the second spiral electrode 87.
[0108] Electrode 85 and 87 may be comprised of any conductive
material known in the art. For example, the electrodes may be
formed of a nitinol tube which has been laser cut and memory set to
the desired spiral profile. Alternatively, electrodes 85 and 87 may
be comprised of a conductive ink which is applied in the desired
pattern to the exterior balloon surface. As an example, the
conductive ink may be comprised of an adhesive binder material
loaded with silver particles. Other conductive materials such as
gold or steel may also be used. In one embodiment, the conductive
ink may be applied in the desired pattern to the balloon surface
using a pen-like applicator and a rotating lathe type fixture. The
application of conductive ink may be between 0.001'' and 0.002'' in
thickness.
[0109] FIG. 15A illustrates an enlarged cross-sectional view of the
spiral electrode assembly 123 positioned on the outer surface of
the balloon. The cross-sectional view is taken along lines A-A of
FIG. 12. Shaft 119 is includes a guidewire lumen 125 and an
inflation lumen 131. The guidewire lumen 125 extends from catheter
hub 3 through shaft 119 exiting at open distal end 139. The
inflation lumen 131 also extends from catheter hub 13 (reference
FIG. 10) but terminates within balloon interior 23 at inflation
lumen dead end 29. In operation, a fluid or other inflation medium
is injected into hub 13 through hub opening 21, directed distally
within inflation lumen 131 and into the balloon interior 23 through
inflation lumen side port 31. This fluid path is also used for
withdrawing fluid to deflate the balloon prior to withdrawal of the
device from the target vessel.
[0110] First spiral electrode 85 is positioned over balloon 19 with
distal electrode collar 89 coaxially surrounding distal balloon
neck 39. In one embodiment, first spiral electrode 85 is of a
positive polarity. Extending proximally from electrode collar 89,
electrode section 91a partially surrounds balloon cone 34 and
comprises the beginning of the first helical turn. The spiral
electrode 85 pattern continues proximally along the surface of
balloon body 35, as illustrated by electrode cross-sections 91b,
91c, 91d, 91e and 91f. The proximal tail 93 of the first electrode
spiral is not visible, but is positioned adjacent to proximal
balloon cone 32. A second or negative spiral electrode 87 includes
a proximal collar 95 that coaxially surrounds balloon proximal neck
37. Extending distally from collar 95, electrode section 97a
partially surrounds proximal balloon cone 32 and comprises the
beginning of the spiral pattern. The negative electrode spiral
pattern continues proximally along the surface of the balloon body
35, as illustrated by electrode cross-sections 97b, 97c, 97d, 97e
and 97f. The distal tail 99 of negative spiral electrode is not
visible, but is positioned adjacent to the balloon cone 34.
[0111] The resulting surface pattern of the combined spiral
electrodes 85 and 87 is a double helix configuration with
alternating polarity electrodes positioned along the balloon
surface. When electrical current of opposite polarity is applied to
electrodes 85 and 87, an electrical field is generated between the
positive spiral electrode 85 and the negative spiral electrode.
With the electrical current flowing from positive to negative, an
electrical field is created between each set of helical turns. As
an example, electrical current will flow from 91a to 97f, from 91b
to 97e and from 91c to 97d. The effect of this electrical field
pattern is that the majority of the balloon surface is within the
active electrical field. This is advantageous in that since the
electrical field encompasses substantially the entire balloon
surface, the resulting ablation zone will uniformly encompass the
vessel wall area corresponding to the balloon surface.
[0112] FIG. 15B illustrates an enlarged cross-sectional view of the
spiral electrode assembly 123 positioned on the outer surface of
the balloon 35 and represent a 90 degree rotation of the
cross-sectional view of FIG. 15A. This shows the electrically
conductive wires 133 and 135 which are not visible in the FIG. 15A
cross-section. Electrically conductive wires 133 and 135 originate
within cable connectors 11 and extend distally within cable lines 9
and enter shaft lumens 127 and 129 through hub 3. Both wires are
comprised of an electrically conductive material such as silver,
gold, copper or other metal. In one embodiment, wires 133 and 135
have a flat profile with dimensions of approximately 0.004'' thick
and 0.015'' wide. In another embodiment the wires may be circular
in cross-sectional diameter. An insulative outer layer may
surrounds the bare wire. The insulative layer may be of a plastic
material such as nylon.
[0113] Each wire has a dedicated lumen within shaft 119, as shown
in FIG. 15B. Electrically conductive wire 133 is coaxially arranged
within lumen 127 and. Electrically conductive wire 135 is coaxially
arranged with lumen 129. The separate, dedicated wire lumens 127
and 129 provide additional assurance that the electrically
conductive wire 133 and 135 which are of opposite polarity when
charged, are electrically isolated from each other for the entire
length of the catheter shaft. Both lumens 127 and 129 terminate
within shaft 119 body forming closed ended lumens so that the
electrically conductive wire elements 53 and 55 and lumens 65 and
66 are not exposed to air or bodily fluids which may compromise the
electrical field.
[0114] Electrically conductive wires 127 and 129 are in connection
with spiral electrodes 85 and 87 at electrode collars 95 and 89
respectively. Electrode wire 133 exits electrode wire lumen 127 at
side port exit 150 just proximal to electrode collar 95. Upon exit,
electrically conductive wire 127 is attached to the outer surface
of collar 95, whereby completing the electrical pathway between the
electrical generator and the spiral electrode 123. Wire 133 is
attached to the spiral electrode collar 95 using either a
conductive epoxy or other attachment method known in the art. In a
similar manner electrode wire 135 exits wire lumen 129 at a side
port exit 152 just distal to the electrode collar 89. Upon exit,
electrically conductive wire 129 is attached to the outer surface
of collar 89, whereby establishing the electrical pathway to the
spiral electrode 85. In an alternative embodiment, electrically
conductive wire 135 may exit wire lumen 129 within the balloon
interior 45. In this embodiment, the insulated wire 135 is
sandwiched between the distal balloon neck 39 and the outer surface
of catheter shaft 119, exiting from the distal end of balloon neck
39 to make contact with electrically conductive collar 89.
[0115] Other embodiments of the electroporation catheter of the
current invention are shown in FIG. 18A-D. FIG. 18A illustrates an
alternative embodiment of the balloon electrode assembly 401
comprising two electrodes 403 and 405 of opposite polarity.
Proximal electrode 403 covers the proximal neck, cone and a portion
of the body of the balloon 407 and in one embodiment is comprised
of a conductive ink coating. Other metallic materials may be used
to manufacture the electrodes. Distal electrode 405 covers the
distal neck, cone and a portion of the balloon 407 body. The
exposed balloon body portion 409 represents the active electrode
zone. In one embodiment, the electrode material is selected so as
to be ultrasonically or fluoroscopically visible allowing the user
to position the active electrode zone 409 within the lesion based
on the location of the proximal electrode leading edge 411 and the
distal electrode tailing edge 413. In a separate embodiment, shown
in FIG. 18B, balloon electrode assembly 421 may include two or more
electrode rings 423 and 425 of opposite polarity. Rings 423 and 425
may be attached to the outer or inner surface of balloon 427, or
may be embedded within the balloon wall. This embodiment, with the
smaller active electrode zone 429, may be advantageous when
treating smaller length lesions. The embodiments of both FIG. 18A
and FIG. 18B are also advantageous in that these designs are easy
to manufacture.
