U.S. patent number 6,041,252 [Application Number 08/476,714] was granted by the patent office on 2000-03-21 for drug delivery system and method.
This patent grant is currently assigned to Ichor Medical Systems Inc.. Invention is credited to Robert M. Bernard, Jeffrey P. Walker.
United States Patent |
6,041,252 |
Walker , et al. |
March 21, 2000 |
Drug delivery system and method
Abstract
A method for delivering a therapeutic agent to a predetermined
location in a host is disclosed, wherein a liposome-encapsulated
therapeutic agent is administered to the host, and an electrical
field which encompasses a predetermined region within the host is
established, such that as the liposome-encapsulated agent is
exposed to the electrical field the release of the agent from the
liposome to the predetermined region is enhanced.
Inventors: |
Walker; Jeffrey P. (San Diego,
CA), Bernard; Robert M. (Rancho Santa Fe, CA) |
Assignee: |
Ichor Medical Systems Inc. (San
Diego, CA)
|
Family
ID: |
23892959 |
Appl.
No.: |
08/476,714 |
Filed: |
June 7, 1995 |
Current U.S.
Class: |
604/20;
435/173.6; 435/285.2; 604/21; 607/72 |
Current CPC
Class: |
A61N
1/0412 (20130101); A61N 1/0476 (20130101); A61N
1/30 (20130101); A61N 1/327 (20130101); A61K
9/0009 (20130101); A61K 9/127 (20130101); A61N
1/0428 (20130101); A61N 1/306 (20130101) |
Current International
Class: |
A61N
1/30 (20060101); A61N 001/30 () |
Field of
Search: |
;604/20-21,49 ;935/52-53
;435/173.6,285.2 ;607/72 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Bockelman; Mark
Attorney, Agent or Firm: Weseman, Esq.; James C. The Law
Offices of James C. Weseman
Claims
We claim:
1. A system for the delivery of electroporation-inducing electrical
fields to a patient comprising:
a plurality of electrodes adapted to be located within a
predetermined three-dimensional space in a patient, said electrodes
arranged to provide at least one reference electrode in
electrically--conductive communication with at least two
geometrically-oriented satellite electrodes; and
electrical pulse generating means for generating
electroporation-inducing electrical fields incorporating control
means connected to at least three of said plurality of electrodes
said control means capable of varying the number and order of
electrodes that participate in each pulse delivered by said pulse
generating means such that a pulse many be delivered to at least
said reference electrode and at least two of said satellite
electrodes simultaneously.
2. A system as recited in claim 1, said control means further
comprising means to control the electrical parameters and temporal
relationship of the electrical pulses applied to each of said
electrodes.
3. A system as recited in claim 2, wherein said control means
comprises a digital computer.
4. A system as recited in claim 2, wherein said control means can
apply electrical pulses of different polarity to selected
electrodes so as to redefine the reference and satellite
relationship as between the plurality of electrodes.
5. A system as recited in claim 1, wherein the electrical pulse
generating means generates an electrical field strength in the
range of approximately 0.4 kV/cm to approximately 1.3 kV/cm in the
tissue of a patient.
6. A system for the delivery of electroporation-inducing electrical
fields to a patient comprising:
a plurality of electrodes adapted to be located within a
predetermined three-dimensional space in a patient, said electrodes
arranged to provide at least one reference electrode in
electrically-conductive communication with at least two
geometrically-oriented satellite electrodes;
electrical pulse generating means for generating
electroporation-inducing electrical fields incorporating control
means connected to at least three of said plurality of electrodes
capable of varying the number and order of electrodes that
participate in each pulse delivered such that a pulse may be
delivered to at least said reference electrode and at least two of
said satellite electrodes simultaneously; and
control means connecting said electrical pulse generating means to
at least three of said electrodes, which control means directs the
electrical parameters and temporal relationship of the electrical
pulses applied to each of said electrodes.
7. A system as recited in claim 6, wherein said control means
comprises a digital computer.
8. A system as recited in claim 6, wherein said control means can
apply electrical pulses of different polarity lo selected
electrodes so as to redefine the reference and satellite
relationship as between the plurality of electrodes.
9. A system as recited in claim 6, wherein the electrical pulse
generating means generates an electrical field strength in the
range of approximately 0.4 kV/cm to approximately 1.3 kV/cm in the
issue of a patient.
10. A system for the delivery of electroporation-inducing
electrical fields to a patient comprising:
a plurality of electrodes adapted to be located within a
predetermined three-dimensional space in a patient, said electrodes
arranged to provide at least one reference electrode in
electrically-conductive communication with at least two
geometrically-oriented satellite electrodes;
electrical pulse generating means for generating
electroporation-inducing electrical fields incorporating control
means connected to at least three of said plurality of electrodes
capable of varying the number and order of electrodes that
participate in each pulse delivered such that a pulse may be
delivered to at least said reference electrode and at least two of
said satellite electrodes simultaneously; and
control means connecting said electrical pulse generating means to
at least three of said electrodes, which control means can apply
electrical pulses of different polarity to selected electrodes so
as to redefine the reference and satellite relationship as between
the plurality of electrodes.
11. A system as recited in claim 10, wherein said control means
comprises a digital computer.
12. A system as recited in claim 10, wherein the electrical pulse
generating means generates an electrical field strength in the
range of approximately 0.4 kV/cm to approximately 1.3 kV/cm in the
tissue of a patient.
Description
TECHNICAL FIELD
This invention relates generally to the delivery of therapeutic
agents to specific locations in patients, and, more particularly,
to effecting such delivery by utilizing electrical fields to
localize the delivery of such agents within the patient.
BACKGROUND OF THE INVENTION
Numerous medical therapies have attempted to treat localized
disease in the body of a patient with techniques designed to direct
the appropriate drug to the affected area and to avoid unacceptable
or toxic side effects to healthy tissue. For example, therapies
have been proposed utilizing liposomes as vehicles to carry the
appropriate drugs to the diseased area.
Liposomes are microscopic particles which are made up of one or
more lipid bilayers enclosing an internal compartment. They are not
normally leaky but can become leaky if a hole or pore occurs in the
membrane, if the membrane is dissolved or degrades, or if the
membrane temperature is increased to the transition temperature,
T.sub.C. The major barrier to the use of liposomes as drug carriers
is making the liposome release the drugs on demand at the target
sites (Science 202:1290 (1978)).
The specific use of applied heat to raise the liposome temperature
to T.sub.C to make them leaky or permeable has been described
(Science 204:188 (1979)). This technique has been proposed in U.S.
Pat. No. 5,190,761 in which a method of activating liposomes to
release their encapsulated drugs in tissue utilizing microwave
radiation is described.
Additionally, it has been proposed that electroporation can be used
to deliver what are normally non-permeable substances into the
interior of tumor cells, thus affecting changes on an intracellular
basis. Attempts to perform this delivery have only recently been
successful (Ceberg et al. (1994)). One difficulty has been the
confinement of the electroporation effect to the desired area.
Widespread electroporation effects have been described in which not
only the diseased area but also normal contralateral and normal
ipsilateral brain.dagger.tissue have been affected (Salford et al.
(1993)).
The currently available methods of electroporation drug delivery as
described in the literature fall short of providing an effective
methodology, due primarily to the inability to limit the scope of
the electroporation effect to the intended target tissue. Under
these circumstances, an unacceptably high level of normal tissue
effect is noted and offsets the potential useful benefits of
electroporation treatment.
In particular, there are a number of applications in tumor therapy,
such as the treatment of glioblastoma multiforme tumors, which
would benefit from a treatment methodology in which the delivery of
a therapeutic agent is highly localized. At the present time, there
is no cure for this uniformly fatal brain tumor which kills over
7,000 U.S. citizens each year.
Therefore, it would be desirable to have available an effective
system or methodology which combines the advantage of selective
drug delivery using a combination of techniques including
electroporation in order to deliver drugs to selected diseased
areas.
DESCRIPTION OF THE PRIOR ART
General references of interest regarding electroporation include,
for example, Guide to Electroporation and Electrofusion, D. C.
Chang et al., Eds., Academic Press, Inc, San Diego, Calif. (1992)
and Electroporation and Electrofusion in Cell Biology, (E. Neumann
et al., Eds., Plenum Press, N.Y. (1982).
Biochemical and Biophysical Research Communications, 194(No. 2):
938 (1993) discusses a new brain tumor treatment combining
bleomycin with in vivo electroporation. A similar article which is
currently in press, Anti-Cancer Drugs 5:463 (1994) also relates
techniques of in vivo electroporation for the purpose of delivering
enhanced boron uptake in gliomas to improve boron neutron capture
therapy.
References pertaining to surfactant treatment of damaged cell
membranes are found in Annals New York Academy of Sciences 720:239
(1994) and in Proc. Natl. Acad. Sci. USA. 89:4525-28 (1992).
Intermittent hypothermic asanguineous cerebral perfusion
(cerebroplegia) is discussed in J. Thorac. Cardiovasc. Surg. 99:878
(1990) and further in J. Thorac Cardiovasc. Surg. 102:85
(1991).
General references of interest regarding liposomes include, for
example, Liposome Technology, Volumes I, II and III, G.
Gregoriadis, Ed., CRC Press, Inc., Boca Raton, Fla. (1985) and
Radiation Research 103:266 (1985).
Biochim. Biophys. Acta 150:333 (1968), discloses the use of
cholesterol to produce a solid phase liposome. Biochim. Biophys.
Acta 164:509 (1977) discloses the effect of cholesterol
incorporation on the temperature dependence of water permeation
through liposome membranes prepared from phosphatidylcholine.
Resealing of electropores is discussed in Proc. Natl. Acad. Sci.
USA 89:4524 (1992) and also in Annal. New York Acad. Sci.
(1992).
Radiation Research 122:161 (1990) and references therein, disclose
the use of heat from a waterbath to release drugs from liposomes
that possess a phase transition temperature (T.sub.C).
DISCLOSURE OF THE INVENTION
The present invention provides a system and method for the
localized delivery of therapeutic agents to patients in need of
such treatment. The invention utilizes a number of aspects which
can be practiced in a variety of combinations to effect such
localized delivery. Such aspects include electropermeabilization,
liposome-mediated drug delivery, localized tissue temperature
control, three-dimensional electrode arrays and convection
enhancement of therapeutic agent concentrations.
In one aspect, the present invention provides a method for
delivering a therapeutic agent to a predetermined location in a
host. The method comprises providing a liposome-encapsulated
therapeutic agent to the host, establishing an electrical field
which encompasses a predetermined region within the host, and
exposing the liposome-encapsulated agent to the electrical field so
as to enhance the release of the agent from the liposome to the
predetermined region.
In the practice of such aspects of the present invention the
release of the contents of both solid and fluid liposomes is
greatly increased by exposure to high voltage transient electrical
fields. It has been shown (Mueller et al. (1983) and Chang et al.
(1992)) that liposomes exposed to brief external high voltage
electrical fields have demonstrated the formation of pores and,
above a critical voltage (E.sub.C), the liposomes will rupture.
These effects can occur either at normal body temperature, over a
wide range of temperatures, or through non-thermal interaction with
non-ionizing electromagnetic radiation at temperatures other than
T.sub.C. Thus, the present invention offers a fast and effective
method for rapid release of liposome encapsulated therapeutic
agents and/or other chemicals into localized areas in cells,
tissues, or organs in the body of a patient.
In accordance with certain aspects of the invention, liposomes may
be made of inexpensive materials and the drug release from these
liposomes can be effected by applying to the predetermined
treatment area an electrical field of intensity sufficient to
effect the release of the drug from the liposomes.
In certain embodiments, the present electroporation effects are
delivered in a manner which utilizes an electrode array which
comprises both central and satellite electrodes located in and
around, e.g, tumoral or diseased tissue.
More specifically, aspects of the present invention relate to the
use of liposomes to deliver drugs or other chemicals to specific
target cells or groups of cells such that the drug or chemical is
released into the target cells (using electroporation and other
techniques) while minimizing entry of said chemicals or drugs into
normal healthy cells. The liposome vesicles are designed to be
employed at temperatures slightly below their phase transition
temperature (T.sub.C).
In additional aspects of the invention, techniques will be employed
to "precondition" the tumor or diseased tissue, in order to
increase the permeabilization effects of the electroporation
pulses.
Additionally, techniques designed specifically to protect the
normal tissue from the effects of electroporation pulses are
provided in certain aspects of the invention, including techniques
such as cerebroplegia, which allows the brain to be cooled to
subthreshold electroporation and low metabolic activity states.
Cerebroplegia provides a second protective mechanism to normal
tissue; removal of the therapeutic agent from normal tissue prior
to electroporation. These techniques will also be employed to
protect healthy tissue from the effect of the electroporation
fields, resulting in a more specific loading of the target tissue
with drugs or chemicals.
Further, aspects of the invention provide the ability to influence
the concentration of administered therapeutic agents via
iontophoretic field application which will influence charged
liposomes and promote adsorption to cell membranes, as well as
influence distribution within diseased tissue.
The present invention will also promote rapid healing of the
electropores utilizing surfactant materials which can be delivered
to the sites of electroporation via either vascular methods or via
liposomes.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphic representation illustrating an electrode array
in accordance with the invention which is positioned into and
around a brain tumor;
FIG. 2 is a graphic representation of a brain tumor in
three-dimensional space;
FIG. 3 is a graphic representation of the electrical field lines
produced by a bipolar electrode array, wherein the field density is
represented by the space between the field lines, with a higher
intensity represented by closely spaced field lines, as seen in the
region directly between the two electrodes. The application of an
electropermeabilization pulse between these two electrodes will
ordinarily provide subthreshold electrical pulses to large areas of
the tumor (widely-spaced lines) thus reducing the effectiveness of
the permeabilization in those areas;
FIG. 4 is a graphic representation of the electrical field lines
produced by a three electrode array, with the satellite electrodes
placed superior and inferior to the tumor mass;
FIG. 5 is a graphic representation of the electrical field lines
produced by a three electrode array, with the satellite electrodes
placed anterior and posterior to the tumor mass, generally as
depicted in FIG. 1. As will be seen, due to the higher density of
electrical field lines in the central portion of the tumor
(tightly-spaced field lines), the application of an
electropermeabilization pulse in this electrode array (as in the
array of FIG. 4) will provide above threshold electrical pulses to
large areas of the tumor, and subthreshold pulses to the healthy
tissue surrounding the tumor, thus increasing the effectiveness of
the permeabilization in the tumor; and
FIG. 6 is a graphic representation of the electrical field lines
produced by a five electrode array, with the satellite electrodes
placed anterior, posterior, superior and inferior to the tumor
mass. As with the array of FIGS. 4 and 5, the effectiveness of the
permeabilization in the tumor will be further enhanced with this
more complex electrode array.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for the localized delivery of
therapeutic agents to patients in need of such treatment. The
invention utilizes a number of aspects which can be practiced in a
variety of combinations to effect such localized delivery.
In one aspect, the present invention provides a method for
delivering a therapeutic agent to a predetermined location in a
host. The method comprises providing a liposome-encapsulated
therapeutic agent to the host, establishing an electrical field
which encompasses a predetermined region within the host, and
exposing the liposome-encapsulated agent to the electrical field so
as to enhance the release of the agent from the liposome to the
predetermined region.
As used herein:
The term "electroporation" refers to a phenomenon wherein the
membrane of a cell exposed to high-intensity electrical field
pulses can be temporarily destabilized, resulting in increased
permeability to exogenous molecules across portions of the
membrane.
The term "electropermeabilization" refers more generally to the
phenomenon of increased permeability following in vivo
electroporation pulses without requiring the formation of physical
pores in the membrane.
