Note: Descriptions are shown in the official language in which they were submitted.
WO93/~1889 PCT/US92/0370~ ~`
2 1 1 ~ O 1 6
TITLE
M~OD FOR HYPE~nE~MIC Po~IATION OF TISSUE
BACKGROUND OF THE INVENTION
Field of the Invention
The present inventi ~ relates to the use of
ultrasonic energy to heat bi~gical tissues and fluids, and
more specifically, to the use ~f hyperthermia
lO potentiators, such as gases, gaseous precursors and per-
fluorocarbons, in combination with ultrasound to facilitate ~.
the selective.heating of the tissues and fluids.
DescriPtion. of the Prior Art
The usefulness of heat to treat various
inflammatory and~arthritic conditions has long been known.
The use of ultrasound to generate such heat for these as well
as other therapeutic purposes, such as in, for example, the
treatment of tumors has, however, been a fairly recent ,:
development.
I
Where the. treatment of înflammation and arthritis
is ooncerned, the use of the ultrasound induced heat serves ~`
to increase blood flow to the affected regi~ns, resulting in
:~ various benefi:cial effects.~ Moreover, when ultrasonic energy
:: :
:
W093/21889 2 1 1 8 0 1 6 PCT/US92/03705
is delivered to a tumor, the temperature of the tumoroustissue rises, generally at a higher rate than in normal
tissue. As this temperature reaches above about 43C, the
tumorous cells begin to die and, if all goes well, the tumor
eventually disappears. Ultrasound induced heat trea~ment of
biological tissues and fluids is known in the art as
hyperthermic ultrasound.
The non-invasive nature of the hyperthermia
ultrasound technique is one of its benefits. Nonetheless, in
employing hyperthermic ultrasound, certain precautions must
be taken. Specifically, one must be careful to focus the
ultrasound energy on only the areas to be treated, in an
attempt to avoid heat-induced damage to the surrounding, non-
targeted, tissues. In the treatment of tumors, for example,
15 when temperatures exceeding about 43C are reached, damage to
the surrounding normal tissue is of particular concern. This
concern with over heating the non-target tissues thus places
limits on the use of hyperthermic ultrasound. Such
therapeutic treatments would clearly be more effective and
20 more widely employed if a way of targeting the desired
tissues and fluidc, and of maximizing;the heat generated in
those targeted tissues, could be devised.
The present invention is dlrected toward improving
the effectiveness and utility of hyperthermic ultrasound by
25 providing agents capable of promoting the selective heating
of targeted tissues and body fluids.
: ~ :
.: . ... ... .
W093/21889 21I80`1`6 PCr~US92/0370s
SUMMARY OF THE INVENTION
The present invention is directed to a method for
heat treating biological tissues and fluids which c~mprises
administering to the tissue or fluid to be treated a thera-
5 peutically effective amount of a hyperthermia potentiator,and then applying ultrasound to that tissue or fluid.
By using the potentiators of the present invention,
hyperthermic ultrasound becomes a better, more selective and
more effective therapeutic method for the treatment of
10 tumors, inflammation, and arthritis, as well as other various
conditions.
BRIF.F DESCRIPTION OF_.THE FIGURES
Figure 1. This figure provides a graph which
15 plots the temperature over time for thrPe different samples
: subjected to ultrasound treatment using a 1.0 megahertz
continuous wave source of ultrasonic energy. Both Sample 1
(multilamellar vesicles composed of egg phosphatidylcholine
and having encapsulated therein CO2 gas) and Sample 3 (a
20 phosphate buffered saline solution pressuri~ed with CO2 gas)
have a 5 imilar increase in temperature over time. Sample 2
(a degassed phosphate buffered saline solution) exhibited a
much lower increase in temperature over:time, as compared
with Samples 1 and 3.
Figure 2. This figure provides a graph which
plots the temperature over time for two different samples
subje~ted to ultrasound treatment using a 1.0 megahertz
continuous wave source of ultrasonic energy. Sample 2 (a
.
WO93/21889 PCT/US92/037~
2118016 -~
-- 4
phosphate buffered saline solution pressurized with COz gas)
shows a much greater increase in temperature over time than
Sample 1 ta degassed phosphate buffered saline solution~.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to a method for
heat treating biological tissues and fluids comprising
administering to the tissues or fluids to be treated a
therapeutically effective amount of a hyperthermia
potentiator, and then applying ultrasound to said tissue or
fluid.
As used hereln the phrase "hyperthermia
potentiator" denotes any biocompatible solid/ liquid or gas
capable of increasing the rate of ultrasound induced heating !~'
in biological tissues and fluids to which it is administered.
Preferably, the hyperthermia potentiator is selected from the
group consisting of gas, gaseous precursors and
perfluorocarbons.
