U.S. patent application number 11/268668 was filed with the patent office on 2006-04-13 for thermal surgical procedures and compositions.
Invention is credited to John D. Belcher, John C. Bischof, Gregory M. Vercellotti.
Application Number | 20060078538 11/268668 |
Document ID | / |
Family ID | 33131709 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060078538 |
Kind Code |
A1 |
Bischof; John C. ; et
al. |
April 13, 2006 |
Thermal surgical procedures and compositions
Abstract
Methods, compositions, and systems useful to enhance a thermal
surgical procedure are described. Compositions include at least one
compound effective to induce an inflammatory response in biological
material identified to undergo a thermal surgical procedure.
Methods and systems include providing compositions of the invention
to biological materials and treating biological materials with an
inflammation inducing composition for a time, amount, and type
effective to induce inflammation in at least a portion of the
biological material.
Inventors: |
Bischof; John C.; (St. Paul,
MN) ; Belcher; John D.; (Minneapolis, MN) ;
Vercellotti; Gregory M.; (Stillwater, MN) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55458
US
|
Family ID: |
33131709 |
Appl. No.: |
11/268668 |
Filed: |
November 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10810956 |
Mar 26, 2004 |
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11268668 |
Nov 7, 2005 |
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60457691 |
Mar 26, 2003 |
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Current U.S.
Class: |
424/85.1 ;
424/85.2; 424/93.4; 424/93.6; 607/1 |
Current CPC
Class: |
A61B 2018/0212 20130101;
A61B 18/04 20130101; A61B 18/06 20130101; A61B 18/02 20130101; A61K
31/00 20130101 |
Class at
Publication: |
424/085.1 ;
424/093.6; 607/001; 424/085.2; 424/093.4 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61K 38/20 20060101 A61K038/20; A61K 35/74 20060101
A61K035/74 |
Goverment Interests
GOVERNMENT FUNDING
[0002] The present invention was made with support from National
Institutes of Health under Grant No. R29CA75284. The U.S.
government may have certain rights in this invention.
Claims
1-6. (canceled)
7. A composition comprising at least one compound effective for
inducing an inflammatory response in biological material identified
to undergo a thermal surgical procedure.
8. The composition of claim 7 wherein the at least one compound
effective for inducing an inflammatory response is selected from
the group consisting of at least one virus, at least one bacterium,
ethanol, cytokines, interleukins, chemokines, oxygen-free radicals,
bacterial lipopolysaccharides, and combinations thereof.
9. The composition of claim 8 wherein the cytokine is selected from
the group consisting of TNF-alpha, truncated versions of TNF-alpha,
and combinations thereof.
10. The composition of claim 8 wherein the interleukin is selected
from the group consisting of IL-beta, IL-8, and combinations
thereof.
11. The composition of claim 7 further comprising a
pharmaceutically acceptable carrier.
12. The composition of claim 11, wherein the pharmaceutically
acceptable carrier is selected from the group consisting of a
saline solution, encapsulation in microbeads, encapsulation in
nanobeads, retroviral gene therapy, impregnated gelfoam, and
combinations thereof.
13. The composition of claim 7 further comprising a compound
selected from the group consisting of a buffering agent, a
chemotherapeutic agent, a salt, a contrast agent, a fluorescent
marker, an impedance metric device, ultrasound contrast agents, and
combinations thereof.
14-31. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application Ser. No. 60/457,691, filed Mar. 26, 2003, the entire
content of which is incorporated herein by reference.
TECHNICAL FIELD
[0003] The present invention relates generally to thermal surgical
procedures.
BACKGROUND OF THE INVENTION
[0004] Thermal surgical procedures, wherein thermal energy is
either withdrawn from and/or delivered to a localized region of
biological material in an effort to destroy the region of
biological material, are known in the art and have been shown to be
an effective treatment of disease, particularly in instances
wherein a patient is unwilling or unable to undergo another form of
surgery. A thermal surgical procedure may include, for example, a
cryosurgical procedure in which thermal energy is removed from
biological material to cool and/or freeze the biological material
in an effort to destroy it. Such procedures have been routinely
used to treat malignancies on the surface of the body and is also
used for treating and managing malignancies of internal organs,
such as kidney and prostate. Also, a thermal surgical procedure can
include a procedure in which thermal energy is added to biological
material to heat the biological material in an effort to destroy
it. The destruction of biological material may or may not result in
ablation of some or all of the biological material. Thermal
surgical procedures are useful in treating diseases of various
tissues, including, for example, carcinomas of the liver, kidney,
and prostate. These techniques are advantageous in that they have
the potential for less invasiveness and lower morbidity as compared
with surgical excision.
[0005] Thermal surgical procedures involving delivery of thermal
energy to increase the local temperature of biological material
above the physiological temperature is known to be an effective
treatment for eliminating malignant tissue. Typical temperatures
for thermal surgical procedures involving the delivery of thermal
energy are at or above 50 degrees Celsius (.degree. C). Typically,
the biological material is heated to elevated temperatures and is
maintained at these temperatures for an interval of several
minutes.
[0006] Additionally, it is known in the art that freezing
biological materials is an effective method for controlling and
destroying the cells and tissues of, for example carcinomas of
various tissues and organs. Cryosurgical techniques, in combination
with monitoring techniques, such as ultrasound and MRI, have
provided effective treatment of a number of internal organs,
including liver, prostate, and kidneys. Results of cryosurgery
involving carcinomas in kidneys suggest that this may prove a
useful technique, particularly for small renal cell carcinomas.
Cryosurgical procedures typically reduce the temperature of the
biological material to temperatures close to or below the
temperature at which the biological material will freeze, often
below 0.degree. C. and as low as -20 to -60.degree. C. Typically
the biological material is cooled to and maintained at these
temperatures for an interval of minutes.
[0007] Nonetheless, there exists clinical evidence of recurrence of
disease in thermal surgically treated patients. This result may be
due to the initial challenge of treating the entire diseased
tissue. For example, in current thermal surgical procedures it is
prudent to take a sufficient surgical margin around diseased tissue
to ensure all of the malignant tissue is removed or destroyed. This
often involves freezing or heating beyond a tumor and invading
surrounding normal tissue. However, care must be taken to not
invade too far beyond the diseased tissues, particularly when
treating biological material near healthy sensitive tissues. In
particular, when treating prostate cancers, which occur principally
in the peripheral zone of the prostate near a number of sensitive
structures, such as the rectum, bladder, external sphincter, and
the cavernosal nerves, a surgeon must be careful to spare the
surrounding tissues from injury. This is particularly important in
treating the prostate where overfreezing into the areas of the
rectum and urethra can cause rectal and urethral fistulas. On the
other hand, if a surgeon is too conservative and underheats or
underfreezes affected tissues, the disease may not be effectively
treated and the likelihood of recurrence of the disease
increases.
[0008] There is a need in the art, therefore, to improve the
clinical application of thermal surgical procedures, including
effectively monitoring of the heating or freezing of the biological
material to more effectively predict the zone of injury,
reproducibly creating and enhancing cell death within the heat
treated area or the cryolesion, and improving definition of the
edge of the heat treated area or the cryolesion to improve the
effectiveness of the kill zone while protecting adjacent normal
tissues.
SUMMARY OF THE INVENTION
[0009] The present invention provides a composition, method,
system, and/or kit for use in a thermal surgical procedure. As used
herein thermal surgical procedures generally include, but are not
limited to, surgical procedures in which thermal energy is either
withdrawn from and/or delivered to a localized region of biological
material in an effort to destroy at least a portion of biological
material.