The terms "drug" and "therapeutic agent" are used interchangeably
to refer to any agent which has a desirable pharmacological action
when administered to a patient.
The term "liposome" refers to a bilayer structure comprised of a
natural or synthetic phospholipid membrane or membranes, and
optionally other membrane components such as cholesterol and
protein, which structure can act as a physical reservoir for drugs.
These drugs may be sequestered in the liposome membrane or may be
encapsulated in the aqueous interior of the vesicle. Liposomes are
generally characterized according to size and to number of membrane
bilayers. The vesicle diameter can be large (>200 nm) or small
(<50 nm) and the bilayer can have a unilamellar, oligolamellar,
or multilamellar membrane.
The term "phase transition temperature (T.sub.C)" refers to the
temperature at which a liposome membrane displays both phase
states, i.e., fluid (liquid) and solid (gel), simultaneously. The
fluid (liquid ) state is characterized by free rotational motion
within the membrane of the hydrocarbon chains of the phospholipids,
whereas the solid (gel) state is associated with restricted
hydrocarbon tail motion. At T.sub.C both phase states coexist and
the liposome membrane becomes naturally permeable or leaky,
resulting in the spontaneous release of encapsulated drug from the
liposome membrane or from the interior space of the liposome. At
temperatures below T.sub.C the bilayer is referred to as being in
the solid or gel state, and at temperatures above T.sub.C the
bilayer is in a liquid or fluid state. To have a phase transition
in the liposome bilayer generally requires the exclusive presence
of highly purified phospholipids of identical fatty acid chain
length and polar head group composition. Mixing phospholipid
species or adding perturbing agents at appropriate concentrations
will obliterate the ability of the bilayer to undergo a phase
transition.
The term "perturbing agent" refers to a natural or synthetic
compound or a combination of compounds which when added to a
liposome membrane obstructs the formation of a phase transition.
The amount of perturbing agent necessary to obstruct the formation
of a phase transition varies according to the stearic nature of the
compound. Usually, but not always, 20-40 percent mole fraction of
the agent is required. Typically a perturbing agent is selected
from natural or synthetic compounds: cholesterol; phospholipids,
for example egg phosphatidylcholine (EPC) or lecithin, egg
phosphatidylglycerol (EPG); inorganic metal compounds and complexes
and proteins such as antibodies. Perturbing agents also include
various compositions of phosphatidylcholine or
phosphatidylglycerol, or phosphatidylethanolamine wherein these
structures are further substituted by aliphatic organic acids
having different carbon chain lengths, e.g. palmitoyl (16 carbons)
and lauryl (12 carbons).
The term "non-phase transition liposome" refers to a liposome that
does not display a phase transition temperature T.sub.C within a
specified temperature range of interest. For example, if a liposome
"A" has a phase transition temperature T.sub.C of 4.degree. C., and
the specified temperature range is from 10-50.degree. C., then,
over this temperature range, it is referred to as a nonphase
transition liposome. In addition, since this particular temperature
range is above the nominal T.sub.C, liposome "A" will be in the
fluid (liquid) phase state at all temperatures between 10.degree.
C. and 50.degree. C.
The term "microinjection of liposomal drug" refers to the technique
of using liposomes that are bound to a target cell surface, or that
have been internalized by the target cell, to directly introduce
drugs into the target cell. This technique is also referred to as
"using liposomes as a cellular-level microsyringe".
THEORETICAL BASIS
Current methods of drug delivery via liposomes require that the
liposome carrier will ultimately become permeable and release the
encapsulated drug. This can be accomplished in a passive manner,
wherein the liposome bilayer membrane degrades over time through
the action of factors inherent in the body. Every liposome
composition will have a characteristic half-life in the circulation
or at other sites in the body.
In contrast to passive drug release, active drug release involves
using an external agent or force to induce a permeability change in
the liposome vesicle. Liposome membranes can be constructed so as
to become destabilized when the environment becomes acidic near the
liposome membrane (Proc. Natl. Acad. Sci. USA 84:7851 (1987);
Biochem. 28:9508 (1989) and references therein). The liposome
membrane can be chemically modified to provide an enzyme as a
coating on the membrane which slowly destabilizes the liposome
(FASEB J. 4:2544 (1990)). However, this technique is limited in
that it does not allow modulation or alteration of drug release to
achieve "on demand" drug delivery.
It has been recognized that a major barrier to the use of liposomes
as drug carriers is the ability of the liposome to release the
drugs on demand at the target sites (Science, 202:1290 (1978)). The
specific use of applied heat to raise the liposome temperature to
T.sub.C to make them permeable has been described (Science, 204:188
(1979)), and addressed in U.S. Pat. No. 5,190,761, in which a
method of activating liposomes to release their encapsulated drugs
in tissue utilizing microwave radiation is described.
A third method to achieve release of active drug is to employ a
liposome having a predetermined phase transition temperature,
T.sub.C, at or above the temperature of the target tissue (see for
example Radiation Res., 112:161 (1990) and references therein).
These liposomes are designed to be employed at temperatures
slightly below their phase transition temperature, T.sub.C, (where
they are naturally permeable) so that in the temperature range of
healthy or normal tissue the liposome membrane is in the solid
(T<T.sub.C) stage. This means that healthy tissue temperatures
will maintain the liposomes below T.sub.C so they will not become
leaky. This mechanism for drug release is capable of "on demand"
drug delivery, since these liposomes experience a greatly increased
membrane permeability at T.sub.C and this effects drug release. To
release drugs from such phase transition liposomes placed in the
body requires the application of heat until T.sub.C is achieved.
Such liposomes are made of highly purified phase transition
temperature phospholipid material (either as a single component or
multi-component mixtures).
"On demand" liposome release can also be obtained utilizing high
voltage electrical fields similar to those found in
electroporation/electropermeabilization. It has been shown (Mueller
et al. (1983) and Chang et al. (1992)) that exposure to brief
external high voltage fields in both solid and fluid liposomes will
promote the formation of pores and, if the electrical field is high
enough, effect rupture. These effects can occur either at normal
body temperature or over a wide range of temperatures. The
electrical fields causing electropermeabilization act to trigger
drug delivery in two ways: (1) by destabilizing the liposome
bilayer so that membrane fusion between the liposome and the target
cellular structure occurs, thus facilitating the direct delivery of
drug into the target cell; and (2) by triggering the release of
drug in high concentrations from liposomes at the surface of the
target cell so that the drugs are driven across the cell membrane
by a concentration gradient. In either case, the direct
cellular-level microinjection of drug into the target cell is
achieved.
A further consideration of liposome-mediated delivery relates to
the potential for controlling the direction and speed of movement
of charged liposomes utilizing subthreshold iontophoretic fields
which are applied from the elements of the electrode array. These
liposomes will contain negative external charges which should cause
them to migrate through the extracellular fluid space towards the
positive pole of the iontophoretic field, thus allowing
differential positioning of the liposomes in vivo. In this aspect
of the invention, a central electrode element in conjunction with
satellite electrodes will act as confining dipoles to limit the
excursion of the electrical field outside the desired area.
Utilization of this aspect of the invention will allow for
increased concentrations of materials in certain areas of the
target body site which may have poor blood distribution, compressed
cytoarchitecture, etc., features well documented in tumors.
Electroporation/Electropermeabilization
The difficulty in transporting a normally nonpermeable active agent
across a membrane can be overcome by utilizing transient high
permeability states induced by transitory high voltage electrical
fields. This transient high permeability state can be used to
increase the transport flux of molecules which may be assisted by a
driving force such as concentration difference or hydrostatic
pressure. Electroporation is characterized by a transient high
permeability state and a decrease in the electrical resistance of
the tissue caused by brief exposure to an abnormally high
trans-tissue potential. The decreased electrical resistance can be
used as an effective means of monitoring electroporation effects.
For example, short electroporation pulses (preferably 10.sup.-6 to
10.sup.-3 seconds) are applied. At a fixed pulse width, the
resistance of the sample will remain unchanged as the voltage
magnitude of the electroporation pulses is increased. Above a
certain threshold, however, the resistance rapidly decreases, with
higher voltage pulses further decreasing the tissue resistance.
Following this, the trans-tissue resistance can gradually recover
to its initial value. The range of transmembrane potentials
associated with electroporation is from approximately 500 to 1500
mV. These values are much higher than the normal physiological
resting potential (approximately 100 mV) and generally above the
magnitude of transmembrane potentials known to result in membrane
rupture (approximately 300 to 600 mV). Thus, the relatively short
duration of the electroporation pulses used to induce
electropermeabilization is a key aspect of this process. It has
been shown that electroporation can be accomplished in multilayer
tissues, including skin and underlying tissue (U.S. Pat. No.
5,019,034). Further, this reference discloses the transport of
molecules across tissue by applying an electrical pulse in order to
cause electroporation and utilizing a driving force to move
molecules across the region. In the specification "driving force"
is defined as including iontophoresis, pressure gradients and
concentration gradients. The reference also discloses the temporary
increase of the permeability of tissue by applying an electrical
pulse of sufficient voltage and duration to a region of tissue to
cause a "reversible electrical breakdown" in the electroporated
region, wherein the region is used as a site of molecular
transport.
Further, a patent to Hofmann (U.S. Pat. No. 5,318,514) details an
apparatus for implanting macromolecules such as genes, DNA or
pharmaceuticals into a preselected surface tissue region of a
patient.
Generally, for cells, electroporation results in non-thermal, short
term membrane changes, with all damage or death occurring only due
to long term osmotic pressure differences, or other physicochemical
imbalances. Cell lysis or cell fusion can occur for some pulse
conditions which induce electroporation. During this process, the
values and changes in values of the electrical impedance between
any pair of electrodes, either during or after any pulse or pulse
series, can be monitored to allow a determination of the occurrence
of decreased electrical resistance for any tissue transport
situation.
Acute electropermeabilization events will also cause short term
reversible changes in the local conductivity and should be
detectable by applying small electrical fields across adjacent
electrodes to determine those areas which have been adequately
treated versus those areas which may require additional
electroporation pulses to induce electropermeabilization.
Electrode Placement
One further aspect of the present invention relates to a system
utilizing the placement of a plurality of electrodes (desirably at
least 7 and less than 15) within or surrounding a predetermined
three-dimensional region in the body. This region can be, for
example, a tumor or other similarly diseased area, or any region in
which the application of the present invention is deemed
desirable.
The basic design of one embodiment of the present electrode array
includes a central reference electrode surrounded by six
geometrically-oriented electrodes (Hexasphere.TM.). These
electrodes are designed to contain the electrical field within the
"sphere" defined by the electrode placement at points in space
equidistant from one to another. This design is considered to be
advantageous in that the electrical fields produced can be oriented
to travel primarily across a hemi-diameter of the preselected
region, e.g. a tumor, and remain "confined" within the substance of
the target body tissue.
This electrode array design may also include synthetic
microcylinder structures which may be used for local delivery of
materials into the extracellular space such as drugs, and hyper- or
hypo-osmotic elements which will facilitate the distribution of the
electrical field and thus enhance the electroporation process.
Another aspect of the invention relates to the "preconditioning" of
the predetermined tissue location in order to maximize the effect
of the electroporation pulses. This preconditioning phase will
generally comprise sub-threshold constant or alternating electrical
field stimulation, using alternating current electrical fields, RF,
ELF fields, and the like. This treatment is designed to increase
the stochastic probability of sites of increased permeability on
cell membranes in the predetermined location in the body, following
the electroporation pulses. This aspect of the invention can also
utilize the delivery of electrically conductive material to the
predefined body site.
A further aspect of this invention is the enhancement of
distribution of the liposome encapsulated material utilizing
iontophoretic field application across the electrode array in
various combinations designed to allow uniform concentration or, in
some cases, deliberate asymmetry in concentration at specified
sites.
A further aspect of the invention involves the use of non-ionic
surfactants or other similar recovery techniques to aid the closure
of pores formed in target body site following electroporation
pulses. This aspect of the invention will aid in retaining the
material delivered via the invention into target cells.
A further aspect of the invention involves the directed migration
of charged liposomes to certain areas of the target body site, as
defined by the subthreshold iontophoretic fields applied utilizing
the electrode array. The liposomes used in this aspect of the
present invention will desirably contain negative charges on the
outer surface, which should cause them to migrate towards the
positive pole of the iontophoretic field, thus potentially allowing
differential positioning of the liposomes in vivo. As a feature of
this aspect of the invention, the central electrode element in
conjunction with the satellite electrodes will act as confining
dipoles to limit the excursion of the electrical field outside the
desired area. This feature will allow for increasing the
concentrations of the liposome-encapsulated materials in certain
areas of the target body site which may have differential blood
distribution, cytoarchitecture, etc.
Yet another aspect of this invention relates to the method of
protecting healthy tissues, such as brain tissues, from the
electroporation pulses delivered to the target body site. There is
a decreased probability of a given electrical field effect causing
electroporation at lower temperatures, i.e. less chance for
membrane destabilization to occur. A process which creates
differential temperatures within the body should increase the
probability that electroporation events will occur in the areas
with higher temperatures, and decrease the probability in areas
which are cooled significantly below body temperature. This
technique of cooling selected tissue, e.g. the brain (otherwise
known as cerebroplegia) and target body site using hypothermic
solution of low conductance liquid is generally performed as
follows (described with reference to the brain):
I Cooled blood or other perfusion solutions are administered via
carotid injection, rapidly cooling the brain or target body site to
10-20.degree. C. This methodology is designed to minimize the
metabolic activity of the brain and to protect the healthy tissue
from the permeabilization effects of electroporation pulses, which
have reduced effect at lower temperatures. Also, cerebroplegia
permits brief disruption of cerebral blood flow without significant
damage to the neural tissue.
II Immediately after the cooling period (1-2 minutes), the cerebral
blood flow will be replaced by hypotonic or other low conductance
solutions in order to diminish the intravascular conductance of
subsequent electroporation pulses. Following this infusion, the
cerebral perfusion is temporarily disrupted (for 10-20 seconds),
during which time:
III The target body site will be differentially heated by
subthreshold electroporation pulses using lower voltage electrical
fields with increased duration;
IV Immediately following the heating of the target body site,
electroporation pulses will be administered via the electrode array
(10-20 seconds); A non-ionic surfactant or other pore closure
recovery method will be employed via circulating this material
through the cerebral circulation, with preferential site of
activity being the target body site;
V A non-ionic surfactant or other pore closure recovery method will
be employed via circulating this material through the cerebral
circulation, with preferential site of activity being the target
body site;
VI Cerebral blood flow will be re-established with gradual rise in
temperature of healthy tissue recovering to normal; and
VII The electrode system will be removed.
In certain aspects, the present invention involves the preparation
of drugs encapsulated in liposomes affected by electroporation
pulses using very brief high voltage electrical fields. The
permeability of liposome membranes depends on many factors which
include their lipid composition, the type of drug, drug
sequestration into the bilayer membrane or into the aqueous
interior compartment, the site of release and other complex
physicochemical properties. It is generally recognized that
undisturbed liposomes are not very permeable, but can be made so by
altering membrane properties.
Thus, the invention provides a novel method of placing a series of
electrodes into the target body site of interest, thereby setting
up geometrically-oriented electrical fields by which to perform
electroporation. As an adjunct to the electroporation, liposomes
encapsulating various compounds, and designed to maximize the
effect of electroporation pulses and deliver drugs to diseased
tissues are also utilized and methods are described to
iontophoretically localize charged liposomes.
