US 20030082101A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082101 A1 Taylor et al. (43) Pub. Date: May 1, 2003

(54) ACCELERATORS FOR INCREASING THE Related U.S. Application Data RATE OF FORMATION OF FREE RADICALS AND REACTIVE OXYGEN SPECIES (60) Provisional application No. 60/296,761, filed on Jun. 11, 2001. (75) Inventors: Kevin Taylor, Mason, OH (US); Jody Mesaros, Mason, OH (US) Publication Classification Correspondence Address: (51) Int. Cl." ...... A61K 51/00; A61K 31/555; BROWDY AND NEIMARK, P.L.L.C. A61K 33/24 624 Ninth Street, N.W. (52) U.S. Cl...... 424/1.11; 514/185; 514/58; Washington, DC 20001 (US) 424/617; 604/20 (57) ABSTRACT (73) Assignee: Cavalier Discovery, Mason, OH The formation of free radicals is enhanced with photody namic agents, Sonodynamic agents, and Systems and thera (21) Appl. No.: 10/166,038 pies utilizing ultrasound by Subjecting the agent to light waves or Sound waves in the presence of a metal, a reduc (22) Filed: Jun. 11, 2002 tant, or a chelate, or mixtures thereof. Patent Application Publication May 1, 2003 US 2003/0082101 A1

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US 2003/0082101 A1 May 1, 2003

ACCELERATORS FOR INCREASING THE RATE 0006 Ultrasonic cavitation (the ultrasound-driven OF FORMATION OF FREE RADICALS AND growth of microbubbles from tiny gas pockets present in a REACTIVE OXYGEN SPECIES Solution, and their Subsequent Violent collapse which pro duces locally extreme temperatures and preSSures inside CROSS REFERENCE TO RELATED these collapsible bubbles) seems to be required for a sono APPLICATION dynamic effect. Although the mechanism of SonoSensitiza tion is not understood, it appears that reactive radical inter 0001. The present application claims priority from non mediates formed from these compounds by ultrasound, provisional Application Serial No. 60/296,761, filed Jun. 11, either as a result of direct pyrolysis in the hot cavitation 2001, the entire contents of which are hereby incorporated. bubbles or after reaction with the OH radicals and H atoms which are produced by Sonnolysis of water, are involved in FIELD OF THE INVENTION cell killing. Formation of peroxyl radicals from DMF and 0002 This invention relates to methods and compositions DMSO has been demonstrated in highly diluted air-saturated which can increase the effectiveness of therapies and pro Solutions of these compounds exposed to 50 kHz ultrasound. ceSSes which involve chemical reactions which produce radicals and reactive oxygen Species. SUMMARY OF THE INVENTION 0.003 Such therapies and processes include, but are not 0007. It is an object of the present invention to overcome limited to, Sonodynamic therapy, high intensity focused the aforementioned deficiencies in the prior art. ultrasound (HIFU) therapies, photodynamic therapy, radia tion therapy for cancer treatment, chemotherapy, waste 0008. It is another object of the present invention to water treatment, treatment of contaminated Soil with ultra achieve increased rates of free radical production from Sound, Sterilization with ultrasound, and polymerization aforementioned therapies and processes. reactions facilitated by ultrasound. Therapies and processes 0009. It is another object of the present invention to which utilize ultrasound are particularly well-Suited to this achieve increased rates of free radical production from invention. Sonodynamic and photodynamic therapy Systems. 0010. It is another object of the present invention to BACKGROUND OF THE INVENTION provide a new class of Sonodynamic therapy agents. 0004 Photodynamic therapy (PDT) involves the use of photoSensitizable compounds for Selective destruction of 0011. It is a further object of the present invention to biological tissue, Such as tumors, using a photosensitizable increase the rate of formation of cytotoxic species from drug which may be linked to a tumor-localizing agent Such existing Sonodynamic Systems and agents by combination as an antibody, followed by exposure of the target region to with the disclosed agents. light. PhotoSensitizable compounds are molecules that are 0012. It is a further object of the present invention to activated by light of a characteristic wavelength, usually provide improved methods for treating patients using Sono from a laser, ultimately resulting in the formation of cyto dynamic or photodynamic therapy. toxic intermediates Such as Singlet oxygen or free radicals. The photoSensitizable