[0116] In yet another embodiment of the invention the balloon
electrode assembly 431 may be comprised of a mesh or woven layer
which includes electrodes as shown in FIG. 18C. Balloon electrode
assembly includes helically wound strands 433, 435 and 429 covering
the surface of balloon 437. Electrode strands 433 and 435
circumferentially surround balloon 437 and are positioned adjacent
to and parallel to each other at an angle of approximately 65
degrees relative to the longitudinal axis of the balloon. Strands
433 and 435 may be of a conductive material such as metallic wire.
Strand 433 may be of a positive polarity and strand 435 may be
negative. Strand 439 is positioned at an opposite angle to strands
433 and 435 and runs circumferentially in a helical pattern around
the balloon surface. Stand 439 may be comprised of a non-conductive
material including high strength polyester, nylon or other material
so as to increase the overall strength of the balloon. In
operation, electrical current will flow from the positively strand
433 to adjacent negatively charged strand 435 creating a tubular
ablation zone extending radially outward from the balloon body.
[0117] FIG. 18D illustrates an embodiment of an electroporation
balloon catheter 441 in which a series of opposite polarity prongs
447 and 449 are present in an alternating pattern across the
balloon body 451. Electrode 443 is comprised of a plurality of
distally extending prongs 447 which may be of a positive polarity.
Interspaced between each positive polarity prong 447 are negatively
charged proximally extending prongs 449 of electrode 445. The
alternating positive and negative polarity electrode prongs create
a ablation zone extending radially outward from the balloon
body.
[0118] The method of using the electroporation balloon catheter of
the current invention to prevent re-stenosis of a vessel will now
be described with reference to FIGS. 16 and 17A-E. To begin the
procedure, access is gained to the vessel using techniques known in
the art such as the Seldinger needle/guidewire access technique as
shown in step 201 of FIG. 16. The electroporation balloon catheter
is inserted into the vessel and advanced to the target lesion
(203). In the various embodiments of the catheter previously
described, the catheter may be inserted directly through an
introducer sheath or may be inserted and advanced over a guidewire
to the target vessel lesion. For non-vascular lumens, a direct
percutaneous stick or cut down procedure maybe used to access the
target area such as a biliary duct.
[0119] FIG. 17A illustrates electrode catheter 10 of FIG. 1A in
position within the lumen 503 of a vessel 501 to be treated prior
to angioplasty. Vessel 501 comprises vessel wall 507 and
endothelial inner layer 509. Also shown is the stenotic region 505,
which extends into the vessel lumen narrowing the luminal diameter
in this area. Electrode balloon catheter device 10 in shown in an
unexpanded state within the vessel 507 lumen 503 with the electrode
assembly 26 positioned adjacent to the stenotic region 505.
Although the longitudinal electrode assembly embodiment 10 of FIG.
1 is shown, any of the described catheter embodiments may used with
this method.
[0120] Once correctly positioned within the anatomical lumen,
electrical connectors 11 of the catheter 10 are connected to an
electrical generator (205). This completes an electrical circuit
between the electrodes and the generator. This step may be
performed at any time prior to applying the electrical pulses to
the device. Treatment protocol parameters such as pulse width,
number of pulses and voltage are set using the generator interface
(207). Typical ranges include but are not limited to a voltage
level of between 100-3000 volts, a pulse duration of between 20-100
.mu.sec, and between 10 and -500 total pulses. By varying
parameters of voltage, number of electrical pulse and pulse
duration, the electrical field will either produce irreversible or
reversible electroporation of the smooth muscle cells comprising
the inner vessel wall or endothelium 509. In one embodiment of the
invention, ten electrical pulses of 3500 V/cm at a frequency of 10
Hz may be used. In another embodiment, 90 electrical pulses of 1750
V/cm at a frequency of 1 Hz may be used. These ranges ensure that
damage caused by Joule heating is avoided.
[0121] In another aspect of the invention, conductance of the
electrical current may be measured during the procedure to
monitoring clinical endpoints. As an example, a successful
treatment may be identified by changes in conductance during the
applied pulses and an overall decrease in conductance. Measuring
conductance will make it possible to calibrate the current,
voltage, and pulse duration parameters to avoid thermal damage and
obtain IRE. The conductance changes when the cells are porated.
[0122] Referring to FIG. 17B, the balloon 19 is then inflated to
dilate the vessel lumen 503 and restore the vessel patency (209).
As balloon 19 is inflated, the electrode assembly 26 surrounding
the balloon is moved into contact with the stenotic region 505 of
vessel wall 507 as shown in FIG. 17B. The stenotic lesion 505 is
pushed radially outward by the force of the balloon enlarging the
vessel lumen 501. The active electrode region of the electrode
assembly 26 is aligned and in contact with the area of the vessel
wall 507 that was forced outwardly. Specifically, electrode collar
55 slides proximally along the distal portion of the catheter shaft
19 as the balloon 19 expands, which causes electrode legs 44, 47,
42 and 59 (not shown) to move radially outward relative to the
longitudinal axis of the shaft 19. The exposed, un-insulated
portions of the plurality of electrode legs come into contact with
the endothelial layer 509 of stenotic lesion 505.
[0123] Once the lumen has been sufficiently enlarged by
angioplasty, electrical pulses of a predetermined pulse width and
voltage are applied across the electrodes. Pulses are applied while
the balloon remains inflated (211). This provides not only contact
between the electrodes and vessel wall, but also ensures that blood
is not present in the electrical field. The conductivity of blood
is known to be higher than the vessel wall. Accordingly, the
treatment may be compromised if a significant amount of blood was
present in the target area of the vessel since the electrical
current would be directed to the bloodstream rather than the vessel
wall.
[0124] Based on the electrical parameters chosen as part of the
treatment protocol, an electrical field gradient is generated
between opposite polarity electrodes of sufficient strength to
non-thermally electroporate the smooth muscle cells in the target
vessel wall 501. The generated electrical field is represented in
FIG. 17C by 505 lines which extend from the expanded balloon 19
through the endothelial layer 509 into the stenotic region 505 of
the vessel wall 507 forming a tubular shaped ablation zone
corresponding to the interior vessel wall segment being treated.
When electrical pulses are administered within the irreversible
parameter ranges, permanent pore formation occurs in the cellular
membrane, resulting ablation of the smooth muscle cells 509 of the
vessel wall. This is the area that is most susceptible to
endothelial proliferation due to vessel wall trauma caused by the
balloon.