Additionally, a method is described which affords protection of
normal tissue using thermal insulation via cerebroplegia
techniques. This will allow normal tissue to establish a
differentially lower temperature than the tumor or diseased tissue
which is heated using subthreshold electroporation pulses, followed
by electroporation pulses designed to both incorporate liposomes
and release liposome contents into the extracellular fluid (ECF)
for uptake into the electroporated cells.
The present invention desirably utilizes liposomes which possess a
phase transition temperature T.sub.C within the temperature range
of interest, generally several degrees below their transition
(T.sub.C) temperatures. Such liposomes are referred to as phase
transition liposomes which will be in the fluid (liquid) phase
state following application of electroporation pulses. Drug
delivery using electrical fields using liposomes at temperatures
corresponding to T.sub.C have been previously described in U.S.
Pat. Nos. 4,801,459 and 5,190,761.
Lipid Components
The liposomes used in this present invention are small unilamellar
vesicles (SUV). The liposomes are formed from standard vesicle
forming lipids, which generally include neutral and negatively
charged phospholipids with or without a sterol, such as
cholesterol. The selection of lipids is generally guided by
considerations of (a) desired liposome size and ease of liposome
sizing, and (b) lipid and water soluble drug release rates from the
site of liposome injection.
Typically, the major phospholipid (PL) components in the liposomes
are phosphatidylcholine (PC), phosphatidylglycerol (PG),
phosphatidylserine (PS) phosphatidylinositol (PI) or egg yolk
lecithin (EYL). PC, PG, PS, and PI having a variety of acyl chain
groups or varying chain length and degree of saturation are
commercially available, or may be isolated or synthesized by well
known techniques. The degree of saturation can be important since
hydrogenated PL (HPL) components have greater "stiffness" than do
unhydrogenated PL components; liposomes made with HPL components
will be more rigid. In addition, less saturated PLs are more easily
extruded, which can be a desirable property, particularly when the
liposomes must be sized below about 0.3 microns, for purposes of
filter sterilization or other formulation requirements. Methods
used in sizing or filter-sterilizing liposomes are discussed
below.
Protective Agent
It is well known that the lipid components of liposomes promote
peroxidative and free radical reactions which cause progressive
degradation of the liposomes. This problem has been discussed at
length in the U.S. Pat. No. 4,797,285. Briefly, the patent
discloses that lipid peroxidative and free radical damage effect
both lipid and entrapped drug components in a liposome/drug
composition. It is noted that the extent of free radical damage to
lipid and drug components was reduced significantly when a
lipophilic free radical quencher, such as alpha-tocopherol
(.alpha.-T) was included in the vesicle-forming lipids. A
significantly greater reduction in lipid damage and drug
modification was observed when the lipid/drug composition was
formulated in the presence of both .alpha.-T and a water soluble,
iron-specific chelator, such as ferrioxamine. Since ferrioxamine
can complex tightly to ferric iron at six coordination sites, it is
likely that the compound acts by inhibiting iron-catalyzed
peroxidation in the aqueous phase of the liposome suspension. The
effectiveness of the two protective agents together suggests that
both iron-catalyzed peroxidative reactions occurring in the aqueous
phase, and free radical reactions being propagated in the lipid
phase are important contributors to lipid peroxidative damage.
Lipophilic free radical scavengers can be used in the composition
employed herein and include the preferable .alpha.-T, an analog or
ester thereof (such as alpha-tocopherol succinate), butylated
hydroxytoluene (BHT), propyl gallate, and their pharmacologically
acceptable salts and analogs. Additional lipophilic free radical
quenchers which are acceptable for parenteral administration in
humans, at an effective level in liposomes, may be used. The free
radical quencher is typically included in the lipid components used
in preparing the liposomes, according to conventional procedures.
Preferred concentrations of the protective compound are between
about 0.2 and 2 mole percent of the total lipid components making
up the liposomes; however higher levels of the protective compound,
particularly .alpha.-T or its succinate analog, are compatible with
liposome stability and are pharmacologically acceptable.
Liposome Formation
The liposome suspension of the invention can be prepared by any of
the standard methods for preparing and sizing liposomes. These
include hydration of lipid films, solvent injection, reverse-phase
evaporation and other techniques such as those detailed in Am. Rev.
Biophys. Bioeng., 9:467 (1980). Reverse-phase evaporation vesicles
(REVs) can be prepared by the reverse-evaporation method as
described in U.S. Pat. No. 4,235,871, incorporated herein by this
reference. The preparation of multilamellar vesicles (MLVs) by
thin-film of a lipid or by an injection technique is described in
U.S. Pat. No. 4,737,923, incorporated herein by this reference. In
known procedures which are generally preferred, a mixture of
liposome forming lipids dissolved in a suitable solvent is
evaporated in a vessel to form a thin film, which is covered by an
aqueous buffer solution. The lipid film hydrated to formation MLVs,
typically with sizes between about 0.1 to 10 microns.
Either the REVs or MLVs preparations can be further treated to
produce a suspension of smaller, relatively homogeneous-size
liposomes, in a 0.1 to 1.0 micron size range. One effective sizing
technique involves extruding an aqueous suspension of the liposomes
through a polycarbonate membrane having a selected uniform pore
size, typically 0.2, 0.4, 0.6, 0.8 or 1 micron as shown in Ann Rev.
Biophys. Bioeng., 9:467 (1980). The pore size of the membrane
corresponds roughly to the largest sizes of liposomes produced by
extrusion through that membrane, particularly where the preparation
is extruded two or more times through the same membrane.
A more recent technique involves extrusion through an asymmetric
ceramic filter, as detailed in U.S. Pat. No. 4,737,323,
incorporated herein by this reference.
Alternatively, the REVs or MLVs preparations can be treated to
produce small unilamellar vesicles (SUVs). Among the advantages of
smaller, more homogeneous-sized liposomes are, for example, the
higher density of liposome packing at a mucosal surface, the higher
likelihood of intact liposomal incorporation into the
electroporated cells, and the higher concentration of liposome
encapsulated drug transported to the target organ. Because of the
small particle sizes, SUVs in suspension can be distributed in the
minute capillary bed of the central nervous system.
One preferred method for producing SUVs is by homogenizing an MLV
preparation, using a conventional high pressure homogenizer of the
type used commercially for milk homogenization. Here the MLV
preparation is cycled through the homogenizer with periodic
sampling of particle sizes to determine when the MLVs have been
substantially converted to SUVs.
The larger liposome vesicles, whether MLVs or LUVs, however, have
other advantages such as, for example, a larger capacity for drug
encapsulation and may therefore be preferred for certain routes of
administration or delivery to specific targets, in particular
target body site outside the central nervous system.
The use of all SUVs, LUVs, MLVs, OLVs, or mixtures thereof, is
contemplated to be within the scope of this invention depending on
intended therapeutic application and route of administration.
A selected drug is encapsulated in the liposomes by using for
example the procedure described in U.S. Pat. No. 4,752,425,
incorporated herein by this reference.
These vesicles can preferably be made by reverse phase evaporation
using chloroform and isopropyl ether. However, the vesicles
prepared in this or any other suitable manner, and for reasons
which become more apparent later, may optionally contain to
radioisotope markers as described in U.S. Pat. No. 5,190,761 and
incorporated by reference herein. Additionally, these liposomes can
contain various boronated compounds, among them BSH, BPA, boronated
porphyrins etc. These compounds are useful in following the release
and quantification of release of liposomes in brain areas which are
subsequently analyzed by inductively coupled plasma-atomic emission
spectroscopy (ICP-AES).
Liposome Sizing and Sterilization
Following liposome preparation, the liposomes may be graded to
achieve a desired size range and relatively narrow distribution of
liposome sizes. A preferred size range is about 30-100 nm. Several
techniques are available for obtaining liposomes of a desired size.
Sonicating a MLV liposome suspension either by bath or probe
sonication produces a progressive size reduction down to SUVs less
than about 0.5 microns in size. Homogenization is another method
which relies on shearing energy to fragment large liposomes into
smaller ones. In a typical homogenization procedure, MLVs are
recirculated through a standard emulsion homogenizer until selected
liposome sizes, typically between about 0.1 and 0.5 microns, are
observed. In both techniques, the particle size distribution can be
monitored by conventional laser beam particle size
discrimination.
The filter sterilization method can be carried out on a high
through-put basis only if the liposomes have been first sized down
to less than or equal to the 0.2-0.4 microns range. The importance
of sterilization for any pharmaceutical product is well understood
and it will be appreciated by using this filtration sizing step the
sterilization will also be achieved at the same time and without
additional steps.
Removing Free Drug
The initial liposome suspension may contain up to 50% or more drug
in free (non-encapsulated) form. The drug can be encapsulated such
that it is sequestered in the liposome bilayer (lipophilic
compounds) or entrained in the liposome internal aqueous region
(hydrophilic compounds). The presence of such free drug may in some
cases be tolerated but in many other cases is undesirable because
these drugs are often toxic in their free state. Therefore, in
order to maximize the advantages of liposome-encapsulated drug and
to minimize the effect of the free drug, it may be important to
remove free drug from the final injectable suspension.
Several techniques are available for removing non-entrapped
compound from a liposome suspension. In one technique, the
liposomes in the suspension are pelleted by high-speed
centrifugation, leaving free compound and very small liposomes in
the supernatant. This approach is followed where several liposome
washings are employed. Another method involves concentrating the
suspension by ultrafiltration, then resuspending the concentrated
liposomes in a drug-free replacement medium. Alternatively,
gel-filtration can be used to separate liposome particles from the
solute molecules.
Following treatment to remove free drug, the liposome suspension is
brought to a desired concentration for administration. Typically,
the liposomes are administered by i.v., i.m., or s.c. injection.
Thus, the liposome may be resuspended in a suitable volume of
injection medium such as saline, or other pharmaceutically
acceptable injectable medium as may be appropriate for the drug
suspension or route of administration. The resuspension is
particularly appropriate where the liposomes have been
concentrated, for example by centrifugation or ultra-filtration, or
concentrating the suspension volume. The suspension is then
sterilized by filtration as described above. These media and other
representative injectable components are well known and set forth
in Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing,
Easton, Pa. 1985.
Non-Phase Transition Liposomes
Liposomes without a reverse transition over a specified temperature
range can be prepared when a suitable perturbing agent is added to
the phospholipid membrane, or when a multicomponent phospholipid
liposome is constructed. Thus, for example, the perturbing agent
cholesterol can be added to the membrane of a liposome displaying a
T.sub.C over a specified temperature range of interest. Such a
membrane is comprised of, for example, a single highly purified
phospholipid. At sufficient concentrations, cholesterol converts
this material essentially into a nonphase transition liposome. The
obliteration of a reverse transition will render liposome membranes
impermeant and highly stable with regard to leakage of drug. In the
method here described using electrical fields as a triggering agent
for liposome drug release, one observes a significant increase in
drug release from nonphase transition liposomes during treatment
with electrical fields.
The use of non-phase transition liposomes as drug delivery vehicles
has several advantages. First, these liposomes are extremely stable
with respect to temperature since they do not exhibit a phase
transition temperature, T.sub.c, at which they become permeable. Of
some benefit is the fact that they can be prepared with very
inexpensive materials, since the use of highly purified
phospholipid is not required. Using liposomes as drug delivery
vehicles, via this technique, has additional advantages. Liposomes
of this type can be prepared to include a broad range of drugs
which may then be usefully administered and/or released to specific
cells, organs or tissue, either intermittently or over a sustained
period of time. Non-phase transition liposomes allow the
administration of relatively high drug doses of relatively toxic
drugs with reduced side effects that are usually associated with
free drug at such high concentrations.
Delivery of Liposomes
There are three main routes by which materials will be delivered
to, e.g., the interstitial spaces of internal tumor areas: (1)
local delivery by injection; (2) intravascular administration
combined with electropermeabilization of selected areas of the
vasculature to increase the unloading of materials to the
interstitial fluid; (3) utilization of liposome-encapsulated
materials to penetrate tumor tissue via the localized disruption of
the blood-brain barrier caused by the tumor. These liposomes would
be allowed to cross the blood-brain barrier and accumulate in
interstitial space of the tumor. The liposomes would carry either
cytotoxic materials or hyperconductive compounds to be distributed
by convection or by convection/diffusion processes in combination
with pressure gradients.
Local injections would be carried out by utilizing either
iontophoretic or simple mechanical injection of specific volumes of
materials designed for cell killing or compounds designed to aid
electrical field propagation within the tumor. Delivery would be
accomplished employing a microtubular system which would be
incorporated within the implanted electrode array. Compounds would
be infused slowly and allowed to move away from the injection
site(s) either by normal convection or diffusion.
The second method involves the vascular (intra-arterial or
intravenous) administration of compounds which would be carried to
the cerebral intravascular spaces, followed by
electropermeabilization pulses which would be delivered to the
selected cerebral vessels. It is expected that this would enhance
the extravasation of compounds from those already leaky capillaries
into the interstitial spaces. This would be of particular benefit
in those tumor types which do not significantly alter the intact
blood-brain barrier.
Liposomes may be administered to persons as a liposome depot at a
tissue site or may be administered directly into the circulation.
Circulating nonphase transition liposomes will not release the drug
unless subjected to an electrical field. In turn, electrical fields
may be selectively directed only to target areas where the drug
release is desired. All other liposomes outside the target area
will not release the drug; liposomes in the general circulation and
liposomes at a distant liposome depot outside of the exposure site
will remain intact until their eventual sequestration by the
reticuloendothelial system in the body. The process of drug release
using electrical fields may be repeated intermittently until all
drug is released from the liposome population.
Driving Forces
In order for the released drugs to effectively penetrate the cells,
it is recognized that some force must move molecules across the
regions of the tissue undergoing electroporation. The driving force
may be electrical, such as iontophoresis, or it may be another
physical or chemical force such as provided by a temperature
gradient, a pressure gradient, or a concentration gradient.
Additionally, the driving force may comprise acoustic or optical
pressure.
Once the compounds have been delivered within tumoral tissue, it is
preferable to evenly distribute the compounds throughout the
interstitial compartment, either for purposes of cell killing or
for purposes of evenly distributing conducting ions for later
electroporation work. There are several natural phenomenon which
mediate distribution as well as several supplementary methods which
might be used to either improve the area of distribution, the
concentration of compounds in a given area or increase the speed of
distribution. The optimal scenario in any therapeutic modality
would be for each tumor cell to have equal access to the treating
agent.
There is a sequential order to the delivery of most blood-borne
molecules to tumor cells. Molecules must be delivered to the
general region of the tumor cells via intravascular transport, then
move across the microvascular walls to the interstitial spaces
where they move through the interstitial matrix either via
convection, diffusion to the tumor cells, or under the influence of
externally applied gradients such as pressure gradients or
electrical fields. The interstitial fluid environment in which
these molecules move in tumors is quite different than normal
tissue. Diffusion is proportional to the concentration gradient in
the interstitium and convection is proportional to the pressure
gradient in the interstitium. In most tumors, there is a
significant heterogeneity in the perfusion within a given tumor,
combining multiple zones of well-vascularized cells with
semi-necrotic regions of intermediate perfusion and possibly one or
more necrotic, avascular regions. In general, the
well-permeabilized regions have low interstitial pressures, leading
to increased extravasation of fluid and macromolecules from the
vasculature. These macromolecules extravasated in the outer zones
of the tumors may then move towards the center by the slow
diffusion processes. opposing this movement centrally is the
movement of molecules by convection which moves in the direction of
high to low pressure, thus centrally towards the periphery and into
normal tissue (Jain (1987)).
