A method for sustained intravascular delivery via electroporation is provided. The method is useful for delivery of therapeutic compositions such as antithrombotic and anticoagulant agents. The invention also provides a catheter apparatus for introducing a composition into at least one cell in a vessel in a subject.

FIELD OF THE INVENTION 
The present invention relates generally to the field of electroporation and 
specifically to a method of sustained intravascular delivery of 
compositions such as antithrombotic and anticoagulant agents. 
BACKGROUND OF THE INVENTION 
For some time now, it has been known 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. 
Treatment of cells by electroporation is carried out by infusing a 
composition into a patient and applying an electric field to the desired 
site of treatment between a pair of electrodes. The field strength must be 
adjusted reasonably accurately so that electroporation of the cells occurs 
without damage, or at least minimal damage, to any normal or healthy 
cells. 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). 
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. 
Iontophoresis uses electrical current to activate and to modulate the 
diffusion of a charged molecule across a biological membrane, such as the 
skin, in a manner similar to passive diffusion under a concentration 
gradient, but at a facilitated rate. In general, iontophoresis technology 
uses an electrical potential or current across a semipermiable barrier. 
Delivery of heparin molecules to patients has been shown using 
iontophoresis (IO), a technique which uses low current (d.c.) to drive 
charged species into the arterial wall. lontophoretic delivery of heparin 
(1000 U/ml) into porcine artery was shown to be safe and well tolerated 
without any change in the coronary angiography or normal physiological 
parameters such as blood pressure and cardiac rhythm. Although heparin in 
varying concentration from 1000 U to 20,000 U/ml results in greater 
concentrations remaining in the vessel after IO delivery compared to 
passive delivery, approximately 1 hour after the delivery of heparin, 96% 
of the drug washes out (Mitchel, et al., ACC 44th Annual Scientific 
Session, Abs.#092684, 1994). It has also been reported that platelet 
deposition following IO delivery of heparin is reduced in the pig balloon 
injury model. .sup.125 I-labeled hirudin has also been delivered 
iontophoretically into porcine carotid artery (Fernandez-Ortiz, et al., 
Circulation, 89:1518, 1994). A local concentration of hirudin can be 
achieved by IO, however, as with the above experiments with heparin, 80% 
of the drug washes out in 1 hour and after three hours, the level is the 
same as for the passive delivery. 
Heparins are widely used therapeutically to prevent and treat venous 
thrombosis. Apart from interactions with plasma components such as 
antithrombin III or heparin cofactor II, interactions with blood and 
vascular wall cells may underlie their therapeutic action. The term 
heparin encompasses to a family of unbranched polysaccharide species 
consisting of alternating 1.fwdarw.4 linked residues of uronic acid 
(L-iduronic or D-glucuronic) and D-glucosamine. Crude heparin fractions 
commonly prepared from bovine and porcine sources are heterogeneous in 
size (5,000-40,000 daltons), monosaccharide sequence, sulfate position, 
and anticoagulant activity. Mammalian heparin is synthesized by connective 
tissue mast cells and stored in granules that can be released to the 
extracellular space following activation of these cells. Overall, heparin 
is less abundant than related sulfated polysaccharides, such as heparan 
sulfate, dermatan sulfate, and chondroitin sulfate, which are synthesized 
in nearly all tissues of vertebrates. Heparin and these other structures 
are commonly referred to as glycosaminoglycans. 
The anticoagulant activity of heparin derives primarily from a specific 
pentasaccharide sequence present in about one third of commercial heparin 
chains purified from porcine intestinal mucosa. This pentasaccharide, 
-.alpha.G1cNR16S.beta.(1-4)G1cA.alpha.(1-4)G1cNS3S6R2.alpha.(1-4)IdoA2S.al 
pha.(1-4)G1cNS6S where R1=--SO.sub.3 -- or --COCH.sub.3 and R2=--H or 
--SO.sub.3 --, is a high affinity ligand for the circulating plasma 
protein, antithrombin (antithrombin III, AT-III), and upon binding induces 
a conformational change that results in significant enhancement of 
antithrombin's ability to bind and inactivate coagulation factors, 
thrombin, Xa, IXa, VIIa, XIa and XIIa. For heparin to promote 
antithrombin's activity against thrombin, it must contain the specifically 
recognized pentasaccharide and be at least 18 saccharide units in length. 
This additional length is believed to be necessary in order to bridge 
antithrombin and thrombin, thereby optimizing their interaction. Other 
polymers found in heparin have platelet inhibitory effects or fibrinolytic 
effects. In clinical development are the low molecular weight heparins 
(LMW). The heparin compounds contain only the specific polymers required 
for antithrombin III activation. They have greater specific antithrombotic 
activity and less antiplatelet activity. They also have the characteristic 
of being easier to dose and being safer. 
A major objective of many biotechnology companies and pharmaceutical 
industries is to find safe, easy and effective ways of delivering drugs 
and genes. Specifically, in the area of cardiology, there has been 
tremendous interest in the delivery of drugs and genes into the arterial 
wall by a variety of means. Brief reviews have appeared on gene transfer 
methods related to cardiology (Dzau, et al., TIBTECH, 11:205, 1993; Nabel, 
et al., TCM, Jan.-Feb, issue:12, 1991). On the viral front, retroviruses, 
despite their high efficiency of transfer, have various limitations, such 
as 1) size (&lt;8 kb), 2) potential for activation of oncogenes, 3) random 
integration and, 4) inability to transfect non-dividing cells. Other viral 
vectors such as adenovirus are efficient but have the potential risk of 
infection and inflammation. HVJ-mediated transfection, although highly 
efficient, can exhibit non-specific binding. Liposomes, which have become 
very popular, are safe and easy to work with, but have low efficiency and 
long incubation times. Recent changes in the formulation of liposomes 
have, however, has increased their efficiency several fold. 