Any and all biocompatible gases may~be employed as
hyperthermia potentiators in the subject method. Preferably,
20 however, the gas~employed is ai~r, carbon dioxide, oxygen,
nitrogen, xenon, a;rgoni neon or helium, or any and all
combinations thereof. Preferably the gas is in the form af
stabilized yas bubbles. The gas~ bubbles~may be stabilized~
by a number of different means well-known to those skilled in
25 the art. In the~most preferred embodiment, the gas employed
as ~he hyperthermia~potentiator is a1r and~the air is ~ `
provided~ n th~e form~of stabilized air bubbles.
WO93J~188~ PCT/US92/0370~
`: ~` 2118016 . :
-- 5
Gaseous precursors can also be employed as
hyperthermia potentiators in the present method. The yaseous
precursors may be of various types, and include te~perature
sensitive, pressure sensitive, photo sensitive, and pH
sensitive gaseous precursors whi~h are designed to form gas
either before or after administration to the biological
tissue or fluid being treated. Such gaseous precursors have
the advantage of being more stable on lonq-term storage than
in many cases the gases themselves, including the stabilized
10 gas bubbles.
The phrase "pH sensitive gaseous precursor", as
used herein, denotes a compound in solid or liquid form
which, when expc ~d to a change in pH, will form a gas. Such
compounds incluc., but are not limited to, metal carbonate
and bicarbonate salts, such as the alkali metal carbonates
and bicarbonates, and the alkaline earth carbonates and
bicarbonates, and mixtures thereof. Exemplary of such
compounds are lithium carbonate, sodium carbonate, potassium
carbonate, lithium bicarbonate, sodium bicarbonate, potassium
20 bicarbonate, magnesium carbonate, calcium carbonate,
magnesium bicarbonate, and the like. Also useful ~as
~enerating compounds are ammonium carbonate, ammonium
. b~icarbonate, ammonium sesquecarbonate, sodium
sesquecarbonate, and the like. These compounds, when
25 dissolved in water, show a pH of greater than about 7,
usually between about 8 and about 12. ~ther p~-activated
gaseous precursors include aminomalonate, which, when
dissoI~r~d~in water, generally shows a:pH of ahout 5 to ~.
W093/21~89 PCT/US92/0370~
2 1 1 8 ~ 1 6 6
The pkal of aminomalonate is 3.32 and the pka2 is 9.83.
Aminomalonate is well known in the art, and its preparation
is described, for example, in Thanassi, Biochemistrv~, Vol. 9,
no. 3, pp. 525-532 (1970), Fitzpatrick et al., Inorqanic
Chemistrv, Vol. 13, no. 3, pp. 568-574 (1974), Stelmashok et
al., Koordinatsionnaya Khimiya, Vol. 3, no. 4, pp. 524-527
(1977). Other suitable pH sensitive gaseous precursors will -
be apparent to those skllled in the art. -
As those skilled in the art would recognize, such ;
compounds can be activated prior to administration, if
desired. Of course, by choosing a gaseous precursor with the
appropriate pKa, one skilled in the art can prepare a
formulation that will form a gas after it has been
administered to the biological tissues or fluids. The pH
sensitive gaseous precursors, for example, may form gas at a
site with lower pH such as in a hypoxic, acidic tumor, or may
simply ~orm a gas upon exposure to physiological pH.
As used herein, the phrase "photo sensitive gaseous
precursor" denotes a light sensitive compound in solid or
20 liquid form which becomes a gas after exposure to such light.
5uitable photosensitive compounds include diazonium compounds
Which decompose to form nitrogen gas after exposure to
ul~traviolet light. Another suitable compound is j ! i
aminomalonate. As one skilled in the~art would recognize, ;
~5 other gaseous precursors may be chosen which form gas after i;i
eXposure to light. Depending upon the application,~exposure ~ ~ '~i
to such light ma~y be necessary prior to administra~ion, or in ,~
.
some instance~s can oocur subsequent to administration.
: . ~
WO93/~1889 PCT/US92/0370~
2 1 1 8 0 1 6
-- 7
As used herein, the phrase "temperature sensitiv~
gaseous precursor" denotes a solid or liquid compound which
forms a gas following a change in temperature. Sui~table
temperature sensitive gaseous precursors are well known to
those skilled in the art, and include, for example,
methylactate, a compound which is in a liquid phase at
ambient temperatures, but which forms a gas at physiological
temperatures. As those skilled in the art would recognize,
such compounds can be activated prior to administration or,
as in the case of methylactate, can be activated upon
administration at physiological temperatures or as a result
of the ultrasound induced hyperthermia.
Of all of the possible gaseous precursors, the most
preferred gaseous precursors for use with the present
invention are those selected from the group consisting of
aminomalonate, sodium bicarbonate, methylactate and diazonium
compounds, including any and all combinations thereof.