[0125] If the electrical generator treatment parameters are set to
deliver electrical pulses within the reversible range therapeutic
agents may be injected through the catheter lumen to the target
lesion site. The agent will be transported to the smooth muscle
cell interior through the transient cellular membrane openings. The
membrane openings will then close retaining the therapeutic agent
within the cell interior. Anti-restenosis drugs such as Paclitaxel
and Vasculast as well as other agents known in the field may be
introduced into the cell.
[0126] Once sufficient electrical energy has been delivered to the
vessel wall 507, the balloon catheter 10 is deflated, causing
distal collar 55 to move distally along the catheter shaft 19. The
plurality of electrodes legs collapse against the deflating balloon
19 as shown in FIG. 17D. The device can then be removed from the
patient (213). Cell death of the smooth muscle cells will occur
within twenty-four hours of the electroporation treatment as
illustrated by the absence of the endothelial layer 509 directly
adjacent to the stenotic lesion 505 in FIG. 17D. The destroyed
smooth muscle cells are subsequently removed by natural body
processes (215). Extra-cellular structures of the vessel including
the elastin/collagen base of the vessel wall are spared allowing
the smooth muscle cells 509 to regenerate in a normal pattern. FIG.
17E illustrates the re-growth of a normal endothelial layer pattern
across the stenotic region 505. Endothelial proliferation, which is
triggered within hours of standard balloon angioplasty, is absent
in the vessel treated with irreversible electroporation.
[0127] Since the voltage pulse generation pattern from the
generator does not generate damaging thermal effect, and because
the voltage pulses only ablate living cells, the treatment does not
damage blood, blood vessel connective tissue or other non-cellular
or non-living materials such as the catheter itself. The
application of energy may be delivered to the vessel wall without
damaging the balloon or other components of the catheter that might
be damaged by temperatures created by a thermal therapy such as
radiofrequency, laser, microwave or cryoplasty.
[0128] In another aspect of the invention, by periodically
administering the electrical pulses according to a predetermined
schedule, native stenotic lesions maybe prevented altogether.
EXAMPLES
[0129] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Example 1
Method
[0130] Eight Sprague-Dawley rats weighting 300-350 grams were used
in this pilot study. All animals received humane care from a
properly trained professional in compliance with both the
Principals of Laboratory Animal Care and the Guide for the Care and
Use of Laboratory Animals, published by the National Institute of
Health (NIH publication No. 85-23, revised 1985).
[0131] Each animal was anaesthetized throughout the procedure. The
left common carotid artery was exposed, and intimal denudation was
performed as previously described. [Maor, et al. "The Effect of
Irreversible Electroporation on Blood Vessels "Technol Cancer Res
Treat. 6, 2007: 255-360; Touchard, et al. "Preclinical Restenosis
Models Challenges and Successes," Toxicologic Pathology, 34, pp.
2006: 11-18.] Briefly, the left external carotid artery was
incised, and a 2F Fogarty arterial embolectomy catheter (Edwards
Lifesciences) was advanced through the incision to the left common
carotid artery. The balloon was inflated and drawn back three
consecutive times. At the end of the procedure the balloon was
deflated, extracted and the left external carotid artery was
ligated.
[0132] Four rats were used as control, and their skin incision was
sutured immediately at the end of the procedure. In the remaining
four rats, a custom made electrode clamp with two parallel disk
electrodes (diameter=5 mm) was applied on the left common carotid
artery, very close to its bifurcation to the internal and external
carotid arteries, at the exact site of intimal damage (see FIG. 1
for further details). The measured distance between electrodes was
approximately 0.3 mm. A sequence of 10 direct current pulses of 115
Volts (i.e. electrical field of approximately 3800 V/cm), 100 .mu.s
each, at a frequency of 10 pulses per second, was applied between
the electrodes using a high voltage pulse generator intended for
electroporation procedures (ECM 830, Harvard Apparatus, Holliston,
Mass.). Current and voltage were recorded by means of special
oscilloscope probes (current probe was AP015 and high voltage probe
was ADP305, both from LeCroy Corp.). From these two signals
conductance was obtained during the pulses. The procedure was
applied in three successive locations along the common carotid
artery. At the end of the procedure the skin incision was sutured
and animals were kept alive for a follow-up period of 28 days until
they were euthanatized. Results obtained are shown in the table of
FIG. 5.
[0133] Animals were euthanized with an overdose of Phenobarbital.
The arterial tree was perfused with 10% buffered formalin for 40
minutes, and the left and right carotid arteries were exposed near
the bifurcation of the internal and external carotid arteries. One
slice of 1 cm from each artery, at the core of the treated area,
was used for histological analysis. Each slice was fixed with 10%
buffered formalin, embedded in paraffin, and sectioned with a
microtome (5-.mu.m-thick). One section was stained with hematoxylin
and eosin. The endothelial layer was assessed by lectin
immunostaining. Each slide was photographed at .times.200
magnification, and the following areas were measured: tunica media
area, neointimal area and lumen area. The unequal variance t-test
method was used to evaluate the statistical difference between the
measured areas of the two different groups.
Results
[0134] All animals survived the procedures. Conductance of the
arterial wall decreased during successive direct current pulses
(FIG. 2a). During the follow-up period there were no signs of
cerebrovascular events (paraplegia, paraparesis, etc.) and there
was no mortality.
[0135] Conductance was measured during IRE pulses and was used to
monitor the successful use of the electroporation device.
Successful IRE was assigned to those cases in which significant
conductance increase was observed during applied pulses, as
depicted in the case shown in FIG. 2. IRE was successful in 3 of
the 4 animals. There were no changes in conductance during the
pulses applied to the fourth animal and this was considered to
indicate unsuccessful IRE (see also FIG. 2). A constant observation
in all successful IRE cases was that, despite conductance increased
during each pulse, the overall conductance for the whole sequence
decreased.
[0136] After 28 days, histological analysis was used to compare the
IRE-treated and the control group (FIG. 3). Measurements of
neointimal area, tunica media area and arterial lumen area are
summarized in Table 1. Compared with control (including the one
unsuccessful IRE animal), successful IRE induced a significant
reduction in the neointima to media ratio (0.57.+-.0.4 vs.
1.88.+-.1.0, P=0.02). In addition, compared with control (excluding
the unsuccessful IRE animal), successful IRE induced a reduction in
neointimal to media ratio that was less significant (0.57.+-.0.4
vs. 1.67.+-.1.0, P=0.06).
[0137] Examples of the endothelial layer in the different animals
are shown in FIG. 3. Endothelial layer seems to have well recovered
in the IRE-treated animals compared with control group animals.
Endothelial integrity was similar in the IRE-treated group to its
appearance in the unharmed right common carotid artery (FIG.
4).
Discussion
[0138] The results demonstrate the ability of IRE to reduce
restenosis. There was reduced neointimal formation following
successful IRE, compared with control animals. Based on
histological analysis, the extra cellular matrix component of the
arterial wall was maintained; there was no evidence of necrosis,
aneurysm formation, or thrombosis, and there was remarkable
recovery of the endothelial layer. Thermal damage to this layer was
avoided.