Catheter delivery systems, with many different balloon configurations, have 
also been used to locally deliver genes and/or drugs. These include: 
hydrogel balloon, laser-perforated (Wolinsky balloon), `weeping,` channel 
and `Dispatch` balloons and variations thereof (Azrin, et al., 
Circulation, 90:433, 1994; Consigny, et al., J Vasc. Interv. Radiol., 
5:553, 1994; Wolinsky, et al., JACC, 17:174B, 1991; Riessen, et al., JACC, 
23:1234, 1994; Schwartz, Restenosis Summit VII, Cleveland, Ohio, 1995, pp 
290-294). Delivery capacity with hydrogel balloon is limited and, during 
placement, the catheter can lose substantial amount of the drug or agent 
to be introduced. High pressure jet effect in Wolinsky balloon can cause 
vessel injury which can be avoided by making many holes, &lt;1 .mu.m, 
(weeping type). The `Dispatch` catheter has generated a great deal of 
interest for drug delivery and it create circular channels and can be used 
as a perfusion device allowing continuous blood flow. 
Gene transfer to endothelium and vascular smooth muscle cells, and 
site-specific gene expression by retrovirus and liposome have been shown 
feasible, and cell seeding of vascular prosthesis and stents have also 
been described (Nabel, et al., JACC, 17:189B, 1991; Nabel et al., Science, 
249:1285, 1990). An ideal method of gene delivery would be intracellular 
introduction of nucleic acid sequences (e.g., plasmid DNA), locally, to 
give high level gene expression over a reasonable period of time. 
SUMMARY OF THE INVENTION 
The present invention provides a method for local and sustained 
intravascular delivery of a composition in a subject by pulsed electric 
field, or electroporation. The mode of delivery described herein allows 
retention of the composition in a vessel in the subject for an extended 
period of time. The method is a catheter-based system for delivery of 
therapeutic agents, for example, directly into the cells of the vessel 
wall. Sustained, high local concentrations of a composition is achieved 
using the method of the invention. 
The method of the invention is useful for intravascular delivery of such 
compositions as antiproliferative, anticoagulative, antithrombotic, 
antirestenoitic and antiplatelet agents. The method is useful for 
cardiologic applications such as treatment of deep-vein thrombosis (DVT), 
unblocking clogged carotid arteries, peripheral arterial disease and 
cardiovascular restenosis, for example. 
The invention also provides a catheter apparatus for introducing a 
composition into at least one cell in a vessel in a subject.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention provides a method for the local, controlled, and 
sustained intravascular delivery of a therapeutic composition to a vessel 
in a subject using electroporation techniques. The method utilizes pulsed 
electric fields and has an advantage of allowing lower concentrations of 
compositions to be utilized as opposed to high dosages typically used with 
passive delivery modalities. 
The method of the invention provides a delivery system that allows 
controlled sustained, high local concentrations of pharmacologic agents to 
be delivered directly at a site without exposing the entire circulation to 
the agent. Pharmacologic approaches to inhibit smooth muscle cells 
migration and proliferation, for example, have been effectively used at 
supraphysiological doses in animal research studies. However, such high 
concentrations may be impractical for clinical use in humans because of 
the risk of systemic side effects and the lack of specific targeting of 
drugs given systemically at such high dosages. This invention is 
clinically relevant for the local treatment of arteries undergoing 
catheter-based interventions, such as angioplasty, atherectomy, 
rotablating or stenting, for example. 
In a preferred embodiment, the invention provides a method for sustained 
intravascular delivery of a composition to a subject. The method includes 
administering the composition to the subject and applying an electrical 
impulse to a vessel via electroporation, wherein the impulse is of 
sufficient strength and time for the impulse to cause electroporation of 
at least one cell in the interior of the vessel such that the composition 
is delivered into the cells in the vessel and is retained in the vessel 
thereby resulting in sustained delivery. In one aspect of the invention, 
iontophoresis can be employed to further deliver the composition to a 
cell, either prior to, simultaneously with or after electroporation. 
The term "sustained" as used herein means that once the composition is 
delivered to the vessel, it is retained in the vessel for a period of time 
of as long as 24 to about 36 hours, and typically for 12 hours. In other 
words, there is no appreciable washout of the composition as compared with 
the concentration of the composition delivered under conventional delivery 
(e.g., passive diffusion or IO). 
The terms "intravascular" and "vessel" mean any artery, vein or other 
"lumen" in the subject's body to which the electric pulse can be applied 
and to which the composition can be delivered. A lumen is known in the art 
as a channel within a tube or tubular organ. Examples of preferred vessels 
in the method of the invention include the coronary artery, carotid 
artery, the femoral artery, and the iliac artery. While not wanting to be 
bound by a particular theory, it is believed that the electric impulse 
applied to the vessel allows the delivery of the composition primarily to 
the cells of the medial region of the vessel, but also to the intima and 
less so to the adventitia. 