The hyperthermia potentiators employed in the
method of the subject invention may also comprise one or more
20 perfluorocarbons, preferably a perfluorocarbon compound
selected from the group consisting of perfluoro- octyliodide,
perfluorotributylamine, perfluorotripropyl- amine and
~erfluorooctlybromide, and any and all combinationsithereof.
Preferably the perfluorocarbons are administered in the form
of an emulsion. Such emulsions are particularly desira~le
when using perfluorocarbons for int~avascular injection to
avoid uptake by the pulmonary vasculature. For such uses,
t~e emulsion p-rt1cAes should be smaller than 5 microns in
WO93~21889 PCT/US92/0370~
211~016 ;~
-- 8
size to allow passage through the pulmonary microcirculation.
I`he art of preparing emulsions is well-known, and the subject
perfluorocarbon emulsions can be prepared in any co~ventional
fashion, such as by those procedures shown in U.S. Patent No.
4,~65,836 for the preparation o~ per~luorocarbon emulsions,
the disclosures of which are incorporated herein by reference
in their entirety.
If desired, the hyperthermia potentiators, such as
the gases, gaseous precursors and perfluorocarbons described
10 herein, may he encapsulated in liposomes prior to
administration, or may be otherwise stabilized. Stabilized
gas bubbles are particularly preferred. The phrase
stabilized gas bubbles, as used herein, refers to any
construct wherein the release of gas bubbles is prevented,
15 constrained or modulated.
Liposomes may be prepared using any one or a
combination of conventional liposome preparatory techniques.
As will be readily apparent to those skilled in the art, such
conventional techni~ues include sonication, chelate dialysis,
20 homogenization, solvent infusion ooupled with extrusion,
freeze-thaw extrusion, microemulsification, a~ well as
others. These techniques, as well a5~otheirs, are discussed,
for example,lin U.S. Patent No. 4,72~8,578, U.K. Paten~ ~
Application G.B. 2193095 A, U.S. Patent No. 4,728,575, U.S.
25 Patent No. 4,737,323, International Application
PCT/US85/01161, Mayer et al., B_ochimica et Biophysica Acta
Vol. 858, pp. 16~1-168 (1986), Hope et al., B?ochimica et
Biophysica Acta, Vol. 812, pp~ 55-65 (1985), U.S. Patent No.
W093l2l~89 PCT/US92/03705
` 2ll8ol6 .i ,
_ 9 _ ~
~,533,254, Mahew et al., Methods In EnzYmoloq~, Vol. 149, pp.
64-77 (1987), Mahew et al., Biochimica et Bio~hvsica Acta,
Vol. 75, pp. 169-174 ~1~84), and Cheng et al., Inve$tiqative
Radioloqy, Vol. 22, pp. 47-55 (1987)) and U.S. Serial No.
428,339, filed Oct. 27, 1989. The disclosures of each of the
foregoing patents, publications and patent applications are
incorporated by reference herein, in their entirety. As a
preferred technique, a solvent free system similar to that
descrlbed in International Application PCT/US85/01161, or
10 U.S. Serial No. 428,339, filed Oct. 27, 1989, is employed in
preparing the liposome constructions. By following these
procedures, one is able to prepare liposomes having
encapsulated therein a gaseous precursor or a solid or liquid
contrast enhancing agent.
The materials which may be utilized in preparing
the liposomes of the present invention include any of the
materials or combinations thereof known to those skilled in
the art`as suitable in liposome construction. The lipids
used may be of either natural or synthetic origin. Such
20 materials include, ~ut are not limited to, lipids such as
cholesterol, cholesterol hemisuccinate, phosphatidyl-
choline, phosphatidylethanolamine, phosphatidylserine,
phosphatidylglycerol, PhosPhatidic acid, phosphatidyl-
inositol, lysolipids, fatty ids, phingomyelin,
glycosphingo1ipids, glucoli~ ds, glycoliplds, sulphatides,
lipids with ether and ester-linked fatty acids, polymerizable
lipids, and combinations thereof. As one s~illed in the art
will recognize, the liposom~_ may be synthesized in the
,
W093/21~89 PCT/US92tO370~
211~016
absence or presence of incorporated glycolipid, complex
carbohydrate, protein or synthetic polymer, using
conventional procedures. The surface of a liposome~may also
be modified with a polymer, such as, for example, with
5 polyethylene glycol ~P~G), using procedures readily apparent
to those skilled in the art.
Any species of lipid may be used, with the sole
proviso that the lipid or combination of lipids and
assoclated materials incorporated within the lipid matrix
should form a bilayer phase under physiologically relevant
conditions. As one skilled in the art will recognize, the
composition of the liposomes may be altered to modulate the
biodistribution and clearance properties of the resulting
liposomes.