In the case of a brain tumor, it is considered desirable that when
electropermeabilization is performed, each tumor cell is porated
and that all cells would have an adequate amount of the therapeutic
agent outside of the cell (and thus able to enter the cell). The
local and vascular delivery systems described above may not be able
to deliver a uniform distribution of the agent, due to regional
variations in blood supply, tumor density, necrotic zones,
variations in interstitial space pressure, etc. It would thus be
highly desirable to influence the distribution of such agents
utilizing a variety of methods as described below.
Convection
From classical physiology, fluid movement across the endothelial
wall is described by Starling's hypothesis:
where
K(x) is Starling's coefficient
p.sub.i =intraluminal hydrostatic pressure
p.sub.e =extraluminal hydrostatic pressure
.pi..sub.i =intraluminal osmotic pressure
.pi..sub.e =extraluminal osmotic pressure
Starling's coefficient is a factor which includes the conductivity
properties of the endothelial wall and other transport properties.
In normal capillaries, it has been shown that the Starling's
coefficient increases from the arteriolar to the venular side by as
much as ten fold. A report by Peterson et al. (1973) has shown that
the endothelial capillary wall in tumors has significantly greater
permeable coefficients than normal vessels.
Darcy's Law describes the relationship between interstitial fluid
velocity and an interstitial pressure gradient: ##EQU1## where
K.sub.t (P.sub.e,x.sub.i) is the Darcy coefficient and is a
function of the interstitial pressure and properties of the medium.
For general purposes, it is usually assumed to be a single property
of the medium, such as porosity.
Convection describes material transport which occurs as a result of
macroscopic movement of the volume element in which the material is
found. There is a normal convective movement of interstitial fluid
within the tissue compartments of both tumor and brain. It is clear
from work by Jain and others that significant movement of
interstitial fluid occurs from areas of high pressure, namely
central tumor areas, to lower pressure areas in the periphery,
resulting in a net outward movement of tumor interstitial fluid
from the interior to the exterior. Therefore, one might exploit
this movement by centrally injecting the desired compound and then
relying on convection to move the materials. There are some
problems with this approach however such as areas of differing
pressure within the tumor and non-uniformity of the interstitial
compartment within the tumor.
In general, the interstitial space in tumors is very large compared
with that in host normal tissues (Peterson (1979)). The
interstitial space of tumors is composed predominantly of a
collagen and elastic fiber network. Interspersed within this
cross-linked structure are the interstitial fluid and
macromolecular constituents (polysaccharides) which form a
hydrophilic gel. Whereas collagen and elastic impart structural
integrity to a tissue, the polysaccharides (glycosaminoglycan and
proteoglycans) are presumably responsible for the resistance to
fluid and macromolecular motion in the interstitium. In several
tumors studied to date, the collagen content of tumors is higher
than that of normal host tissue. On the other hand, hyaluronate and
proteoglycans are, in general, present in lower concentrations in
several tumors studied to date than in normal host tissue.
Therefore, the large interstitial space and low concentrations of
polysaccharides suggest that values for interstitial hydraulic
conductivity and diffusivity should be relatively high in tumors.
Some experimental work supports this. Tumor transport coefficients
with values an order of magnitude higher than those of several
normal tissues should favor movement of macromolecules in the tumor
interstitium.
Various experiments have attempted to quantity fluid movement in
tumor and normal tissue. In other papers describing bulk movement
of interstitial fluid., fluid loss has been measured at 0.14-0.22
ml/hr per gm of tissue in four different rat mammary carcinomas.
This fluid leakage leads to a radially outward interstitial fluid
velocity of 0.1-0.2 .mu.m/sec at the periphery of a 1 cm `tissue
isolated` tumor. The radial outward velocity is an order of
magnitude lower in a tumor grown in the subcutaneous tissue or
muscle. A macromolecule at the tumor periphery has to overcome this
outward convection to penetrate into the tumor by diffusion. The
relative contribution of this mechanism of heterogeneous
distribution of macromolecules in tumors is, however, smaller than
the contribution of heterogeneous extravasation resulting from
elevated pressure and necrosis. It is also apparent from
experimental studies that large molecules move mainly by
convection.
Diffusion
Diffusion is the movement of molecules from an area of high
concentration to an area of lower concentration. Molecular
diffusion results from the random motion of the molecules of the
material and depends upon the molecular weight of the material,
concentration gradient, and other factors. Diffusion of materials
along concentration gradients is also well described in tumor and
normal neural tissue and can be relied upon for some degree of
distribution of materials which are injected in concentrated
amounts. High molecular weight compounds have low diffusivity in
brain or tumor and for low molecular weight compounds, capillary
loss and metabolism often underlie the restricted distribution.
Diffusion is unaffected by pressure gradients. Small molecules such
as oxygen and conventional chemotherapeutic drugs which have MW
lower than 2,000 daltons leave blood vessels and migrate through
normal tissue mainly by diffusion.
The time required for a molecule with diffusion coefficient D to
move by diffusion across distance L is approximately L.sup.2 /4D.
For diffusion of IgG in tumors, this time is of the order of 0.5 hr
for a distance of 100 .mu.m, .apprxeq.2 days for a distance of 1
mm, and .apprxeq.7-8 months for a distance of 1 cm. Consider a
hypothetical tumor that is uniformly perfused, has nearly zero
interstitial pressure, and has exchange vessels .apprxeq.200 .mu.m
apart. In such a tumor, IgG would reach uniform concentration
approximately lhr post injection, provided the plasma concentration
remains constant. In a normal tissue with the value of D lower by
an order of magnitude, it would take .apprxeq.10 hours to reach
uniform concentration. In a more realistic scenario, the tumor
vessels are .apprxeq.200 .mu.m apart and uniformly perfused, but
the interstitial pressure in the center is increased such that
fluid extravasation, and hence, convective transport of
macromolecules across the vessels have stopped. In such as case,
the only way macromolecules can extravasate in the center is by the
slow process of diffusion across vessel walls. Also, they can reach
the center from the periphery (where interstitial pressure is near
zero) by interstitial diffusion. If the distance is 1 cm from
center to periphery, it would take months to travel this distance.
If, as a result of elevated interstitial pressure and cellular
proliferation, the central vessels have collapsed completely, then
there is no delivery of macromolecules by blood flow to the
necrotic center. In such as case, there are no molecules available
for extravasation by diffusion across a vessel wall, and
consequently the central concentration would be even lower.
Mathematical modeling conducted by Jain's group (Jain (1994))
postulates that a continuously supplied monoclonal antibody of
molecular weight 150,000 daltons could take several months to reach
a uniform concentration in a tumor that measured 1 cm in radius and
had no blood supply at its center.
Pressure Gradients
The enhancement of convective fluid movement utilizing small
amounts of continuous pressure has been demonstrated by several
studies, increasing flow by at least an order of magnitude. Bobo et
al. (1994) explored the use of pressure gradients to enhance
convection volume of distribution (V.sub.d) of materials in cat
brain. The V.sub.d of the infusion concentration increased linearly
with the infusion volume. Immediately after the completion of
infusion of 600 .mu.l of solution, approximately 50% of the cat
hemisphere had received .gtoreq.1% of the concentration of .sup.111
In-Tf in the infusion. Since the normal rate of diffusion of
.sup.111 In-Tf over the three hours of infusion would be
negligible, this distribution was felt to be the result of
convection. The rates of infusion during the experiments proved
significant, as infusion rates greater than a few microliters per
minute produced leakage of the infusion solution out of the cannula
tract and lowered the infusion pressure. The CNS is normally able
to remove fluid from the interstitial space in edematous white
matter at about 0.3-0.5 .mu.l min.sup.-1, equivalent to
approximately 2.5 .mu.l/min per hemisphere in the cat. Two hour
infusions spread .sup.111 In-Tf .apprxeq.1.5 cm and sucrose
.apprxeq.2.0 cm in an anterior-posterior direction immediately
after completion of the infusion. Although predominantly
distribution in white matter immediately after infusion, .sup.111
In-Tf showed increasing penetration of gray matter over the next 24
hours. Sucrose was extensively distributed into gray matter by two
hours. In these experiments, the infusion of .sup.111 In-Tf
solutions occurred at concentrations that were nearly five orders
of magnitude greater than the reported tissue-averaged density of
receptors, thus avoiding the problem of .sup.111 In-Tf binding to
receptors and being internalized by cells.
With regards to the side effects of infusion, all of the
interstitial brain infusions of the study were well tolerated and
were not associated with any hemodynamic instability during the
infusions. Two chronic animals demonstrated transient lethargy and
weakness that resolved within 24 hr. Structural studies by
Marmaroue et al. have demonstrated that myelinated axons remained
spatially related via oligodendroglial processes despite the
expansion of the extracellular space and there was orderly
reconstitution of the tissue as the edema resolved, leaving only a
mild fibrillary astrocytosis. In a variety of models, cerebral
edema does not cause neurologic dysfunction as long as intracranial
pressure does not appreciably elevate. Furthermore, evidence
suggests that even when edema is severe enough to cause neurologic
dysfunction, deficits related to edema are reversible. Thus,
evidence suggests that cerebral edema per se does not alter brain
function as long as there are no associated herniations of cerebral
tissue, significant elevation of intracranial pressure, or
reduction of cerebral blood flow below the normal range.
Therefore, the possibility exists of utilizing pressure gradients
within extravascular space of tumors, which is significantly
increased above normal tissue, to allow spread of materials over
1.5-2.0 cm of the interstitial compartment from each
electrode/injection sites.
Positioning of the Electrical Field
It is logical to begin consideration of the application of an
electrical field to a biological tissue by considering the site of
origination of the electrical field to be applied. In reviewing the
pertinent literature regarding in vivo brain electroporation or
electropermeabilization, only bipolar electrode configurations have
been used to "bridge" across the tissue to be electropermeated. In
recent work by Ceberg et al. (1994), the results demonstrated that
the electropermeabilization effect in brain tissue extended well
beyond the expected and desired confines of the theoretical
electrical field lines. Both electrode sites were located outside
of the primary tumor tissue, potentially allowing for significant
current flow into adjacent normal tissue.
In order to provide equal distribution of electropermeabilization
voltages, electroporation pulses should ideally be distributed in
uniform fashion throughout the target body site, minimizing the
flow of current in retrograde fashion up the electrode tract.
Subsequent refinements in the electrode design may provide for a
facilitation of closure of the brain tissue around the electrode as
it penetrates the brain, thus creating a natural barrier to the
flow of current. Additionally, secondarily coating the surface of
the electrode would also aid in creating a resistance to retrograde
current flow, as would the use of appropriate dielectric
insulators. Additional measures for prevention of electrode tract
flow include physical barriers such as collars or balloon devices
which would fit around the shaft of the electrodes.
The electrodes will be placed by two methods: (1) stereotaxic
placement or (2) direct placement. Prior to therapy all tumor
patients undergo at least one form of routine imaging study (MRI or
CT) to localize and differentiate diseased from healthy tissue.
Recent advanced imaging techniques combine these techniques with
stereotaxic coordinate systems which enable the precise
localization of target body sites within three-dimensional space.
In addition, valuable information may be gained as to the detailed
internal architecture of the tumor such as variations in tumor
density and vascular supply. This information should prove useful
in directing the strength and number of electroporation pulses
which in turn determine the magnitude of the
electropermeabilization field within different regions of the
tumor. By pursuing this rationale, it should prove beneficial to
permeabilize areas of the tumor, such as the central necrotic
region, which are inherently more resistant to the electroporation
pulses effects.
The first placement method involves utilization of stereotaxic
imaging information to precisely locate the electrodes both within
and around a tumoral or diseased area of the brain. The general
method would be similar to other methods described in the
literature which involve delivery of the electrodes to their
desired location utilizing either hand-held instrumentation or
mechanical drive systems which are linked to the three-dimensional
imaging coordinates. It is anticipated that for the purposes of the
acute implementation, electrode immobilization provided by normal
frictional forces would be adequate and not require positional
stabilization.
The second method involves the use of hand-held or guided
instrumentation during surgical biopsy procedures. In this
procedure, electrode insertion would be verified by intraoperative
radiological methods and although potentially less accurate, would
nonetheless may be more widely utilized as dictated by efforts
toward cost effectiveness.
Iontophoretic Fields
There are a number of aspects of the invention which will utilize
iontophoretic or electrophoretic fields. The first involves
enhancement of distribution of the hyperconductive materials which
will be moved throughout the interstitial compartment via constant
or alternating field application. Low intensity electrical fields
(Chang et al. (1992)) have been proven useful for electroporation
and also useful for cell fusion. The application of low-intensity
AC field has resulted in a dielectrophoretic process resulting in
the formation of pearl chains. This low intensity field results in
alignment and positioning of cells such that their membranes are
perpendicular to the electrical fields where conditions for fusion
are most suitable. Also related is the fact that AC fields are also
particularly important when fusing enucleated oocytes to cells with
reduced diameters since the polarization caused by the AC field
will aid in bringing their membranes into contact.
A further aspect involves the "preconditioning" of the target
tissue (utilizing sub-threshold DC, AC, RF, or ELF electrical
fields) in order to maximize the effect of the electroporation
pulses. This treatment is designed to increase the stochastic
occurrence of increased permeability sites in cell membranes of the
target body site. This aspect of the invention may also rely upon
the delivery of electrically conductive material to the target body
site as defined above.
Use of Electrical Fields To Trigger Drug Release
The present invention is typically used in the following manner: A
suspension of liposomes with encapsulated drug is prepared in
sterile pharmaceutical formation suitable for i.v., i.m., s.c., or
any other route of injection administration.
The suspension is then administered to the patient in need of
treatment and the liposomes are subsequently treated with a safe
but effective dose of electrical field. "Safe" in this context
means that it does not heat the tissue to hyperthermic (43.degree.
C.) or supra-hyperthermic (>43.degree. C.) temperature levels
that may cause tissue damage.
The liposomes may be injected as localized depots or may be
injected to circulate freely in the blood stream with the potential
to be targeted to specific tissue sites and localize at a site of
interest. The latter case is termed targeted drug delivery and the
bound liposomes are treated with the electrical field to trigger
localized drug release at the target site.
The electrical fields causing electroporation act to trigger drug
delivery in two ways: (1) by destabilizing the liposome bilayer so
that membrane fusion between the liposome and the target cellular
structure occurs, thus facilitating the direct delivery of drug
into the target cell; and (2) by triggering the release of drug in
high concentrations from liposomes at the surface of the target
cell so that the drugs are driven across the cell membrane by a
concentration gradient upon via the created electropores. In either
case, the direct cellular-level microinjection of drug into the
target cell is achieved.
The electrical field source is then placed, desirably via the
HexasphereTM electrode array, into the tissue of desired
localization of the drug delivery. Although the liposomes are
delivered systemically, with some exception, the localized field
effect serves to constrain the electroporation effect to the
geometrically-oriented area as defined by the electrodes. Thus, the
liposomes in this area are treated and will release encapsulated
drug as they circulate through this local electrical field. The
patient can be treated with the field for a single treatment or be
treated at different time periods (i.e. multiple doses) using a
number of intermittent applications of the field.
The specific process of targeted drug delivery using liposomes via
the present method has several unique advantages. The liposomes
affinity for the target cell results in adsorption or binding to
the target cell resulting in an extraordinarily high concentration
of encapsulated drug at the surface of the target cell. A typical
target cell has a diameter of approximately 7 .mu.m (7,000 .mu.m).
This is large compared to the size of a liposome vesicle (having a
typical diameter of 100 nm). Approximately 450 million liposome
vesicles can be bound to the surface of such a target cell, and
each liposome vesicle can be loaded with drug at a high
concentration (>100 mM). This situation represents the most
effective means for bringing high concentrations of drug to the
surface of a target cell. Using electrical fields via the method
provided, the problem of releasing drug from these bound liposomes
can be overcome.
