U.S. patent application number 10/783161 was filed with the patent office on 2005-09-01 for method of treatment using electroporation mediated delivery of drugs and genes.
Invention is credited to Dev, S.B., Gilbert, Richard A., Hayakawa, Yasuhiko, Heller, Richard, Hofmann, Gunter A., Jaroszeski, Mark J..
Application Number | 20050192542 10/783161 |
Document ID | / |
Family ID | 23856229 |
Filed Date | 2005-09-01 |
United States Patent
Application |
20050192542 |
Kind Code |
A1 |
Dev, S.B. ; et al. |
September 1, 2005 |
Method of treatment using electroporation mediated delivery of
drugs and genes
Abstract
A method for in vivo electrotherapy, or electroporation-mediated
therapy, using a needle array apparatus is provided. Treatment of
tumors with a combination of electroporation using the apparatus of
the invention, and a chemotherapeutic agent, caused regression of
tumors in vivo.
Inventors: |
Dev, S.B.; (San Diego,
CA) ; Hofmann, Gunter A.; (Dan Diego, CA) ;
Gilbert, Richard A.; (Tampa, FL) ; Hayakawa,
Yasuhiko; (Chiba, JP) ; Heller, Richard;
(Tampa, FL) ; Jaroszeski, Mark J.; (Tampa,
FL) |
Correspondence
Address: |
DANIEL M. CHAMBERS
Biotechnology Law Group
658 MARSOLAN AVE
SOLANA BEACH
CA
92075
US
|
Family ID: |
23856229 |
Appl. No.: |
10/783161 |
Filed: |
February 20, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10783161 |
Feb 20, 2004 |
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10213514 |
Aug 6, 2002 |
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6763264 |
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10783161 |
Feb 20, 2004 |
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08467566 |
Jun 6, 1995 |
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5702359 |
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08467566 |
Jun 6, 1995 |
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08042039 |
Apr 1, 1993 |
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5439440 |
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Current U.S.
Class: |
604/200 |
Current CPC
Class: |
A61N 1/327 20130101;
A61N 1/0502 20130101; A61N 1/325 20130101; C12M 35/02 20130101 |
Class at
Publication: |
604/200 |
International
Class: |
A61M 005/24; A61M
005/28 |
Claims
1. canceled
2. A method of electroporating a portion of the body of a patient
to introduce molecules into tissues therein, comprising: a.
providing an array of multiple opposed pairs of electrodes
connected to a generator, wherein at least two pairs of electrodes
are activated simultaneously, at least one of said pairs of
electrodes having a needle configuration for penetrating tissue; b.
inserting a first pair of said needle electrodes, into selected
tissue for introducing molecules into the tissue; c. positioning a
second pair of electrodes of said array of electrodes in conductive
relation to said selected tissue so that said tissue is between
said first and second opposed pairs of electrodes; and d. applying
electric pulses to at least two different pairs of electrodes to
generate an electric field of sufficient strength to cause in vivo
electroporation of the tissue.
3. A method for the delivery of molecules to a predetermined tissue
of a subject, comprising: a. providing an array of electrodes
comprising at least two pairs of opposed electrodes, wherein at
least one of said electrodes has a needle configuration for
penetrating said tissue; b. contacting said tissue with said array
such that the electrode(s) penetrate said tissue; C. administering
said molecules to said tissue; and d. applying at least one
electric pulse simultaneously to at least two of the pairs of
electrodes sufficient to electroporate cells of said tissue and
thereby allow said molecules to be delivered to said tissue.
4. A method according to claim 3, wherein the array comprises at
least three pairs of opposed electrodes and at least one of said
pairs is not pulsed when at least two of the pairs of electrodes
are pulsed simultaneously.
5. A method according to claim 3, wherein the molecules are
selected from the group consisting of a chemotherapeutic agent, a
polynucleotide, and a polypeptide.
6. A method according to claim 5, wherein the chemotherapeutic
agent is bleomycin.
7. A method according to claim 2, wherein the molecules are
administered by a method selected from the group consisting of
intratumoral, systemic, and local administration.
8. A method according to claim 2, wherein the predetermined tissue
is selected from the group consisting of pancreas, lung, heart,
kidney, muscle, breast, colon, prostate, thymus, testis, and
ovary.
9. A method for the delivery of molecules to a predetermined tissue
of a subject, comprising: a. providing an array of electrodes
comprising at least two pairs of opposed electrodes, wherein at
least one of said electrodes has a needle configuration for
penetrating said tissue; b. contacting said tissue with said array
such that the electrode(s) penetrate said tissue; c. administering
said molecules to said tissue; d. applying at least one electric
pulse simultaneously to at least two of the pairs of opposed
electrodes sufficient to electroporate cells of said tissue using
an electric field of predefined strength and thereby allow said
molecules to be delivered to said tissue.
10. A method according to claim 9, wherein the electric field of
predefined strength is created by delivering electric pulse(s) of a
voltage suitable to generate said electric field.
11. A method according to claim 10, wherein the voltage is
determined based on the distance between the electrodes of the
electrode pair to be energized.
12. A method according to claim 9, wherein the array comprises at
least three pairs of opposed electrodes and at least one of said
pairs is not pulsed when at least two of the pairs of electrodes
are pulsed simultaneously.
13. A method according to claim 9, wherein the molecules are
selected from the group consisting of a chemotherapeutic agent, a
polynucleotide and a polypeptide.
14. A method according to claim 9, wherein the molecules are
introduced by a method selected from the group consisting of
intratumoral, systemic, and local administration.
15. A method according to claim 13, wherein the chemotherapeutic
agent is bleomycin.
16. A method according to claim 9, wherein the tissue is selected
from the group consisting of pancreas, lung, heart, kidney, muscle,
breast, colon, prostate, thymus, testis, and ovary.
17. A method according to claim 8, wherein the electric field of
predefined strength has a field strength of from about 0.2 kV/cm to
about 20 kV/cm.
18. A method according to claim 8, wherein the electrode array is
in a configuration selected from the group consisting of a
substantially square, rectangular, and circular pattern.