[0139] Atherosclerosis, arterial remodeling and restenosis
following angioplasty are complex processes, in which the arterial
wall in general, and the vascular smooth muscle cells in
particular, play a role. [Ward, et al. "Arterial Remodeling
Mechanisms and Clinical Implications," Circulation. 102, 2000:
1186-1191; Davies, et al. "Pathobiology of intimal hyperplasia," Br
J. Surg. 81, 1994: 1254-69; Lusis, et al. "Atherosclerosis,"
Nature. 407, 2000: 233-241.]
[0140] Results provided here show that compared with non-IRE
treated controls, there is significant decrease in neointimal
formation 28 days after intimal damage in IRE-treated arteries. In
a previous study we showed that in the same model, IRE induced
significant reduction in the VSMC population without apparent
damage to elastic fibers. [Maor, et al. "The Effect of Irreversible
Electroporation on Blood Vessels "Technol Cancer Res Treat. 6,
2007: 255-360.] Clarke et al. ["Apoptosis of vascular smooth muscle
cells induces features of plaque vulnerability in atherosclerosis,"
Nat Med. 12, 2006: 1075-1080] investigated the role of VSMC per se
in vascular disease. Using transgenic mice expressing human
diphtheria toxin receptor on all VSMCs, they showed that apoptosis
of 50-70% of the VSMC population in normal arteries induced no
endothelial loss, inflammation, reactive proliferation, thrombosis,
remodeling or plaque formation.
[0141] The results provided here show that by selectively
destroying the VSMC population without affecting the extracellular
matrix, the specific non-thermal IRE ablation method described here
significantly reduces the potential ability of neointimal
formation, without significant damage to arterial function and
overall structure.
[0142] To date, different methods to ablate or stop the
proliferation of cells in the different layers of the arterial wall
have been suggested. These methods include cryoplasty,
brachytherapy, photodynamic therapy, drug-eluting stents and
genetic manipulations using gene therapy. [Tanguay, et al.
"Percutaneous endoluminal arterial cryoenergy improves vascular
remodelling after angioplasty," Thromb. Haemost. 92, 2004:
1114-1121; Yiu, et al. "Vascular Smooth Muscle Cell Apoptosis
Induced by `Supercooling` and Rewarming" J Vasc Interv Radiol. 17,
2006: 1971-1977; Fava, et al. "Cryoplasty for Femoropopliteal
Arterial Disease: Late Angiographic Results of Initial Human
Experience," J Vasc Interv Radiol. 15, 2004: 1239-1243; Laird, et
al. "Cryoplasty for the Treatment of Femoropopliteal Arterial
Disease Results of a Prospective, Multicenter Registry," J Vasc
Interv Radiol. 16, 2005: 1067-1073; Samson, et al. "CryoPlasty
Therapy of the Superficial Femoral and Popliteal Arteries: A Single
Center Experience," Vasc. Endovascular Surg. 40, 2007: 446-450;
Lagerqvist, et al. "Long-Term Outcomes with Drug-Eluting Stents
versus Bare-Metal Stents in Sweden," N Engl J. Med. 356, 2007:
1009-1019; Leon, et al. "Localized Intracoronary Gamma-Radiation
Therapy to Inhibit the Recurrence of Restenosis after Stenting," N
Engl J. Med. 344, 2001: 250-256; Waksman, et al. "Two-year
follow-up after beta and gamma intracoronary radiation therapy for
patients with diffuse in-stent restenosis," Am. J. Cardiol. 88,
2001: 425-428; Teirstein, et al. "New Frontiers in Interventional
Cardiology: Intravascular Radiation to Prevent Restenosis,"
Circulation. 104, 2001: 2620-2626; Salame, et al. "The Effect of
Endovascular Irradiation on Platelet Recruitment at Sites of
Balloon Angioplasty in Pig Coronary Arteries," Circulation. 101,
2000: 1087-1090; Cheneau, et al. "Time Course of Stent
Endothelialization After Intravascular Radiation Therapy in Rabbit
Iliac Arteries," Circulation. 107, 2003: 2153-2158; Waksman, et al.
"Intracoronary photodynamic therapy reduces neointimal growth
without suppressing re-endothelialisation in a porcine model,"
Heart. 92, 2006: 1138-1144; Mansfield, et al. "Photodynamic
therapy: shedding light on restenosis." Heart. 86, 2001: 612-618;
Stone, et al. "A Polymer-Based, Paclitaxel-Eluting Stent in
Patients with Coronary Artery Disease," N Engl J. Med. 350, 2004:
221-231; Moses, et al. "Sirolimus-Eluting Stents versus Standard
Stents in Patients with Stenosis in a Native Coronary Artery," N
Engl J. Med. 349, 2003: 1315-1323; Makinen, et al. "Increased
Vascularity Detected by Digital Subtraction Angiography after VEGF
Gene Transfer to Human Lower Limb Artery: A Randomized,
Placebo-Controlled, Double-Blinded Phase II Study," Mol Ther. 6,
2002: pp. 127-133; Hedman, et al. "Safety and Feasibility of
Catheter-Based Local Intracoronary Vascular Endothelial Growth
Factor Gene Transfer in the Prevention of Postangioplasty and
In-Stent Restenosis and in the Treatment of Chronic Myocardial
Ischemia. Phase II Results of the Kuopio Angiogenesis Trial (KAT),"
Circulation. 2003: 01.1
[0143] The IRE methodology disclosed and described here is
different from and has advantages over these other methods for
reducing restenosis. The nature of the IRE mechanism alone is to
produce only nanoscale defects in the cell membrane. [Chen, et al.
"Membrane electroporation theories: a review." Med Biol Eng Comput.
44, 2006: 5-14.] In the absence of thermal damage, IRE does not
affect connective tissue, the extracellular matrix, nor does it
denaturizes proteins. [Maor, et al. "The Effect of Irreversible
Electroporation on Blood Vessels." Technol Cancer Res Treat. 6,
2007: 255-360; Lee, et al. "Distinguishing Electroporation from
Thermal Injuries in Electrical Shock By MR Imaging." Conf Proc IEEE
Eng Med Biol Soc. 6, 2005: 6544-6546.] Therefore, the integrity of
the extracellular matrix is retained during the process. The
extra-cellular matrix plays an important role in arterial
remodeling and in the elastic properties of the arterial wall. [Li,
et al. "Elastin is an essential determinant of arterial
morphogenesis," Nature. 393, 1998: 276-280.] One explanation for
the absence of aneurysm formation in accordance with the present
invention may be that IRE does not damage elastin or collagen
within the arterial wall. One of the problems with an
intra-arterial stent is the intense extra cellular formation in the
later stages of restenosis, probably due to the mechanical damage
caused by the stent. [Chung, et al. "Enhanced extracellular matrix
accumulation in restenosis of coronary arteries after stent
deployment," J Am Coll Cardiol. 40, 2002: 2072-2081.1 Because
electrical fields can either produce IRE or not, without any
gradual modalities of damage, the margins of the treated region are
well delineated and do not extent beyond the area of application of
the IRE field. Therefore, with IRE the effect can be achieved only
in the area of interest, without collateral damage. The use of the
IRE method of the present invention is a non-pharmacological
method, and therefore there is less concern regarding allergic
reaction or drug safety.