Method of In Vivo Electroporation
Electroporation is a phenomenon in which the membrane of a cell
exposed to high-intensity electrical field pulses can be
temporarily destabilized in specific regions of the cell. During
the destabilized period, the cell membrane is highly permeable to
exogenous molecules present in the surrounding interstitial or
extracellular spaces.
The phenomenon of electroporation has been described as a threshold
dependent phenomenon, in that the threshold field strength
(E.sub.C) for electroporation is a "point of no return". If the
electric field E (.gtoreq.E.sub.C) is maintained, the electropores
induced by the supercritical field increase in number and size
until, at a supercritical number density and pore size, the
membrane ruptures. If electroporation pulses of short duration
.DELTA.t are applied, the field is already switched off before
rupture can occur. It is therefore appropriate to view membrane
electroporation as being characterized by critical values for the
extent (.xi.) of structural rearrangement, for the field strength
(E.sub.C), and for the pulse duration (.DELTA.t.sub.C). The primary
requirement for the onset of electroporation is that the threshold
.xi..sub.c has to be reached. The minimum field strength to attain
the critical value .xi..sub.C is the critical field strength
E.sub.C. Once the threshold .xi..sub.C is reached
(E.gtoreq.E.sub.C), the actual electroporation starts and proceeds
unidirectionally until the rupture threshold .xi..sub.r is
attained, i.e. where the membrane ruptures. If the field is reduced
below E.sub.C or switched off before .xi..sub.r is reached, the
pores reseal such that the original membrane state appears to be
completely restored. Since the threshold .xi..sub.C is attained
faster at a higher field strength, the minimum pulse duration
.DELTA.t.sub.C that is required for the onset of the
electroporation process decreases as the applied external field
increases.
Sudden non-thermal rupture (irreversible mechanical breakdown)
occurs in bilayer membranes exposed to a transmembrane potential,
U, in the approximate range 200 .ltoreq.U.ltoreq.500 mV for a
relatively long time (i.e., .DELTA.t.gtoreq.10.sup.-4 sec). Larger
but shorter duration U results in non-damaging, more rapid
discharge of the membrane. Typical square wave pulse
characteristics which cause electroporation are a pulse width in
the range of from 10.sup.-7 to 10.sup.-4 sec, and amplitudes at the
membrane in the range of 500 to 1500 mV.
A recent paper by Prausnitz et al. (1994) noted that the actual
electrical field in electroporation may be up to 10% less than the
nominal electrical field, perhaps due to voltage drops at the
electrode interface. Therefore, although only nominal electrical
fields are generally reported in the literature, differences
between nominal and actual electrical fields are probably present
in many electroporation protocols.
The recovery process in cells versus artificial bilayers is much
slower and strongly temperature dependent in cells. It has also
been reported that: (1) The greater the applied field strength, the
larger the probe molecules which can permeate into the treated
cells preceding cell lysis; (2) The longer the pulsed electric
field, the larger the probe molecules can permeate; (3) Pulsed
electric field treatment in a higher-ionic-strength medium (e.g.
saline, leads to creation of small pores, and in a
lower-ionic-strength medium, e.g., isotonic sucrose, to bigger
pores when identical pulsed electric fields were used; and (4)
Pulsed electric field treatment at higher temperatures leads to a
lower critical voltage, implying that the induced pores could be
larger (Kinosita et al. (1977)).
However, Prausnitz et al. (1994) noted that longer pulses were less
effective than multiple pulses for maximizing transport while
minimizing damage. Furthermore, multiple pulsing enhanced uptake
strongly at lower electrical field strengths, but weakly at higher
field strengths. This suggests the existence of a transport maximum
beyond which additional pulses can not increase uptake. It follows
that more pulses at moderate E lead to the same uptake as fewer
pulses at higher E. However, pulses at larger E are generally
associated with lower cell viability (Chang et al. (1992)).
Multiple pulses at moderate E may maximize transport and cell
viability. Further work demonstrated a comparison of the effects of
multiple pulses and single pulses having the same time integral of
electrical field strength (INT) where INT is defined by ##EQU2##
where E.sub.o is the peak field strength,
t is time, and
.tau. is the decay time constant. D1573713
For multiple pulses,
The flux of molecular transport through a tissue is a function of
the product of the tissue permeability, the driving force and the
area of the tissue. Two mechanisms of electroporation-mediated
permeability are utilized in the present invention:
(1) transient electropores: Within a few msec after the cessation
of the electrical field, these pores partially decrease in diameter
and adopt a stable configuration. The pores allow translocation of
various molecules (influx of exogenous molecules and efflux of
cytosolic compounds) by two slightly different mechanisms. Large
molecules and even macromolecules are assumed to cross only the
transient electropores. They must be present in the extracellular
medium during the electroporation pulses and efficient
electroporation requires long electroporation pulses or a large
number of pulses and an E generally greater than the absolute or
E.sub.C. The amount of compound electroincorporated is inversely
related to the molecular weight.
(2) long-lasting electropores: Created after shorter
electroporation pulses, these electropores are only efficient for
ions and small or intermediate size molecules. It is possible to
add the exogenous molecules to the electroporation cells after
electrical field delivery as well as before. Concentrations on both
sides of the plasma membrane are roughly equilibrated.
With respect to resealing, the higher the temperature, the more
rapid the resealing. There is about one order of magnitude between
37.degree. C. and 20.degree. C., and another between 20.degree. C.
and 4.degree. C. Also, the larger the electropores formed, the
longer the time necessary to recover the initial membrane
impermeability. Resealing appears to be also a function on ionic
strength, osmotic pressure, the presence of membrane perturbing
agents and integrity of the cytoskeleton.
It has been shown (Andreason et al. (1989)) that electroporation
using a single high voltage square wave pulse was not effective for
gene treatment. However, following this pulse with a series of low
voltage pulses allowed gene transfer to occur and yielded
significantly greater efficiency of transfection. Electroporation
using the same series of low voltage pulses without the initial
high voltage pulse did not result in detectable electroporation.
Additionally, the viability of cells following electroporation
appears to be greater than that observed with exponentially
decaying waves. The effectiveness of complex series of pulses
suggests that the mechanism of electroporation may depend on the
exact characteristics of the electroporation pulses, rather than
simply membrane effects.
The cells of a tissue are connected to each other, in particular
through gap-junctions, establishing an electric continuum which
results in a great difference as to what happens when an electrical
field is applied on a cell suspension. Some studies (Maurel et al.
(1989)) showed that monolayer threshold of electroporation was
lower than suspension cells of the same type.
The work of Kinosita et al. (1977) and Rols et al. (1992) suggests
that the electroporation phenomenon can be described as a three
step process of: (i) induction of transient permeated structures
for electrical field intensities greater than a threshold value
E.sub.C ; (ii) expansion of these permeated structures which is
related to the slope dP/dE of the permeabilization curve; and (iii)
resealing of the electropores.
Resealing of field-induced membrane perturbations is a prerequisite
for entrapment of membrane impermeable substances, and the
restorative kinetics are highly dependent on temperature. At
physiological temperatures, the resealing is very rapid (few
minutes). At low temperatures (4-10.degree. C.), resealing is very
slow. Resealing properties also depend on .DELTA.t and E.sub.C. To
overcome the nonuniform permeability pattern in the membrane, it is
important to apply several consecutive pulses. Time interval on the
order of 1-2 seconds to allow sufficient time to reseal the lipid
bilayer structure of the biological membrane.
The procedures involved in using electroporation via the
Hexasphere.TM. electrode array revolve around placing the array
such that the electrical fields produced are oriented such that
they travel across the hemi-diameter of the tumor or diseased area
and remain confined within the substance of the target body site.
Concomitant with this is the need to electrically isolate each
electrode with respect to one another in order to drive the
electroporation pulses "through" tissue rather than permit
retrograde transmission back up along the electrode tracts. The
electrodes will be spaced using stereotaxic equipment, with the
core electrode(s) placed within the tumor or diseased material.
Following this, the remaining electrodes will be placed into
satellite positions, for example as illustrated in FIG. [?].
Techniques to insure proper placements of the satellite and core
electrodes will involve imaging studies performed prior to the
procedure, or else via direct operative placement at the time of
biopsy or debulking procedures. The electrodes will typically be
placed with the aid of a stereotactic instrument through burr holes
in the skull, drilled down to the dura mater. Due to the anatomic
difficulty in approaching the inferior surface of a predetermined
area in the brain, the Hexasphere.TM. electrode array will
desirably be oriented such that the inferior most electrode points
will be placed at 45.degree. angles with respect to the coronal
plane using the vertical meridian as reference.
At this point, the tumor will have threshold electroporation pulses
applied, likely in an alternating fashion utilizing different
electrode sites in order to allow for complete distribution of
current density to all parts of the tumor. As the
electropermeabilized or electroporated cells spill the cytoplasmic
contents, the conductivity will significantly increase, allowing
subsequent pulses even greater effect on the cell population. The
specific sequencing of the pulses may prove important in allowing
complete coverage of the tumoral area. Following placement of the
electrodes and subsequent to the preconditioning phase of the
procedure, electroporation pulses (8-12) can be delivered using
standard electroporation units (e.g. Instrument Research Co.).
These pulses will consist of rapid serial square-waved pulses of
approximately 400-1300 V/cm, with pulse duration of ranging from 10
.mu.sec to 1 msec delivered at one pulse per second. The intensity
of the electrical pulses will be checked by a digital storage
oscilloscope connected to the electric pulse generator. This set of
stimulus parameters has been experimentally used by Ceberg et al.
(1994) as well as Salford et al. (1993) and in human clinical
trials. The initial sequence of electroporation pulses may be
followed by a second or even third series of pulses, dependent upon
conditions.
The occurrence of electroporation effect can be detected by
monitoring the tissue for a decrease in electrical resistance,
which, along with an enhanced tissue permeability, is the
characteristic effect of electroporation. Therefore, some measure
of the effectiveness of the electroporation pulses may be
appreciated by measuring the relative conductance between
electroporation electrodes following the treatment pulses. In other
words, prior to the first series, a series of smaller brief pulses
can be delivered between electrodes in serial fashion to determine
the pre treatment conductance. Following the treatment pulses,
follow-up measurements may help determine the success of the
electroporation pulses by documenting the presence of an increased
conductance due to large ionic shifts as a result of the
poration.
There are a number of technical issues which must be considered
when contemplating electroporation of a tissue as a whole, rather
than tissues in suspension or culture. The overall effectiveness of
electroporation is dependent upon the spread of the electrical
field through the tissue and the voltage potential each cell
membrane sees. Sources of heterogeneous electroporation within a
cell population include:
cell size, shape and orientation
non-uniformity of electrical field
cell-cell separation
tissue heterogeneity (perturbation of local field by tissue)
membrane composition (varies within cell population)
Because of the above mentioned properties, it is useful to consider
other methodologies to insure the uniformity of the applied
electrical fields throughout the target body site tissue.
Use of Cerebroplegia
The use of intermittent hypothermic cerebral perfusion
(cerebroplegia) has been reported in the literature as a
concomitant procedure to surgeries involving intracardiac repair in
infants, aortic arch replacement, chronic pulmonary embolectomy,
and selected neurosurgical and vascular surgical procedures. In
general, these procedures have involved cooling the entire body
using cardiopulmonary bypass procedures in which the subjects have
been maintained at low core hypothermic conditions (<20.degree.
C.) over 1-2 hours. Cerebroplegia is aimed at cooling the brain
only through selective perfusion of the brachiocephalic arteries
with cool blood or fluids (6-12.degree. C.) (analogous to cold
blood cardioplegia).
The perfusion equipment utilized include basically a heat exchanger
which allows blood derived from the general circulation (or
perfusion fluid) to be cooled to 6.degree. to 12.degree. C. A
perfusion line distributes the perfusate to the brachiocephalic
arteries and special cannulae are available in several diameters to
perfuse the carotid arteries.
Perfusion solutions will likely consist of either cooled blood
which has been slightly heparinized, or asanguineous oxygenated
solution (NIH cardioplegic solution; Robbins et al. (1990)),
consisting of 0.45 normal saline with 2.5% dextrose, mannitol,
sodium bicarbonate, lidocaine, nitroglycerine and calcium chloride.
This solution also has 300 ml of oxygen added to produce a PO.sub.2
>600 mm Hg (oxygen content=1.5 mmol/dl) at pH 8.0.+-.0.1.
Additionally, several other hypotonic solutions, can be utilized
for brief time periods to introduce minimally conductive solutions
into the cerebral vasculature. The primary value of these solutions
is to provide oxygen delivery to the tissues and clear metabolic
by-products. Crystalloid has some obvious advantages such as
simplicity of delivery. However, blood clearly provides superior
buffering capacity and oxygen free radical scavenging properties
and should generally be preferred.
The patient is then prepared and anesthetized, with continuous
monitoring of electroencephalogram, rectal and nasal temperatures.
The subjects will have T shunts placed in the carotid arteries
bilaterally.
Thereafter, patient cooling is initiated. In small animals, this
will consist of cooling (with cooling pads and ice packs to the
periphery) down to nasopharyngeal temperature of 12-15.degree. C.
In large animal subjects and humans, this will consist primarily of
cardiopulmonary bypass with cooling to 20.degree. C. When proper
core temperatures are achieved, both carotid arteries are
cannulated and held by means of purse-string sutures. The proximal
limb of the carotid shunt will be occluded with the delivery of
solution into the distal carotid artery through the side port of
the shunt. The proximal limb is then opened to establish cerebral
reperfusion at the conclusion of the period.
It has been shown that the administration of cerebroplegia solution
maintained ATP and CrP at significantly higher levels and Pi at a
lower concentration, for all points during the cerebroplegia
period. It has also been demonstrated that cerebroplegia produces
significantly higher values of intracellular pH throughout the
arrest periods.
The cold perfusion solution is then initiated and maintained until
the electroencephalogram demonstrates total disappearance of
activity (generally 3-16 minutes in humans). After this time (flat
electroencephalogram) the cerebroplegia will be converted from a
continuous flow to an intermittent flow. The pressure in the
carotid arteries will be maintained to approximately 30-40 mm Hg,
which approximates normal arterial pressures in the rodent
population, versus 60-70 mm Hg in humans.
After the appropriate procedures have been performed, the carotid
solution is gradually rewarmed and then bypass is discontinued. The
cannulae are removed carefully to prevent the introduction of air
bubbles.
Of additional benefit is the use of selective calcium antagonists
and prostaglandin derivatives as protective agents during the
hypothermic ischemia periods.
The small amount of oxygen delivered to the brain at reduced
temperatures and corresponding reduced metabolic demands is
sufficient for the maintenance of high-energy phosphates. Also, the
intermittent delivery of an alkalotic solution could neutralize and
washout ischemic metabolic by-products, resulting in a less
acidotic cellular environment.
Strategy to Seal Cell Membranes Post Electroporation
A further aspect of this invention involves the use of recovery
agents such as non-ionic surfactant or other similar agents to aid
the closure of pores, electropores or cell membrane defects formed
in target body site following electroporation pulses. This
technique will aid in retaining the material delivered via the
liposomes into target cells.
Biological lipid membranes are supermolecular assemblies of
biological surfactants that spontaneous aggregate in an aqueous
environment. During an ultrastructural examination of
electroporated cell membranes, Chang and others (Chang et al.