19. A method for the delivery of molecules to a predetermined
tissue of a subject, comprising: a. providing an electrode
apparatus for the application of at least one electric pulse,
wherein said electrode apparatus comprises: i. at least two pairs
of needles for penetrating said predetermined tissue, wherein each
needle has a lumen for insertion of an electrode; ii. at least two
pairs of electrodes, wherein each electrode is configured for
insertion into the tissue through the lumen of one of said needles;
and iii. a power generator to apply an electric potential of
sufficient strength across at least two of the pairs of the
electrodes to generate an electric field for electroporation of
cells of the tissue; and b. applying at least one electric pulse
simultaneously to at least two of the pairs of electrodes
sufficient to electroporate cells of said tissue and thereby allow
said molecules to be delivered to said tissue.
20. A method according to claim 19, wherein the array comprises at
least three pairs of opposed electrodes and at least one of said
pairs is not pulsed when at least two of the pairs of electrodes
are pulsed simultaneously.
21. A method according to claim 19, wherein said needles are
tubular.
22. A method according to claim 19, wherein said needles are
removed and the electrodes are left in said tissue.
23. A method according to claim 19, wherein said needles are
nonconductive.
24. A method according to claim 19, wherein each of the electrodes
comprises an elongated insulated conductor with a conductive tip at
its distal end.
25. A method according to claim 19, wherein the needles are
connected to a source of molecules and are used to inject a
solution comprising said molecules and a carrier into the
tissue.
26. A method according to claim 19, wherein the electrode apparatus
produces electrical pulses between electrodes according to a set of
predetermined electrode configurations.
27. A method according to claim 19, wherein the electrode apparatus
further comprises an identification circuit to determine the number
of electrodes in the electrode apparatus and desired electrical
signal parameters for the application of one or more electric
pulses.
28. A method according to claim 19, wherein the power generator
includes a control module that controls at least one electrical
treatment parameter selected from the group consisting of an
electrode voltage set point, pulse length, pulse shape, pulse
number, and an electrode switching sequence.
29. A method according to claim 28, wherein the control module
directs the power generator to generate at least one electric pulse
having a voltage that depends on a spacing of the electrodes in the
electrode apparatus.
30. A method according to claim 28, wherein the control module
directs the power generator to generate at least one electric pulse
according to the number of electrodes in the electrode
apparatus.
31. A method according to claim 19, wherein electric field strength
produces by the pulse(s) between the electrodes ranges from about
10 V/cm to about 1300 V/cm.
32. A method according to claim 19, wherein each pulse produced by
the power generator has a pulse width from about 10 microsecond to
about 100 millisecond.
33. A method according to claim 19, wherein at least one needle in
the electrode apparatus is configured for transit of a
solution.
34. A method according to claim 19, wherein each electrode
comprises a base portion that makes electrical contact with the
electrode apparatus and a distal tip portion that is exposed to
deliver electricity to tissue, wherein the electrode is covered
with a layer of electrically insulating material between the base
portion and the distal tip portion.
35. A method for the delivery of molecules to a predetermined
tissue of a subject, comprising: a. providing an electrode
apparatus for the application of at least one electric pulse,
wherein said electrode apparatus comprises: i. at least two pairs
of needle electrodes for penetrating said predetermined tissue,
wherein at least one of said needle electrodes is used to deliver a
solution containing said molecules to said tissue, and wherein at
least two pairs of said needle electrodes is used to deliver at
least one electric impulse; and ii. a power generator to apply an
electric potential of sufficient strength across at least two of
the pairs of the electrodes to generate an electric field for
electroporation of cells of the tissue; and b. applying at least
one electric pulse simultaneously to at least two of the pairs of
electrodes sufficient to electroporate cells of said tissue and
thereby allow said molecules to be delivered to said tissue.
36. A method according to claim 35, wherein the electrode apparatus
comprises at least three pairs of opposed electrodes and at least
one of said pairs is not pulsed when at least two of the pairs of
electrodes are pulsed simultaneously.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation-in-Part of
co-pending application Ser. No. 08/467,566, filed on Jun. 6, 1995,
which is a Continuation-in-Part of co-pending application Ser. No.
08/042,039 filed on Apr. 1, 1993.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the treatment of ailments
in humans and other mammals, and more particularly, to an improved
method and apparatus for the application of controlled electric
fields for in vivo delivery of genes and pharmaceutical compounds
into live cells of a patient by electroporation.
[0003] In the 1970's it was discovered that electric fields could
be used to create pores in cells without causing permanent damage
to them. This discovery made possible the insertion of large
molecules into cell cytoplasm. It is known that genes and other
molecules such as pharmacological compounds can be incorporated
into live cells through a process known as electroporation. The
genes or other molecules are mixed with the live cells in a buffer
medium and short pulses of high electric fields are applied. The
cell membranes are transiently made porous and the genes or
molecules enter the cells. There they can modify the genome of the
cell.
[0004] Electroporation has been recently suggested as one approach
to the treatment of certain diseases such as cancer. For example,
in the treatment of certain types of cancer with chemotherapy it is
necessary to use a high enough dose of a drug to kill the cancer
cells without killing an unacceptable high number of normal cells.
If the chemotherapy drug could be inserted directly inside the
cancer cells, this objective could be achieved. Some of the best
anti-cancer drugs, for trample, bleomycin, normally cannot
penetrate the membranes of certain cancer cells. However,
electroporation makes it possible to insert the bleomycin into the
cells.
[0005] One therapeutic application of electroporation is for cancer
treatment. Experiments on laboratory mammals have been carried out
and reported as follows: Okino, M., E. Kensuke, 1990. The Effects
of a Single High Voltage Electrical Stimulation with an Anticancer
Drug on in vivo Growing Malignant Tumors. Jap. Journal of Surgery.
20: 197-204. Mir, L. M., S. Orlowski, J. Belehradek Jr., and C.
Paoletti. 1991. Electrochemotherapy Potentiation of Antitumor
Effect of Bleomycin by Local Electric Pulses. Eur. J. Cancer. 27:
68-72. Clinical trials have been conducted and reported by Mir, L.
M., M. Belehradek, C. Domenge, S. Orlowski, B. Poddevin, et al.