[0144] IRE, and electroporation in general, produces nano-scale
defects in the cell membrane and thereby facilitates unimpeded ion
transport across the membrane. [Chen, et al. "Membrane
electroporation theories: a review." Med Biol Eng Comput. 44, 2006:
5-14.] Therefore, successful IRE results in immediate changes in
the passive electrical properties of the tissue that can be
measured and employed as a feedback mechanism for real time control
of the technique. In fact, within the context of reversible
electroporation, such strategy has been described previously for
individual cells [Huang et al. "Micro-electroporation: improving
the efficiency and understanding of electrical permeabilization of
cells," Biomed. Microdevices. 3, 1999: 145-150], cell cultures
[Pavlin, et al. "Effect of Cell Electroporation on the Conductivity
of a Cell Suspension," Biophys. J. 88, 2005: 4378-4390] and
tissues. [Davalos, et al. "A Feasibility Study for Electrical
Impedance Tomography as a Means to Monitor Tissue Electroporation
for Molecular Medicine," IEEE Trans. Biomed. Eng. 49, 2002:
400-403; Cukjati, et al. "Real time electroporation control for
accurate and safe in vivo non-viral gene therapy,"
Bioelectrochemistry. 70, 2007: 501-507.]
[0145] A common, and expected, observation in previous studies in
which in vivo conductance has been measured during the application
of a sequence of high voltage pulses, either for reversible or for
irreversible electroporation [Ivorra, et al. "In vivo electrical
impedance measurements during and after electroporation of rat
liver," Bioelectrochemistry. 70, 2007: 287-295; Pavselj, et al.
"The course of tissue permeabilization studied on a mathematical
model of a subcutaneous tumor in small animals," IEEE Trans.
Biomed. Eng. 52, 2005: 1373], is that electrical conductance
increases during the sequence and not only within the pulses. The
only exception seems to be the skeletal muscle under IRE. In that
particular case, conductance measured at the pulses is quite
constant during the whole sequence. In accordance with the
methodology of the present invention, conductance decreases during
the sequence of pulses. With the understanding that we are not
bound to a particular theory or explanation, we believe that a
plausible hypothesis is that IRE pulses cause contraction of the
arteries [Jackson, et al. "Regional variation in
electrically-evoked contractions of rabbit isolated pulmonary
artery," Br J. Pharmacol. 137, 2002: 488-496] and that such
contraction results in an increase of the impedance of the
arteries, particularly of the smooth muscle tissue. [Liao, et al.
"The Variation of Action Potential and Impedance in Human Skeletal
Muscle during Voluntary Contraction," Tohoku J. Exp. Med. 173,
1994: 303-309; Shiffman, et al. "Electrical impedance of muscle
during isometric contraction," Physiol. Meas. 24, 2006:
213-234.]
[0146] The results provided here show a failure to induce IRE in
one of the animals. This may have been caused by not applying the
electrodes properly to the artery so that the resulting electrical
contact was not good enough over the artery. Direct
short-circuiting of the electrodes or through plasma or saline
solution does not seem plausible because it would have caused
larger conductivity than the measured conductivity during the
pulses (FIG. 2).
[0147] Successful IRE depends on parameters such as electric field
magnitude, pulses length and frequency. The reason for choosing the
particular electrical parameters used in this study are consistent
with the mode of application of IRE of the present invention. These
are electrical parameters that were assessed to be high enough to
ensure irreversible electroporation [Davalos, et al. "Tissue
Ablation with Irreversible Electroporation," Ann. Biomed. Eng. 33,
2005: 223-231; Edd, et al. "In vivo results of a new focal tissue
ablation technique: irreversible electroporation." IEEE Trans
Biomed Eng. 53, 2006: 1409-15; Miller, et al. "Cancer Cells
Ablation with Irreversible Electroporation," Technol Cancer Res
Treat. 4, 2005: 699-705; Rubinsky, "Irreversible electroporation in
medicine." Technol. Cancer Res Treat. 6. 2007: 255-60; Rubinsky, et
al. "Irreversible electroporation: a new ablation
modality--clinical implications." Technol Cancer Res Treat. 6,
2007: 37-48; Ivorra, et al. "In vivo electrical impedance
measurements during and after electroporation of rat liver,"
Bioelectrochemistry. 70, 2007: 287-295; Maor, et al. "The Effect of
Irreversible Electroporation on Blood Vessels" Technol Cancer Res
Treat. 6, 2007: 255-360; Touchard, et al. "Preclinical Restenosis
Models Challenges and Successes," Toxicologic Pathology, 34, pp.
2006: 11-18; Dev, et al. "Intravascular Electroporation Markedly
Attenuates Neointima Formation After Balloon Injury of the Carotid
Artery in the Rat." J Interven Cardiol. 13, 2000: 331-338] but
which do not cause damaging levels of Joule heating. We used a
sequence of 10 direct current pulses of 115 Volts (i.e. electrical
field of approximately 3800 V/cm), 100 .mu.s each, at a frequency
of 10 pulses per second. These parameters where partially based on
previous reports that showed successful tumor cell ablation with
IRE. [Miller, et al. "Cancer Cells Ablation with Irreversible
Electroporation," Technol Cancer Res Treat. 4, 2005: 699-705;
Rubinsky, et al. "Irreversible electroporation: a new ablation
modality--clinical implications." Technol Cancer Res Treat. 6,
2007: 37-48; Al-Sakere, et al. "Tumor Ablation with Irreversible
Electroporation." PLoS ONE. 2, 2007: e1135.] Since the arterial
wall has different morphology, and since we did not have data
regarding the specific susceptibility of vascular smooth muscle
cells to IRE, we used an electrical field that was higher than any
previous report but low enough not to produce thermal damage within
the constraints of the treated tissue dimensions. Those skilled in
the art will be able to follow the results provided here to show
the relation between conductance measurements during the procedure
and IRE efficiency.
[0148] The examples described here used rodent carotid artery
model. This model is an acceptable animal model of restenosis
[Touchard, et al. "Preclinical Restenosis Models: Challenges and
Successes," Toxicologic Pathology, 34, pp. 2006: 11-18;
Narayanaswamy, et al. "Animal Models for Atherosclerosis,
Restenosis, and Endovascular Graft Research," J Vasc Interv Radiol.
11, 2000: 5-17], but it is important to clarify that our
experiments were performed on arteries that were not
atherosclerotically changed. However, we believe these results can
be readily applied to humans to show the efficacy of IRE in
atherosclerotically changed arteries.