compound acts either at the cell 0013. It is yet another object of the present invention to Surface, or is internalized, ultimately destroying the mem achieve increased rates of free radical production from brane at the cell Surface or on cellular organella, respec Sonodynamic and photodynamic Systems. tively, leading to cell death. In cancer treatment the tumor 0014. It is still another object of the present invention to destruction is believed to proceed via one or both of the provide a method for combining Sonodynamic therapy and following two Suggested mechanisms: the intravascular photodynamic therapy to enhance the effects of both thera pathway, i.e., collapse of blood vessels with which hamper pies. blood perfusion to the tumor and deprive the tumor of oxygen and nutrients, and/or the parenchymal tumor path 0015 According to the present invention, the rate of ways in with which the tumor is destroyed by direct necrotic formation of free radicals from Sonodynamic and photody effects on the tumor cells. One of the severe problems with namic Systems can be increased by adding at least one photodynamic therapy is post-treatment Sensitivity to Sun activator which can be a transition metal, a reducing agent light, which required that the patients remain out of direct (reductant), or a transition metal chelator (chelant) to a light for Several weeks after photosensitizable compounds photodynamic or Sonodynamic agent prior to irradiating have been administered. with the appropriate exogenous energy. This method can be used to increase the formation of free radicals in chemical or 0005 Sonodynamic therapy (SDT) is relatively newer biological Systems, including in production of polymers, than photodynamic therapy, and is based upon the Synergis wastewater or Soil treatment, treatment of patients, etc. tic effect of drugs and ultrasound in producing cytotoxic effects on tissues, particularly on tumors. The cytotoxicity of 0016. The addition of an accelerator, which is at least one SDT can be enhanced by the presence of Sonosensitizable of a transition meal, a reductant, or a chelant, provides faster compounds, i.e., agents with which can emit Single oxygen free radical production as well as enhanced radical produc or free radicals in response to irradiation by ultrasound. tion via the addition of more chemical pathways which Some photoSensitive compounds, Such as porphyrin and generate radicals. porphryinyl analogs, have been found to be SonoSensitizable 0017. A transition metal chelating compound can be agents in cultures of tumor cells. A problem with Some added to the combination of metal and reductant to further Sonodynamic therapies is that the Sonodynamic agent is accelerate the production of toxic free radicals by lowering cytotoxic in the absence of ultrasound. the redox potential of the metal allowing the metal to react US 2003/0082101 A1 May 1, 2003 more easily. These chelating compounds also promote pro diseased tissues. The quinone containing species interacts duction of free radicals by maintaining iron in a Soluble with ultrasound to form Semiquinone radical, and the Semi form. quinone radical mobilizes transition metals Such as iron from biological Sources. Upon application of ultrasound, 0.018. The present invention is able to take advantage of iron interacts with hydrogen peroxide generated from the the Fenton and Fenton-type reactions, which involves the action of ultrasound on water and oxygen, resulting in the reaction of hydrogen peroxide with a transition metal to production of hydroxyl ion and hydroxyl ion radical. The produce hydroxyl radical and hydroxyl radical ion. Accord Semiquinone radical then Serves as a reductant which accel ing to the present invention, ultrasound can be used to erates the reaction of the metal by reducing it back to an accelerate the Fenton reaction in Vivo. active Species after it has reacted with the hydrogen perOX 0.019 Bicarbonate ion can be added to the compounds ide. claimed in the patent to further Stimulate radical production. Related reference: Stadtman, E. R. Fenton chemistry. The 0026 Quinone compounds can also accelerate radical Journal of Biological Chemistry. Vol 266 pp 17201-17211 productions by: (1991). The bicarbonate can be any bicarbonate salt that 0027) 1... chelating iron produces bicarbonate ion in the reaction medium, including alkali metal bicarbonates, ammonium bicarbonates, etc. 0028 2. generating Superoxide by redox cycling, 0020. The present invention is able to take further advan and tage of Fenton and Fenton-type reactions by adding an 0029. 