The composition delivered by the method of the invention includes any 
composition which would have a desired biological effect at the site of 
electroporation. For example, preferred compositions include 
antithrombotic, antirestenoitic, antiplatelet, and antiproliferative 
compositions. Other compositions include platelet receptor and mediator 
inhibitors, smooth muscle cell proliferation inhibitors, growth factor 
inhibitors, GpIIb/IIa antagonists, agents that inhibit cell adhesion and 
aggregation, agents that block thromboxane receptors, agents that block 
the fibrinogen receptor, etc. Specific examples of such compositions 
include heparin (including high and low molecular weight and fragments 
thereof), hirulog, tissue plasminogen activator (tPA), urokinase, 
streptokinase, warfarin, hirudin, angiotensin converting enzyme (ACE) 
inhibitors, PDGF-antibodies, proteases such as elastase and collagenase, 
serotonin, prostaglandins, vasoconstrictors, vasodialators, angiogenesis 
factors, Factor VIII or Factor IX, TNF, tissue factor, VLA-4, 
growth-arrest homeobox gene, gax, L-arginine, GR32191, sulotroban, 
ketanserin, fish oil, enoxaprin, cilazapril, forinopril, lovastatin, 
angiopeptin, cyclosporin A, steroids, trapidil, colchicine, DMSO, 
retinoids, thrombin inhibitors, antibodies to von Willebrand factor, 
antibodies to glycoprotein IIb/IIIa, calcium chelation agents, etc. Other 
therapeutic agents (e.g., those used in gene therapy, chemotherapeutic 
agents, nucleic acids (e.g., polynucleotides including antisense, for 
example c-myc and c-myb), peptides and polypeptides, including antibodies) 
may also be administered by the method of the invention. 
The therapeutic composition can be administered alone or in combination 
with each other or with another agent. Such agents include combinations of 
tPA, urokinase, prourokinase, heparin, and streptokinase, for example. 
Administration of heparin with tissue plasminogen activator would reduce 
the dose of tissue plasminogen activator that would be required, thereby 
reducing the risk of clot formation which is often associated with the 
conclusion of tissue plasminogen activator and other thrombolytic or 
fibrinolytic therapies. 
Compositions used in the method of the invention include biologically 
functional analogues of the compositions described herein. For example, 
such modifications include addition or removal of sulfate groups, addition 
of phosphate groups and addition of hydrophobic groups such as aliphatic 
or aromatic aglycones. Modifications of heparin, for example, include the 
addition of non-heparin saccharide residues such as sialic acid, 
galactose, fucose, glucose, and xylose. When heparin is used as the 
composition, it may include a fragment of naturally occurring heparin or 
heparin-like molecule such as heparan sulfate or other glycosaminoglycans, 
or may be synthetic fragments. The synthetic fragments could be modified 
in saccharide linkage in order to produce more effective blockers of 
selectin binding. Methods for producing such saccharides will be known by 
those of skill in the art (see for example: M. Petitou, Chemical Synthesis 
of Heparin, in Heparin, Chemical and Biological Properties, Clinical 
Applications, 1989, CRC Press Boca Raton, Fla. D. A. Lane and V. Lindahl, 
eds. pp. 65-79). 
The composition administered by the method of the invention may be a 
mixture of one or more compositions, e.g., heparin and tPA. Further, 
compositions such as heparin may include a mixture of molecules containing 
from about 2 to about 50 saccharide units or may be homogeneous fragments 
as long as the number of saccharide units is 2 or more, but not greater 
than about 50. 
Where a disorder is associated with the expression of a gene (e.g., IGF-1, 
endothelial cell growth factor), nucleic acid sequences that interfere 
with the gene's expression at the translational level can be delivered. 
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. 
Preferably the subject is a human, however, it is envisioned that the 
method of sustained in vivo delivery of compositions via electroporation 
as described herein can be performed on any animal. 
Preferably, the therapeutic composition is administered either prior to or 
substantially contemporaneously with the electroporation treatment. The 
term "substantially contemporaneously" means that the therapeutic 
composition and the electroporation treatment are administered reasonably 
close together with respect to time. 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. The composition can be 
administered at any interval, depending upon such factors, for example, as 
the nature of the clinical situation, the condition of the patient, the 
size and chemical characteristics of the composition and half-life of the 
composition. 
The composition administered in the method of the invention can be 
administered parenterally by injection or by gradual perfusion over time. 
The composition can be administered intravenously, intraperitoneally, 
intramuscularly, subcutaneously, intracavity, or transdermally, and 
preferably is administered intravascularly at or near the site of 
electroporation. 
Preparations for administration include sterile aqueous or non-aqueous 
solutions, suspensions, and emulsions. Examples of non-aqueous solvents 
are propylene glycol, polyethylene glycol, vegetable oils such as olive 
oil, and injectable organic esters such as ethyl oleate. Aqueous carriers 
include water, alcoholic/aqueous solutions, emulsions or suspensions, 
including saline and buffered media. Vehicles include sodium chloride 
solution, Ringer's dextrose, dextrose and sodium chloride, lactated 
Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient 
replenishers, electrolyte replenishers (such as those based on Ringer's 
dextrose), and the like. Preservatives and other additives may also be 
present such as, for example, antimicrobials, anti-oxidants, chelating 
agents, and inert gases and the like. Further, vasoconstrictor agents can 
be used to keep the therapeutic composition localized prior to pulsing. 
In another embodiment, the invention provides a catheter device 100 useful 
in the method of the invention that can be modified as described herein, 
as shown in FIGS. 1, 6, and 7. The catheter may be, for example, a 
modified Berman catheter (Arrow International, Inc., Reading, Pa.). One of 
skill in the art will know of other balloon catheter devices for 
endoluminal electroporation mediated drug delivery that can be modified 
according to the present invention. 
The catheter 100 may include at least one inflatable balloon 102 near the 
distal end of the catheter 100, and at least one inflation port 104 for 
inflating each balloon 102, in a conventional manner. The catheter 100 
also includes a first electrode 110 and a second electrode 112 that are 
coupled by wires to a voltage source generator 114, which may be, for 
example, an ECM 600 exponential generator from BTX, a division of 
Genetronics, Inc., San Diego, Calif. The first electrode 110 is preferably 
placed close to at least one infusion opening 120. In one embodiment, the 
infusion openings 120 may be coincident with the first electrode 110, such 
that the first electrode 110 completely surrounds at least one infusion 
opening 120. 