[0149] Our electrodes were clamping the artery on its outer
surface, but this does not imply that this method will be used as
an invasive procedure. Previous reports have already demonstrated
the ability to design and use intra-vascular devices in order to
induce reversible electroporation of the arterial wall. [Dev, et
al. "Intravascular Electroporation Markedly Attenuates Neointima
Formation After Balloon Injury of the Carotid Artery in the Rat." J
Interven Cardiol. 13, 2000: 331-338.] Those skilled in the art will
understand that similar designs can be used to achieve IRE on
humans using intra-vascular devices.
[0150] The invention provides in vivo, long-term results of a new
non-thermal, non-pharmacological strategy to attenuate neointimal
formation following intimal damage. Importantly, the invention
provides for the treatment of restenosis following coronary
angioplasty and the delivery of that treatment with real time
control.
[0151] The method of the invention can be used in preventing and/or
ablating coronary and peripheral restenosis process, while also
playing a role in attenuating atherosclerotic processes in
clinically important locations, such as coronary, carotid and renal
arteries.
Example 2
Summary of Method and Results
[0152] 33 Sprague-Dawley rats were used to compare NTIRE protocols.
Each animal had NTIRE applied to its left common carotid using
custom-made electrodes. The right carotid artery was used as
control. Electric pulses of 100 microseconds were used. Eight IRE
protocols were compared: 1-4) 10 pulses at a frequency of 10 Hz
with electric fields of 3500, 1750, 875 and 437.5 V/cm and 5-8) 45
and 90 pulses at a frequency of 1 Hz with electric fields of 1750
and 875 V/cm. Animals were euthanized after one week. Histological
analysis included VSMC counting and morphometry of 152 sections.
Selective slides were stained with elastic Van Gieson and Masson
trichrome to evaluate extra-cellular structures. Most efficient
protocols were 10 pulses of 3500 V/cm at a frequency of 10 Hz and
90 pulses of 1750 V/cm at a frequency of 1 Hz, with ablation
efficiency of 89.+-.16% and 94.+-.9% respectively. Extra-cellular
structures were not damaged and the endothelial layer recovered
completely.
SUMMARY CONCLUSION
[0153] NTIRE is a promising, efficient and simple novel technology
for VMSC ablation. It enables ablation within seconds without
causing damage to extra-cellular structures, thus preserving the
arterial scaffold and enabling endothelial regeneration. This study
provides scientific information for future anti-restenosis
experiments utilizing NTIRE.
Method
[0154] Thirty three Sprague-Dawley rats weighting 160-280 grams
were used in this study. All animals received humane care from a
properly trained professional in compliance with both the
Principals of Laboratory Animal Care and the Guide for the Care and
Use of Laboratory Animals, published by the National Institute of
Health (NIH publication No. 85-23, revised 1985).
[0155] Animals were anaesthetized with an intramuscular injection
of Ketamin and Xylazine (90 mg/Kg and 10 mg/Kg, respectively). The
left common carotid artery of each animal was exposed and a custom
made electrode clamp with two parallel disk electrodes was applied
on the left common carotid artery as previously described. (Maor E,
Ivorra A, Leor J, Rubinsky B. Irreversible Electroporation
Attenuates Neointimal Formation After Angioplasty. Biomedical
Engineering, IEEE Transactions on. 2008; 55(9):2268-2274) The
custom made electrode clamp consists of two printed circuit boards
(1.5 mm thickness) with disk electrodes (diameter=5 mm) made of
copper (70 .mu.m thickness) plated with gold (manufacturing process
by Sierra Proto Express, Sunnyvale, Calif.).
[0156] Animals were divided to eight different groups (FIG. 24).
All groups had their left common carotid artery treated with NTIRE
and their right common carotid artery used as a control. NTIRE was
performed by applying short electric pulses between the electrodes
using a high voltage pulse generator intended for electroporation
procedures (ECM 830, Harvard Apparatus, Holliston, Mass.). Current
and voltage were recorded by means of special oscilloscope probes
(current probe was AP015 and high voltage probe was ADP305, both
from LeCroy Corp.). From these two signals conductance (defined as
current/voltage) was obtained for each pulse (mean value of the
last 10 .mu.s of the pulse). The procedure was repeated in three
successive locations along the common carotid artery, thus treating
approximately 1.5 cm of the left common carotid artery. At the end
of the procedure the skin incision was sutured closed and the
animals were kept alive for a follow-up period of 7 days.
[0157] All pulses were 100 .mu.s in length. The number of pulses,
the applied electric field, and the frequency of the pulses
differed between the groups as summarized in FIG. 24.
Histological Assessment
[0158] Animals were euthanized with an overdose of Phenobarbital
followed by bilateral chest dissection. Gross inspection of carotid
arteries was used to identify arterial wall integrity or
intraluminal massive thrombus formation. The arterial tree was
perfused with 10% buffered formalin, and the left and right carotid
arteries were harvested near the bifurcation of the internal and
external carotid arteries. The treated area was cut to two or three
consecutive slices. One section from each slice was used for
histological analysis. Each slice was fixed with 10% buffered
formalin, embedded in paraffin, and sectioned with a microtome
(5-.mu.m-thick). Sections were stained with hematoxylin and eosin.
Each section was photographed at .times.200 magnification, and the
following parameters were quantitatively evaluated: number of VSMC
nuclei in each of the three layers of the Tunica Media, total area
of the Tunica Media, and the average thickness of the Tunica Media
based on 5 different measurements in each section. VSMC
concentration was calculated by dividing the total number of nuclei
by the measured area of the Tunica Media. The paired t-test method
was used to evaluate the statistical difference between the
measured areas of the control versus IRE-treated groups.
[0159] In addition, selected sections were stained with elastic Van
Gieson (EVG) and Masson trichrome in order to evaluate the
extra-cellular elastic and collagen fibers, respectively.
Immunostaining with CD31 and CD34 antibodies (Pathology Services
Inc., Berkeley, Calif.) was used to evaluate the endothelial
layer.
Results
[0160] All 33 animals survived the procedure. During follow-up
period, there were no cases of infection, bleeding at IRE-treated
arteries, thrombosis or animal mortality.
NTIRE VSMC Ablation Efficiency
[0161] Results of all eight groups are summarized in FIGS. 25 and
26. Best NTIRE ablation results were achieved in Groups 1 and 7
(FIG. 24). Group 1 had 89.+-.16% reduction in the number of VSMC
compared with control (24.+-.34 vs. 208.+-.40, P<0.001) and
Group 7 had 94.+-.9% reduction in the number of VSMC compared with
control (13.+-.21 vs. 213.+-.33, P<0.001). An example of
complete ablation of the entire arterial wall is shown in FIGS. 28
and 30.