1991. Electrochemotherapy, a novel antitumor treatment: first
clinical trial. C.R. Acad. Sci. Paris. 313: 613-618.
[0006] This treatment is carried out by infusing an anticancer drug
directly into the tumor and applying an electric field to the tumor
between a pair of electrodes. The field strength must be adjusted
reasonably accurately so that electroporation of the cells of the
tumor occurs without damage, or at least minimal damage, to any
normal or healthy cells. This can normally be easily carried out
with external tumors by applying the electrodes to opposite sides
of the tumor so that the electric field is between the electrodes.
The distance between the electrodes can then be measured and a
suitable voltage according to the formula E=V/d can then be applied
to the electrodes (E=electric field strength in V/cm; V=voltage in
volts; and d=distance in cm). When internal tumors are to be
treated, it is not easy to properly locate electrodes and measure
the distance between them. In the aforementioned parent
application, there is disclosed a system of electrodes for in vivo
electroporation wherein the electrodes may be inserted into body
cavities. In a related U.S. Pat. No. 5,273,25 a syringe for
injecting molecules and macromolecules for electroporation utilizes
needles for injection which also function as electrodes. This
construction enables the subsurface placement of electrodes. It
would be desirable to have an electrode apparatus having electrodes
that can be inserted into or adjacent tumors so that predetermined
electric fields can be generated in the tissue for electroporation
of the cells of the tumor.
[0007] Studies have also shown that large size nucleotide sequences
(up to 630 kb) can be introduced into mammalian cells via
electroporation (Eanault, et al., Gene (Amsterdam), 144(2): 205,
1994; Nucleic Acids Research, 15(3): 1311, 1987; Knutson, et al.,
Anal. Biochem., 164: 44, 1987; Gibson, et al., EMBO J., 6(8): 2457,
1987; Dower, et al., Genetic Engineering, 12: 275, 1990; Mozo, et
al., Plant Molecular Biology, 16: 917, 1991), thereby affording an
efficient method of gene therapy, for example.
SUMMARY OF THE INVENTION
[0008] Accordingly, it is a primary object of the present invention
to provide an improved apparatus that can be conveniently and
effectively positioned to generate predetermined electric fields in
pre-selected tissue.
[0009] It is another principal object of the present invention to
provide an improved apparatus that provides an effective and
convenient means for positioning electrodes into tissue for the
injection of therapeutic compounds into the tissue and application
of electric fields to the tissue.
[0010] In accordance with a primary aspect of the present invention
an electrode apparatus for the application of electroporation to a
portion of the body of a patient, comprises a support member, a
plurality of needle electrodes adjustably mounted on said support
member for insertion into tissue at selected positions and
distances from one another, and means including a signal generator
responsive to said distance signal for applying an electric signal
to the electrodes proportionate to the distance between said
electrodes for generating an electric field of a predetermined
strength.
[0011] Another aspect of the invention includes needles that
function for injection of therapeutic substances into tissue and
function as electrodes for generating electric fields for portion
of cells of the tissue.
[0012] In yet another aspect of the invention is provided a
therapeutic method utilizing the needle array apparatus for the
treatment of cells, particularly tumor cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a side elevation view, in section of a needle
assembly in accordance with a preferred embodiment of the
invention.
[0014] FIG. 2 is a bottom view of the embodiment of FIG. 1.
[0015] FIG. 3 is an assembly drawing showing a perspective view of
an alternate embodiment of the invention.
[0016] FIG. 4 is a perspective view of the embodiment of FIG. 3
shown assembled.
[0017] FIG. 5 is a perspective view of a selector switch for the
electrode assembly of FIG. 4.
[0018] FIGS. 6a-6b is a diagrammatic illustration of selected
contact positions of the switch of FIG. 5.
[0019] FIG. 7 is a perspective view of a further embodiment of the
invention.
[0020] FIG. 8 is a perspective view of a still further embodiment
of the invention.
[0021] FIGS. 9a-9d is a top plan view, illustrating a preferred
form of electrodes and sequence of use.
[0022] FIGS. 10a and 10b show the tumor volume after 43 days of ECT
with bleomycin in Panc-3 xenografted nude mice. (D=drug;
E=electroporation)
[0023] FIG. 11 is an illustration of tumor growth of Panc-3 cells
after ECT with bleomycin in nude mice.
[0024] FIGS. 12a and 12b show the tumor volume after 20 and 34 days
of ECT with bleomycin, respectively, in non-small cell lung
carcinoma (NSCLC) xenografted nude mice. (D=drug;
E=electroporation)
[0025] FIG. 13 shows the tumor volume after 34 days of ECT with
bleomycin in non-small cell lung carcinoma (NSCLC) xenografted nude
mice. The arrow indicates retreatment of one mouse at day 27.
(D=drug; E=electroporation)
[0026] FIGS. 14a and 14b show pre-pulse dosing with
neocarcinostatin in Panc-3 and NSCLC, respectively, in the nude
mouse model.
[0027] FIGS. 14c and 14d show post-pulse dosing with
neocarcinostatin in Panc-3 in the nude mouse model.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] As used herein the term "molecules" includes pharmacological
agents, genes, antibodies or other proteins. One human therapeutic
application of electroporation consists of infusion of an
anticancer drug and electroporation of the drug into the tumor by
applying voltage pulses between electrodes disposed on opposite
sides of the tumor, called electrochemotherapy (ECT). The present
invention was devised primarily for enabling ECT such as that
reported by Okino and Mir et al to be carried out on non-surface
tumors such as those inside the body. However, it may be utilized
for other therapeutic applications.
[0029] Referring to FIG. 1 of the drawings, a needle assembly in
accordance with preferred embodiment of the invention is
illustrated and designated generally by the numeral 10. The needle
assembly comprises an elongated tubular support body 12 which is
preferably in the form of a hollow stainless steel shaft. A center
needle mount 14 is mounted on the lower end of the shaft 12 and has
a central bore 16 for receiving and guiding a center needle 18. The
shaft 12 includes a needle exit slot 20 through which the needle
electrode 18 extends from the interior thereof to the exterior
where it is secured by a clamp 22 to the outside of the tube
12.
