TECHNICAL FIELD
[0001]
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
[0002]
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.
[0003] 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)).
[0004] 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.
[0005]
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\tissue have been
affected (Salford et al. (1993)).
[0006] 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.
[0007] 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.
[0008] 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
[0009]
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).
[0010] Biochemical and
Biophysical Research Communications, 194(No. 2):938 (1993) discusses a
new brain tumor treatment combining bleomvcin 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.
[0011]
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).
[0012]
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).
[0013]
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).
[0014] 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.
[0015]
Resealing of electropores is discussed in Proc. Natl. Acad. Sci. USA
89:4524 (1992) and also in Annal. New York Acad. Sci. (1992).
[0016]
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
[0017]
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.
[0018]
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.
[0019]
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.
[0020] 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.
[0021] 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.
[0022]
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).
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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
[0027]
FIG. 1 is a graphic representation illustrating an electrode array in
accordance with the invention which is positioned into and around a
brain tumor;
[0028] FIG. 2 is a graphic representation of a brain tumor in
three-dimensional space;
[0029]
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;
[0030] 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;
[0031] 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
[0032] 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
[0033]
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.
[0034] 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.
[0035] As used herein:
[0036]
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.
[0037] 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.
[0038]
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.
[0039] 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.
[0040] 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. A. 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.
[0041] 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).
[0042] 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.
[0043] 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
[0044] 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.
[0045]
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.
[0046] 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.
[0047] 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).
[0048] "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.
[0049] 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
[0050]
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.
[0051]
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.
[0052]
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.
[0053]
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
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060]
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.
[0061] 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): [0062] 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. [0063] 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: [0064] III The target body site will be
differentially heated by subthreshold electroporation pulses using
lower voltage electrical fields with increased duration; [0065] IV
Immediately following the heating of the target body site,
electroporation pulses will be administered via the electrode array
(10-20 seconds); [0066] 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; [0067] VI Cerebral blood flow will be re-established
with gradual rise in temperature of healthy tissue recovering to
normal; and [0068] VII The electrode system will be removed.
[0069]
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.
[0070] 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.
[0071] 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.
[0072] 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
[0073]
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.
[0074]
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
[0075]
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.
[0076] 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
[0077] 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.
[0078]
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 (1930). 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.
[0079] 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.
[0080]
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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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
[0086]
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.
[0087] 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 or 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
[0088]
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.
[0089] 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.
[0090] 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
[0091]
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.
[0092] 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
[0093]
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.
[0094] 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.
[0095] 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.
[0096] 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
[0097]
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.
[0098] 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.
[0099] 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)).
[0100] 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
[0101]
>From classical physiology, fluid movement across the endothelial
wall
is described by Starling's
hypothesis:j=.rho.K(x)[(p.sub.i-P.sub.e)-(.pi..sub.i-.pi..sub.e)] where
[0102] K(x) is Starling's coefficient [0103] p.sub.i=intraluminal
hydrostatic pressure [0104] p.sub.e=extraluminal hydrostatic pressure
[0105] .pi..sub.i=intraluminal osmotic pressure [0106]
.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.
[0107]
Darcy's Law describes the relationship between interstitial fluid
velocity and an interstitial pressure gradient: v i = - K t .function.
( P e ' .times. x i ) .times. .differential. p e .differential. x i
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.
[0108] 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.
[0109] 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.
[0110] 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
[0111]
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.
[0112] 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 1 hr 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.
[0113]
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
[0114]
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.111In-Tf in the infusion. Since the normal rate
of diffusion of .sup.111In-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.111In-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.111In-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.111In-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.111In-Tf binding to receptors and being internalized by
cells.
[0115] 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.
[0116]
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
[0117]
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.
[0118] 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.
[0119] 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.
[0120]
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.
[0121] 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
[0122]
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.
[0123]
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
[0124]
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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] The electrical field source
is then placed, desirably via the Hexasphere.TM. 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.
[0129] 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 nm). 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
[0130]
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.
[0131]
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.
[0132] 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.
[0133] 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.
[0134] 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)).
[0135]
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 INT = .intg. 0 .infin. .times. E 0 .times. e - t / .tau.
.times. d t = E o .times. .tau. where [0136] E.sub.o is the peak field
strength, [0137] t is time, and [0138] .tau. is the decay time
constant. [0139] For multiple pulses,INT=.SIGMA.E.sub.o.pi.
[0140]
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:
[0141] (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.
[0142]
(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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148]
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.
[0149]
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.
[0150] 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.
[0151]
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: [0152] cell size, shape and orientation [0153] non-uniformity
of electrical field [0154] cell-cell separation [0155] tissue
heterogeneity (perturbation of local field by tissue) [0156] 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
[0157] The use of
intermittent hypothermic cerebral perfusion (cerebroplegia) has been
resorted 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).
[0158] 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.
[0159] 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.
[0160] 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.
[0161]
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.
[0162]
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.
[0163]
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.
[0164]
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.
[0165] 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.
[0166] Of additional
benefit is the use of selective calcium antagonists and prostaglandin
derivatives as protective agents during the hypothermic ischemia
periods.
[0167] 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
[0168]
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.
[0169]
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.
[0170]
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
[0171]
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.sup.++-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.
[0172] 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.
[0173] 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.