[0162] While ten pulses of 3,500 V/cm were efficient, ten pulses of
lower electric fields had a minor ablation effect (1,750 V/cm,
group 2: 167.+-.66 vs. 214.+-.38, P=0.05) or no effect at reducing
VSMC population (Groups 3 & 4, 875 and 437.5 V/cm
respectively). Increasing the number of pulses with electric field
of 1,750 V/cm improved the ablation efficiency (VSMC population
reduction of 22.+-.30%, 86.+-.16% and 94.+-.9% with 10, 45 and 90
pulses respectively). Similar trend of increasing efficiency was
also apparent with an electric field of 875 V/cm (63.+-.29% and
79.+-.17% with 45 and 90 pulses, respectively), but efficiency
values were not high enough even with 90 pulses (49.+-.40 vs.
236.+-.31, P<0.001).
[0163] Sub-analysis of ablation efficiency at the three separate
layers of the Tunica Media showed that the best results were
achieved in the outer layers of the Tunica Media, and most VSMC
that survived NTIRE were located in the inner most layer (FIG. 29).
For example, in the case of Group 7, no VSMC nuclei could be
located in the outer layer in all sections evaluated. All 6%
surviving VSMC in this group were located in the inner layer of the
Tunica Media (FIG. 30).
[0164] Electric conductance changed during the application of NTIRE
(FIG. 31). The conductance measured during the last pulse was lower
compared with conductance measured during the first pulse in all
groups except Groups 3 & 4 (two groups with no significant
NTIRE ablation effect). For the most effective protocols,
conductance was reduced by 31.+-.6% and 32.+-.20% for Groups 1 and
7, respectively.
NTIRE Effect on Other Arterial Wall Components
[0165] Successful NITRE ablation of VSMC induced a reduction in
media thickness: 25.+-.17% reduction in group 1 (45.+-.10 vs.
59.+-.8 .mu.m) and 27.+-.7% reduction in Group 7 (37.+-.4 vs.
51.+-.6 em). No change in media thickness was induced in the two
non-successful NTIRE groups (61.+-.9 vs. 58.+-.7 .mu.m in Group 3,
61.+-.9 vs. 60.+-.6 .mu.m in Group 4).
[0166] Endothelial cells of treated arteries were similar in number
and morphology to those of non treated control arteries, but were
negative to both CD31 and CD34 antibodies (data shown only for CD34
staining, see bottom row in FIG. 32). EVG stain demonstrated intact
elastic fibers and preserved vessel wall, similar to that of
control arteries (middle row, FIG. 32). Masson Trichrome stain
demonstrated minor fibrosis in perivascular areas, with collagen
being the dominant component of the Tunica Media following the
complete loss of VSMC population (top row, FIG. 32).
Discussion
[0167] This is the first large scale, in-vivo survival experiment
to evaluate and compare the effect of different NTIRE protocols on
VSMC population. The results show that NTIRE can achieve efficient
ablation of VSMC within seconds, without damaging extra-cellular
components.
[0168] Current study results are supported by previous studies by
our group. In a preliminary study evaluating NTIRE effect on blood
vessels, a 87% reduction in VSMC concentration after 28 days was
observed following NTIRE with similar parameters to those of Group
1 (10 pulses, 100 .mu.sec, 10 Hz, 3800 V/cm). (Maor E, Ivorra A,
Leor J, Rubinsky B. The Effect of Irreversible Electroporation on
Blood Vessels. Technology in Cancer Research and Treatment. 2007;
6(4):255-360) Parameters similar to Group 1 have also been shown to
significantly reduce neointimal formation following angioplasty in
rodent carotid injury model. (Maor E, Ivorra A, Leor J, Rubinsky B.
Irreversible Electroporation Attenuates Neointimal Formation After
Angioplasty. Biomedical Engineering, IEEE Transactions on. 2008;
55(9):2268-2274).
[0169] Our results are also supported by the work of Al-Sakere et
al. (Al-Sakere B, Andre F, Bemat C, et al. Tumor Ablation with
Irreversible Electroporation. PLoS ONE. 2007; 2(11):e1135) In their
in-vivo study with sarcoma tumor, best tumor ablation using
irreversible electroporation was achieved with the use of 80
electroporation pulses of 100 .mu.s at 0.3 Hz with an electrical
field magnitude of 2,500 V/cm. Their most efficient protocol was
the one with the largest number of pulses and the highest electric
field evaluated, similar to the results presented here. Based on
these results, it seems that irreversible electroporation is
limited only by the joule heating effect. As long as there is no
thermal damage to extra-cellular structures, increase in electric
field magnitude and pulse number will be translated to larger
ablation volume and better ablation efficiency.
[0170] For Groups 1 and 7, where best ablation efficiency was
observed, around 10% of the VSMC population survived the ablation.
Further analysis of the results demonstrated 100% efficiency in the
outer layers of the Tunica Media, with all surviving cells located
in the inner most layers of the arterial wall (FIGS. 29 and 30).
The most probable explanation for this phenomenon is the nature of
the electric field. We assumed uniform electric field between the
two electrodes, but since the arterial tissue is not homogeneous
with respect to its electric properties, the actual electric field
in the inner most area of the artery might have been lower than
expected. A better design with a more uniform electric field might
allow NTIRE to achieve higher ablation efficiencies compared with
those reported in this study. Another plausible explanation is the
proximity of surviving cells to the oxygenated blood of the carotid
artery. The availability of oxygen and nutrient might have a
protective effect that reduces the vulnerability of these cells to
the stress insult caused by the damage to the cell's membrane.
[0171] Our results show that reduction of the electric field
magnitude can be compensated by increasing the number of NTIRE
pulses. Ten pulses of 3500 V/cm achieved similar effect to 90
pulses of 1750 V/cm. However, decreasing the electric field even
more to 875 V/cm caused a decrease in NTIRE efficiency even with
the use of 90 pulses. This observation may be important in future
NTIRE device designs, where intervention time could be reduced by
increasing trans-electrode electric potential.
[0172] A common observation in previous electroporation studies,
either reversible or irreversible, is that electrical conductance
measured at the pulses increases during the sequence of pulses.
(Ivorra A, Miller L, Rubinsky B. Electrical impedance measurements
during electroporation of rat liver and muscle. In: 13th
International Conference on Electrical Bioimpedance and the 8th
Conference on Electrical Impedance Tomography; 2007:130-133.
Available at: http://dx.doi.org/10.1007/978-3-540-73841-1.sub.--36
[Accessed Oct. 21, 2008]) The only exception to this seems to be
for skeletal muscle under NTIRE. (Ivorra A, Miller L, Rubinsky B.