[0030] The upper end of the electrode 18 may be secured to a screw
24 for connection to an electrical circuit. The lower end of the
tubular holder 12 includes threats 26 for threatably receiving a
collar 28 for mounting a plurality of needles and a stop collar 30
for stopping or locking the collar 28 in position.
[0031] A plurality of needles 32 are mounted in grooves 34 equally
spaced around the outer surface of the needle collar 28. This
provides a circular array of equally spaced needles, eight in
number in the illustrated embodiment. The needles are held in place
by a band clamp 36, having the ends clamped together by a screw or
nut and bolt 38 which also serves as an electrical connection for
the needles. The band clamp 36 directly engages and holds the
needles in place.
[0032] This electrode assembly is designed to apply electrical
energy to living tissue when the needles are inserted into the
tissue. The center needle 18 acts as one electrode, such as an
anode or cathode, and the other or annular arrangement of needles
32 functions as the opposite electrode. All of these needles are
held in fixed positions when the clamps are installed and secured.
One or more of the needles may be cannular or tubular in form for
injecting molecules of genes, pharmaceutical or other substances
into the tissue.
[0033] In operation the center needle should be adjusted in order
to achieve the desired tissue penetration. This is done by
releasing the pressure of the center needle clamp 22 and sliding
the center needle 18 outwardly or inwardly, as seen in FIG. 1, so
that it extends from the center needle guide 14 to desired
penetration distance. The needle is then clamped in position.
Thereafter annular needles 32 are adjusted to achieve the desired
penetration into the tissue. This can be accomplished by releasing
the pressure of the band clamp 36 and sliding the needles 32 into
the desired position. Minor adjustments can also be made by moving
the needle collar 28 toward and away from the end of the shaft 12.
A therapeutic substance may be injected into the tissue through one
or more of these needles or by a separate means.
[0034] After all needles are adjusted to the proper penetration,
the shaft 12 is grasped and the needles are inserted into the
tissue to the desired depth. Thereafter, a suitable pulse generator
is connected to the electrode assembly and the appropriate voltage
applied to the electrodes. A suitable quantity of therapeutic
substance such as genes or molecules of a suitable chemical or
pharmaceutical for treatment of the tissue is injected into the
tissue before the voltage is applied.
[0035] A modification to this electrode assembly could include a
solid non-penetrating electrode (not shown) in place of the center
needle. The non-penetrating center electrode could be any suitable
shape conductor such as a button or plate attached to the end of
the shaft 12 to contact the surface tissue. The annular needle
arrangement would be adjusted to penetrate the tissue at the
desired depth when the center electrode is resting on a tissue
surface. Electrical energy would flow from the penetrating needles
through the tissue and to the central electrode on the surface.
These arrangements can be utilized to treat near surface rumors
where the circular array of electrodes are designed to encircle the
tumor. The central electrode is positioned such that the electrical
energy flows through the tumor to the central electrode.
[0036] Other advantages of this electrode assembly are that all
needles 18 and 32 can be independently adjusted to achieve the
desired penetration. The needle 28 collar can also be adjusted to
position it from the end of the shaft 12 so that insertion of the
center and annular needles can be directly observed. In addition,
the needle collar 28 can have any size or configuration to encircle
the tissue area to be treated.
[0037] Referring to FIGS. 3 and 4 an alternate embodiment of a
circular array needle electrode assembly is illustrated and
designated generally by the numeral 40. This needle assembly
comprises a circular array of needles 42 through 52, which are
mounted in equally spaced relation in a hub 54 mounted on an
elongated cylindrical shaft 56. The hub 54 is preferably of a
suitably selected diameter to provide the desired diameter of the
arrays to position around a tumor or other tissue to be treated.
One or more of the needles may be hollow to enable the injection of
molecules of a therapeutic substance, as will be more fully
described hereinafter.
[0038] An electrical connector socket assembly comprises a body
member 58 having a central opening or bore 60 for receipt of shaft
56 and, an annular array of a plurality of sockets 62 through 72
for receipt of the ends of needles 42 through 52. The sockets 62
through 72 electrically connect the needles to leads 74 through 84
which connect to a distributing switch, as will be subsequently
described.
[0039] The electrical connector socket 58 fits onto shaft 56 with
the end of the needles extending into the electrical sockets 62
through 72 for connecting to the leads 74 through 84. The shaft 56
which mounts the needle array hub 54 and the socket assembly 58
mounts onto a holder 86 adapted to be held in the hand. The holder
86 has an elongated cylindrical configuration adapted to be held in
the hand for manipulation. The holder 86 has a forward socket and
including a forwardly extending tubular shaft 88 having a bore 90
into which shaft 56 extends while the shaft 88 extends into a bore
(not shown) within the connector member 58. The shaft 56 extends
into bore 90 and has a annular groove or recess 92 which is engaged
by a retainer latch which comprises a transverse plug 94 in a bore
96 biased to one side and including a bore 98 in which the annular
slot 92 extends and is retained in the holder. A spring 102 mounted
in bore 96 biases plug 94 to the latched position. The shaft 56 may
be released for removal by pressing on end 100 of plug 94.
[0040] The holder when assembled as shown in FIG. 4 may be grasped
in the hand and the needles inserted into a selected tissue area.
The needles 42-52 are preferably spaced and positioned to surround
the selected tissue of treatment. One or more of the needles 42-52,
as previously explained, may be hollow to enable the injection of
the desired therapeutic substance. The electrode leads 74-84 are
then connected in a preferred arrangement to a rotatable switch
assembly, as shown in FIG. 5, which enables the selection of
opposed pairs of the needles for activation or the application of
the electrical potential.
[0041] The switch assembly designated generally by the numeral 104
comprises a stationary housing 106 which, in the illustrated
embodiment, is generally cylindrical in configuration and in which
is mounted a rotor 108 with spaced contacts 110 and 112 connected
by a pair of conductors 114 and 116 to a pulse power generator 115.
The rotor contacts 110 and 112 are positioned within housing 106 to
engage annular contacts 118, 120, 122, 124, 126 and 128 to which
leads 74-84 are connected.