In addition, the size of the vesicles can be
adjusted by a variety of procedures including filtration,
sonication, homogenization and similar methods to modulate
liposomal biodistribution and clearance. To increase
internal aqueous trap volume, the vesicles can be subjected
20 to repeated cycles of freezing and thawing.
The liposomes empIoyed may be of varying sizes, but
preferably have a mean outer diameter between about 30
nanometers and about 10 microns. As is known to those
skilled in the art, vesicle size influences biodistribution
and, therefore, different size vesicles are selected for
various purposes. For intravascular use, for example,
vesicle si~e is generally no larger than abou~ 2 microns, and
generally no smaller than about 30~nanometers, in mean outer
WO93/21889 2 i 18 016 PCT/US92/03705
diameter. For non-vascular uses, larger vesicles, e.g.,
between about 2 and about 10 micron mean outside diameter may
be employed, if desired.
The lipids employed may be selected to optimize the
particular therapeutic use, minimiæe toxicity and maximize
shelf-life of the product. Neutral vesicles composed of
either saturated or unsaturated phosphatidyl~ choline, with
or without sterol, such as cholesterol, function quite well
as intravascular hyperthermia potentiators to entrap gas and
10 perfluorocarbons. To improve uptaXe by cells such as the
reticuloendothelial system tRES), a negatively charged lipid
such as phosph~tidylglycerol, phosphatidylserine or similar
materials is added. For even gre~ter vesicle stability, the
liposome can be polymerized using polymerizable lipids, or
15 the surface of the vesicle can be coated with polymers such
as polyethylene glycol so as to protect the surface of the
vesicle from serum proteins, or gangliosides such as GM1 can
be incorporated within the lipid matrix. Vesicles or `
micelles may also b prepared with attached receptors or
antibodies to facilitate their targeting to specific cell
types such as tumors.
The gas, gaseous precursors, perfluorocarbons, and
other hyperthermia potentiators can be encapsulatedlby the
liposome by being added to the medium in which the liposome
is being formed, in~accordance with conventional protocol.
,.
Where gases are concerned, the~procedures preferably employed
are those techniques f3r encapsulating gases within a
liposome described in applicant's copendlng application U.S.
WO93/~18~9 PCT/US9~/03705
2118016
- 12 -
Serial No. 569,828, filed on August 20, lsso, the disclosures
of which are hereby incorporated by reference in their
entirety herein.
It should be noted that where pH sensitive gaseous
5 precursors are encapsulat~d in liposomes, ionophores should
be incorporate~ into the liposome membrane so that the -
gaseous precursors can more efficiently produce gas when
exposed to a pH gradient. Indeed, it has been found that
although liposomes are not impermeable to protons or
10 hydroxide ions, the permeability coefficient of liposomes is
generally so very low that it often takes weeks or months to
dissipate a pH gradient. Providing a more rapid transport of
hydrogen ions or hydroxide ions across a liposome membrane in
order to activate pH-modulated gaseous precursors is
necessary. The incorporation of ionophores in the liposome
membrane, in accordance with the present invention, provides
the necessary means of transporting the activating ions. By
increasing the rate of hydrogen or hydroxide ion flux across
the liposome membrane, such ionophores will increase the rate
20 within the liposome of gas formation from the pH-activated -
gaseous precursor.
~ s used herein, the phrase "ionophore-containing
liposome" denotes a liposome havinglincorporated in the
membrane thereof an ionophore. The term "ionophorP", as used
25 herein, denotes compounds which are capable of facilitating
the transport of ions across the liposome membrane to effe t
a change in pH inside the liposome membrane, and include
WO93t21889 PCT/US92/03705 ii
`` 2118016
- l3 -
compounds commonly referred to as proton carriers and channel
formers.
Suitable ionophores include proton carrier~ such as
nitro-, halo- and oxygenated phenols and carbonylcyanide
5 p11enylhydraæones. Preferred of such proton carriers are
carbonylcyanide, p-trifluoromethoxyphenylhydrazone (FCCP),
carbonylcyanide M-chlorophenylhydrazone (CCCP),
carbonyl~yanide phenylhydrazine (CCP), tetrachloro-2-
trifluoromethyl benzimidazole (TTFB), 5,6 dichloro-2-
l0 trifluc-omethyl benzimidazole (DTFB), and Uncoupler 1799
Suitable channel formers include gramicidin, alamethicin,
filipin etruscomycin, nystatin, pimaricin, and amphotericin.