[0010] The compositions of the present invention include one or
more compounds that can effectively induce an inflammation response
in biological materials involved in the thermal surgical procedure.
A "compound" as used herein, may include a single constituent or a
combination of two or more constituents. Furthermore, a
"composition" as used herein may include only one compound or
combination of two or more compounds.
[0011] Biological materials that may be treated using the
compositions, methods, and systems of the present invention
include, but are not limited to, cells, tumor cells, tissue, tumor
tissues, tissues of internal organs such as liver tissue, prostate
tissues, breast tissue, and kidney tissues. In addition, biological
materials may also include, but are not limited to vascular
tissues, gastrointestinal tissues, muscle tissues, including
myocardium, tissues of the skin, and connective tissues.
Combinations of these biological materials in situ are possible,
and treatment of some biological materials to the exclusion of
others is also contemplated.
[0012] The present invention may be used in the treatment of
various cancers and/or tumors such as, but not limited to, prostate
cancer, liver cancer, kidney cancer, breast cancer, uterine
fibroids, as well as any other tumor or tissue where thermal
surgical procedures have typically been used or which may be found
useful in the future. The present invention may also be useful in
the treatment of benign prostatic hypertrophy (BPH), or treatment
of stenosis of the urethra. In addition, the present invention may
also be useful in treating any number of autoimmune and chronic
inflammatory disorders, where the associated tissues involved in
the disorder are predisposed to injury from cooling or heating.
Examples include, but are not limited to, rheumatoid arthritic
syndrome, emphysema, pulmonary hypertension and cardiac failure,
Crohn's disease, neurological disorders that display
neuroinflammatory disease, ulcerative colitis, and other known
autoimmune diseases.
[0013] In addition, the present invention may also be useful in any
number of interventional procedures that are currently used to
treat individuals. For example, the present invention may be useful
in procedures that utilize cooling or heating to destroy biological
materials. Thus, the present invention may be used in conjunction
with thermal surgical procedures performed on myocardial tissue for
treating rhythm irregularities of the heart. Further, the present
invention may be used in preventing restenosis of arteries treated
with angioplasty, atherectomy, or other procedures for opening
occlusions in the vasculature.
[0014] In one aspect, the present invention includes a method of
performing a thermal surgical procedure, wherein the method
includes: identifying biological material to undergo the thermal
surgical procedure; contacting the biological material with an
inflammation inducing composition, wherein inflammation is induced
in at least a portion of the identified biological material; and
adjusting the temperature of the identified biological material,
wherein at least a portion of the biological material is destroyed
after undergoing the thermal surgical procedure. The temperature
may be adjusted above a physiological temperature of the biological
material, a thermosurgical procedure, or the temperature may be
adjusted to below a physiological temperature, as in a cryosurgical
procedure. It is contemplated that a thermal surgical procedure may
also include both a thermosurgical procedure and a cryosurgical
procedure, either on the same identified biological material with
the procedures performed at separate times, or on separate sites of
the identified biological material.
[0015] In another aspect, the present invention includes a
composition that includes at least one compound effective for
inducing an inflammatory response in biological material that is
identified to undergo a thermal surgical procedure. The composition
may include a single constituent as the active ingredient, or may
include a combination of active ingredients. Furthermore, the
composition may also include such optional constituents as a
physiological carrier and/or a buffering agent.
[0016] In a further aspect, the present invention provides a method
of performing a thermal surgical procedure for biological material,
wherein the thermal surgical procedure may be a thermosurgical
procedure, a cryosurgical procedure, or any combination thereof.
The thermal surgical procedure includes: identifying biological
material to be treated prior to a thermal surgical procedure;
contacting the biological material with an inflammation inducing
composition for a time, amount and type effective to induce
inflammation in at least a portion of the biological material,
wherein inflammation is induced in at least a portion of the
identified biological material; and adjusting the temperature of
the identified biological material, wherein at least a portion of
the biological material is destroyed after undergoing the thermal
surgical procedure.
[0017] The present invention additionally provides a system for
inducing inflammation in biological material identified to undergo
a thermal surgical procedure. This system generally includes: a
composition including at least one compound effective for inducing
inflammation in at least a portion of the biological material; and
means for delivering the composition to a least a portion of the
biological material. The composition may include a single active
ingredient or more than one active ingredient, and may further
include optional constituents, such as a pharmaceutically
acceptable carrier and/or a buffering agent.
[0018] In an additional aspect, the present invention provides
methods of treating diseases, such as cancer. A method of treating
cancer is disclosed which includes: identifying a localized region
of a mammal comprising biological material further including
cancer; providing to at least a portion of the biological material
a composition comprising as an active ingredient at least one
compound for a time, amount and type effective to induce
inflammation in at least a portion of the biological material,
thereby providing inflamed biological material; and applying a
thermal surgical procedure to at least a portion of the inflamed
biological material. The thermal surgical procedure may be a
thermosurgical procedure, a cryosurgical procedure, or a
combination thereof.
[0019] Additional diseases may be treated by the methods and
compositions of the present invention. For example, the present
invention includes a method of treating a disease that includes:
identifying a localized region of a mammal comprising biological
material typical of the disease; providing to at least a portion of
the biological material a composition comprising as an active
ingredient at least one compound for a time, amount and type
effective to induce inflammation in at least a portion of the
biological material, thereby providing inflamed biological
material; and
[0020] applying a thermal surgical procedure to at least a portion
of the inflamed biological material.
[0021] The present invention further discloses a kit for use in a
thermal surgical procedure. Such kit includes, generally: a thermal
surgical probe adapted to transfer thermal energy; and a
composition comprising at least one compound effective for inducing
an inflammatory response in biological material identified to
undergo a thermal surgical procedure.
[0022] The above summary of the present invention is not intended
to describe each embodiment or every implementation of the present
invention. Advantages, together with a more complete understanding
of the invention, may become apparent and appreciated by referring
to the following detailed description of illustrative embodiments
taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF FIGURES
[0023] FIG. 1 shows one embodiment of a system according to the
present invention.
[0024] FIG. 2 shows one example of a relationship of temperature
versus distance from the center of an ice ball according to the
present invention.
[0025] FIG. 3 shows a cross-sectional view of one example of an ice
ball according to the present invention.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0026] In the following detailed description of certain
illustrative embodiments, reference is made to drawings that form a
part hereof, and in which are shown by way of illustration, certain
embodiments through which the invention may be practiced. It is to
be understood that other embodiments may be utilized and processing
steps/structural changes may be made without departing from the
scope of the present invention.
[0027] As will be discussed below, the present invention provides
methods, compositions, and systems for use in treating biological
materials including, for example, cells, tissues, and combinations
thereof, with a thermal surgical procedure. The present invention
generally includes an inflammation inducing composition that can be
used to treat the biological material that is the subject of the
thermal surgical procedure, wherein the biological material to
undergo a thermal surgical procedure may be identified, in whole or
a portion thereof, and is treated with an inflammation inducing
composition of the present invention for a time, in an amount, and
of a type effective to induce at least some inflammation in at
least a portion of the identified biological material. The present
invention further includes methods and systems for inducing such
inflammation. The inflammation inducing composition of the present
invention can be used to induce an inflammatory response in the
biological material, where the induced inflammatory response may
provide for an enhancement of destruction of the biological
material during the thermal surgical procedure.