The first electrode 110 is preferably made of an electrically conductive 
material that is biologically compatible, e.g., biologically inert, with a 
subject. Examples of such material include silver or platinum wire wrapped 
around or laid on or near the surface of the catheter 100; a plated or 
painted coating of conductive material, such as silver paint, on some 
portion of the catheter 100; or a region of the catheter 100 that has been 
made conductive by implantation (during or after manufacture, such as by 
ion implantation) of electrically conductive materials, such as powdered 
metal or conductive fibers. The conductor need not be limited to metal, 
but can be a semiconductor or conductive plastic or ceramic. For ease of 
manufacture, the embodiments illustrated in FIGS. 6 and 7 use conductive 
silver paint for the first electrode 110 as a coating on approximately 2.5 
cm of the length of the catheter 100 near the infusion ports 120. 
The second electrode 112 similarly comprises an electrically conductive 
material, and can be of the same or different type of conductive material 
as the first electrode 110. In the embodiment shown in FIG. 6, the second 
electrode comprises a silver plate 112a configured to be applied to a 
portion of the body of a subject such that an electric field sufficient to 
cause electroporation of at least one cell in a vessel is generated when 
voltage from the voltage source 114 is applied to the first electrode 110 
and the second electrode 112. The second electrode, when placed 
externally, is preferably placed on bare skin (e.g., shaved abdominal 
muscle of the subject), preferably using a conductive gel for better 
contact. FIG. 7 shows that the second electrode 112 may be a conductive 
guide wire for the catheter 100. 
The first electrode 110 and the second electrode 112 are coupled to the 
voltage source 114 by conductors, which may be, for example, silver or 
platinum wires, but can be any conductive structure, such as flexible 
conductive ink within the catheter 100 for connecting the first electrode 
110. 
The infusion ports 120 can be made during or after manufacture of the 
catheter 100, and can be placed on one or both sides of the first 
electrode 110, or within the bounds of the first electrode 110. 
In an alternative embodiment, the second electrode 112 may be formed in a 
manner similar to the first electrode 110 and positioned between the first 
electrode 110 and the infusion openings 120, or positioned with the 
infusion openings 120 between the first electrode 110 and the second 
electrode 112. Other configurations of the first electrode 110 and the 
second electrode 112 can be utilized, such as interdigitated electrodes 
with infusion openings 120 nearby or between the interdigitated "fingers" 
of the electrodes, or as concentric rings with the infusion openings 
within the centermost ring, between the centermost and outermost ring, 
and/or outside of the outermost ring. Additional configurations are within 
the scope of the present invention so long as they provide a structure 
that, when supplied by voltage from the voltage source 114, generates an 
electric field sufficient to cause electroporation of at least one cell in 
the vessel. 
In operation, the catheter 100 is positioned so that a balloon 102 
traverses or crosses a stenotic lesion, for example, and the balloon 102 
is inflated to expand the vessel (e.g., an artery or vein), thereby 
dilating the lumen of the vessel. A therapeutic composition is delivered 
into the vessel via the infusion openings 120, and at least during part of 
the time before, during, or after infusion occurs, electrical pulses from 
the voltage source 114 are applied to the first electrode 110 and second 
electrode 112 so as to cause electroporation of at least one cell in the 
vessel. Following delivery of the therapeutic composition to such cell, 
the catheter may be withdrawn, unless additional composition delivery and 
electroporation is desired. 
The methods described above are also applicable with metallic stents. The 
stent itself forms one set of electrodes while a guide wire acts as the 
second electrode. Stents, on their own, or coated with heparin, are useful 
for reduction of restenosis. Such results can be further augmented when 
combined with pulsed electric fields. This would be particularly suitable 
for angioplasty where a stent is deployed. (For detailed review, see de 
Jaegere, P. P. et al., Restenosis Summit Proc. VIII, 1996, pp 82-109). 
Stent implantation, along with local delivery of antirestenotic drugs by 
pulsed electric fields reduces the restenosis rate. Besides a normal 
stent, a retractable or biodegradable stent can also be used with this 
mode of delivery. 
In another aspect of the invention, the described method is useful for 
bypass grafts. These can include aortocoronary, aortoiliac, aortorenal, 
femoropopliteal. In the case of a graft with autologous or heterologous 
tissue, the cells in the tissue can be electroporated, ex vivo, with a 
nucleic acid encoding a protein of interest. Since electroporation is 
relatively fast, a desired nucleic acid can be transferred in a saphenous 
vein, e.g., outside the body, while the extracorporeal circulation in the 
patient is maintained by a heart-lung machine, and the vein subsequently 
grafted by standard methods. Where synthetic material is used as a graft, 
it can serve as a scaffolding where appropriate cells containing a nucleic 
acid sequence of interest that has been electroporated, ex vivo, can be 
seeded. 
The method of the invention can be used to treat disorders by delivery of 
any composition, e.g., drug or gene with a catheter, as described herein. 