Other sultable proton carriers include the ~ollowing
compounds which preferably axhibit selectivity for cations,
but will also transport protons and/or hydroxide ions:
valinomycin, enniatin (type A, B or C), beauvericin,
: mo~omycin, nonactin, monactin, dinactin, trinactin,
tetranactin, antamanide, nigericin, monensin, salinomycin,
narisin, mutalomycin, carriomycin, dianemycin, septamycin, A-
204 A, X-206, X-537 A (lasalocid), A-23187 and dicyclohexyl
18-crown-6. Such ionophores are well known in the art and
are described, for example in Jain t al., Introduction to
. Bioloqical Membranes, (J. Wiley and Sons, N.~Y. 1980)1,
especially pp.:192-231, and Metal Ions In Bioloqical SYst ms,
ed. H. Sygel, Vol. l9, "Antibiotics And Their Complexes"
(De~ker, N.Y. 1985), disclosures of each of which are
incoFporated herein by reference in their entirety. The
WO93t21889 P~T/US92/0370~
o l 6 i ` .
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ionophores may be used alone or in combination with one --
another.
To incorporate ionophores into the liposom~
membrane, the ionophores, which are lipophilic, are simply
added to the lipid mixture, and the liposomes are prepared in
the usual fashion. They may also, if desired, be added after
the liposome has been formed, and will spontaneously
intercalate into the membrane.
Other methods of stabilizing the compounds of the -
invention, particularly the gases, are well known. For
example, a material may be formulated as a closed membrane-
bounded structure encompassing the enclosed gas bubble, ,-
examples of which incIude, but are not limited to polyme~ic
microcapsules prepared by a variety of methodologies such as
15 those disclosed in U.S. Patent No. 4,8~8,734, polymer
mixtures such as those described in U.S. Patent No.
4,466,442, and albumin microspheres such as those disclosed
in U.S. Patent No. 4,718,433, the disclosures of each of
which are incorporated herein by reference in their entirety. ~-
20 Such structures prevent or constrain the release of gas
because either the entrapped gas bubble cannot physically
pass through the intact membrane and/or the membranes have an
! i intrinsically low~permeabilit~ to the entrapped gasO
Materials may also be formulated as a macroreticulated porous
structures which serve to physically entrap the ~as bubble
within a highly cross-linked matrix. Examples of such
systems include, but are not limited to, cross-linked dextran
beads, sllica aerogels or cross-linked proteinaceous
. .
WOg3/21889 PCT/US92/03705
`` ~118016
- 15 - ~
structures. The nature o~ the cross-link may be physical,
i.e., non-covalent, as in the physical entwining of long
polymer fibers, or else may be chemical, i.e., cova~nt, as
in, for axample, the glutaraldehyde cross-linking of
synthetic polyaminoacid chains. Such macroreticulated
systems may be formulated as a hollow shell or as a filled
structure. Micelle structures of lipids may also be
employed. Finally, a material may be prepared for which the
gas has a naturally high affinity and is either absorbed onto
10 the surface or is soluble within the material of the
structure. An example of the former includes, but is not
limited to, carbon particles or low surface-tension
surfactant particles onto which many gases absorb. Examples
of the latter include an oil in water emulsion or coacervate,
or silicone particles in which a gas such as nitrogen may
preferentially dissolve. Such materials might preferably be
prepared under high pressure, or over a certain range of
temperature, in order to maximize th~ amount of gas either
absorbed to or dissolved within ~he material.
The hyperthermic potentiators of the present
invention are administered to a biological tissue or to
biological fluids, whereupon ultrasound is then applied to
; the biological matter. The methods of the inventionlare
partic:ularly useful when employed in relation to such
biological matter as tumor tissue, muscle tissue or blood
fluids.
Where the usage is in vivo, administration may be
carried out in various fashions, such as ntravascularly,
.
::
WO93/218~9 pCT/1J~92~03705
2118016 16 - `
intralymphatically, parenterally, subcutaneously,
intramuscularly, intraperikoneally, interstitially,
hyperbarically or intratumorly uising a variety of dosage
forms, the particular route of administration and the dosage
S used being dependent upon the type of therapeutic use sought,
and the particular potentiating agent employed. A gaseous
hyperthermic potentiator, for example, may be injected
directly into a tumor, with or without stabilization. To
deliver the air bubbles to the tumor site using an
intravascular administrative route, however, the air bubbles
are preferably stabilized to avoid uptake by the pulmonary
circulation. Where intraarterial injection of gas is used
for delivery to a tumor, the air bubbles need not be as
stable as in the case of peripheral intravascular injection. -~
lS Perfluorocarbons are preferably administered either
intravascularly or~interstitially. Typically, dosage is
initiated at lower levels and increased until the desired
temperature increase effect is achieved. In tumors with a
principal dominant arterial supply such as the kidney, these
20 hyperthermic potentiating agents may be administered intra-
arterially.