[0174]
During the application of a subthreshold (E<E.sub.C) electrical
field, there are a number of subcritical membrane changes which occur,
namely from to .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>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
sequenceM.fwdarw.(P.sub.HO).sub.r.fwdarw.(P.sub.HO)r.sub.c.fwdarw.(P.sub.-
HI)r.sub.c.fwdarw. . . .
P.sub.CR where
sequence of membrane changes
from the poreless state M to hydrophobic (P.sub.HO) to hydrophilic
(P.sub.H) pores, the return transition P.sub.HO.fwdarw.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.
[0175] 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.DELTA..PHI.(E)=-1.5 f(.lamda.) a E|cos
.delta.| where .delta. is the angle between the membrane site
considered and the direction of E. The conductivity factor f(.lamda.)
is a function of the specific conductances or conductivities of the
external solution (.lamda..sub.0.gtoreq.10.sup.-4 S cm.sup.-1), of the
cell interior (.lamda..sub.i=10.sup.-2 S cm.sup.-1), and of the
membrane (.lamda..sub.m.apprxeq.10.sup.-7 S cm.sup.-1), respectively
and of the ratio d/a.
[0176] Usually,
.lamda..sub.m<<.lamda..sub.i, .lamda..sub.0 and d<<a such
thatf(.lamda.)=[1+.lamda..sub.m(2+.lamda..sub.i/.lamda..sub.0)/(2
.lamda..sub.id/a)].sup.-1 From this, it is readily seen that an
increase in the external ionic strength leading to an increase in
.lamda..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 .lamda..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 .lamda..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.
[0177] 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 .lamda..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.
[0178] 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 100V. 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 preconditioning. This
estimate is based on diffusion studies which indicate this order of
magnitude of time.
[0179] 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.
[0180]
Another aspect of the preconditioning phase will be the use of a single
electroporation pulse followed by a series of smaller subthreshold
pulses.
[0181] 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
[0182]
Iontophoresis involves the application of an electromotive force to
drive or repel oppositely charged ions through tissue. Positively
charged ions are driven S 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. 3,9,91,755, 4,141,359, 4,398,545, and
4,250,878 disclose examples of iontophoretic devices and some
applications thereof.
[0183] 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.
[0184] 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:J.sub.i=-D.sub.i.gradient.C.sub.i-z.sub.iF
u.sub.jC.sub.i.gradient..PHI.+C.sub.iv where [0185] J.sub.i=flux of
species i (moles/cm.sup.2-sec) [0186] D.sub.i=diffusion coefficient of
i (cm.sup.2/sec) [0187] .gradient.=the gradient operator [0188]
C.sub.i=concentration of species i [0189] z.sub.i=number of charges per
molecule of species i [0190] F=Faraday's constant (96,500 coulombs/mole
of charge) [0191] u.sub.i=mobility of species i (velocity/force) [0192]
.PHI.=electrical potential (volts) [0193] 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.
[0194] 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
0.5-0.7V. It is likely that there will be hindrance to high molecular
weight compounds in the brain extracellular microenvironment
[0195]
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.
[0196]
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.
[0197] 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
[0198] 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
[0199]
Brain tumors present a unique challenge to provide methods to
selectively destroy tumor cells while preserving normal brain tissue.
[0200]
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.
[0201] 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:
[0202] 1. The work of Peterson et al. (1973) has shown that the
endothelial wall in tumors is significantly more permeable than normal
vessels. [0203] 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. [0204] 3. Vascular
compression might occur followed by the development of central
necroses. When the blood flow has stopped, the capillary endothelial
cells die rapidly. [0205] 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%. [0206] 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. [0207]
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. [0208] 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)). [0209] 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.
[0210] 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)). [0211]
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. [0212] 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. [0213] 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)). [0214] 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. [0215] 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
[0216] 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 mode-rate pleomorphism, increased proliferative
activity and variable vascular proliferation; and glioblastoma
multiforme (GBM) in which there is tumor necrosis.
[0217] 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.
[0218] 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.
[0219]
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
[0220] 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.
[0221] 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.
[0222]
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.
[0223] 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
[0224]
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.
[0225] 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. High-grade 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
[0226]
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
[0227]
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
[0228]
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.
[0229]
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.
[0230] 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
[0231]
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.
[0232]
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.
[0233]
Brachytherapy has been combined with hyperthermic treatment immediately
prior to the loading of .sup.125I 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
[0234]
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.
[0235] 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.
[0236] 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.
[0237] 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 or acetazolamide to
decrease CSF production, thereby reduce the bulk flow and turnover of
CSF.
[0238] 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.
[0239]
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).
[0240]
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.
[0241]
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.
[0242]
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.
[0243] Low-grade tumors had a HpD uptake of 8 times normal tissue.
[0244]
Boron neutron capture therapy is predicated on the preferential
accumulation of boron (.sup.10B) 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, namelv
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.
[0245]
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 nigh 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
[0246]
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
[0247]
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
[0248]
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.
[0249] 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 "ion 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
[0250]
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: [0251] Hypothermia
allows temporary interruption in the blood flow. [0252] Lower
temperature raises poration threshold in normal brain. [0253] 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. [0254] 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. [0255] A decrease in cerebral blood flow will
enhance the effective arterial concentration in that slow blood flow
allows higher tissue drug extraction.
[0256] 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.
[0257]
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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