For example, patients with peripheral arterial disease, e.g., critical 
limb ischemia (Isner, J. M. et al, Restenosis SummitVIII, Cleveland, Ohio, 
1996, pp 208-289) can be treated as described herein. Both viral and 
non-viral means of gene delivery can be achieved using the method of the 
invention. These include delivery of naked DNA, DNA-liposome complex, 
ultraviolet inactivated HVJ (haematoagglutanating virus of Japan) liposome 
vector, delivery by particle gun (e.g., biolistics) where the DNA is 
coated to inert beads, etc. Various nucleic acid sequences encoding a 
protein of interest can be used for treatment of cardiovascular disorders, 
for example. The expression of the growth factors PDGF-B, FGF-1 and 
TGF.beta.1 has been associated with intimal hyperplasia, therefore, it may 
be desirable to either elevate (deliver sense constructs) or decrease 
(deliver antisense) such gene expression. For example, whereas PDGF-B is 
associated with smooth muscle cell (SMC) proliferation and migration, 
FGF-1 stimulates angiogenesis and TGF .beta.1 accelerates procollagen 
synthesis. 
Any composition that inhibits SMC proliferation and migration, platelet 
aggregation and extracellular modeling is also desirable for use in the 
electroporation-mediated delivery method of the invention. Such 
compositions include interferon-.gamma. which inhibits proliferation and 
expression of .alpha.-smooth muscle actin in arterial SMCs and non-protein 
mediators such as prostaglandin of the E series. 
Examples of other genes to be delivered by the method of the invention 
includes Vascular endothelial growth factor (VEGF) and endothelial 
specific mitogen, which can stimulate angiogenesis and regulate both 
physiologic and pathologic angiogenesis. 
Administration of the composition in the method of the invention may be 
used for ameliorating post-reperfusion injury, for example. When treating 
arterial thrombosis, induction of reperfusion by clot lysing agents such 
as tissue plasminogen activator (tPA) is often associated with tissue 
damage. 
Administration of the composition by the method of the invention, alone or 
in combination with other compositions, for example that may be 
administered passively, is useful in various clinical situations. These 
include but are not limited to: 1) acute arterial thrombotic occlusion 
including coronary, cerebral or peripheral arteries; 2) acute thrombotic 
occlusion or restenosis after angioplasty; 3) reocclusion or restenosis 
after thrombolytic therapy (e.g., in an ishemic tissue); 4) vascular graft 
occlusion; 5) hemodialysis; 6) cardiopulmonary bypass surgery; 7) left 
ventricular cardiac assist device; 8) total artificial heart and left 
ventricular assist devices; 9) septic shock; and 10) other arterial 
thromboses (e.g., thrombosis or thromboembolism where current therapeutic 
measures are either contraindicated or not effective). 
The method of the invention is also useful for the treatment of microbial 
infections. Many microbes, such as bacteria, rickettsia, various 
parasites, and viruses, bind to vascular endothelium and leukocytes. Thus, 
the method of the invention may be used to administer a composition to a 
patient to prevent binding of a microbe which uses a particular receptor 
(e.g., selectin) as its binding target molecule, thereby modulating the 
course of the microbial infection. 
The method of the invention can be used to treat vasculitis by 
administering to a patient a composition described above. Tissue damage 
associated with focal adhesion of leukocytes to the endothelial lining of 
blood vessels is inhibited by blocking the P- and L-selectin receptors, 
for example. 
The dosage ranges for the administration of the compositions in the method 
of the invention are those large enough to produce the desired effect in 
which the symptoms of the disease/injury are ameliorated. The dosage 
should not be so large as to cause adverse side effects. Generally, the 
dosage will vary with the age, condition, sex and extent of the disease in 
the patient and can be determined by one of skill in the art. The dosage 
can be adjusted by the individual physician in the event of any 
complication. When used for the treatment of inflammation, 
post-reperfusion injury, microbial/viral infection, or vasculitis, or 
inhibition of the metastatic spread of tumor cells, for example, the 
therapeutic composition may be administered at a dosage which can vary 
from about 1 mg/kg to about 1000 mg/kg, preferably about 1 mg/kg to about 
50 mg/kg, in one or more dose administrations. 
Controlled delivery may be achieved by selecting appropriate 
macromolecules, for example, polyesters, polyamino acids, polyvinyl 
pyrrolidone, ethylenevinylacetate, methylcellulose, 
carboxymethylcellulose, protamine sulfate, or lactide/glycolide 
copolymers. The rate of release of the therapeutic composition may be 
controlled by altering the concentration of the macromolecule. 
Another method for controlling the duration of action comprises 
incorporating the composition into particles of a polymeric substance such 
as polyesters, polyamino acids, hydrogels, polylactide/glycolide 
copolymers, or ethylenevinylacetate copolymers. Alternatively, it is 
possible to entrap the composition in microcapsules prepared, for example, 
by coacervation techniques or by interfacial polymerization, for example, 
by the use of hydroxymethylcellulose or gelatin-microcapsules or 
poly(methylmethacrolate) microcapsules, respectively, or in a colloid drug 
delivery system. Colloidal dispersion systems include macromolecule 
complexes, nanocapsules, microspheres, beads, and lipid-based systems 
including oil-in-water emulsions, micelles, mixed micelles, and liposomes. 
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 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. 
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 BTX, a division of 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 ECM 
600 signal generator, the electroporation pulse length is set by selecting 
one often timing resistors marked R1through 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). 
The application of an electrical field across the cell membrane results in 
the creation of transient pores which are critical to the eletroporation 
process. The ECM 600 signal generator provides the voltage (in kV) that 
travels across the gap (in cm) between the electrodes. This potential 
difference defines what is called the electric field strength where E 
equals kV/cm. Each cell has its own critical field strength for optimum 
electroporation. This is due to cell size, membrane make-up and individual 
characteristics of the cell wall itself. For example, mammalian cells 
typically require between 0.5 and 5.0 kV/cm before cell death and/or 
electroporation occurs. Generally, the required field strength varies 
inversely with the size of the cell. 
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 maximum 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 LVM mode, by a simultaneous combination of resistors parallel to 
the output and a bank of seven selectable additive capacitors. 