Electrical impedance measurements during electroporation of rat
liver and muscle. In: 13th International Conference on Electrical
Bioimpedance and the 8th Conference on Electrical Impedance
Tomography; 2007:130-133. Available at:
http://dx.doi.org/10.1007/978-3-540-73841-1.sub.--36) In that
particular case, conductance measured at the pulses remains quite
constant during the whole sequence and it can be explained as a
saturation effect of the electroporation phenomenon. However, the
fact that conductance decreases during the sequence is quite
surprising as it contradicts what would be expected in a simple
electroporation model: electroporation increases cell membrane
permeability to ions and therefore its conductance should also
increase. We do not have a definitive explanation for the
conductance decrease observed here. We consider that a plausible
hypothesis is that NTIRE pulses cause a contraction of the arteries
by stimulating the vascular smooth muscle cells and that such a
contraction results in an increase in the electrical impedance of
the arteries. (Liao T J, Nishikawa H. The variation of action
potential and impedance in human skeletal muscle during voluntary
contraction. Tohoku J Exp Med. 1994; 173(3):303-9; Shiffman C A,
Aaron R, Rutkove S B. Electrical impedance of muscle during
isometric contraction. Physiol Meas. 2003; 24(1):213-34; Jackson V
M, Trout S J, Cunnane T C. Regional variation in
electrically-evoked contractions of rabbit isolated pulmonary
artery. Br J. Pharmacol. 2002; 137(4):488-496) Another explanation
could be based on the fact that electroporation disturbs the
osmotic balance of the cells and causes cell swelling which in turn
can result in a decrease of the conductance. (Ivorra A, Miller L,
Rubinsky B. Electrical impedance measurements during
electroporation of rat liver and muscle. In: 13th International
Conference on Electrical Bioimpedance and the 8th Conference on
Electrical Impedance Tomography; 2007:130-133. Available at:
http://dx.doi.org/10.1007/978-3-540-73841-1.sub.--36 [Accessed Oct.
21, 2008]) Nevertheless, we believe that such swelling cannot be
manifested as fast as would be required here in order to explain
the conductance decrease during the sequence, particularly in
Groups 1 and 2 (sequence duration=1 second).
[0173] NTIRE is not the first method to address the challenge of
VSMC ablation. Several alternative technologies have been studied,
and some have become a common clinical practice for the treatment
of post-angioplasty and in-stent restenosis. These technologies
include: cryoplasty (Fava M, Loyola S, Polydorou A, et al.
Cryoplasty for Femoropopliteal Arterial Disease: Late Angiographic
Results of Initial Human Experience. J Vasc Interv Radiol. 2004;
15(11):1239-1243), brachytherapy (Leon M, Teirstein P, Moses J, et
al. Localized Intracoronary Gamma-Radiation Therapy to Inhibit the
Recurrence of Restenosis after Stenting. N Engl J Med. 2001;
344(4):250-256), photodynamic therapy (Waksman R, Leitch I,
Roessler J, et al. Intracoronary photodynamic therapy reduces
neointimal growth without suppressing re-endothelialisation in a
porcine model. Heart. 2006; 92(8):1138-1144), radiofrequency
ablation (Taylor G W, Kay G N, Zheng X, Bishop S, Ideker R E.
Pathological Effects of Extensive Radiofrequency Energy
Applications in the Pulmonary Veins in Dogs. Circulation. 2000;
101(14):1736-1742), drug-eluting stents (Stone G, Ellis S, Cox D,
et al. A Polymer-Based, Paclitaxel-Eluting Stent in Patients with
Coronary Artery Disease. N Engl J. Med. 2004; 350(3):221-231) and
molecular-based therapies (Aubart F C, Sassi Y, Coulombe A, et al.
RNA Interference Targeting STIM1 Suppresses Vascular Smooth Muscle
Cell Proliferation and Neointima Formation in the Rat. Mol Ther.
2008. Available at: http://dx.doi.org/10.1038/mt.2008.291 [Accessed
January 6, 2009]).
[0174] However, delayed re-endothelialization (Cheneau E, John M,
Fournadjiev J, et al. Time Course of Stent Endothelialization After
Intravascular Radiation Therapy in Rabbit Iliac Arteries.
Circulation. 2003; 107(16):2153-2158), economic impact (Weintraub W
S. The Pathophysiology and Burden of Restenosis. The American
Journal of Cardiology. 2007; 100(5, Supplement 1):S3-S9) and late
in-stent thrombosis (Lagerqvist B, James S, Stenestrand U, et al.
Long-Term Outcomes with Drug-Eluting Stents versus Bare-Metal
Stents in Sweden. N Engl J. Med. 2007; 356(10): 1009-1019; Costa
Mass., Sabate M, van der Giessen W J, et al. Late Coronary
Occlusion After Intracoronary Brachytherapy. Circulation. 1999;
100(8):789-792) are some of the major concerns with all of the
current VSMC ablation modalities. We believe NTIRE should be
further investigated as an alternative to current modalities, since
it has two major advantages.
[0175] First, its non-pharmacological nature can overcome
biological phenomena such as cellular adaptation or acquired
drug-resistance, thus achieving higher local efficiency. The non
pharmacologic nature also guarantees an accurate local effect that
depends entirely on electric field distribution and does not induce
collateral damage to adjacent structures.
[0176] Second, its ultra short duration can decrease intervention
time in the clinical setting of primary percutaneous intervention
(PCI). It enables one to minimize obstruction of blood flow to
viable myocardial tissue during the ablation procedure. Moreover,
short intervention duration will enable prompt and full endothelium
recovery by immediate recruitment of circulating progenitor
endothelial cells. Incomplete neointimal coverage has been
demonstrated as a probable cause for late stent thrombosis in
patients with drug-eluting stents6, as well as a reason for
brachytherapy failure. (Waksman R, Bhargava B, Mintz G S, et al.
Late total occlusion after intracoronary brachytherapy for patients
with in-stent restenosis. Journal of the American College of
Cardiology. 2000; 36(1):65-68)
[0177] The complete endothelial regeneration observed in this
report can be attributed to two properties of NTIRE. First, the
ultra short duration of the modality enabled immediate repopulation
of the endothelium by either endothelial cells from adjacent non
treated areas, or by adherence of progenitor endothelial cells from
the circulation. Second, the non-thermal nature of this modality
minimized the insult to extra cellular components of the
endothelial layer, probably creating a more comfortable environment
for cellular regeneration.
[0178] Endovascular NTIRE has clinical potential for both the
prevention and the treatment of restenosis following angioplasty.
Due to its short duration and high efficiency NTIRE can become a
preventive treatment immediately before stent deployment. It might
also prove to be a valuable tool for the effective treatment of
in-stent restenosis several weeks following the angioplasty.
[0179] All animals were evaluated after a follow-up period of one
week. This was based on our previous study, where ablation
efficiency was evident by the complete loss of VSMC population as
early as one week following NTIRE. (Maor E, Ivorra A, Leor J,
Rubinsky B. The Effect of Irreversible Electroporation on Blood
Vessels. Technology in Cancer Research and Treatment. 2007;
6(4):255-360).
CONCLUSION
[0180] This study provides scientific proof and justification
irreversible electroporation as a promising non-thermal, non
pharmacological, ultra short modality for the treatment of VSMC
proliferation and the clinical problem of in-stent restenosis.
[0181] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
* * * * *
References