[0028] The methods and compositions of the present invention may be
useful in treating several diseases, including cancer. Cancers that
may potentially be treated by the present methods and compositions
include, but are not limited to, cancers of internal organs, such
as prostate, liver, and kidney, cancers of bone and cartilage, skin
cancer, oral cancer, musculoskeletal cancers, breast cancer,
gynecological cancers including uterine fibroids. Other diseases
that may benefit from the methods and compositions of the present
invention include, for example, benign prostatic hypertrophy,
stenosis of the urethra, rheumatoid arthritic syndrome, emphysema,
pulmonary hypertension, cardiac failure, Crohn's disease,
neurological disorders displaying neuroinflammatory disease,
ulcerative colitis, and gynecological disorders.
[0029] Inflammation is understood herein to typically involve a
complex series of events which may include, but is not limited to,
dilatation of arterioles, capillaries and venules, with increased
vessel permeability and blood flow, exudation of fluids through the
vessel walls, including plasma proteins and leucocytic migration
into the inflammatory focus. Further, on a molecular level adhesion
molecules are typically upregulated in endothelial cells which slow
down (by rolling) and capture (by adhesion) leucocytes to the
vessel walls in the area of inflammation.
[0030] Without wishing to be bound by any particular theory, it is
believed that an induced inflammatory response in biological
materials intended to undergo a thermal surgical procedure may
augment the effectiveness of that procedure. It is further believed
that by controlling the extent and degree of the induced
inflammatory response in the biological material, the degree to
which the thermal surgical procedure will be successful may be
significantly influenced. Using compounds that, for example, induce
non-destructive inflammation in biological material provides
beneficial changes in the effectiveness of thermal surgical
procedures as compared to untreated biological material. Thus, the
present invention provides improvements in typical thermal surgical
procedures by providing what is believed to be a controllable and
reproducible technique to accentuate the injury and death of
biological material undergoing the thermal surgical procedure.
[0031] Thermal surgical procedures have been shown to be effective
treatment modalities for several tumor tissues. For example,
cryosurgical procedures are known to be effective treatments for
eliminating malignant tissue. In cryosurgical procedures, thermal
energy is removed from at least a portion of the biological
material so as to decrease the local temperature below a
physiological temperature of the biological material. A
physiological temperature of the biological material is generally
understood to be that temperature at which the physical mechanisms
of living organisms and/or parts thereof are able to function.
Cryosurgical procedures reduce the temperature of the biological
material to temperatures close to and/or below the temperature at
which the biological material will freeze. Typical temperatures for
cryosurgical procedures include those at or below 0.degree. C., and
may further include temperatures at or below approximately
-20.degree. C., going down to at or below -60.degree. C. The
biological material may be cooled to and maintained at these
temperatures for, e.g., an interval of minutes, or any other
suitable period of time, to ensure effectiveness of the
treatment.
[0032] While in a cryosurgical procedure it may be preferred to
take a sufficient surgical margin around the malignant tissue to
ensure that all tumor tissue has been removed or destroyed, often
requiring freezing beyond the tumor into normal tissue, the present
invention is believed to reduce the potential side effects of
normal tissue damage during cryosurgery, and to maximize the tumor
destruction at the edge of the cryosurgical ice ball, strategies to
both protect (e.g., normal) and sensitize (e.g., tumor) cells to
freezing. Protecting and/or sensitizing tissues from temperatures
experienced within the ice ball may allow surgeons to functionally
increase the surgical margin while decreasing damage to surrounding
tissues. Also, increasing the efficiency of tissue destruction
within the ice ball may increase the confidence that an increased
number of, e.g., tumor cells are killed near the periphery of the
tissue of interest while decreasing the chances of over-freeze
damage into adjacent normal tissues, such as, e.g., the rectum, in
prostate cryosurgery.
[0033] It is also believed that the present invention may provide
for better assessment of the actual location of cell and tissue
death in the ice ball formed during a cryosurgical procedure.
Assessment of this location can be based in part on the region of
biological material undergoing an inflammatory response induced by
the use of the inflammation inducing composition of the present
invention. Use of the inflammation inducing composition of the
present invention may allow for a greater percentage of cell and/or
tissue destruction during the cryosurgical procedure.
[0034] Thermal surgical procedures that deliver thermal energy to
biological material, understood herein as "thermosurgical
procedures" are also known to be effective treatments for
eliminating malignant tissue. In these thermosurgical procedures,
thermal energy is supplied to at least a portion the biological
material so as to increase the local temperature above a
physiological temperature of the biological material. For example,
typical temperatures for these thermosurgical procedures generally
include those at or above 50.degree. C. It is noted that by the
methods and composition of the present invention, it may be
possible to perform effective thermosurgical procedures at
temperatures below 50.degree. C., such as temperatures no greater
than about 40.degree. C., thereby preventing injury to adjacent
tissues. The biological material may be heated to and maintained at
these temperatures for, e.g., an interval of minutes, or any other
suitable period of time, to ensure effectiveness of the
treatment.
[0035] As with cryosurgery, it is typically considered to be
beneficial to take a sufficient surgical margin around the
biological material of interest through the use of the heat to
ensure that all the biological material of interest has been
removed or destroyed. However, taking a sufficient margin around
the biological material typically requires heating beyond the
biological material of interest into normal tissue. To minimize the
potential side effects of normal tissue damage during a
heat-delivering thermal surgical procedure, and to maximize the
destruction at the edge of the heated biological tissue, strategies
to both protect (e.g., normal) and sensitize (e.g., tumor) cells to
heating are also desirable.
[0036] The present invention is believed to sensitize tissues to
and/or protect tissues from temperatures experienced at either the
edge of the ice ball or the edge of the heated biological material.
As a result, surgeons performing thermal surgical procedures
according to the present invention could potentially functionally
increase the surgical margin while decreasing damage to surrounding
tissues by increasing the efficiency of tissue destruction at the
edge of the ice ball or the edge of the heated biological tissue
and may also obtain better assessment of the actual location of
cell and tissue death in the ice ball formed during a cryosurgical
procedures and/or the actual location of cell and tissue death at
the edge of the heated biological tissue formed during the thermal
surgical procedure.
[0037] Improvement of the assessment of the actual location of cell
and/or tissue death, according to the present invention, is
believed to be based in part on the region of biological material
undergoing an inflammatory response induced by the use of the
inflammation inducing composition of the present invention. Use of
the inflammation inducing composition of the present invention is
believed to provide a greater percentage of cell and/or tissue
destruction during the thermal surgical procedures contemplated by
the present invention.
[0038] Generally, the compositions of the present invention may be
used in a localized region of a mammal involved in the thermal
surgical procedure. Typically, the composition includes as an
active ingredient at least one compound effective to induce at
least some inflammation in at least a portion of the biological
material of interest, such as any native or artificial tissue of a
mammal, where the at least one compound can be effective to induce
inflammation in at least a portion of the localized region of the
native or artificial tissue of the mammal.
[0039] A composition of the present invention typically induces a
non-destructive inflammation within the biological material of
interest, either localized to the entire region of the thermal
surgical site or localized to one or more portions of the thermal
surgical site, prior to, during, and/or after the thermal surgical
procedure. For example, a composition of the present invention may
be used to induce an inflammatory response in a localized region of
the biological material prior to or concurrent with a thermal
surgical procedure. Furthermore, a composition of the present
invention may be used in a localized region of a mammal to treat
biological material, for a time, and in an amount and using a type
of composition effective to induce inflammation in the material,
that has previously been identified and has undergone a thermal
surgical procedure.