[0042] Referring to FIGS. 6a, b and c, the rotor 108 has an
internal portion having contacts 110 and 112 each of which bridge
between two contacts 118-128 to which the leads 74 through 84 are
connected to connect the source of power. The internal contacts 110
and 112 rotate with the rotor 108 and can be selectively positioned
in conductive relation with pairs of the internal contacts 118-128
to thereby activate opposed pairs of the needle electrodes. This
enables the operator to selectively position the electrodes
surrounding a selected tissue and to selectively apply the
direction of the electrical field as desired for optimum treatment.
The rotor 108 enables the field to be selectively generated around
or across the tissue from all directions.
[0043] Referring to FIG. 7 an alternate embodiment of an electric
field generating array of parallel adjustably positionable
electrodes, as disclosed in the parent application, is illustrated.
The electrode assembly designated generally by the numeral 130
includes a pair of spaced apart arrays 132 and 134 of conductive
needle electrodes 136 and 138 mounted on a dielectric carrier or
support member 140. The needle array 132 is held in a fixed clamp
142 which allows the needles 136 to be adjusted in depth relative
to the support 140.
[0044] The needles 138 are mounted in a moveable clamp 146 which is
adjustably mounted on support member 140 by a clamp screw 148. The
needles 136 and 138 are each provided with a penetration stop 144.
The gap spacing clamp screw 148 secures the clamp 146 in selected
positions on the support 140. A gap spacing sensor 150 senses the
distance between the needle arrays 132 and 134 and generates a
signal that is sent to the pulse generator via conductor cable 152.
A pulse generator is connected to the needle electrodes by means of
cables 154 and 156.
[0045] Referring to FIG. 8, details of a needle holder or template
for various arrangements for establishing a spaced pair or parallel
arrays of needles is illustrated. This embodiment comprises a base
holder member 158 having a plurality of adjacently positioned
parallel slots 160 into which selected needles 162 and 164 may be
positioned in selected spaced relation. This holder may serve to
mount a pair of oppositely polarized needle electrodes 162 and 164,
as illustrated. These can be selectively positioned in selected
space relationship to be disposed on opposite sides of a selected
tissue. The needles are clamped into the slots by a clamp or plate
159. In addition, the holder may be used in combination with an
additional holder for provision of multiple arrays on opposite
sides of a selected tissue. The illustrated needles may be
connected by conductors 166 and 168 to a suitable pulse
generator.
[0046] Referring to FIGS. 9a through 9d, an additional aspect of
the invention is illustrated. As more clearly illustrated, the
combination electrodes may take the form of separate needles 170
and 172 which may be first inserted into or beside a selected
tissue area such as on opposite sides of a tumor 194 as
illustrated. Thereafter the needles may be connected to a syringe
or other source of molecules and used to inject a selected
molecular solution into the tissue area. The needles may be
non-conductive and a pair of electrodes 176 and 178, as illustrated
in FIG. 9b, are selectively fed through the bore or lumen of the
respective needles into the tissue, as illustrated, and thereafter
the needle is removed, as shown in FIG. 9c. The electrodes 176 and
178 are each provided with an elongated insulated conductor 180 and
182 with conductive tips 184 and 186.
[0047] A pair of conductors 188 and 190 from a suitable power
generator may then be connected to the ends of the conductors of
the electrodes by micro clamps 192 and 194, as shown in 9d, and an
electric potential applied across the electrodes. This generates a
field in the tissue and electroporates the cells of the selected
tissue, such as a tumor or the like. This electroporation enables
the selected molecules to enter the cells of the tissue and more
efficiently kill or alter the cells as desired. This form of needle
and electrode may be used with any or all the above described
assemblies.
[0048] These needle electrode assemblies, as above described,
enable the in vivo positioning of electrodes in or adjacent to
subsurface tumors or other tissue. While the focus of the present
application has been on electrochemotherapy, the embodiment of the
subject invention may be applied to other treatments, such as gene
therapy of certain organs of the body.
[0049] The nature of the electric field to be generated is
determined by the nature of the tissue, the size of the selected
tissue and its location. It is desirable that the field be as
homogenous as possible and of the correct amplitude. Excessive
field strength results in lysing of cells, whereas a low field
strength results in reduced efficacy. The electrodes may be mounted
and manipulated in many ways including but not limited to those in
the parent application. The electrodes may be conveniently
manipulated on and by forceps to internal position.
[0050] The waveform of the electrical signal provided by the pulse
generator can be an exponentially decaying pulse, a square pulse, a
unipolar oscillating pulse train or a bipolar oscillating pulse
train. The electric field strength can be 0.2 kV/cm to 20 kV/cm.
The pulse length can be ten .mu.s to 100 ms. There can be one to
one hundred pulses. Of course, the waveform, electric field
strength and pulse duration are also dependent upon the type of
cells and the type of molecules that are to enter the cells via
electroporation.
[0051] The various parameters including electric field strengths
required for the electroporation of any known cell is generally
available from the many research papers reporting on the subject,
as well as from a database maintained by Genetronics, Inc., San
Diego, Calif., assignee of the subject application. The electric
fields needed for in vivo cell electroporation, such as ECT, are
similar in amplitude to the fields required for cells in vitro.
These are in the range of from 100 V/cm to several kV/cm. This has
been verified by the inventors own experiments and those of others
reported in scientific publications. The first in vivo application
of pulsed electric fields in the chemotherapy field to treat tumors
was reported in 1987 by Okino in Japan.
[0052] Pulse generators for carrying out the procedures described
herein are and have been available on the market for a number of
years. One suitable signal generator is the ELECTRO CELL
MANIPULATOR Model ECM 600 commercially available from GENETRONICS,
INC. of San Diego, Calif., U.S.A. The ECM 600 signal generator
generates a pulse from the complete discharge of a capacitor which
results in an exponentially decaying waveform. The electric signal
generated by this signal generator is characterized by a fast rise
time and an exponential tail. In the signal generator, the
electroporation pulse length is set by selecting one of ten timing
resistors marked R1 through R10. They are active in both High
Voltage Mode (HVM) (capacitance fixed at fifty microfarads) and Low
Voltage Mode (LVM) (with a capacitance range from 25 to 3,175
microfarads).