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. 
The waveforms of the voltage pulse provided by the generator in the power 
pack can be an exponentially decaying pulse, a square pulse, a unipolar 
oscillating pulse train or a bipolar oscillating pulse train, for example. 
Preferably, the waveform used for the method of the invention is an 
exponential pulse. The voltage applied between the at least first and 
second electrode is sufficient to cause electroporation of the vessel such 
the composition delivered to the vessel is retained for a period of time, 
as described above. The field strength is calculated by dividing the 
voltage by the distance (calculated for 1 cm separation; expressed in cm) 
between the electrodes. For example, if the voltage is 500 V between two 
electrode faces which is 1/2 cm apart, then the field strength is 
500/(1/2) or 1000 V/cm or 1 kV/cm. Preferably, the amount of voltage 
applied between the electrodes is in the range of about 10 volts to 200 
volts, and preferably from about 50 to 90 volts. 
The pulse length can be 100 microseconds (.mu.s) to 100 millisecond (ms) 
and preferably from about 500 .mu.s to 10 ms. There can be from about 1 to 
10 pulses applied to an area or group of cells. The waveform, electric 
field strength and pulse duration are dependent upon the exact 
construction of the catheter device and types of molecules in the 
composition to be transferred to the cells or vessel via electroporation. 
One of skill in the art would readily be able to determine the appropriate 
pulse length and number of pulses. 
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. 
EXAMPLE 1 
ENDOLUMINAL INJECTION OF FLUORESCEINATED HEIN AND PULSED ELECTRICAL 
STIMULATION OF THE CAROTID ARTERY IN A SPONTANEOUSLY BREATHING RABBIT 
1. Methods 
Experiments were performed in 12 New Zealand white rabbits of either sex 
(2.5-3.4 kg) preanesthetized with xylazine (2 mg.kg.sup.-1) and ketamine 
(50 mg.kg.sup.-1) intramuscularly and an injection of alphachloralose (30 
mg.kg.sup.-1) intraveneously through an ear vein. A supplemental dose of 
10 mg.kg.sup.-1 chloralose was given every hour. The anesthetic state was 
maintained such that the toe-pinching reflex and corneal reflexes were 
absent. 
All experiments were conducted in accordance with the guidelines adopted by 
American Physiological Society on the use of animals for research. 
Animals were placed supine and strapped on the surgical table. The trachea 
was intubated to allow spontaneous breathing of ambient air. 
Electrocardiogram (EKG) of the animal was obtained by using Lead II in 
differential mode. End-tidal CO.sub.2 tension was monitored by a CO.sub.2 
analyzer (Datex, Puritan-Bennett). Body temperature was kept at the 
38-38.5.degree. C. range by radiant heating. 
2. Surgical Preparation and Experimental Protocol 
A longitudinal incision in the cervical region was made in the rabbit to 
expose the common carotid arteries on both sides. Approximately 6 cm in 
length of carotid artery on each side was isolated from the surrounding 
tissue and vagosympathetic nerve trunk. The caudal end of the carotid 
artery on one side was transiently occluded with a vascular clip at the 
junction between the neck and chest. A small incision was then made at the 
rostral end of the artery just below transversus vein) to push an 
electroporator catheter (FIG. 1) through this incision. After insertion of 
the catheter, the catheter balloon was repeatedly inflated for 30 seconds 
inside the arterial lumen in order to denude the endothelial lining. An 
indelible ink mark was placed on the inflated portion of the artery. The 
balloon was then deflated and the catheter tip was held just above the 
vascular clip. 
A 0.2 ml of freshly prepared diluted heparin (1 mg. of fluoresceinated 
heparin (F-heparin) with an activity of 167 unit/mg Molecular Probe, 
Inc.! dissolved in 4 ml) was injected through the one port of a double 
lumen catheter over a period of about 10 seconds. The catheter was then 
pulled out of the artery and the vascular clip was taken off from the 
caudal end to restore blood flow in the artery. Exactly the same procedure 
was adopted for the contralateral carotid artery (test artery). The only 
exception was that for the test artery, the carotid artery was stimulated 
intraluminally using a platinum or silver electrode. Two platinum or 
silver wires were coiled around the catheter just above the balloon for a 
length of about 10 mm with an interelectrode distance of 2 mm-3 mm. 
Lead II EKG was differentially amplified and the output was continuously 
monitored on an osciloscope (Tektronix) and recorded on a Gould TA-2000 
thermal-array recorder for evaluation. 1-12 hours after heparin injection, 
both carotid arteries were excised and immediately flash frozen in 
isopentane pre-chilled in liquid nitrogen. Arteries were stored in 
-70.degree. C. until further processing. 
Arterial segments were subsequently freeze sectioned (10 micron) 
transversely. Microscopic slides containing arterial sections were 
observed under a Zeiss confocal laser (argon-krypton) scan microscope (LSM 
410 Invert), (excitation at 495 nm and emission at 515 nm) to obtain video 
image (magnification 40 times) of fluorescence. Subsequently, control and 
test samples were compared by analyzing fluorescence intensity by Line 
Intensity Scan at different depths of the arterial wall using commercially 
obtained software (Image 1:Universal Imaging Corp.). 
3.Protocols of Pulsed Stimulation 
The luminal wall of the carotid artery was stimulated through bipolar 
platinum or silver electrodes, which were laid against the luminal surface 
sufficiently without damage. Pulsed activation of the luminal surface was 
obtained using an exponential pulse generator (Model ECM 600, BTX, a 
division of Genetronics, Inc., San Diego, Calif.). Four pulses of 50-60 V 
amplitude with a pulse width of .about.500 .mu.s were applied over a 
period of 60 seconds. This protocol was adopted either for the left or 
right carotid artery. 