For in vivo uiage, the patient can be any type of
mammal, but most preferably is a human. Thei method of the
invention is particularly useful in the treatment of tumors,
25 Yarious in~lammatory conditions, and arthritis, especially in
the treatment of tumors. The stabilized bubbles, gaseous ~ ¦
precursQrs and perfluorocarbons accumulate in tumo~s,
particularly ln the brain, because of the leaky capillaries
W093~2l889 2 1 1 8 0 1 6 PCT/~JS92/037~ :
- 17 -
and delayed wash-out from-the diseased tissues. Similarly,
in other regions of the body where tumor vessels are leaky,
the hyperthermic potentiating agents will accumulat~.
The hyperthermic potentiators of the present
invention may be used alone, or in combination with one
another, such as in using perfluorocarbons in combination
wi~n gases. In addition, the potentiators of the invention
may be employed in combination with other therapeutic and/or
diagnostic agents. In tumor therapy applications, for
example, the hyperthermic potentiators may be administered in
combination with various chemotherapeutic a~ents.
Any of the various types of ultrasound imaglng
devices can be employed in the practice of the invention, the
particular type or model of ~he device not being critic~l to
15 the method of the invention. Preferably, however, devices ;
specially designed for administering ultrasonic hyperthermia
are preferred. Such devices are described U.S. Patent Nos.
4,620,S~6, ~i,658,828 and 4,586,512, the disclosures of each
of Which are hereby incorporated herein by reference in their
entirety.
Although applicant does not intend to be limited to
any particular theory of operation, the hyperthermic
p~tentiators employed in the methods of the present invention
are believed to possess their excellent results because of
2S the following scientific postulatesO
Ultrasonic energy may either be transmitted ~hrough
1,
a tissue, reflected or~absorbed. It is believed that the
potentiators of the invention Serve to increase the
: ,
:
WO93/21889 PCT/US92/0370~
21 1~ 01~ ;
- 18 -
absorption of sound energy within the biological tissues or
fluids, which results in increased heating, thereby
increasing the therapeutic effectiveness of ultrasonic
hyperthermia.
Absorption of sound is believed to be increased in
acoustic regions which have a high degree of ultrasonic
heterogeneity. Soft tissues and fluids with a higher degree
of heterogeneity will absorb sound at a higher rate than
tissues or liquids which are more homogeneous acoustically.
10 When so~nd encounters an interface which has a different
acoustic impedance than the surrounding medium, there is
believed to be both increased reflection of sound and
increased absorption of sound. The degree of absorption of
sound is believed to rise as the difference between the
acoustic impedances between the two tissues or structures
comprising the interface increases.
Intense sonic energy is also believed to cause
cavitation and, when cavitation occurs, this in ~urn is
thought to cause intense loc~l heating. Gas bubbles are
believed to lower the cavitation threshold, that is,
accelerate the process of cavitation during sonication.
Since gas bubbles and perfluorocarbons have high
acoustic impedance difPerences between liquid~s and soft
tissues, as well as decrease the cavitation threshold, the
gas bubbles and perfluorocarbons may act to increase the rate
oP absorption of ultrasonic energy and effect a conversion of
that energy into local heat. Additionally, the low thermal
conductivity of gas may serve to decrease local heat
WO93/~1889 PCT/VS92/~370~
2 1 1 8 0 1 6
- 19 -
dissipation, with the result that there is both an increase
in the rate of heating and an increase in the final
equilibrium temperature.
The potentiators of the present invention may serve
5 `to increase the acoustic heterogeneity and generate
cavitation nuclei in tumors and tissues thereby acting as a
potentiator of heating in ultrasonic hyperthermia. Because
the gases and perfluorocarbons create an acoustic impedance
mismatch between tlssues and adjacent fluids, the
perfluorocarbons and gas bubbles act similarly and increase
the absorption of sound and conversion of the energy into
heat.
The following examples~are merely illustrative of
the present invention and should not be considered as
limiting the scope of the invention in any way. These
examples and equivalents thereof will become more apparent to
those versed in the art in light of the present disclosure,
and the accompanying claims.
In all of the examples which~follow, a 1.0
20 megahertz oontinuous wave ultrasonic transducer tMedco Mark
IV Sonlator) was used to apply the ultrasonic energy.
Degassing of the solution, that is, removal of the gas from
~, the solution, wa~ accomplished by ~using standar~ va¢cum
procedures.
Exampl~es 1 through 7 are actual examples o~ ~he
invention. Examples~ 8 through 16 are prophetic examples
,.
meant to be illustrative of how the invention would operate -
~under the specified conditions.
: ' ; .
W~93/21889 2 1 1 8 ~ 1 ~ PCT/usg2/03705
~ . .