[0040] While not wishing to be bound by a particular theory, it is
believed that the composition of the present invention may induce
this non-destructive inflammation by altering the behavior of
vascular endothelial cells present in biological material. In
particular, it is believed that the inflammation induced by the
compositions of the present invention may injure the
microvasculature of the biological tissue prior to the thermal
surgical procedure. It is also believed that this induced
inflammatory injury may precondition the microvasculature so that
it "shuts down" after the thermal surgical procedure. In
combination with the thermal surgical procedure, it is believed
that the use of the composition of the present invention may
provide a more effective destruction of the biological material
undergoing a thermal surgical procedure by enhancing the effects of
the procedure than would be provided by the thermal surgical
procedure performed alone without benefit of inflaming the
biological material.
[0041] Without being held to any particular theory, it is believed
that through an understanding of the nature of cell injury, it may
be possible to accentuate the mechanisms of injury utilizing
targeted molecular adjuvants such as those described herein. It is
further believed that two biophysical changes that occur in cells
during freezing, osmotic dehydration of cells and intracellular ice
formation (IIF) may be linked to cell injury. At low cooling rates,
as the freezing propagates extracellularly, the solute
concentration outside the cell begins to rise, causing osmotic
dehydration of the cells. As the solutes become concentrated within
the cells, the high concentration of solute has been hypothesized
to injure the cell in several ways including damage to the
enzymatic machinery and destabilization of the cell membrane.
[0042] The second biophysical response, IIF, is believed to occur
when the cooling rate is sufficiently rapid to trap water within
the cell. In this case, the cell cannot osmotically equilibrate
with the extracellular space. As a result, the cytoplasm cools and
ice ultimately nucleates within the cell, the ice crystals causing
injury to the organelles and membranes.
[0043] Damage due to solute effects is believed to typically happen
at relatively low cooling rates when the cells have sufficient time
to dehydrate substantially completely. IF damage, on the other
hand, is believed to typically occur at relatively high cooling
rates, when the water is trapped inside the cells. This results in
an "inverse U curve" of cell viability with low viability at
extremely high and low cooling rates, and high viability at cooling
rates between the extremes. This cooling rate behavior is highly
cell-type dependent with the cooling rate that yields maximum
viability (i.e. the top of the inverse U) ranging over many orders
of magnitude 1-1000.degree. C./minute.
[0044] Cellular injury mechanisms may depend on the thermal history
that a cell experiences during freezing. This thermal history is
defined by four thermal parameters: cooling rate (CR), end (or
minimum) temperature (ET), time held at the minimum temperature
(hold time, HT), and thaw rate (TR), all of which have been linked
to injury. It has been found in AT-1 tumor cells that ET and HT are
the most injurious in cell suspensions. However, each cell type
investigated typically has a unique thermal threshold where AT-1
cells can survive to -80.degree. C. and ELT-3 uterine fibroid cells
can survive only to -30.degree. C. with other thermal parameters
being similar, and, as indicated above, if the rates of cooling and
heating (CR, TR) are sufficiently high, cell damage irrespective of
the ET and HT may be obtained.
[0045] Treating biological material by contacting the material with
the compositions of the invention, thereby inducing inflammation in
the localized region of the material, is also believed to cause
various changes in the biological material. For example, it is
believed that such treatment may be effective in changing a
destruction point of the biological material in a localized region
of a mammal. As used herein, the "destruction point" is understood
to mean the temperature at which the biological functions of the
biological material undergoing the thermal surgical procedure are
rendered irreversibly inoperative, either during the procedure or
shortly thereafter, preferably within three days or less, more
preferably within two days or less, even more preferably within one
day or less, and still more preferably within 12 hours or less of
the procedure. In some instances, the functions of the biological
material may be rendered irreversibly inoperative within 2 hours or
less of the procedure, and preferably within one hour or less of
the procedure. In other words, the destruction point is the
temperature, the hold time at a given temperature, and/or the rates
of heating and/or cooling at which cell death results in the
localized region of the biological material. These temperatures can
include those that are below normal physiological temperatures and
those that are above normal physiological temperatures, and the
destruction point is variable, depending upon the biological
materials of interest.
[0046] Without being held to a particular theory, it is believed
that so treating the biological material induces more effective
killing at the edge of the treated area, such as through
endothelial injury and microvascular shut-down post freeze at the
edge of the ice ball in a cryosurgical procedure. One may be able
to visualize the edge of the ice ball using monitoring techniques
such as NMR, CT, or ultrasound. Therefore, by using an inflammatory
agent to provide destruction of biological material out to the edge
of the ice ball, it may be possible to visualize the region of
injury intraoperatively rather than post-operatively in follow-up,
thus potentially increasing the control and effectiveness of the
thermal surgical technique.
[0047] The inflammatory inducing compounds of the present invention
are considered herein to be adjuvants that enhance the thermal
surgical procedure. As used herein, to "enhance a thermal surgical
procedure" is considered to include, but not be limited to,
increasing a percentage of cell death in the localized region of
the biological material within a given time period as compared to
untreated regions of the biological material.
[0048] Alternatively, a thermal surgical procedure may be enhanced
by attempting to control the injury. This may be accomplished in a
cryosurgical procedure, for example, through the use of any of a
class of compounds used to diminish the injury, rather than to
augment it. These compounds are cryoprotective agents and include,
for example, glycerol, dimethylsulfoxide, various sugars, various
alcohols, and various polymers such as PVP and HES, may enhance the
surgical procedure by, in essence, "sculpting" the ice ball from
the outside rather than controlling it from within the ice
ball.
[0049] The compositions of the present invention include at least
one compound, and may include more than one of any of a number of
compounds capable of inducing some degree of inflammation in at
least a portion of the treated (i.e., contacted) biological
material of a mammal, wherein the material is identified to undergo
a thermal surgical procedure. For example, the compounds may
include, but are not limited to, one or more viruses, one or more
bacteria, ethanol, cytokines such as Tissue Necrosis Factor-alpha
(TNF-alpha) or truncated versions of TNF-alpha, bacterial
lipopolysaccarides (LPS), interleukins such as IL-1 beta and IL-8,
chemokines which recruit white blood cells, oxygen-free radicals,
and combinations thereof.
[0050] Selection of the one or more inflammatory inducing compounds
may take into consideration the individual effects and traits of
the compound. TNF-alpha, for example, is known to promote
inflammation, endothelial injury, and apoptosis, and may be used
alone or in combination with other compounds to provide the desired
benefit. TNF-alpha is produced by a number of different cell types,
macrophages, tumor and stromal cells and is thought to be
responsible for manifestation of autoimmune and chronic
inflammatory disorders. As discussed hereinbelow, in one embodiment
of the invention, TNF-alpha may be directly injected into the
biological material of interest (e.g. a tumor) in order to increase
the efficacy of the thermal surgical procedure. Alternatively, in a
further embodiment, cells that produce TNF-alpha might be directed
to, or injected into, the biological material of interest to
increase the efficacy of the thermal surgical procedure through
inflammation of the biological material.
[0051] There are various methods for delivering the composition to
the biological material of interest, such methods being an issue of
selecting an appropriate drug delivery system. One method includes
the addition of one or more carriers, particularly carriers that
may have specific receptors for the tumor or tissue of interest.
Thus, the inflammation inducing compositions of the present
invention may optionally include a pharmaceutically acceptable
carrier for delivery to the material of interest, which carrier may
also optionally have specific receptors for the tumor or tissue of
interest.