4. Observation and Data Analysis 
During pulse stimulation of the carotid artery, mild twitching of the 
cervical region could be seen, but no appreciable change was observed in 
EKG dynamics over the entire experimental duration. 
Green fluorescence heparin of the arterial wall could be distinctly seen in 
the microscopic slide preparations (in different layers of the arterial 
wall). Confocal scan image of the arterial wall showed penetration of 
F-heparin in both control and test samples. However, it was evident that 
the flourescent-intensity in the test sample was much stronger and went 
into the deeper region of the arterial wall (FIGS. 2-5). 
The pulsed electrical stimulation facilitated introduction of small amount 
(.about.50 ug) of F-heparin effectively to the deeper region of arterial 
wall in a physiologically normal experimental animal. Heparin was mostly 
present in the media but also in the intima of the vessel wall. However, 
the intensity dropped significantly towards the adventitia. It is possible 
that only the portion of the electrode making contact with the luminal 
wall shows more fluorescence than the adjacent space. From the tissue 
sectioning, it is not possible to say which portion of the tissue 
sectioning of the luminal wall sample had contact with the electrodes. 
However, it is possible that if some sections in the test sample show 
greater penetration and intensity than the others, those sections probably 
were in contact with the luminal wall. Also, the fluorescent image could 
not ascertain if balloon inflation of the bilateral arteries had equal 
degree of endothelial denudation, the variation in which could alter the 
penetration of F-heparin among the samples. 
FIG. 1 shows a schematic of the catheter used in the above examples. One of 
the problems of working with fluoresceinated heparin is that there is 
considerable amount of autofluorescence from the collagen and elastin of 
the tissue sample. In absolute terms of fluorescent intensity, these tend 
to distort the real pattern of the fluorescence in the vessel wall due to 
heparin alone. However, in the present examples, in every case, it is 
clear that the relative fluorescent intensity was always stronger in the 
treated vessel that was pulsed compared to the non-pulsed artery. All the 
photographs had identical magnification (40.times.) and the brightness and 
contrast were set to the same level for photography (FIGS. 2-5). All 
epifluorescence images were monitored in Sony videocon monitor attached to 
a Hamamatsu CCD camera. 
However, by processing the samples at higher pH (9.0), it was possible to 
considerably reduce or even eliminate the interfering autofluorescence. 
The photos of FIGS. 2-5 indicated that the local delivery of heparin in 
the vessel completely washes out in two hours, whereas heparin delivery in 
the pulsed artery was sustained for at least 12 hours. 
EXAMPLE 2 
FIG. 6 shows another configuration for a catheter useful in the method of 
the invention, whereby conductive silver paint or a similar conductive 
material is placed around the catheter covering a length of approximately 
2.5 cm. This portion of the catheter is attached to a silver wire which, 
in turn, is connected to one terminal of a generator, e.g., ECM 600 
exponential generator (BTX, a division of Genetronics, Inc., San Diego, 
Calif.). The second electrode is placed externally and is placed on the 
abdominal muscle, preferably using a gel for better contact (FIG. 6, 
shaved area). This second electrode, serving as the anode, is in turn 
connected to the other terminal of the generator. 
Another embodiment of the catheter comprises one electrode positioned 
between two balloons and a guidewire acting as a second catheter. Such a 
configuration is shown in FIG. 7. This catheter was used in the following 
experiment. Three rabbits weighing .about.4 Kg were anesthetized with 
xylazine (0.1 ml/kg) and ketamine (0.5 ml/kg i.m.). General anesthesia was 
maintained with .alpha.-chloralose (30 mg/Kg. i.v.). Intubation was 
endotracheal, as described in example 1. A femoral artery in the leg on 
one side of the rabbit was exposed. A 5F sheath was introduced and the 
catheter was pushed under fluoroscopic guidance to the right or left 
carotid artery. A series of x-rays, FIG. 8, panels a-c, show successful 
deployment of the catheter (panel a, insertion). Radiocontrast fluid was 
infused (panel b) allowing confirmation of the catheter position, the 
patient artery, the balloon and the built-in radiopaque marker, as well as 
presence of the dye in the side branches. After balloon inflation, (panel 
c) 1 ml of fluoresceinated heparin (concentration 1 mg dissolved in 2 ml: 
biological activity of heparin as per manufacturer: 167 U) was infused 
between the occluded segment via the drug port and the artery pulsed 
immediately with the balloons in the inflated condition. Initially, field 
parameters tested were .about.60 V and four pulses each of .about.600 
.mu.s pulse length. With these settings, very little uptake of heparin was 
observed in the treated artery. In a subsequent experiment, voltage and 
pulse length were changed to 57 V and 22 ms, respectively. As before, four 
pulses were delivered from ECM 600 pulse exponential generator. The 
balloon was deflated immediately afterwards with the catheter taken out, 
but the sheath was left behind to avoid bleeding from the nicked femoral 
artery. Two hours after infusion of F-heparin, both arteries (treated and 
the contralateral untreated artery) were taken out for processing. 
Microscopic images of the treated artery showed massive uptake of the 
heparin. The fluorescent image of the artery was extremely intense, and 
the separated arterial sections could not be discerned. Although the 
control artery also shows fluorescence, visually it was much weaker. 
Although heparin was not delivered into the control artery, it is obvious 
that there was systemic circulation from infusion of heparin in the 
treated artery- part of which must have been taken up by the control 
artery. In addition, fluorescence due to collagen and elastin was also 
present. However, both autofluorescence correction at higher pH, as 
described previously, and computer subtraction of the fluorescence from 
the control artery from that of the treated artery, showed deep 
penetration and uptake of the F-heparin in the pulsed artery. 