- 20 -
EXAMPLES
Example l
A cooled degassed solution of phosphate buf~ered
saline tPBS) was sub~ected to ultrasonic hyperthermia.
5 Another equal volume of standard PBS was pressurized in a
commercial soda syphon with carbon dioxide. The pressure was
released and the solution was then subjected to ultrasound
with identical parameters as for the previously describe~
solution of PBS. The gassed solution reached a significantly
10 higher temperature than the degassed solution. These results
are illustrated in Figure 1.
Example 2
Gas bubbles of nitrogen were passed through a
standard solution of PBS. A degassed solution of PBS was
15 prepared. Ultrasound energy was applied to each solution,
during which time the temperature was measured with a
thermometer. The solution containing gas bubbles (Sample 2)
reached a significantly higher temperature than the degassed
solution (Sample 1). The results in this example are shown
in Figure 2, and are qualitatively similar to those observed
in Example l.
In both Examples 1 and 2, it should be noted that
the ultrason,ic,hyperthermia was commenced immediately a~ter
gasing the solutions. When ultrasonic hyperthermia was
25 delayed more than five minutes after the gasing step, the
resultant temperature was only slightly greater than for the
deqassed PBS. This is attributed to the relatively rapid
W093/t1889 ~ PCT/US92/03705
118016
- 21 -
decay of the non-stabilized gas bu~bles in solution. Example
Liposomes encapsulating gas were prepared v1a a
pressurization process as previously described in applicant's
copending application, U.S. Serial No. 569,823, filed August
20, 1990. A liposome without gas was als- ~repared. The two
samples were exposed to ultrasonic energy as described above.
The results revealed improved heating for the liposomes that
encapsulated the gas similar to that shown in Figure 2. The
10 gas, whether or not entrapped in an outer stabilizing
covering such as a liposome, serves to potentiates the
heating.
The advantage of using liposomes or other such
stabilizing methods is that in vivo the stabilized bubbles
15 may perhaps he more readily directed to si.tes, e.g., tumors
than unencapsulated bubbles. Note that the nonencapsulated
bubbles as described in Examples 1 and 2 were only stable for
several minutes in solution, whereas the liposomal bubbles
will have a much longer stablilty.
Exa~le 4
Albumin microspheres were prepared as previously
described U.S. Patent No. 4,718,433 to encapsulate air. Two
sol~ ions of PBS were prepared, one containinq albumin
microspheres encapsulating gas and the~other containin~ a
solution of the same concentration of albumin in degassed
PBS. The concéntration of albumin in both cases was 1%.
r~,
Ultrasonic energy was then applied as~in Example 1. The
~ solution containing the gas filled albumin microspheres
: .
.
WO93/2188~ 2 1 1 8 D 1 ~ PCl/US92/0370~
- 22 -
reached a si~nificantly higher temperature than the solution
of albumin without gas. The temperature increase observed
for the gassed solution was similar to that observed~for the
samples containing gas described in Examples 1 through 3.
Example 5
Stabilized air bubbles were prepared as previously
described using a mixture of the polymers polyoxyethylene and
polyoxypropylene as in U.S. Patent No. 4,466,442 in solution.
Ultrasonic energy was applied. Again, the temperature
10 measurements showed a higher temperature for the solution
containing the stabilized air bubbles.
ExamPle 6
A solution containing emulsions of perfluoro-
octylbromide (PFOB~ was prepared as described in U.S. Patent
No. 4,865,836 ~Sample 1), and the solution was exposed to
ultrasonic hyperthermia. Additionally, a second solution of
PFOB emulsion was prepared following the same procedures,
except that this second solution was gassed with oxygen as
described in U.S. Patent No. 4,927,623 (Sample ~). Sample 2
20 was then exposed to ultrasonic hyperthermia. The Samples 1
and 2 containing the PFOB both achieved a higher temperature
upon ultrasound treatment than the degassed PBS of Examples 1
! and 2~ In a~ddition, Sample 2 reached an even higher
temperature with ultrasonic hyperthermia than Sample 1.
Example 7
A tissue equivalent ph~ntom was prepared using low
temperature agar gel with a 50C gelling temperature. A
phantom was prepared from degassed PBS and 4% agar gel.
WO93/21889 2 118 0 16 ` ~ PCT/US92/03705
- Z3 -
Another phantom was prepared, but in this case the liquid gel
was pressurized with nitrogen gas at 180 psi for 24 hours in
a custom built pressurization chamber at 52C. The,pressure
was released over a period of 5 seconds thus forming
5 microbubbles in the liquid yet ~iscous ael. Both gel samples
(degassed and that containing micro~ub~ s~ were allowed to
gel and to cool to 37C. The samples were then exposed to
ultrasonic energy as above and the temperatures recorded.
The sample containing microbubbles again had a much higher
ra'te of heating than the gel prepared from the degassed
solution.