[0052] As used herein, a pharmaceutically acceptable carrier may
include, but is not limited to, liquid solvents in which the
inflammation inducing compound can be at least partially suspended
and/or diluted, such as a saline solution, and any other carrier
which may provide for direct interstitial injection in liquid
suspension, IV or IP injection, impregnation of the composition
into microbeads or nanobeads to be injected locally or systemically
and then targeted, gelfoam, retroviral DNA injections (gene
therapy), etc., and combinations thereof.
[0053] Pharmaceutically acceptable carriers may also optionally
include buffering agents, as are known, to ensure the resulting
inflammation inducing composition has a pH value within a range
acceptable for physiological use. Such agents may include, but are
not limited to phosphate buffered solutions.
[0054] The inflammation inducing compositions of the present
invention may also include further components to provide additional
benefits. For example, additional components may include, but are
not limited to, a composition to further enhance cell and tissue
destruction by cryosurgery. U.S. Pat. No. 5,654,279 to Rubinsky et
al. provides one example of possible additional additives. An
additional example includes additives that may provide for eutectic
freezing in the biological material 12 as provided in U.S. Ser. No.
10/461,763 entitled CRYOSURGERY COMPOSITIONS AND METHODS, filed on
Jun. 13, 2003 (Atty. Docket 110.01920101). In addition,
chemotherapeutic agents can also be introduced with the
inflammation inducing composition.
[0055] As discussed in more detail below, the location and/or
extent to which the inflammation inducing composition may be
infused into the tissue can be monitored through any number of
known techniques. The inflammation inducing compositions may,
therefore, optionally include compounds to assist in visualization
and monitoring. For example, compounds and/or solutions that may
enhance ultrasonic imaging, fluoroscope, MRI, impedance technique
(e.g., U.S. Pat. No. 4,252,130 to Le Pivert), etc., can be added to
the inflammation inducing composition to allow for visualization of
the location of the inflammation inducing composition. Examples
include, but are not limited to, contrast agent added with salt
(i.e., hypaque) and/or the inflammation inducing compounds, salt
and/or the inflammation inducing compounds tagged with a
fluorescent marker, ultrasound contrast agents, and use of an
impedance metric device to see how impedance changes locally with
infusion.
[0056] The time interval for treating the biological material with
the composition of the present invention prior to performing a
thermal surgical procedure can range from, e.g., a matter of
minutes, hours, or days, depending on the composition and
biological material of interest, and the required time interval is
measured according to the effectiveness of the kill. However, it is
currently believed that improved effects may be provided if at
least about one hour or more elapses between delivery of the
composition to the biological material and the thermal surgical
procedure. As an example, it may be possible to use a four (4) hour
time interval for treating biological material with the composition
of the present invention prior to performing the thermal surgical
procedure. This time may, however, change depending upon any number
of factors, including but not limited to, the type and location of
the biological material, the inflammatory composition used (and/or
its delivery system), and the existing physiological state of the
biological material.
[0057] Without being held to any particular theory, it is believed
that the inflammatory response typically should be sufficiently
activated within the endothelium of the microvasculature to give
the augmented injury response after cryosurgery or thermosurgery.
The inflammatory response as measured by adhesion molecule
production within endothelium (VCAM and ICAM) may typically take
several hours to peak. However, in certain instances it may be true
that much shorter times, such as on the order of minutes, will
provide the desired response.
[0058] The methods of the present invention include methods of
performing a thermal surgical procedure in which the biological
material identified to undergo the procedure is contacted with an
inflammation inducing composition of the present invention, and the
temperature of the biological material is adjusted. The temperature
is adjusted such that the material is either cooled to below or
heated to above a physiological temperature to destroy at least a
portion of the material. The composition selected is a type such
that, for a specified time and amount of the composition,
inflammation is induced in at least a portion of the biological
material, and such inflammation is induced. Such treatment of the
material (i.e., contact with the composition) may occur either
during the surgical procedure, before the procedure, after the
procedure, or any combination thereof.
[0059] Treatment of the biological material is considered to
include, but not be limited to, one or a combination of means for
delivering the composition to at least a portion of the identified
biological material. Such delivery means may include, but not be
limited to, introduction of the composition into one or more
locations of the biological material through the use of hypodermic
needles, introduction via of one or more needles integrated into or
attached to a cryoprobe, introduction via diffusion, and
introduction via iontophoresis (or any other use of electric fields
to drive solution flow in tissues), direct interstitial injection
in liquid suspension, IV or IP injection, impregnation of the
composition into microbeads or nanobeads to be delivered locally or
systemically and then targeted, retroviral DNA injections (gene
therapy), etc., and combinations thereof.
[0060] Alternatively, the delivery means could involve
incorporation of the composition into a gel or foam for topical
use, or incorporated into an implantable material to be used before
or after the thermal surgical procedure, for example, a gelfoam, a
tissue engineered collagen, fibrin based product, etc.
[0061] Biological materials to be treated according to the methods
and systems of the present invention typically have a destruction
point, that is, a temperature, or the hold time at a given
temperature, and/or the rates of heating and/or cooling at which
cell death results in the localized region of the biological
material, with the temperature typically above or below
physiological temperatures, that is, temperatures at which the
biological functions of the biological material undergoing the
thermal surgical procedure are rendered irreversibly inoperative
(i.e., cell death in the localized region of the biological
material). Without being held to any particular theory, it is
believed that the present invention operates, at least in part, to
change the destruction point of, for example, a localized region of
a mammal that includes biological materials identified to undergo a
thermal surgical procedure.
[0062] The present invention is believed to provide improved
assessment of the actual location of cell and/or tissue death in
identified biological materials. Such assessment is aided through
the use of known monitoring techniques that may locate and/or
determine the extent to which the inflammation inducing composition
has been infused into the tissue including, for example, ultrasonic
imaging, fluoroscope, MRI, and impedance techniques, particularly
when used in conjunction with a composition including an imaging
enhancing compound such as vascular perfusion contrast agents.
Thus, by being able to visualize, for example, the edge of an ice
ball during a cryosurgical procedure, the edge of the injury during
the procedure may be monitored during the procedure, providing
improved intraoperative imaging and injury assessment.
[0063] The present invention further provides systems for inducing
inflammation in at least a portion of biological material intended
to undergo a thermal surgical procedure that includes a composition
of the present invention and means for delivering the omposition to
the biological material. Such delivery means may include, for
example, delivery of the composition through a catheter (e.g.,
through a lumen, in a balloon or other chamber positioned at a
desired location, etc.), delivery via a needle, and delivery via a
thermal surgical probe adapted to transfer thermal energy, such as,
e.g., a cryoprobe.
[0064] The composition may be delivered either directly to the site
of the thermal surgical procedure, or may be delivered to another
location, such as, e.g., a site adjacent to the location of the
thermal surgical procedure, or may be delivered systemically when
appropriate. Further, the inflammation inducing composition may be
delivered to the biological material before, during, and/or after a
thermal surgical procedure.
[0065] Systems of the present invention may further include the use
of a thermal energy transfer means that provides thermal energy to
biological material (a heat source) or that removes thermal energy
from biological material (a heat sink). Such means may include, for
example, thermal surgical probes, catheters, implantable devices,
etc. An effective means of providing and/or removing thermal energy
in the methods and systems of the present invention may be a
thermal surgical probe, wherein the probe is effective for either
removing thermal energy from or supplying thermal energy to,
depending on the type of thermal surgical procedure contemplated,
at least a portion of the biological material of interest at a rate
to provide heating or cooling, resulting in at least partial
destruction of biological material at the location of the thermal
surgical procedure. Such probes may include, for example,
catheters, hollow needles, cryoprobes, implantable devices,
etc.