[0053] The ECM 600 signal generator has a control knob that permits
the adjustment of the amplitude of the set charging voltage applied
to the internal capacitors from 50 to 500 volts in LVM and from
0.05 to 2.5 kV in the HVM. The amplitude of the electrical signal
is shown on a display incorporated into the ECM 600 signal
generator. This device further includes a plurality of push button
switches for controlling pulse length, in the Low VM mode, by a
simultaneous combination of resistors parallel to the output and a
bank of seven selectable additive capacitors.
[0054] The ECM 600 signal generator also includes a single
automatic charge and pulse push button. This button may be
depressed to initiate both charging of the internal capacitors to
the set voltage and to deliver a pulse to the outside electrodes in
an automatic cycle that takes less than five seconds. The manual
button may be sequentially pressed to repeatedly apply the
predetermined electric field.
[0055] Preferably, the therapeutic method of the invention utilizes
a square wave pulse electroporation system. For example, the
ElectroSquarePorator (T820), also available from GENETRONICS, INC.,
can be used.
[0056] Square wave electroporation systems deliver controlled
electric pulses that rise quickly to a set voltage, stay at that
level for a set length of time (pulse length), and then quickly
drop to zero. This type of system yields better transformation
efficiency for the electroporation of plant protoplast and
mammalian cell lines than an exponential decay system.
[0057] The ElectroSquarePorator (T820) is the first commercially
available square wave electropomtion system capable of generating
up to 3000 volts. The pulse length can be adjusted from 5 .mu.sec
to 99 msec. The square wave electroporation pulses have a gentler
effect on the cells which results in higher cell viability.
[0058] The T820 ElectroSquarePorator is active in both the High
Voltage Mode (HVM) (100-3000 volts) and the Low Voltage Mode (LVM)
(50-500 volts). The pulse length for LVM is about 0.3 to 99 msec
and for HVM, 5 to 99 .mu.sec. The T820 has multiple pulsing
capability from about 1 to 99 pulses.
[0059] Mir and others have used square wave pulses for
electrochemotherapy, which allows the insertion of chemotherapeutic
agents into cancerous tumors. Mice were injected with a low dose of
bleomycin. The cancerous tumors were then electroporated resulting
in the reduction or complete remission of the tumors (Mir, L. M.,
Eur. J. Cancer, 27(1): 68, 1991).
[0060] Saunders has compared the square wave with exponential decay
pulses in the electroporation of plant protoplast. Square wave
electroporation produced higher transformation efficiency than the
exponential decay pulses. He also reported that the optimization of
electroporation parameters is much easier with square wave pulses
since sufficient transformation efficiency can be produced over a
larger range of voltages (Saunders, Guide to Electroporation and
Electrofusion, pp. 227-247, 1991).
[0061] The therapeutic method of the invention includes
electrotherapy, also referred to herein as electroporation-mediated
therapy, using the apparatus of the invention for the delivery of
macromolecules to a cell or tissue. As described earlier, the term
"macromolecule" or "molecule" as used herein refers to drugs (e.g.,
chemotherapeutic agents), nucleic acids (e.g., polynucleotides),
peptides and polypeptides, including antibodies. The term
polynucleotides include DNA, cDNA and RNA sequences.
[0062] Drugs contemplated for use in the method of the invention
are typically chemotherapeutic agents having an antitumor or
cytotoxic effect. Such drugs or agents include bleomycin,
neocarcinostatin, suramin, and cisplatin. Other chemotherapeutic
agents will be known to those of skill in the art (see for example
The Merck Index). The chemical composition of the agent will
dictate the most appropriate time to administer the agent in
relation to the administration of the electric pulse. For example,
while not wanting to be bound by a particular theory, it is
believed that a drug having a low isoelectric point (e.g.,
neocarcinostatin, IEP=3.78), would likely be more effective if
administered post-electroporation in order to avoid electrostatic
interaction of the highly charged drug within the field. Further,
such drugs as bleomycin, which have a very negative log P, (P being
the partition coefficient between octanol and water), are very
large in size (MW=1400), and are hydrophilic, thereby associating
closely with the lipid membrane, diffuse very slowly into a tumor
cell and are typically administered prior to or substantially
simultaneous with the electric pulse. Electroporation facilitates
entry of bleomycin or other similar drugs into the tumor cell by
creating pores in the cell membrane.
[0063] It may be desirable to modulate the expression of a gene in
a cell by the introduction of a molecule by the method of the
invention. The term "modulate" envisions the suppression of
expression of a gene when it is over-expressed, or augmentation of
expression when it is underexpressed. Where a cell proliferative
disorder is associated with the expression of a gene, nucleic acid
sequences that interfere with the gene's expression at the
translational level can be used. This approach utilizes, for
example, antisense nucleic acid, ribozymes, or triplex agents to
block transcription or translation of a specific mRNA, either by
masking that mRNA with an antisense nucleic acid or triplex agent,
or by cleaving it with a ribozyme.
[0064] Antisense nucleic acids are DNA or RNA molecules that are
complementary to at least a portion of a specific mRNA molecule
(Weintraub, Scientific American, 262: 40, 1990). In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of the mRNA, since the cell will not
translate a mRNA that is double-stranded. Antisense oligomers of
about 15 nucleotides are preferred, since they are easily
synthesized and are less likely to cause problems than larger
molecules when introduced into the target cell. The use of
antisense methods to inhiiit the in vitro translation of genes is
well known in the art (Marcus-Sakura, Anal. Biochem., 172: 289,
1988).
[0065] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher, et al., Antisense Res. and Dev., 1(3): 227, 1991;
Helene, C., Anticancer Drug Design, 6(6): 569, 1991).
[0066] Ribozymes are RNA molecules possessing the ability to
specifically cleave other singles-tranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences which encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:
3030, 1988). A major advantage of this approach is that, because
they are sequence-specific, only mRNAs with particular sequences
are inactivated.
[0067] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334: 585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0068] The present invention also provides gene therapy for the
treatment of cell proliferative or immunologic disorders mediated
by a particular gene or absence thereof. Such therapy would achieve
its therapeutic effect by introduction of a specific sense or
antisense polynucleotide into cells having the disorder. Delivery
of polynucleotides can be achieved using a recombinant expression
vector such as a chimeric virus, or the polynucleotide can be
delivered as "naked" DNA for example.