(1992)) demonstrated that stable structural defects occur in cell
membranes. Their studies demonstrated pore diameters in the range
of 100 nm. It is theoretically possible for a surfactant molecule
to fill a 100 nm diameter defect in the cell membrane. The physics
of membrane formation are such that it is favorable for surfactants
to formation sheets across such defects. Therefore, it appears that
the problem of restoring integrity to a damaged cell membrane is
equivalent to the problem of achieving a high enough concentration
of the correct surfactant at the site of damage. These compounds
must not be toxic. Work by Lee et al. (1992) has found that one
such surfactant (Poloxamer 188) was able to seal
electropermeabilized skeletal muscle fiber cell membranes by
placing it into the solution in which the cell was contained. This
material is a reverse tri-block copolymer that has hydrophilic ends
and a `hydrophobic` center. It is known to adhere to cell
membranes. In vivo administration of this compound into the
circulation of a rat demonstrated successful repair of
electroporation damaged muscle tissue membrane in an island flap
model. This was reported to be the first direct demonstration of
membrane repair in vivo. An adequate supply of surfactant molecule
present in the extracellular spaces, by incorporating the
surfactant material into liposomes which are preloaded into the
electroporation treatment area, should prove beneficial in
obtaining the desired result. The material has also been
demonstrated to perform successfully within a 30 minute period of
time.
Another well established membrane recovery technique in
electroporation has been well studied and is related to the
temperature at which the post electroporation membrane resides. By
cooling the membrane, the permeabilization effect persists much
longer as compared to permeabilization found at increasing
temperatures. Therefore, manipulation of the rate at which the post
cerebroplegia brain is rewarmed will influence the duration of the
electropermeabilization effect and the resealing rate of the cell
membrane.
"Pre-conditioning" for Electroporation Effects
Another aspect of this invention is enhancement of the conductance
of the electrical field throughout the target body site utilizing
liposome-encapsulated particular materials designed to allow
application of similar electrical fields throughout the target body
site as defined by the electrode array. Local delivery of
electrically conductive solutions (e.g. ionic solutions,
Fe++-containing solutions, etc.) designed to facilitate the spread
of the electrical field throughout the interstitial spaces of the
tissue defined by the outline of the array would assist in
preconditioning the predefined region and create a more uniform
field conductance, thus maximizing the electroporation effect.
This aspect of the invention will involve the use of subthreshold
electroporation pulses which will be used to influence the
distribution of free ions throughout tumor interstitial fluids.
There are a number of factors which suggest that subthreshold
electrical field application may influence the overall
electroporation treatment pulses. First, even very small field
intensities may cause electroporation, provided the field
application is long enough. Second, if a second electroporation
pulse hits the membrane patch which resides, not in the closed
membrane state, but in a partial electropore state, the change
induced by the second pulse is facilitated, because the pore
transitions have already been facilitated by the first pulse.
Third, the existence of transient aqueous pores can be consistent
with the known behavior of bilayer membranes at low E. Fourth, low
intensity electrical fields (Chang et al. (1992) page 320) have
been proven useful for electroporation and also useful for cell
fusion. The application of low-intensity AC field has resulted in a
dielectrophoretic process resulting in the formation of pearl
chains. This low intensity field results in alignment and
positioning of cells such that their membranes are perpendicular to
the electrical fields where conditions for fusion are most
suitable. Also related is the fact that AC fields are also
particularly important when fusing enucleated oocytes to cells with
reduced diameters since the polarization caused by the AC field
will aid in bringing their membranes into contact. The energy
needed to form an aqueous pore is reduced as the transmembrane
voltage is increased by application of an external electrical field
(Weaver (1993) page 428) which raises the possibility that the
pre-conditioning effects may in fact consist of one large
electroporation pulse followed by a series of smaller pulses
(Weaver (1993) pp. 429-430)). Further support for this notion stems
from the fact that the signature of electroporation is a tremendous
increase in electrical conduction which is measured and is believed
to be due to ionic conduction through transient aqueous pores.
During the application of a subthreshold (E<E.sub.C) electrical
field, there are a number of subcritical membrane changes which
occur, namely from .xi..sub.o to .xi..sub.C, which represent
reversible structural rearrangements such as the increase in number
and size of hydrophobic defect sites and micropores in the bilayer.
The minimum field strength to attain the critical value .xi..sub.C
is the critical field strength E.sub.C. Once the threshold
.xi..sub.C is reached (E.gtoreq.E.sub.C), electroporation starts.
In fact, the data suggests that interfacial polarization precedes
the structural transitions (Neumann (1987)). It is further
elaborated by Neumann (at page 82) that the return to the closed
membrane state M after switching off the field occurs in the
absence of the external field. Given the sequence ##EQU3## where
sequence of membrane changes from the poreless state M to
hydrophobic (P.sub.HO) to hydrophilic (P.sub.HI) pores, the return
transition P.sub.HO P.sub.HI (involving reorientation of the wall
lipids) may face major activation barriers. Thus, if now a second
pulse hits the membrane patch in the P.sub.HI state, the change
induced by the second pulse is facilitated, because the transitions
P.sub.HO P.sub.HI have already been caused by the first pulse.
Pre-conditioning also describes an alteration of the interstitial
fluid milieu in order to enhance the electroporation given a
particular value of E.sub.C. Therefore, conductivity of the
electroporation pulse and its relationship to ionic interfacial
polarization is necessarily considered. For low conductivity
membranes of thickness d of cells of radius a, the stationary value
is given by
where .delta. is the angle between the membrane site considered and
the direction of E. The conductivity factor f(.lambda.) is a
function of the specific conductances or conductivities of the
external solution (.lambda..sub.0 .gtoreq.10.sup.-4 S cm.sup.-1) ,
of the cell interior (.lambda..sub.i .apprxeq.10.sup.-2 S
cm.sup.-1), and of the membrane (.lambda..sub.m .apprxeq.10.sup.-7
S cm.sup.-1), respectively and of the ratio d/a.
Usually, .lambda..sub.m <<.lambda..sub.i, .lambda..sub.0 and
d<<a such that
From this, it is readily seen that an increase in the external
ionic strength leading to an increase in .lambda..sub.0 will
increase .DELTA..phi.. This is consistent with the notion that the
interfacial polarization is associated with ion accumulations at
the interfaces of the membrane. Therefore, efforts to increase the
.lambda..sub.0 of the interstitial fluids will increase the effect
of the electroporation pulses, thus allowing either more
electroporation at a given value of E.sub.C or else the same effect
at decreasing levels of E.sub.C. The present invention proposes to
deliver liposomes with hypertonic materials within them to the
tumoral or diseased sites; then at subthreshold values of E.sub.C,
effect release of the materials which will diffuse into the
interstitial medium and cause a relative increase in
.lambda..sub.0. Then, during the electroporation phase, more
effective electroporation will result, causing increased number of
cells to undergo electropermeabilization and thus be susceptible to
the liposomes containing the drug compound to be delivered.
Thus, the present invention includes the delivery of materials
which will aid in the pulse conduction through the interstitial
compartment. This may be accomplished utilizing liposomes or may
involve the already described local delivery methods of injection
followed by distribution utilizing electrical field influence upon
charged particles. Alternatively, this increase in ionic strength
in the interstitial fluid might be accomplished by first using
liposome-mediated delivery of hypertonic materials to the tumoral
or diseased sites; then at subthreshold values of E.sub.C, bring
about the release of the materials which will diffuse into the
interstitial medium and cause a relative increase in
.lambda..sub.0. Thus, during the electroporation phase, a more
effective pulse propagation will result, resulting in an increased
number of cells which undergo reverse electrical breakdown.
One method to accomplish this would be the serial application of a
low voltage field across all elements of the array. The range of
voltages would be 10 to 100 V. Consideration is also given to the
possible utilization of an AC field which may be phase shifted in
order to provide some asymmetry of the field duration, thus pulling
ionic elements in one direction. The duration of the pulses will
most likely be in the 0.5-5 second range. Multiple pulses will be
required with some interval between each pulse ranging from 500
msec to 5 sec. It is anticipated that it will take on the order of
minutes but less than 2 hours to accomplish the pre-conditioning.
This estimate is based on diffusion studies which indicate this
order of magnitude of time.
The application of the subthreshold electroporation pulses will
desirably be computer driven and allow variation of the appropriate
signal strength and duration, and also the number and order of
active electrodes which will participate in each pulse.
Furthermore, as suggested by Chang in U.S. Pat. No. 5,304,486, the
fields (both AC and pulsed RF) may be generated by synthesizing the
required electrical wave with a digital computer and amplifying the
waveform using a power amplifier. It is anticipated that emphasis
may be placed on those electrodes which will effectively
concentrate or orient an electrical field in those areas which are
deemed to require increased distribution of the facilitory ions due
to increased density or decreased blood supply. The exact
specifications of intensity of voltage, duration of pulses, number
and orientation of pulses will be more fully elucidated as
subsequent data are accumulated.
Another aspect of the preconditioning phase will be the use of a
single electroporation pulse followed by a series of smaller
subthreshold pulses.
This would involve the inclusion of liposomes filled with
hyperconductive solutions used to preposition ionic compounds into
the tumoral areas via the microcirculation and leaky capillaries.
Once these are in place, a single electroporation pulse large
enough to cause rupture of the liposomes would facilitate the
selective delivery of hypertonic medium directly to the site of the
target body site, thus enabling local specificity. As examples,
hypertonic saline might be encapsulated in liposomes and delivered
to the target body site via leaky capillaries. At this point, a
single preconditioning electroporation pulse would be delivered
which would rupture the liposomes, thus releasing contents into the
extravascular (interstitial) spaces. Once this has been
accomplished, then the preconditioning pulses would be used to
effect migration of ionic elements throughout the interstitial
fluid as described above.
Iontophoretic Field Application
Iontophoresis involves the application of an electromotive force to
drive or repel oppositely charged ions through tissue. Positively
charged ions are driven into the tissue at the anode while the
negatively charged ions are driven into the tissue at the cathode.
Therefore, at least two electrodes are used. One electrode, called
the active or donor electrode, is the electrode from which the
ionic substance is delivered into the body by iontophoresis. The
other electrode, called the counter or return electrode, serves to
close the electrical circuit through the body. If the ionic
substance to be delivered into the body is positively charged, i.e.
a cation, then the anode will be the active electrode and the
cathode will serve to complete the circuit. If the ionic substance
to be delivered is negatively charged (i.e. an anion), then the
cathode will be the active electrode and the anode will be the
counter electrode. Alternatively, the anode and the cathode may be
used to deliver drugs of opposite charge into the body.
Iontophoretic devices have been known since the early 1900's. U.S.
Pat. Nos. 3,991,755, 4,141,359, 4,398,545, and 4,250,878 disclose
examples of iontophoretic devices and some applications
thereof.
Iontophoretic delivery devices can also be used to deliver an
uncharged drug or agent into the body. This is accomplished by a
process called electroosmosis which is the transdermal flux of a
liquid solvent (e.g. the liquid solvent containing the uncharged
drug) induced by the presence of an electrical field imposed across
tissue by the donor electrode. Furthermore, iontophoretic devices
generally require a reservoir or source of the agent to be
delivered.
This aspect of the invention relates generally to the
electrokinetic mass transfer of charged molecules or liposomes to
particular regions of tissue based upon the desired overall
interstitial fluid distribution. It is considered desirable to have
an effective way of delivering the desired compounds without
risking harm to the tissue structure from direct electrical contact
and to avoid exposure of healthy tissue from the effect of the
iontophoretic field. Care must be taken to avoid current flow along
the path of least resistance into an area of tissue weakness,
resulting in a localized burn. This pattern of current flow is also
known as tunneling. Current carried through the liquid reverse
(interstitial fluid) is carried by ions (ionic conduction). In
order for current to flow, it is necessary for electrical charge to
be transferred to chemical species in solution by means of
oxidation and reduction charge transfer reactions at the electrode
surfaces. The Nernst-Planck equation describes the movement of
ionic species in mass transport. The first term describes the flux
due to passive diffusion, which is proportional to the
concentration gradient of species i. The second term describes the
flux due to the electromigration or electrodiffusion, where the
driving force is the gradient of electrical potential. The third
term describes the flux due to convection, where the mechanism of
transport is the movement of material by bulk fluid flow which is
determined by the magnitude and direction of the bulk fluid
velocity vector:
where
J.sub.i =flux of species i (moles/cm.sup.2 -sec)
D.sub.i =diffusion coefficient of i (cm.sup.2 /sec)
.gradient..sub. =the gradient operator
C.sub.i =concentration of species i
z.sub.i =number of charges per molecule of species i
F=Faraday's constant (96,500 coulombs/mole of charge)
u.sub.i =mobility of species i (velocity/force)
.PHI.=electrical potential (volts)
v=velocity vector
It is the sum of the fluxes resulting from these three processes,
passive diffusion, electromigration and bulk fluid flow resulting
from electroosmosis, which define electrotransport. Electroosmosis
is defined as the volume flow of solvent through a charged membrane
when an electrical field is imposed across that membrane.
In this device, the core and satellite electrodes will be used as
iontophoretic devices with application of low voltage constant
electrical fields across varying configurations in order to
thoroughly distribute the various charged particles (including
charged liposomes and other macromolecules including concentration
ionic solutions for the improvement of intratumoral conductivity)
throughout the tumoral or diseased tissue. DC currents in the micro
to milliampere range will be utilized and the likely source of the
constant current would likely be an appropriate field effect
transistor and a variable resistor. These controllers are
commercially available and normally consume only about 0.5-0.7 V.
It is likely that there will be hindrance to high molecular weight
compounds in the brain extracellular microenvironment
Given a contiguous interstitial compartment it is reasonable to
argue that either constant or pulsed electrical fields could be
used to induce the migration of particulates to a given direction,
thus allowing control of the distribution of ionic or otherwise
designated materials which are locally injected. Thus materials
would be "pulled" or "pushed" from one area of the tumor to the
next, to introduce a desired pattern of uniformity or
concentration.
Alternatively, the use of iontophoretic or pulsed fields can be
employed to influence the migration of charged liposomes within
interstitial fluid, again concentrating materials in particular
locations. It has been demonstrated that constant electrical fields
can increase the adsorption of liposomes to cell walls, thus
increasing the likelihood of incorporation or of fusion following
electroporation pulses. Also of use in this regard would be the
utilization of phase transition temperature-specific liposomes for
the purpose of controlled release at the appropriate
temperature.
The following examples serve to illustrate certain preferred
embodiments and aspects of the present invention and are not to be
construed as limiting the scope thereof.
EXPERIMENTAL
In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); mM (millimolar);
.mu.M (micromolar); N (Normal); mol (moles); mmol (millimoles);
.mu.mol (micromoles); nmol (nanomoles); kg (kilograms); gm (grams);
mg (milligrams); .mu.g (micrograms); ng (nanograms); L (liters); dl
(deciliters); ml (milliliters); .mu.l (microliters); vol (volumes);
V (volts); mV (millivolts); cm (centimeters); mm (millimeters);
.mu.m (micrometers or microns); nm (nanometers); hr (hours); sec
(seconds); msec (milliseconds); .mu.sec (microseconds); and
.degree. C. (degrees Centigrade).
Example 1
Brain Tumor Therapy
Brain tumors present a unique challenge to provide methods to
selectively destroy tumor cells while preserving normal brain
tissue.
There are a number of features which distinguish tumor from healthy
tissue. It should be recognized that tumors are often unique from
one another, even in the same subclass of cell type and that two
tumors of the same type, age and size may have quite different
internal structure and composition.
In spite of the differences between tumor cells, there are a number
of generalizations which can be made to describe significant
distinctions between tumor and normal cells:
1. The work of Peterson et al. (1973) has shown that the
endothelial wall in tumors is significantly more permeable than
normal vessels.