[0066] The present invention may include a kit for use in a thermal
surgical procedure. The kit may include a thermal surgical probe
that is adapted to transfer thermal energy as appropriate, either
by removing thermal energy for use in a cryosurgical procedure, or
by supplying thermal energy, for use in a thermosurgical procedure.
The kit preferably includes a composition that includes at least
one compound effective for inducing an inflammatory response in
biological material identified to undergo a thermal surgical
procedure. Such compositions may include one or more of compounds
that are selected from the group of at least one virus, at least
one bacterium, ethanol, cytokines, interleukins, chemokines,
oxygen-free radicals, bacterial lipopolysaccharides, and any
combination thereof. If a cytokine is selected for use, it may be
preferred that the cytokine used is TNF-alpha, truncated versions
of TNF-alpha, and any combination thereof. If an interleukin is
selected for use, it may be preferred that the interleukin used is
IL-beta, IL-8, and any combination thereof. The composition may
further include an optional pharmaceutically acceptable carrier
and/or any of the optional constituents previously discussed. The
probe that is used is any that may be adapted for use in a thermal
surgical procedure such as, but not limited to, a catheter, a
hollow needle, a cryoprobe, an implantable medical device, etc.
[0067] As an example, FIG. 1 shows one possible embodiment of a
system 10 according to the present invention for inducing
inflammation in biological material 12 that includes a portion 14
that has been identified to undergo a thermal surgical procedure.
As discussed herein, the biological material 12 can include a
tissue, including cells, intended to undergo, at least in part, a
thermal surgical procedure. The portion 14 of biological material
12 can have a similar cell and/or tissue structure as the
surrounding segment of biological material 12. Alternatively, the
portion 14 can have one or more morphologically distinct cell
and/or tissue structures as compared to the remaining segment of
the biological material 12. In one example, the portion 14 can be a
tumor.
[0068] The inflammatory state of the portion 14 of the biological
material 12 can be changed relative to the remaining segment of the
biological material 12 through the use of the inflammatory inducing
composition of the system 10 of the present invention. The
biological material 12 may be treated with the inflammation
inducing composition of the present invention for a time, in an
amount and of a type effective to induce inflammation in at least a
portion of the identified biological material. In one example, the
inflammation inducing composition can be one or more of the
compounds for inducing an inflammatory response as discussed
herein.
[0069] The portion 14 of the biological material 12 to be treated
with the inflammation inducing composition as a part of the thermal
surgical procedure may be identified by any number of known
techniques. For example, tumor structures may be identified through
tissue structure, biological markers, ultrasound, or any number of
other techniques. Furthermore, the location and/or extent to which
the inflammation inducing composition has been infused into the
tissue (e.g., the portion 14 in FIG. 1) can also be monitored
through any number of techniques, and one or more identification
and/or monitoring techniques may be used as required.
[0070] Once identified, the inflammation inducing composition can
be delivered to the portion 14 of the biological material 12. In
one possible embodiment, the composition can be delivered through
the use of delivery device, such as, e.g., a catheter 16. In
general, the catheter 16 includes a lumen, where the inflammation
inducing composition can move through the lumen of the catheter 16
and into the biological material 12 in which the inflammatory
response is desired. The catheter 16 of the present invention may
also include a needle at a distal end of the catheter 16 for
delivering the inflammation inducing composition. Alternatively,
the catheter 16 can further include a trocar in the lumen of the
catheter 16 to facilitate delivering a portion of the catheter 16
to the biological material 12 in which the inflammatory response is
desired. U.S. Pat. No. 5,807,395 provides some examples of
catheters 16 that may be suitable for injecting the inflammation
inducing composition of the present invention.
[0071] The system 10 may also include one or more probes 18, where
the probes 18 can remove and/or deliver thermal energy from the
location for thermal surgical procedure at a rate sufficient to
cause biological material 12 at the location for thermal surgical
procedure to undergo cooling or heating. In one embodiment, heat
may be removed at a rate sufficient to cause cooling of the tissue
surrounding the probe at a 1-100.degree. C. per minute rate. In an
additional embodiment, thermal energy may be supplied at a rate
sufficient to cause heating of the tissue surrounding the probe at
a 1-100.degree. C. per minute rate. In some embodiments, the
catheter 16 (or other device) used to deliver the inflammation
inducing composition may also be used to deliver or remove thermal
energy, as discussed herein.
[0072] Other rates are also contemplated in the methods and systems
of the present invention, depending on the circumstances of use.
During a cryosurgical procedure, for example, intracellular ice
formation may occur at higher rates, typically greater than about
30.degree. C./minute, more typically greater than about 50.degree.
C./minute. This mechanism of injury may occur proximate the
cryosurgical probe; however, as rates typically decrease quickly
moving away from the probe, the effects of a broad range of rates
may be more strongly felt proximate the probe, while the rates in
the outer areas of the biological material being treated may be
lower (e.g., in the range of 1-10.degree. C./minute) regardless of
the rate at which the probe, or other device, is cooled or
heated.
[0073] One or more probes 18 may be used to cool and/or heat the
biological material 12 at a rate effective to destroy at least the
portion 14 of the treated biological material. In a further
embodiment, for example, when the biological material 12 is cooled
with the probe 18, an ice ball is formed. The ice ball formation
typically originates proximate the tip of each probe 18. As thermal
energy is removed from the tissue, the ice ball grows. Visualizing
the size of the ice ball formation may assist in determining the
extent, or amount, of tissue and cell material killed during a
thermal surgical procedure. Visualization of the size of the ice
ball may be accomplished, e.g., through the use of hypaque with
fluoroscopy, ultrasonic imaging, MRI, gadolinium with MRI,
impedance techniques, or other applicable techniques.
[0074] FIG. 2 depicts one example of the relationship of
temperature versus distance from the ice ball center. Line 100
illustrates the distance from the center of the ice ball (e.g., the
location of the probe) where cell death will typically occur for
biological material that has not been treated with the inflammation
inducing composition. As will be noted, the temperature at the
distance where the cell death is suggested to occur within tumors
is between approximately -20.degree. C. to approximately
-60.degree. C. in the depicted example. In contrast, when the
biological material is treated with the inflammation inducing
composition as described herein, the distance from the center of
the ice ball (e.g., the location of the probe) where cell death
will typically occur may be increased along with the temperature at
which this cell death occurs. This is illustrated by line 120.
Thus, the inflammation inducing composition may effectively
increase the distance from the probe, or other device, for which
cell death will typically occur without a corresponding increase in
the diameter of the ice ball.
[0075] In addition to increasing the volume in which cell death
will typically occur, the use of the inflammation inducing
composition is believed to enable a change in the size or extent of
the ice ball such that the use of the composition may, for example,
reduce the size of the ice ball. Although not wishing to be bound
by any particular theory, it is believed that this may be due, at
least in part, to the preconditioned state of the microvasculature
of the biological material caused by the introduction of the
inflammation inducing composition. A reduction in the size of the
ice ball formation coupled with the increase in the volume within
which cell death will typically occur in the cryosurgical ice ball
results in an ice ball with a size that more closely correlates to
the volume in which the actual cell death occurs. Further, the
inflammation composition is believed also to enable a change of the
temperature at which cell death occurs (the "destruction point") by
augmenting the injury zone such that it more closely matches the
ice ball or, alternatively, by altering the phase change
temperature, thereby potentially decreasing the ice ball size for a
given probe operation.