The above was repeated but in this case liposomes
entrapping gas were placed in the~gel and the gel again
cooled to 37C. Ultrasonic heating again showed an improved
rate of heating. The purpose of the tissue e~uivalent
phantom was to demonstrate how the bubbles might potentiate
heating in tissues, e.g., a tumor.
Example_8
Two rats bearing C2 clonal derived epithelial
carcinoma are treated with ultrasonic therapy. In one of
these rats, 2 cc o~ nitrogen gas is injected into
approximately 4 cc of tumor volume. Hyperthermia is
,administered t,o both rats and the intra-tumoral temperature
monitored. The rat treated with an interstitial injection of
nitrogen has a higher tumor temperature.
; Example 9
One group of rabbits bearing VX2 carcinoma of the
brain are treated with ultrasonic hyperthermia while the
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- 24 -
tumor temperature and the temperature of the surrounding
tissue is monitored with a probe. A volume of 3 to 5 cc of
perfluorooctybromide emulsion is injected into a sec~nd group
of rabbits in the carotid artery ipsilateral to the brain
tumor, while m~nitoring the tumor and surrounding tissue.
The rabbits treated with the PFOB show increased tumor
temperatures and a more selective heating of the brain tumor
as compared to the normal tissue.
Example l0
The same experiment as in Example 9 is repeated
using a 3 cc injection of liposomes encapsulating gas. Again
temperature measurements of tumor and normal tissue show
increased temperature ln the tumor relative to normal tissue
of the animal treated with the gas filled liposomes.
Example ll
A solution of liposomes encapsulating the gaseous
precursor methylactate is prepared and suspended in PBS. A
control solution of PBS and the solution containing the
liposomes encapsulating methylactate is heated with
20 ultrasound and the temperature measured. The temperature of
the ~olution containing the liposomes encapsulating
methylactate ha5 a biexponential rate of heating re~lecting
, , the improvemen~ in heating efficienc~ past the point at wh;i`ch
gas is formed from the gaseous precursor.
~ .
Example l2
In a patient with cancer of the kidney, the left
femoral artery is catheterlzed using standard technique. The
renal artery is catheterized and l0 cc of a 1% solution of
: ,
WO~3/218~9 PCT/VS92/0370~ ~
``` 211~iO~6 ` ~
- 25 -
sonicated album_~ microspheres entrapping gas is injecte~
into the renal artery. Therapeutic ultrasound is used to
heat the tumor and the microbubbles of ~as delivered ~to the
tumor cause improved tumor heating.
Example 13
Example 12 is repeated in another patient but in
~his case gas bubbles encapsulated in the tensides
polyoxyethylene and polyoxypropylene are used to embolize the
kidney. Again therapeutic ultrasound is applied to the
lO kidney and the result is improved heating of the tumor.
Exam: ~ 14 -
Example 13 is repeated but this time using
liposomes encapsulating both chemotherapy an~ carbon dioxide
gas. Again hyperthermia is applied to the tumor using j,~
lS ultrasound and not only is there improved tumc_ heating, but
also improved tumor response caused by the interaction of
simultaneous heating and chemotherapy.
Example 15
Small liposomes, less than about 100 nm diameter,
20 are prepared to entrap nitrogen gas under pressure. Phase
sensitive lipids are selected wlth gel to liquid crystal-
line transition temperature of 42.5C. These are
administered intravenously to a patient with glioblastoma
multiforme, which is a usually deadly brain tumor. -~
25 Ultrasonic hyperthermia iis applied to the region of the brain
tumor through a skull flap~which has been previously made
surgically. The microbubbles entrapped in the liposomes
accumulate in the patient's tumor because of the leakiness of
WO~3~21889 PC~/VS9~/03705
2 1 1 8
- 26 -
the tumor vessels. The mlcrobubbles are excluded from the
normal brain because of the integrity of the blood-brain
barrier. The ultrasonic energy raises the tumor temperature
to 42.S degrees centigrade and the liposomes underwent phase
transition allowing the bubbles to expand. The intratumoral
bubbles increases the effectiveness of heating in the tumor
by the therapeutic ultrasound.
Example 16
Air bubbles are entrapped in lipid monolayers as
previously described in U.S. Patent No. 4,684,479. In a
patient with glioblastoma multiforme, these lipid monolayer
stabilized air bubbles are administered I.V. every day for 7
days during daily treatments with ultrasonic hyperthermia.
The stabilized air bubbles accumulate in the patient's tumor
and the patient has improved response to treatment with
ultrasonic hyperthermia.
Various modifications in addition to those shown
and described herein will be apparent to those skilled in the
art from the foregoing description. Such modifications are
also intended to fall within the scope of the appended
claims.
.. I ; I