2. The extravascular compartment and the interstitial space are
much larger in tumors than in normal tissues (Peterson (1979)). A
recent article by Jain (1994) states that tumor cells often occupy
less than half the volume of a tumor, with blood vessels comprising
1-10% of the volume and the extracellular matrix, a collagen-rich
environment, occupying the remainder.
3. Vascular compression might occur followed by the development of
central necroses. When the blood flow has stopped, the capillary
endothelial cells die rapidly.
4. The extravascular space in human gliomas and meningiomas showed
a large extracellular space: 20-40% in gliomas and 13-15% in
meningiomas (Bakay, (1970); Rauen et al. (1967); Peterson et al.
(1979)), while that in normal brain tissue was 6-7%.
5. The vascular volume in tumors seems to remain rather stable
during growth. However, central necroses develop during growth
after human brain tumors have reached a certain diameter (1-3 cm).
This central necrosis is probably due to compression of vessels by
increasing tumor cell masses or to a more rapid growth of tumor
cell mass versus vascular endothelial cell proliferation. Tannock
(1970) demonstrated a difference in the turnover times between
endothelial (50-60 hours) and neoplastic cells (22 hours).
Additionally, hypoxia, anoxia, and glucose depletion in the growing
tumor caused by the absence of a sufficient neovascularization and
general rarefaction of the terminal vascular bed might explain the
development of necrotic areas in large tumors.
6. Morphological studies of blood vessels in human brain tumors
showed fenestration, widened intercellular junctions, increased
pinocytotic vesicles and infolding of the luminal surface, all of
which suggest an increase in the transvascular transport of
different materials. Most experimental data confirm a high
permeability of the tumor capillary wall for large protein
molecules. This is probably explained by morphological changes in
tumor vessels as observed. It is also evident that the transport of
large molecules across the tumor capillary wall is based on a
passive diffusion, and concentrations of active drugs sufficient
for a therapeutic effect are difficult to achieve. Normal passage
of molecules across the blood vessel walls takes place across or
between endothelial cells which line the vessel walls in a single
layer. Molecules leave the vessels by either diffusion or
convection except for cells such as white blood cells which leave
the blood vessels by attaching to endothelial walls and deforming
themselves to "squeeze through" the spaces between endothelial
cells and thus gain access to the matrix. Once cells are in the
interstitial matrix, they migrate by attaching to the matrix and
crawling through it. This movement is influenced by the cells'
adhesive properties and deformability. Certain molecules can
facilitate or hamper cell motility and influence the direction of
migration.
7. The vascular space of solid tumors becomes smaller as the tumor
mass grows. In general, as the tumor increases in size, the
vascular surface area decreases. The reduction of the vascular bed
is accompanied by a widening of the vessel diameter (Vaupel et al.
(1971); Vaupel (1974); Vogel (1965); Himas et al. (1974)), an
increase in vessel length (Vaupel et al. (1971); Vaupel (1974);
Jirtle et al. (1978)) and a broadening of the distance between
tumor capillaries (Vaupel et al. (1971); Vaupel (1974); Vogel
(1965)). In DS-carcinosarcoma, the mean intercapillary distance
ranges are increased 3-fold during tumor growth from 3-12 gm. Also,
the general rareification of the terminal vascular bed in
DS-carcinosarcoma is accompanied by a 10-fold increase in the
vascular flow resistance within the tissue when a tumor grows from
4-10 gm (Vaupel (1975)).
8. Tumor tissue exhibits a remarkable lack of homogeneity of blood
vessel distribution and thus inhomogeneity in the supply of oxygen
and nutrients to different parts of the tumor. This will certainly
have an important effect on the manner in which materials are
transported from the capillary to the tumor cell. This difference
applies not only when comparing tumor center and peripheral areas,
but also within neighboring parts of the superficial layers. Some
regions of the superficial tumor may be absolutely ischemic (Vaupel
(1977)). Regurgitation and intermittent circulation, i.e. periods
of pre-stasis or stasis followed by resumption of blood flow
sometimes in a direction opposite to the previous one are probably
the `normal` features of the intravascular transport system of
neoplastic tissues. It is also estimated that in some tumor types,
arterio-venous (AV) shunt perfusion represents up to 30% of the
total perfusion.
9. There is also a pronounced tissue acidosis in tumor tissue which
causes erythrocyte membranes to stiffen, reducing erythrocyte
flexibility and fluidity and leading to a reduction of the
microcirculation in malignant tumors. (Vaupel et al. (1976)).
10. Work in hyperthermia suggests that the preferential damage to
tumor cells seen in this form of treatment may be mediated by
differences in the tissue O.sub.2 concentration of both
tissues.
11. Due to the extreme tortuosity and number of vessels in the
tumor, there is often a significant slowing of blood flow in the
tumor which is accompanied by an abnormally high viscosity. The
slowed flow often contributes to poor penetration of drugs such as
chemotherapeutic agents. This may however, be turned into an
advantage in that the accumulated drug which is trapped in this
"reservoir" can slowly release drug gradually into neighboring
regions of a tumor.
12. There is often an abnormally high pressure in the interstitial
matrix which can slow the passage of large molecules across the
vessel walls into the interstitial space. The pressure measurements
also indicate that the pressure in tumor blood vessels is higher
than it is in normal capillaries. It is believed that this
elevation results mainly from the direct and indirect compression
of the vessels by the proliferating tumor cells (Jain (1994)).
13. Gullino (1974) documented that approximately 10% of the blood
fluid leaving a solid tumor oozes out from its periphery rather
than draining via a vein. This oozing fluid migrates into the
matrix of the normal cells carries drug molecules out and away from
the tumor.
14. "The extent of liposome transport to the interstitium would be
improved, however, if the permeability of nonleaky tumor vessels
could somehow be increased." (Jain (1994)).
A. Brief Anatomy of Brain Tumors
This example is directed to the application of the present
invention to malignant intracranial neoplasms, more specifically
astrocytomas of which glioblastoma multiforme are a particularly
lethal subclass. Gliomas constitute the majority of all primary
brain tumors and occur more commonly in adults. There are three
classes of astrocytomas: (low-grade) astrocytoma (LGA) which
demonstrates mild hypercellularity and pleomorphism; anaplastic
astrocytoma (AA) with moderate pleomorphism, increased
proliferative activity and variable vascular proliferation; and
glioblastoma multiforme (GBM) in which there is tumor necrosis.
Most astrocytomas are believed to begin as LGA, with potential
evolution into AA and GBM as a result of dedifferentiation over
time. Thus, regional heterogeneity is a finding of all the
astrocytomas, leading to sampling errors and misdiagnosis. In this
light, the current recommended technique for biopsy diagnosis is to
sample stereotactic needle aspirates from one particular axis of
the tumor so that representative samples are obtained from
superficial, deep and central areas of the tumor.
The anatomy of these tumors may be characterized as diffusely
infiltrating, expansile or as a combination of an expansile core
and an infiltrating corona. The outer margins of the corona are
usually poorly differentiated from normal tissue, making it
difficult to separate out tumor from normal tissue in treatment
settings. In some cases, this expansile tumor pushes normal tissue
aside, showing a narrow rim of invasive cells which may form a
cleavage plane for surgical resection. Low-grade astrocytomas tend
to infiltrate diffusely and may not form a discrete mass. In
contrast, AA and GBM form an expanding and infiltrating mass with a
gradient of infiltrating cells extending away from the main mass.
The primary pattern of spread is along the white matter tracts with
generally less involvement of gray matter. Infiltrating tumor cells
are usually accompanied by edema which may facilitate invasion.
Individual cells may infiltrate a long distance from the main tumor
mass and may produce secondary tumor masses. These multicentric
astrocytomas can be tracked during autopsy proceedings quite often
by following a trail of individual infiltrating cells. AA and GBM
may also spread via seeding through the CSF with possible
widespread subependymal and subarachnoid dissemination. To
summarize, astrocytomas do not grow as spheres; instead their
contours are highly irregular as their white matter extensions
conform to the barriers of cortical convolutions and deep unclear
structures.
One particular approach to this problem involves the sequential
application of procedures which are designed to functionally
isolate the target area of the brain while protecting normal neural
tissue from treatment effects. Ultimately, specific measures are
taken to "open" the cell membranes of the tumor cells, thus
permitting entry of a desired therapeutic compound or agent which
will be used to effect cell death.
Electrode Placement
The placement of electrodes within the predetermined area in the
present invention is important to the overall success in achieving
electroporation of the target region with maximal sparing of
healthy tissue.
In the example of brain, integral to the placement strategy is the
fact that the central electrode will be placed within the tumoral
or diseased area in order to maximize the penetration of the target
body site by the electrical field. It is considered desirable to
ensure that the current flow is distributed in fairly uniform
fashion throughout the target body site, and not allow for reflux
of current in a retrograde manner along the electrode pathway which
creates a disturbance in the blood-brain barrier. It is anticipated
that appropriate design of the electrodes will facilitate closure
of the tissue around the electrode, thus creating a natural barrier
to the flow of current. Alternatively, coating substances on the
surface of the electrode could aid in creating a resistance to
current flow, as could the use of dielectric materials which could
impede current flow. Also of use will be physical barriers, such as
collar or balloon devices which would fit around the shaft of the
electrode.
The electrodes will be placed by two methods: (1) stereotaxic
placement or (2) direct placement. Prior to therapy, all patients
will have some type of imaging study done to localize and
characterize diseased tissue such as tumors within healthy tissue.
Among the more advanced imaging techniques combine the imaging with
stereotaxic coordinate systems which enable the precise
localization of target body site within 3-D space. It is
anticipated that such a coordinate system will be utilized in order
to create a physical 3-D map of the tumor area, in addition to
demonstrating internal variations in density, blood supply, etc.
This information will be used to determine the best placement for
the central electrode which will be placed in such a way as to
allow access to the more dense areas of the tumor, thus insuring
some flow of the electrical fields throughout the denser areas.
The second method would involve direct placement of the electrodes
during surgical procedures, most likely resection or debulking
procedures during which the electrodes would be placed in areas
where the tumor either had been resected. Electrodes would consist
of stainless steel, platinum, or platinum iridium electrodes which
are coated with dielectric materials, for example Teflon.
Diagnostic Imaging Studies
Light Microscopy--A fairly uniform but nonspecific finding in
tumors is the failure of local blood-brain barrier, which allows
leakage of contrast material (contrast enhancement) into
parenchymal tissues. Endothelial cells of cerebral capillaries have
fused membranes, called tight junctions, which are the most
important feature in regulating capillary permeability in the
brain. The capillaries of normal brain are impermeable to
intravascular injected contrast agents. Capillaries of tissues
outside the nervous system are fenestrated with discontinuities in
their basement membranes, with wide intercellular gaps permitting
the passage of protein molecules from the lumen of the capillary
into the extravascular space. The blood-brain barrier interfaces
are not found in some regions of the brain. These areas include the
choroid plexus, pituitary gland, cavernous sinus, pineal gland and
dura. Capillaries in these areas are fenestrated and allow the
diffusion of contrast material into the extracellular space and
exhibit normal enhancement following the intravenous injection of
contrast agents.
Tumors often stimulate the formation of capillaries in their
tissue. Tumor capillaries in gliomas may have near-normal features
with an intact blood-brain barrier. These areas of tumor will not
enhance. In other more malignant gliomas, there is stimulation of
capillaries the endothelia of which are fenestrated with poorly
functioning or nonexistent blood-brain barrier. Metastatic brain
lesions have non-CNS capillaries that are similar in to the tissue
of origin, therefore possessing fenestrations and therefore
enhancement under IV contrast conditions. This finding is also
noted in other conditions such as infarction and infection.
Highgrade tumors enhance owing to absence or deterioration of the
blood-brain barrier, whereas well-differentiated tumors generally
have intact blood-brain barrier and do not enhance. In general,
these areas of enhancement are correlated well with a highly
cellular and mitotically active neoplasm with proliferating
vascular cells. Typically, a decreasing gradient of tumor cells
extends away from the enhancing area into the surrounding edema.
The majority of cells are within 2 cm of the original lesion. In
GBM, it has been demonstrated that the microscopic infiltration of
tumor was often over 2 cm from the enhancing rim.
Magnetic Resonance Imaging
The MRI is generally more sensitive to regions of edema than CT.
The extent of T2-signal abnormality is currently the most accurate
imaging study of the extension of tumor cell infiltration in
primarily gliomas. Studies have demonstrated that tumor cells in
high-grade astrocytomas are found even slightly beyond the region
of high T2-signal intensity, thus beyond the areas defined by CT.
An exception here is that gray matter or subarachnoid spread is not
detected well by MRI. It is also clear that isolated tumor cells
may infiltrate without eliciting edema, thus making them
undetectable by MRI. The identification of these individual
infiltrating tumor cells seems likely to remain beyond the range of
detection by any radiological method.
Angiography
Angiography can demonstrate the vascular supply to a tumor and the
positional relationship of the major intracerebral vessels, both
arterial and venous, to the tumor mass. In many instances the
angioarachitecture of the tumor may suggest the correct
pathological diagnosis.
C. Surgical Treatment of Malignant Brain Neoplasms
Surgery is almost never the sole modality for treatment of
malignant brain neoplasms but it is often combined with other
treatment modalities within the context of a total treatment plan.
It is now safely possible to remove the greater portion of glial
tumors from virtually every location in the cerebral hemispheres as
well as from many sites within the ventricles and in close
proximity to the thalamus and basal ganglia. A radical excision of
a glioma may be said to be the removal of its enhancing rim as well
as the tissue defined by that boundary. However, this still leaves
the scattered nest of malignant cells that extend for variable
distances into the surrounding neuropil. Immediate benefits to
surgical resection include: mechanical cytoreduction which produces
a rapid cell kill, removes resistant cells and prolongs survival;
amelioration of symptoms via improved neurological status and
reduction of increased intracranial pressure; potentiation or
facilitation of radiotherapy, chemotherapy and immunotherapy; and
diagnostic precision with extensive tissue sampling, and tissue
culture; improve the susceptibility of remaining cells by
increasing access of drugs and biologicals to the remaining
mass.
The mating of MRI/CT with traditional stereotactic frames to
produce image-based stereotaxy is used to determine the
three-dimensional coordinates of any point inside the head in
relation to the stereotactic space delimited by the frame. These
coordinates are used to control the entry of various micrometer
driven instruments to any intracranial location. The patient is
fitted initially with a CT and MRI-compatible stereotactic
headframe (COMPASS system), which is applied under local anesthesia
and mild sedation. This is secured to the skull by carbon fiber
pins. These procedures are carried out under local anesthesia and
employ small puncture holes in the skin along with twist-drill
holes in the skull. Experimental techniques currently utilize such
delivery methods for facilitation of entry of biopsy instruments,
endoscopes, catheters for delivery of interstitial radiation or
microwave hyperthermia, laser light for photoactivation
chemotherapy, endoscopic laser ablation and catheter deposition of
immunological reagents and other biologicals.
Another recently developed treatment modality employs stereotactic
localization with focused beam ionizing radiation for the
noninvasive destruction of small intracranial lesions. This
technique, called radiosurgery, enables the neurosurgeon to deliver
very intense radiation to a very sharply delineated area, thus
destroying only the tumor and sparing the normal tissue.
Instrumentation includes the Leksell Gamma Knife, cyclotron or
synchrocyclotron instruments, modified linear accelerators.