[0076] FIG. 3 illustrates what is believed to occur with respect to
the size of an ice ball according to the methods, compositions, and
systems of the present invention. The ice ball is shown generally
at 150. Probe 160 is used to remove thermal energy from the
biological material so as to create the ice ball 150. When the ice
ball is formed in biological material not treated with the
inflammation inducing composition, a kill zone 170 surrounds the
tip of the probe 160, within a boundary 180 of the ice ball 150. As
FIG. 3 illustrates the boundary 180 of the ice ball 150 is located
at a distance 190 from the edge of the kill zone 170.
[0077] In contrast, when the biological material is treated with
the inflammation inducing composition according to the present
invention, the kill zone 200 may thereby be enlarged as compared to
kill zone 170. In addition, the boundary 210 of the ice ball 150
may be reduced as compared to the boundary 180. Thus the size of
the ice ball may be reduced and the size of the kill zone within
the ice ball may be increased due to administration of the
inflammation inducing composition of the present invention. One
potential beneficial result of these changes in kill zone and ice
ball size is that the kill zone may more closely correlate with the
size of the ice ball. This may allow surgeons to more closely
predict the actual kill zone created during the thermal surgical
procedure and more effectively treat diseased tissues while
preserving adjoining normal tissues from injury.
[0078] For example, during cryosurgery of the prostate and many
other organs such as liver, kidney or brain, ultrasound or MRI can
be used to monitor the extent of the cryosurgical ice ball and it
is used at some level to predict the outcome of the procedure. The
ice ball boundary, however, is typically at a temperature of
approximately -0.5.degree. C., while thresholds of prostate cancer
destruction are reported anywhere from approximately -20.degree. C.
to -60.degree. and in some instances even lower (Hoffmann N,
Bischof J. 2002. Urology 60 (Supplement 2A): 40-9; Saliken J,
Donnelly B, Rewcastle J C. 2002. Urology (Supplement 2A): 26-33).
Thus, while monitoring is useful for imaging the ice ball and
predicting likely outcome of the surgery, it may not assist in the
outcome that not all of the tissue that is frozen is also
effectively treated. In some tissues, such as liver and sometimes
kidney, the ice ball may be allowed to progress into a margin of
normal tissue beyond the tumor. However, this is not the case with
prostate since overfreezing into sensitive adjacent structures such
as the rectum and urethra can cause complications such as rectal
and urethral fistulas. On the other hand if the surgeon is too
conservative and under freezes by keeping the ice ball solely
within the prostate, then cancer which often exists under the
prostate capsule at the edge of the gland may not be effectively
treated leading in some cases to recurrence of disease. One
approach of the present invention may include the use of
cryosurgical adjuvants in the form of both an inflammation inducing
composition and a eutectic freezing point changing agent (such as,
e.g., those described in U.S. patent application Ser. No.
10/461,763, entitled CRYOSURGERY COMPOSITIONS AND METHODS, filed
Jun. 13, 2003 (Attorney Docket No. 110.01920101) may be used
together. The combination may provide inflammation to the
biological material to both increase freeze destruction (salts and
TNF-alpha) and reduce the temperature at the edge of the ice ball.
The combination may improve the effectiveness and predictability of
the kill zone while preserving normal tissues from excessive and/or
unnecessary injury.
[0079] A composition including the cytokine TNF-alpha was used to
increase the threshold temperature of destruction after cryosurgery
in human prostate cancer (LNCaP grown in nude mice) to a mean
temperature above 0.degree. C. The local use of TNF-alpha to
pre-inflame prostate cancer increased the ability of freezing to
destroy the cancer. Thus, monitoring techniques such as ultrasound,
CT, MR, and others which focus on the edge of the ice ball may, in
the presence of TNF-alpha, also be capable of predicting the
outcome of the treatment by measuring the edge of the injury at the
same time that the edge of the ice ball is measured.
[0080] Pre-inflammation of biological tissue may be a mechanism
useful in accentuating vascular injury during thermal surgical
procedures. In addition, there may be a role for the endothelium in
shutting down the microvascular supply to prostate cancers (Dunning
AT-1) grown in Copenhagen rats fitted with dorsal skin fold
chambers (DSFC) (Hoffmann N, Bischof J. 2002. Urology 60
(Supplement 2A): 40-9). Data from these studies indicates that the
tumor could under some freeze/thaw conditions survive freezing to
-80.degree. C. and below in vitro, but that moderate freezing and
thawing to about -20.degree. C. leads to vascular stasis and
histological necrosis by ischemia as assessed at day 3 after the
freeze in both the cancer and normal rat skin in vivo.
[0081] The accentuation of the vascular mechanism of injury has
been approached by focusing on inflammation. The role of the
endothelium suggested the possibility of creating a pre-existing
non-destructive inflammation within the tissue prior to the freeze.
The cytokine TNF-alpha is known to upregulate NF-kB and various
adhesion molecules within endothelium and has also recently been
used in the DSFC (Fukumura D et al. 1995. Cancer Research 55:
4824-9). As discussed herein, local TNF-alpha delivery is an
effective way to achieve pre-inflammation prior to a thermal
surgical procedure.
EXAMPLES
[0082] The following are examples are provided to illustrate the
present invention and are not intended to limit the present
invention thereto in any manner
Example 1
[0083] The dorsal skin flap chamber (DSFC) of male athymic nude
mice was seeded with the tumor LNCaP Pro 5 human prostate cancer,
inflamed with TNF-alpha, subjected to cryosurgery, and assessed
according to the following procedure.
[0084] The dorsal skin of a male athymic nude mouse was sandwiched
between two identical anodized aluminum frames, a 19 millimeter
(mm) by 22 mm chamber was mounted onto the mouse by three screws,
the skin was attached to the chamber with 4-O silk using suture
holes, and the skin on the side of the viewing region was removed,
exposing the dermis containing the microvasculature on the opposite
side of the skin.
[0085] To provide chambers having tumors, approximately
5.times.10.sup.6 LNCaP Pro 5 human prostate tumor cells were mixed
with MATRIGEL matrix (BD Biosciences, Bedford, Mass.) as described
by Lim et al. (Prostate, 22:109-118 (1993)). Approximately 30 .mu.l
of the cell suspension was applied to the surface of the
microvascular bed immediately after the initial chamber
implantation and the tumor was allowed to grow for 10 days, the
skin reaching a total thickness of about 450 micrometers (.mu.m)
and the tumor extending approximately 10 mm in diameter.
[0086] A local application of 20 .mu.l of a 10 ng/ml TNF-alpha
(total application of 0.2 ng) was applied to the tumor tissue
within the DSFC for about 15 minutes, after which the TNF-alpha
solution was wicked off and the tissue was covered with a glass
window. After four (4) hours, the mouse was anesthetized,
TNF-alpha-induced inflammation was measured by observation of
leukocyte rolling, and thereafter cryosurgery was immediately
performed in the inflamed tissue.
[0087] The cryosurgery was performed with an argon-cooled, 5 mm
diameter cryoprobe (EndoCare, Irvine, Calif.) activated for a
cooling time of 5 minutes and a target temperature of -160.degree.
C., which corresponded to an average external probe end temperature
of about -125.degree. C. Type "T" thermocouples (Omega Tech. Corp,
Stamford, Conn.), having a 0.5 mm bead diameter, were inserted into
the tissue and were used to measure the average external probe end
temperature. The temperature at each thermocouple was recorded
using a HYDRA DATA LOGGER SERIES 2 (Fluke, Everett, Wash.).