[0069] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
When the subject is a human, a vector such as the gibbon ape
leukemia virus (GALV) can be utilized. A number of additional
retroviral vectors can incorporate multiple genes. All of these
vectors can transfer or incorporate a gene for a selectable marker
so that transduced cells can be identified and generated.
[0070] Therapeutic peptides or polypeptides may also be included in
the therapeutic method of the invention. For example,
immunomodulatory agents and other biological response modifiers can
be administered for incorporation by a cell. The term "biological
response modifiers" is meant to encompass substances which are
involved in modifying the immune response. Examples of immune
response modifiers include such compounds as lymphokines.
Lymphokines include tumor necrosis factor, interleukins 1,2, and 3,
lymphotoxin, macrophage activating factor, migration inhibition
factor, colony stimulating factor, and alpha-interferon,
beta-interferon, and gamma-interferon and their subtypes.
[0071] Also included are polynucleotides which encode metabolic
enzymes and proteins, including antiangiogenesis compounds, e.g.,
Factor VIII or Factor IX.
[0072] The macromolecule of the invention also includes antibody
molecules. The term "antibody" as used herein is meant to include
intact molecules as well as fragments thereof, such as Fab and
F(ab').sub.2.
[0073] Administration of a drug, polynucleotide or polypeptide, in
the method of the invention can be, for example, parenterally by
injection, rapid infusion, nasopharyngeal absorption, dermal
absorption, and orally. In the case of a tumor, for example, a
chemotherapeutic or other agent can be administered locally,
systemically or directly injected into the tumor. When a drug, for
example, is administered directly into the tumor, it is
advantageous to inject the drug in a "fanning" manner. The term
"fanning" refers to administering the drug by changing the
direction of the needle as the drug is being injected or by
multiple injections in multiple directions like opening up of a
hand fan, rather than as a bolus, in order to provide a greater
distribution of drug throughout the tumor. As compared with a
volume that is typically used in the art, it is desirable to
increase the volume of the drug-containing solution, when the drug
is administered (e.g., injected) intratumorally, in order to insure
adequate distribution of the drug throughout the tumor. For
example, in the EXAMPLES herein, one of skill in the art typically
injects 50 .mu.l of drug-containing solution, however, the results
are greatly improved by increasing the volume to 150 .mu.l.
Preferably, the injection should be done very slowly and at the
periphery rather than at the center of the tumor where the
intertitial pressure is very high.
[0074] Preferably, the molecule is administered substantially
contemporaneously with the electroporation treatment. The term
"substantially contemporaneously" means that the molecule and the
electroporation treatment are administered reasonably close
together with respect to time. The administration of the molecule
or therapeutic agent can at any interval, depending upon such
factors, for example, as the nature of the tumor, the condition of
the patient, the size and chemical characteristics of the molecule
and half-life of the molecule.
[0075] Preparations for parenteral administration include sterile
or aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Carriers for occlusive dressings can
be used to increase skin permeability and enhance antigen
absorption. Liquid dosage forms for oral administration may
generally comprise a liposome solution containing the liquid dosage
form. Suitable forms for suspending the liposomes include
emulsions, suspensions, solutions, syrups, and elixirs containing
inert diluents commonly used in the art, such as purified water.
Besides the inert diluents, such compositions can also include
adjuvants, wetting agents, emulsifying and suspending agents.
Further, vasoconstrictor agents can be used to keep the therapeutic
agent localized prior to pulsing.
[0076] Any cell can be treated by the method of the invention. The
illustrative examples provided herein demonstrate the use of the
method of the invention for the treatment of tumor cells, e.g.,
pancreas and lung. Other cell proliferative disorders are amenable
to treatment by the electroporation method of the invention. The
term "cell proliferative disorder" denotes malignant as well as
non-malignant cell populations which often appear to differ from
the surrounding tissue both morphologically and genotypically.
Malignant cells (i.e., tumors or cancer) develop as a result of a
multi-step process. The method of the invention is useful in
treating malignancies or other disorders of the various organ
systems, particularly, for example, cells in the pancreas and lung,
and also including cells of heart, kidney, muscle, breast, colon,
prostate, thymus, testis, and ovary. Preferably the subject is
human.
[0077] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLES
[0078] The following examples illustrate the use of
electrochemotherapy (ECT) of a poorly differentiated human
pancreatic tumor (Panc-3) xenografted subcutaneously on the left
flank of nude mice. The single treatment procedure involved
injection of bleomycin (0.5 units in 0.15 ml saline)
intratumorally, using farming, as described herein followed by
application of six square wave electrical pulses, ten minutes
later, using proprietary needle array electrodes arranged along the
circumference of a circle 1 cm in diameter. Needle array of
variable diameters (e.g., 0.5 cm, 0.75 cm and 1.5 cm can also be
used to accommodate tumors of various sizes. Stoppers of various
heights can be inserted at the center of the array to make the
penetration depth of the needles into the tumor variable. A
built-in mechanism allowed switching of electrodes for maximum
coverage of the tumor by the pulsed field. The electrical
parameters were: 1300 V/cm and 6.times.99 .mu.s pulses spaced at 1
sec interval.
[0079] Results showed severe necrosis and edema in nearly all the
mice at the treatment site. While there was a substantial reduction
in the tumor volume (after a slight initial increase due to edema)
of the mice in the treated group (D+E+; D=Drug, E=Electrical
field), those in the control group (D+E-) increased dramatically.
Nearly complete tumor regression was observed in 90% of the mice
treated by ECT after 28 days. No response was seen in 10% of the
mice. A complete regression with no palpable tumor has been
observed in 60% of the cases 77 days after the initial treatment.
However, there was tumor regrowth in 20% of the mice 35 days after
treatment but at a much slower growth rate compared to the control.
This observation has been linked to incomplete treatment of large
primary tumors where the needle depth was lower than the Z
dimension of the tumor. Histological analysis of tumor samples
showed necrotic tumor cell ghosts in D+E+group compared to a
mixture of viable and necrotic cells in D+E-group. Preliminary
studies with human non-small cell lung cancer (NSCLC) tumors
xenografted onto nude mice have also shown very encouraging results
with ECT treatment with bleomycin.
