CA2118016A1 - Methods for providing localized therapeutic heat to biological tissues and fluids - Google Patents

Abstract

Gas, gaseous precursors and perfluorocarbons are presented as novel potentiators for ultrasonic hyper-thermia. The gas, gaseous precursors and perfluorocarbons which may be administered into the vasculature, interstitially or into any body cavity are designed to accumulate in cancerous and diseased tissues. When therapeutic ultrasonic energy is applied to the diseased region heating is increased because of the greater effectiveness of sound energy absorption caused by these agents.

Description

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 :: :
:

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.

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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 -~

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 . :

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.

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WO93/~1889 PCT/US92/0370~

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 ;~

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 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 ` .

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, .

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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~ :

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~ ;

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~

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.

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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 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~

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 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 W093/21889 2 1 1 8 0 1 ~ PCT/US92/03705 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 ` ~

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 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.

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Claims (12)

- 27 - What is claimed is:

1. A method for heat treating biological tissues and fluids which comprises:
(i) administering to the tissue or fluid to be treated a therapeutically effective amount of a hyper-thermia potentiator; and (ii) applying ultrasound to said tissue or fluid.

2. A method of Claim 1 wherein said hyper-thermia potentiator is selected from the group consisting of gas, gaseous precursors and perfluorocarbons.

3. A method of Claim 2 wherein said hyperthermia potentiator is gas.

4. A method of Claim 3 wherein said gas is selected from the group consisting of air, carbon dioxide, oxygen, nitrogen, xenon, argon, neon and helium.

5. A method of Claim 3 wherein said gas consists of stabilized gas bubbles.

6. A method of Claim 5 wherein said stabilized gas bubbles consist of stabilized air bubbles.

7. A method of Claim 2 wherein said hyper-thermia potentiator is a gaseous precursor.

8. A method of Claim 7 wherein said gaseous precursor is selected from the group consisting of amino-malonate, sodium bicarbonate, methylactate and diazonium compounds.

9. A method of Claim 2 wherein said hyper-thermia potentiator is a perfluorocarbon.

10. A method of Claim 9 wherein said perfluoro-carbon is selected from the group consisting of perfluoro-octyl iodide, perfluorotributylamine, trifluoropropylamine or perfluorooctlybromide.

11. A hypothermia potentiator for use in heat treating biological tissues and fluids using ultrasound.

12. The use of a hypothermia potentiator in the manufacture of a product for heat treating biological tissues and fluids using ultrasound.