[0088] After the cryosurgery the probe was turned off and the
tissue was allowed to thaw passively at room temperature.
[0089] The vasculature was imaged using a 70 kD fluorescein
isothiocyanate (HITC)-labeled dextran (Molecular Probes, Eugene
Oreg.). At 3 days post-treatment, 0.05 ml of a solution of
FITC-labeled dextran (10 mg dextran/mi PBS (Gibco BRL,
Gaithersburg, Md.)) was injected into the tail vein of the mouse.
The dorsal skin flap chamber was then illuminated with a mercury
lamp and a FITC signal-enhancing filter (.lamda.=470-490 nm) to
view the contrast fluorescence. A Silicon Intensified Transmission
camera (Hamamatsu, North Central Instruments, Twin Cities, Minn.)
was used to detect the fluorescent signal, and the signal was
recorded with a JVC S-VHS video recorder (JVC Company of America,
Aurora, Ill.).
[0090] Hstological analysis of the entire tissue was performed at
day 3 post-cryosurgery according to Hoffmann et al. (ASME J.
Biomechanical Engineering 123:310-316 (2001)), and images of the
histology were taken on an Olympus BX-50 upright microscope (Leeds
Precision Instruments, Minneapolis, Minn.). The end temperature of
the cryolesion and the thermal parameters within the cryosurgical
ice ball were calculated according to the methods of Chao et al.
(Cryobiology, 2004 (in press)).
[0091] The above example was repeated a total of 9 times to provide
data for TNF-alpha-treated tumor tissue, and was repeated a total
of 13 times without TNF-alpha treatment.
Results:
[0092] The area of vascular injury was observed with FITC-labeled
dextran. The results showed a substantially complete destruction of
the vasculature in the center of the lesion and an abrupt change to
normal patency moving radially outward. It was determined that
regions of vascular stasis lead directly to tissue necrosis.
Further, the edge of the static zone (i.e., the zone of vascular
stasis) at day 3 post cryosurgery in tissues inflamed with
TNF-alpha was at a radius greater than that for tissues that were
not inflamed with TNF-alpha (r=3.81.+-.0.29 mm in LNCaP Pro 5 tumor
tissues without TNF-alpha treatment and r=4.07.+-.0.34 mm in
tissues with TNF-alpha treatment). Additionally, the edge of the
static zone extended beyond the edge of the ice ball for LNCaP Pro
5 tumor tissues that were treated with TNF-alpha, whereas the edge
of the static zone stayed within the edge of the ice ball in
inflamed normal skin tissues that were treated with TNF-alpha
(Example 2, below).
[0093] The minimum temperature required for causing necrosis was
3.5.+-.6.9.degree. C. in TNF-alpha-treated LNCaP Pro 5 tumor
tissue. Compared to tissues without TNF-alpha treatment, where the
minimum temperature required for causing necrosis was
-16.5.+-.4.3.degree. C. in LNCaP Pro 5 tumor tissue, the results
indicate that the local use of TNF-alpha can increase the threshold
temperature of cryo-destruction by more than 10.degree. C.
Example 2
[0094] The procedure according to Example 1 was repeated, with the
exception that the biological material treated was normal nude
mouse hypodermis (i.e., normal skin).
[0095] Example 2 was repeated a total of 9 times to provide data
for TNF-alpha-treated normal tissue, and was repeated a total of 14
times without TNF-alpha treatment.
Results:
[0096] Similar, results were obtained in normal nude mouse
hypodermis without tumors. Inflammation induced by TNF-alpha moved
the edge of the static zone closer to the edge of the cryosurgical
ice ball (r=3.99.+-.0.13 mm in tissues treated with TNF-alpha, as
compared with r=3.13.+-.0.39 mm in tissues without TNF-alpha
treatment), and the minimum temperature required for
cryo-destruction was -9.8.+-.5.8.degree. C. in TNF-alpha-treated
normal skin as compared with -24.4.+-.7.0.degree. C. in untreated
normal skin.
[0097] Cell suspensions of human endothelial cells (MVECs) were
used to assess the enhancement of direct cellular injury (DCI) by
use of an adjuvant, wherein the adjuvant used is TNF-alpha.
Example 3
[0098] Measuring Enhancement of DCI in MVEC Endothelial, MCF-7
Breast Cancer and LNCaP Pro 5 Prostate Cancer cells by TNF-alpha
addition.
[0099] Human dermal microvascular endothelial cells (MVEC) were
prepared and grown as adherent monolayers as described by Gupta et
al., Experimental Cell Research, 230:244-251 (1997), maintained in
a 37.degree. C./5% CO.sub.2/95% humidified air environment in
T-flasks pre-coated with 1% gelatin in MCDB 131 medium supplemented
with 20% heat-inactivated human male serum, hydrocortisone, cAMP,
L-glutamine, heparin, endothelial cell growth supplement (Vec Tec,
Schenectady, N.Y.) and antibiotics. LNCaP cells were cultured as
adherent monolayers in DMEM/F12 medium supplemented with 10% FBS,
antibiotics and dihydrotesterone (DHT). MCF-7 cells were grown in
similar medium with the following exception: 5% (rather than 10%)
FBS, antibiotics and insulin (no DHT). Cells were subcultured by
rinsing with Hank's balanced salt solution (HBSS), followed by
light trypsinization, enzyme neutralization, and reseeding.
[0100] One to three days prior to TNF-alpha exposure and/or
freezing, cells were reseeded onto 96-well plates for
apoptosis/necrosis assay and onto T-flasks for EMSA or Western
blots, as monolayers or in Petri dishes as collagen gels (live-dead
assay). Each sample was then exposed to medium with TNF-alpha at
concentrations from 10 nanogram per milliliter (ng/ml) to 1
microgram per milliliter (.mu.g/ml) for 4 to 48 hours.
[0101] Control and experimental FIT groups were then assessed for
cell viability at varying time points starting 3 hours after
intervention. Cell viability was measured microscopically by a
fluorescent dye assay (cell viability assay) by an
apoptotic/necrotic assay.
[0102] The fluorescent dye assay was used to assess the plasma
membrane integrity of cells immediately before (control) and after
F/T using Hoechst 33342 and propidium iodide (PI). Each dye has
affinity to nucleic acid, i.e. all cells regardless of viability
take up Hoechst and only plasma membrane compromised cells take up
PI. Cells were incubated with 9 .mu.M Hoechst 33342 and 7 .mu.M PI
for 15 minutes at 37.degree. C., placed on a microslide,
cover-slipped and the percentage of dead cells/field determined at
200.times. using a fluorescent microscope (Olympus BX-50, Tokyo,
Japan).
[0103] The apoptosis/necrosis assay was used to map
thermal/adjuvant conditions that triggered apoptosis. The cells
were stained with fluorescent Annexin V.
[0104] The fluorescent dye assay was performed with at least 5
representative fields and a total of 100-200 cells/sample were
counted. All samples were measured in four or six replicates and
the resulting values were averaged.
Results:
[0105] TNF-alpha was shown to increase cryosensitivity of the MVEC,
MCF-7 and LNCaP Pro 5 cells in vitro. The action of the TNF-alpha
predominantly inflamed cells, and the inflamed cells exhibited
increased necrosis in vitro.
[0106] All references identified herein are incorporated by
reference in their entirety as if each were incorporated
separately. This invention has been described with reference to
illustrative embodiments and is not meant to be construed in a
limiting sense. Various modifications of the illustrative
embodiments, as well as additional embodiments of the invention,
will be apparent to persons skilled in the art upon reference to
this description.
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