Example 1
[0080] The tumor cell line Pant-3, a poorly differentiated
adenocarcinoma cell line of the pancreas, was supplied by
Anticancer, Inc., San Diego. For ECT experiments, tissue taken from
the stock mice, where the tumor line was maintained, was thawed and
cut into very small pieces about 1 mm each, and 8-10 pieces were
surgically xenografted in a subcutaneous sac made in left flank of
nude mice, and then closed with 6.0 surgical suture. After the
average tumor size reached about 5 mm, mice with palpable tumors
were divided randomly, 10 mice for control group (D+E-; D=Drug,
E=Electric field) and 10 mice for ECT treatment, namely bleomycin
injection followed by pulsing (D+E+) from a BTX Square Wave T820
Generator. The tumor dimensions were measured and the tumor volume
calculated using the formula:
(II/6).times.a.times.b.times.c
[0081] where a, b, and c are, respectively, the length, width and
thickness of the tumor. 0.5 units Bleomycin (Sigma Chemicals) was
dissolved in 0.15 ml of 0.9% NaCl and was injected in each mice
intratumorally by fanning for both the control (D+E-) and the
treated (D+E+) groups. Ten minutes after the injection, each mouse
in the D+E+group was pulsed from a BTX T820 square wave
electroporator with a set of needle array electrodes as described
in the present invention. Electrical parameters used were as
follows: field strength 1300 V/cm, 6 pulses of 99 .mu.s each, at 1
sec interval.
[0082] The mice were monitored every day for mortality and any
signs of a diseased state were noted. The tumor dimensions were
measured at regular intervals and tumor growth
regression/progression monitored. Another set of nude mice with
xenografts of non-small cell lung cancer line was also treated by
the same procedure as for the Panc-3 tumors.
[0083] FIGS. 10a and 10b show the analysis of the tumor volume
determined over a 43 day period after ECT using bleomycin for the
Panc-3 tumors. There was a dramatic difference between the
untreated and treated mice in terms of tumor volume. There was
essentially no detectable tumor after approximately 24 days of
treatment. The results of FIG. 10 are also summarized in Table 1
below. An illustration of the actual regression of the tumor is
shown in FIG. 11.
1TABLE 1 ELECTROCHEMOTHERAPY OF PANC-3 TUMORS IN NUDE MICE Tumor
Tumor Tumor Tumor Days after volume volume volume volume treatment
(mm.sup.3) C1 (mm.sup.3) C2 (mm.sup.3) T1 (mm.sup.3) T2 0 138.746
148.94 123.11 178.37 1 206.979 179.82 210.95 252.72 8 394.786
451.787 104.55 211.11 15 557.349 798.919 113.21 226.966 18 939.582
881.752 161.73 246.91 24 1391.057 1406.98 41.56 47.2228 28 1628.631
1474.21 0 0 35 2619.765 2330.31 0 0 38 2908.912 2333.967 0 0 43
3708.571 5381.759 0 0 Cell Line: poorly differentiated human
pancreatic tumor (panc3) Mouse model: nude mouse Transplant:
subcutaneous xenograft Control mice: C1 and C2 Treated mice: T1 and
T2
[0084] The Panc-3 experiment was repeated using a non-small cell
lung cancer cell line (NSCLC), 177 (Anticancer, San Diego, Calif.).
The results were similar to that found with bleomycin and Panc-3 as
shown in FIGS. 12a and 12b. In one experiment, a tumor that had
recurred was retreated at day 27 (FIG. 13) and after 7 days, there
was no evidence of tumor.
[0085] The Panc-3 and NSCLC models were utilized with the drug
neocarcinostatin (NCS) following the same procedures as outlined
above. As shown in FIGS. 14a and 14b, pre-pulse dosing with NCS in
a manner similar to that used for the bleomycin studies, was not
effective in reducing tumor size at all. It was believed that due
to the low isoelectric point of NCS, electrostatic interaction
prevented the drug from entering the tumor cell. Therefore, the
experiment was repeated by pulsing first and injecting NCS
post-pulse.
[0086] FIG. 14c shows the initial tumor volume (I) as compared to
the final tumor volume (F) at day 13 for 7 mice treated (Mouse ID
1-7). In several of the mice (ID 1, 2, 4, and 7), an increase in
tumor volume was observed, but appeared to be due to edema.
However, as shown in FIG. 14d, when a separate group of 5 mice were
examined at day 23, all mice showed a marked reduction in tumor
volume.
[0087] A comparison of FIGS. 14a and b with 14c and d indicated
that post-pulse with NCS was more effective than pre-pulse
administration for NCS.
SUMMARY
[0088] The present Examples illustrate that a poorly differentiated
Pancreatic cancer (Panc-3) and Non-small cell lung cancer (NSCLC)
xenografted subcutaneously onto nude mice can be effectively
treated by the electrochemotherapy protocol using bleomycin or NCS
and needle array electrodes. Other similar chemotherapeutic agents
can also be effective using the method of the invention.
[0089] The results show a complete regression of Panc-3 tumors was
achieved in 60% of the treated group with no palpable tumor seen
even 77 days after the single treatment. Partial regression (80%
reduction in tumor volume) was observed in 30% of cases, while only
10% did not respond (Table 2).
[0090] Histological studies clearly showed severe necrosis of the
tumor region for the group subjected to ECT whereas no necrosis was
apparent in the control group. Intratumoral drug injection with
larger volume of bleomycin, combined with fanning to maximize
uniform drug distribution throughout the tumor volume, was found to
be very effective as compared to the conventional mode of injecting
the drug prior to pulsing.
2TABLE 2 Electrochemotherapv of Panc-3 with Bleomycin Days after
treatment 28 35 57 77 CR (100%) 6 6 6 6 PR (80%) 3 NR (%) 1 1 1 1
Death 2* Tumor regrowth 2 Retreatment 2 Histology 1 Number of mice
treated: 10 CR: Complete Regression PR: Partial Regression NR: No
Response *1 mice died after retreatment 1 mice died after 64 days
survival
[0091] Although the invention has been described with reference to
the presently preferred embodiment, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
* * * * *