A similar catheter (as depicted in FIG. 7) was also used for a gene marking 
experiment in a rabbit carotid artery. A New Zealand white rabbit weighing 
3.5 Kg was anesthetized with ketamine/xylene cocktail (IM). Intubation was 
with halothane @1%. After a midline incision, the right common carotid was 
isolated with silk ligature. 5F sheath was placed into right common 
carotid over the guidewire after an initial scissor nick in artery. 014" 
Schneider guidewire was placed through the sheath into the left iliac 
artery. The electroporation (EP) catheter was advanced over the wire to 
left iliac artery. 50% contrast injections with the balloon inflated 
through the infusion port guided placement to avoid side branches. The 
infusion sleeve was flushed with saline and the balloons inflated 2 atom. 
Plasmid (150 .mu.l) (a standard marker gene, lacZ, driven by a CMV 
promoter) was injected into the infusion port followed by saline. The 
iliac was pulsed from a BTX ECM 600 exponential pulse generator. Three 
pulses were given at approximately 10 sec intervals at 76 V and 758 .mu.s. 
For the control artery, balloons were deflated and the wire placed down the 
right iliac. The procedure was as described above, except that no pulse 
was applied. The dwell time was .about.30 secs. After the procedure, the 
balloons were deflated and catheters and wires removed. The carotid was 
ligated proximal and distal to the entry site and the incision was closed 
in 2 layers. 1500 units of heparin were given after the sheath was in 
place. 
The plasmid DNA was electroporated into the rabbit iliac artery (catheter 
was guided through to the iliac via the carotid as described above) and 
gene expression was confined five days later using standard x-gal 
processing of the artery. In contrast, the control artery did not show 
detectable gene expression. 
EXAMPLE 3 
For further drug delivery studies, the same protocol will be followed as 
described in detail in Example 1. Forty New Zealand white rabbits will be 
used for these studies. Time points of approximately 2 hours and 24 hours 
(group 1) will be tested with balloon catheters as described herein. 
Twenty animals, ten animals in each of the time points of group 1, will be 
used. Both the left and the right arteries will serve as the treated (T) 
and the control (C). These will be chosen randomly but the number for the 
T and C will be the same. An ECM 600 pulse generator, which delivers 
exponential pulses and was used to generate the results described above, 
will also be used for these experiments. 
Ten animals will be tested with square wave pulses from a BTX T820 Square 
Wave Pulser and arteries will be excised after two hours for subsequent 
studies. The arteries which will serve as T and C will be randomized. BTX 
T820 delivers square wave pulses where the number of pulses, the voltage 
and the pulse length can be adjusted. The voltage is about 60 V and the 
pulse parameters are: four pulses delivered at 1 Hz each of 40 ms (based 
on studies with the BTX T820 on rat vascular smooth muscle cell 
experiments in vitro). Square wave pulses have been known to be gentler to 
some cells. In this group, there will be five arteries in each of the 
treated and control category. The inflammatory response of the vessel due 
to balloon inflation as well as application of the pulsed electric field 
is also evaluated. 
Twenty rabbits will be used where the catheter will be introduced either 
percutaneously or via a small incision in the femoral. This would give 
results on twenty treated and twenty control arteries. Arteries will be 
processed after eight hours. The ECM 600 will be used to deliver 
exponential pulses. An endoluminal balloon catheter used herein has one 
electrode between two balloons whereas the guide wire will serve as the 
second electrode (one design). To facilitate proper viewing of the 
balloons in the inflated and the deflated position under fluoroscopic 
guidance, radio-opaque markers will be put in appropriate positions. 
Calculations suggest that there will be enough field penetration into the 
arteries to deliver drugs although the electrodes are not in direct 
contact with the arteries. 
For each of the specific aims given above, electric field plots will be 
generated using a commercially available software package EMP (Field 
Precision, Albuquerque, N. Mex.). This package solves Poisson's equation 
is solved numerically by finite elements methods. The initial parameters 
are electrode geometry, resistivities of the artery from the lumen side 
and the connective tissue side and the range of field strength to be 
investigated. 
The amount of heparin left in the vessel will be determined in each case 
following a procedure recommended by Molecular Probe. An InSpeck 
Microscope Image Intensity Calibration Kit will be used. First, the 
microscope will be calibrated with the beads (microsphere) provided in the 
kit and the fluorescein-heparin solution will be equilibrated to the 100% 
microsphere. Alternatively, for different size microsphere, the available 
figures for "fluorescein equivalent per microsphere" can be used. 
The protocol for reduction of autofluorescence due to collagen and elastin 
from the arterial wall of the isolated rabbit carotid artery is as 
follows: Tris-buffered glycerol is prepared (90 ml glycerol and 5 ml of 
0.5M Tris-HCl, pH 9.0). This is dispensed in 19 ml aliquots in glass 
scintillation vials and stored 4.degree. C. 2% n-propyl gallate (npg: 
anti-fading substance) is prepared in tris-buffer (2 mg npg and 1.0 ml of 
0.5M tris-HCl, pH 9.0) is prepared fresh and protected from light. 1 ml of 
the 2% npg solution is added to 19 ml of tris-buffered glycerol and the 
solution is protected from light. This is the solution used to mount 
arterial sections on to the microscopic glass slides. Precaution needs to 
be taken that the solution is discarded on discoloration. All images will 
be obtained at 40.times. magnification under immersion oil (Plan-Neofluor 
objective). Identical brightness and contrast will be set for all 
photographs. 
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.