D. Interstitial Radiation Therapy of Tumors
Brachytherapy (also known as interstitial radiation therapy) refers
to treatment of tumors with radiation sources placed directly
adjacent or into tumors. Advantages of this include the fact that
the radiation emitted from a localized source implanted in tissue
decreases rapidly with distance, owing to the inverse square law
and to attenuation of the radiation as it passes through tissue.
Additionally, low-dose-rate radiation tends to make proliferating
tumor cells remain in G.sub.2, a radiosensitive phase of the cell
cycle during which RNA is synthesized prior to cell division.
Normal, noncycling neuronal cells tent to remain in G.sub.1, a
radioresistant phase of the cell cycle. Another advantage of this
therapy is that hypoxic cells (which might be found in the dense,
central areas of a tumor mass) are less resistant to low-dose-rate
radiation than to high-dose-rate radiation.
Tumors selected for implantation are supratentorial, unifocal,
well-circumscribed lesions smaller than 5-6 cm in diameter.
Patients undergo tumor resection 2-4 weeks prior to
brachytherapy.
Brachytherapy has been combined with hyperthermic treatment
immediately prior to the loading of .sup.125 I and a second
treatment after unloading the sources. The same catheters are used
but the catheters are placed more peripherally, about 3-5 mm within
the boundary of the contrast-enhancing tumor mass, evenly spaced
about 1.2-2.0 cm apart from each other. In addition, one to three
extra catheters are implanted for multipoint thermometry.
E. Chemotherapy
Chemotherapeutic agents are designed to affect the cell at the most
vulnerable time in the cell cycle. These four stages are as
follows: G.sub.1 (protein synthesis); S (DNA replication); G.sub.2
(RNA synthesis) and M (mitosis). Following these stages, cells such
as neurons and glial cells are said to be post mitotic (G.sub.0).
As a general rule, chemotherapy agents are most effective during S
phase and as most tumor cells are not in the S phase at any given
time, only a portion of tumor cells are killed through the
administration of a single cycle of chemotherapy. Therefore, agents
are administered in multiple cycles to kill cells as they enter the
correct cell cycle phase.
Chemotherapy relies on physical properties which exist in the tumor
tissue which allow for penetration by these agents. The most
effective chemotherapeutic agents for the CNS are highly
lipid-soluble which allow relatively free access to the entire CNS
and permit agents to reach not only the tumor mass, but also the
malignant cells located at a distance from the main mass. The
normal blood-brain barrier is created by tight cellular junctions
and a lack of fenestrations of the brain capillary endothelial
cells and basement membrane. Those non-ionized chemotherapeutic
agents with high lipid solubility are able to cross the vascular
barrier and enter the brain. Other chemicals may gain access to the
brain by crossing vascular endothelial cells through nonspecific
adsorptive transcytosis or receptor-mediated transcytosis. Some CNS
areas have access to the intravascular compartment and include the
pineal body, posterior pituitary, tuber cinereum, wall of the optic
recess, area postrema, subfornical and commissural organs and the
choroid plexus. Similarly, areas of the brain may allow breakdown
of the normal blood-brain barrier due to trauma, vasculitis,
radiation and infection in addition to infiltrating tumors.
Common modes of delivery include oral and intravenous routes.
Intra-arterial treatment is an effective means of delivering high
concentrations of chemotherapy directly to the region of interest
while potentially reducing the risk of systemic toxicity. However,
clinical trials have demonstrated multiple complications including
depression of consciousness, paresis due to thromboembolic events,
loss of visual acuity due to ocular toxicity, aphasia and white
matter changes in the brain. Another delivery method for
chemotherapeutic agents includes a synthetic wafer impregnated with
a lipid-soluble N-(2-chloroethyl)-N-nitrosuoreas (CNUS)
specifically known as Carmustine (BCNU). This wafer, which is
placed on surgical resection sites, is formed utilizing a
polyanhydride polymer and is designed to allow BCNU to slowly
diffuse away from the polymer wafer into the interstitial
compartment.
Intrathecal administration using cisternal or intraventricular
injection (Ommaya reservoir) has been used to deliver larger
molecular weight or polar drugs to tumor cells, bypassing the
blood-brain barrier. However, drug penetration into the parenchymal
tissue is often limited. For example, intrathecal administration of
methotrexate penetrated to a depth of 3.2 mm at 1 hour. Drug
distribution in the CSF is influenced by several factors, including
bulk CSF flow, diffusion through the extracellular spaces of the
brain and spinal cord, transport across the choroid plexus, removal
by CSF absorption and diffusion from the extracellular space in the
capillaries of the CNS. A related approach to maintain CSF drug
levels would be to decrease CSF clearance. The normal mechanisms of
drug clearance include CSF reabsorption, diffuse bulk CSF flow,
transport across cell membranes, and absorption into capillaries.
Probenecid, an inhibitor of the active transport of methotrexate,
has been used clinically to prolong CSF levels of this drug,
presumably by inhibiting the drug's active transport across the
choroid plexus. Consideration has also been given to the use of
acetazolamide to decrease CSF production, thereby reduce the bulk
flow and turnover of CSF.
Intratumoral delivery methods have also been explored, primarily
utilizing the Ommaya reservoir or an adapted tumor cyst device
which permits direct installation of several chemotherapeutic
agents into tumors. There are a number of technical limitations
including the fact that water-soluble drugs are likely to diffuse
slowly throughout the extracellular space. More lipid-soluble
agents are likely to diffuse back across the barrier into the
systemic circulation. Therefore, either of these limitations will
require a large drug dose to overcome the diffusion problem.
Harbaugh (1989) has described intratumoral chemotherapy through an
external catheter infusion method. He also proposed the utilization
of such devices for delivery of other therapeutic agents such as
those which might be used in the treatment of Parkinson's or
Alzheimer's disease.
Liposome mediated delivery has been used as a method of selective
drug delivery and transport to tumor tissue. Early work has
demonstrated the successful the incorporation of bleomycin and
vincristine into liposomes of 0.1-15 .mu.m diameter (Firth et al.
(1984)). Experimentation in rats demonstrated a much slower release
over time for the liposome delivered drugs versus "free"
chemotherapeutic agents. MTX/cholesterol liposomes have been
studied in primates and have demonstrated a higher average brain
concentration than injection of free drug (Stewart (1984)). More
recent research utilizing liposome-mediated delivery includes the
work of Wowra et al. (1992); Gennuso et al. (1993); Fukuda et al.
(1989); and Shibata et al. (1990).
Blood-brain barrier disruption chemotherapy has been attempted
utilizing hyperosmolar iodinated contrast agents or compounds such
as hyperosmolar mannitol, urea or arabinose to reversibly breach
this barrier, temporarily opening the tight junctions and allowing
transient unregulated entry of circulating substances into the CNS.
A paper by Morantz et al. (1994) stated "When the use of
lipid-soluble agents is not possible or if greater access to the
brain parenchyma and tumor is desired, techniques of blood-brain
barrier disruption are employed. This usually involves the
intra-arterial infusion of mannitol . . . ", Neuwelt (see Morantz
et al. (1994) pp. 776-777 for bibliography) has applied the
observation to human and animal treatment. The state of the
blood-brain barrier within any tumor is highly variable, even to
within regions of a given tumor. There are several competing
phenomenon which tend to rapidly reverse any advantage gained by
partial or total breakdown of the blood-brain barrier in the region
of the tumor. Given the fact that compounds diffuse from areas of
high concentration to areas of low concentration, to the point of
equilibrium. Therefore, even if a tumor has complete absence of a
blood-brain barrier, because the barrier remains intact in the
surrounding brain parenchyma, any immediate increased concentration
of drug to the tumor rapidly diffused out to equilibrate with the
remaining CNS (the "sink effect"). The technique of blood-brain
barrier disruption provides an increased and more uniform drug
delivery, decreases the tendency toward rapid diffusion and thereby
allows tumor exposure to a higher concentration of drug for longer
time period. However, this also exposes the normal CNS to a much
higher concentration of chemotherapeutic agents.
The technique as detailed by Neuwelt involves opening the
blood-brain barrier in the distribution of one circulation in the
brain (carotid or vertebral artery). The exact distribution of
disruption, therefore, is dependent on the flow, as determined by
these vessels and the circle of Willis. One then selects the
appropriate arterial distribution pertinent to tumor location. To
obtain reversible disruption of the blood-brain barrier, a
hyperosmolar saturated solution of 25% mannitol is injected at
sufficient rate and volume to replace blood flow. This infusion
must continue for approximately 30 seconds, at which time the
threshold event of disruption occurs. Disruption is documented
utilizing either Evans blue or the use of iodinated contrast agents
and/or radioisotopes. The procedure is performed under general
endotracheal anesthesia. Patients undergo retrograde
catheterization of the femoral artery (Seldinger technique) and the
selected artery is cannulated. Blood-brain barrier disruption
allows for nonselective entry (for a period of approximately 30
minutes) of substances previously disallowed from the CNS and
tumor. The use of blood-brain barrier disruption in cases of
cerebral lymphoma have been most impressive given the often diffuse
nature of this disease.
Photodynamic therapy involves exposure of a tumor to a
photosensitizer such as a hematoporphyrin derivative (HpD) after
which the tumor is exposed to light of an appropriate wave length
to activate the sensitizer. This therapy relies on the selective
tumor uptake of hematoporphyrin derivatives by the tumor compared
to the surrounding normal brain. The HpD compound is infused
preoperatively and at surgery the patient's tumor is exposed to
light (630 nm) by an argon dye laser. The mechanism of cell
necrosis may be related to activated free radicals, with damage to
blood vessels and cell membranes. The mechanisms of HpD
localization in tumor remain to be elicited. Uptake in various
tumor types is variable, with glioblastomas demonstrating the
highest uptake which was 30 times that in normal brain tissue.
Low-grade tumors had a HpD uptake of 8 times normal tissue.
Boron neutron capture therapy is predicated on the preferential
accumulation of boron (.sup.10 B) in conjunction with sufficiently
high thermal neutron fluxes at the tumor site. The disintegration
of the boron atom which is precipitated by collision with a slow
neutron yields ionizing radiation of a very short diameter of
travel, namely the approximate diameter of a cell. The slow neutron
is several thousand times more likely to interact with a boron
nucleus than with the nucleus of any element of human tissue. This
therapy has been known and tried for many years with mixed results
and has recently experienced a resurgence in it's popularity and
research focus.
Drug rescue techniques are also employed in order to attempt to
deliver high concentrations of cytotoxic drug to tumor and to
increase the duration of tumor exposure to that drug. However, dose
limitation is frequently due to extraneural side effects. The
rescue technique might include administration of an antidote either
concomitant with or sequentially to the administration of a
chemotherapeutic drug. Other agents might be administered to
protect certain areas of the body such as the use of mannitol to
protect against nephrotoxicity or the use of systemic thiosulfate
with cisplatin to protect against nephrotoxicity and reduce
thrombocytopenia. Autologous bone marrow transplant has been used
with BCNU and other drugs. Other attempts combine an "isolated
perfusion" approach which uses an extraction hemoperfusion column
or dialysis variation to remove the drug from the systemic
toxicity. Similar novel approaches use the formation of antibody
against a particular chemotherapeutic agent to bind and inactivate
the drug. Such a method is particularly applicable to brain tumor
treatment for which systemic toxicity is limiting, and systemically
administered antibody can bind peripheral drug, yet has only
limited access to CNS drug. Another application of monoclonal
antibody (MAb) is the conjugation of the antibody to an enzyme to
form a relatively high molecular weight molecule. The conjugate can
be delivered across the blood-brain barrier with osmotic disruption
where it binds to surface antigen and the barrier returns to a
predisrupted condition. A low molecular weight prodrug capable of
being activated to the cytotoxic agent by the antibody-bound
enzymes is given, resulting in localized drug treatment.
F. Enhancement of Electrical Conductivity of Tumor
The internal architecture of a given tumor is thought to be highly
variable, both within a given tumor and across the spectrum of
other tumors of the same cytological origins. Therefore, the
internal environment within the tumor is likely to be highly
variable with respect to its ability to propagate electrical
fields, and therefore equally variable with respect to the
likelihood of electropermeabilization at any given site. In order
to maximize the internal conductivity of the tumor, selective
delivery and distribution of highly conductive materials throughout
the interstitial space of the tumor, thus enhancing conductivity,
is considered desirable.
G. Thermal Isolation of Tumor
Research indicates that there exists a direct correlation between
temperature and the electropermeabilization threshold. There
appears to be a direct positive effect on the likelihood of pore
formation with increasing temperature. Conversely, decreasing the
temperature at which electropermeabilization occurs results in a
decreased likelihood of poration events at the membrane level.
Therefore: (1) if a temperature gradient were to be established
between normal and tumor tissue, with the tumoral tissue at a
higher temperature than normal tissue: (2) if the electrical
properties of both tissues were equal; then a simultaneous
electrical field applied across both areas would result in a net
increase in electroporation in the tumoral tissue. This
differential electropermeabilization would increase as the
temperature differential increased, although the linearity of such
a relationship is yet to be established. Additionally, cooling the
normal brain tissue would have significant protective effect both
to the administration of other drugs or conductive materials as
well as well as in the protection of normal brain tissue from the
potential for seizure induction. The purpose of the cerebroplegia
also extends beyond protection of the normal tissue by temporary
interruption of the blood flow to the brain for periods of time for
up to one hour. During this time either complete cessation of flow
or intermittent pulsed flow can maintain the low metabolic
requirements of the brain.
H. Heating of the Tumor
To create a temperature differential driving force as previously
described, tumor tissue will be heated utilizing high voltage brief
yet intense pulses. By increasing the duration of electroporation
pulses, subthreshold poration fields can effect rapid heating of
the intratumoral area, particularly as the rate of heating is a
function of field strength.
It has been demonstrated that for high voltage fields, heating of
10.sup.3 -10.sup.5 .degree. C./sec can be reached. It is at this
point that utilization of phase-transition-temperature liposomes
results in the release of the contents of the liposomes as
temperatures within tumoral tissue approach the critical threshold
for liposome rupture within the interstitial space. This creates an
"on demand" rupture of liposomes and distribution of their contents
in the interstitial space adjacent to the cells which will be
porated.
I. Blood Replacement by Hypoconductive Material
Once the cerebroplegia has been initiated, one can briefly replace
the contents of the vascular tree with hypoconductive media. The
vascular tree itself may provide a significant avenue of conduction
of the electropermeabilization pulses, thereby carrying the
electropermeabilization effect away from the tumoral area into the
distribution of the normal brain tissue. It appears that replacing
the vascular tree with hypoconductive medium would substantially
eliminate the vascular tree as a likely conduit of the current,
thereby confining the field to the predetermined region, namely the
interstitial space. The feasibility of temporarily replacing the
intravascular contents in the brain is based from work done in
blood-brain barrier disruption studies which involve the injection
of 25% mannitol solutions at a high rates of infusion, completely
replacing the blood flow for up to 30 seconds. The key points of
this technique include:
Hypothermia allows temporary interruption in the blood flow.
Lower temperature raises poration threshold in normal brain.
Hypotonic or hypoconductive medium in the vascular tree inhibits
the spread of electroporation pulses via the vascular tree, thus
directing the current into a higher resistance pathway, namely the
interstitial space.
The injection of the hypoconductive medium will replace or wash out
left over substances from the previous steps including liposomes,
or other hyperconductive medium which might have remained in the
vascular space.
A decrease in cerebral blood flow will enhance the effective
arterial concentration in that slow blood flow allows higher tissue
drug extraction.
All patent publications cited in this specification are herein
incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity and
understanding, it will be apparent to those of ordinary skill in
the art in light of the teaching of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
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