3. releasing iron from biological sources. additional transition metal, adding a chelant, and/or adding a reductant. The chelant preferably reduces the reduction 0030) A new Sonodynamic drug is presented where a potential of the transition metal, and the reductant preferably chelant is added to the diseased tissues. Upon application of has a reduction potential which permits reduction of the ultrasound, iron from biological Sources is mobilized and transition metal or transition metal complex to a lower will interact with hydrogen peroxide generated from the oxidation number. Free radical production is also promoted action of ultrasound on water and oxygen, resulting in the by the chelating compounds, which maintain the iron in a production of hydroxyl ion and hydroxyl ion radical. The Soluble form. chelant, for example EDTA, accelerates the reaction of the metal by reducing its redox potential and allowing it to react 0021. The present invention takes advantage of the cor more easily with hydrogen peroxide, and/or by chelating the relation between known Fenton activity of a Substance and oxidized metal and maintaining it in a State that can be the ability to accelerate free radical production during expo reduced back to an active form of the metal, for example Sure to ultrasound. For example, a compound that exhibits oxalate. Additionally, the chelating compounds promote Fenton activity in an enzymatic System or a radiolytic production of free radicals by maintaining iron in a Soluble System is also able to accelerate radical production in an form. ultrasound System of the present invention. 0031 Compounds that stimulate the production of hydro 0022. The effectiveness of existing Sonodynamic drugs gen peroxide in the body can be used along with the process can be improved by taking advantage of the Fenton reaction of the present invention to enhance free radical production. by adding more transition metal, adding a chelant, and/or Examples of these Substances include but are not limited to adding a reductant to reduce the metal. 3-amino-1,2,4-triazole, 6-formylpterin, Sinuline, Systemin, 0023) A new Sonodynamic drug is presented where a methyl jasmonate, thrombin; Substance P, Sn-1,2-dio reductant Such as ascorbic acid is added to the diseased ctanoylglycerol, ionomycin; formylmethionyl-leucyl-phe tissues. Upon application of ultrasound, iron from biological nylalanine; interferon gamma; poly-L-histidine, and 6-hy Sources is mobilized and interacts with hydrogen peroxide droxydopamine. generated from the action of ultrasound on water and 0032 Macrophage/Neutrophil stimulators can be used oxygen, resulting in the production of hydroxyl ion and along with the ultrasound process of the present invention to hydroxyl ion radical. The reductant accelerates the reaction enhance production of free radicals. Examples of these of the metal by reducing it back to an active species after it Stimulators include but are not limited to polysaccharides has reacted with the hydrogen peroxide. Such as sizofiran, fucosamine and krebiozen; leucokinins 0024. A new Sonodynamic drug is presented where a Such as tuftsin, granulocyte-macrophage colony-stimulating quinone or a quinone containing Species is added to the factorS Such as RegramoStim, SargramoStim, Milodistim, diseased tissues. The quinone containing species interacts Molgramostin, TAN 1511, and TAN 1031A; phorbol esters with ultrasound to form Semiquinone radical, and the Semi Such as phorbol 12-myristate 13-acetate; cytokines Such as quinone radical acts as a transition metal reductant. Upon interferon, interleukin, and tumor necrosis factor; immuno application of ultrasound, iron from biological Sources is modulatorS Such as betafectin; and other compounds Such as mobilized and will interact with hydrogen peroxide gener DMPO, Formylated peptides, and opsonified Zymosan. ated from the action of ultrasound on water and oxygen, resulting in the production of hydroxyl ion and hydroxyl ion 0033 Compounds that deactivate catalase in vivo can be used along with the ultrasound therapy of the present radical. The reductant accelerates the reaction of the metal invention. Among the compounds that deactivate catalase in by reducing it back to an active Species after it has reacted Vivo are interleukin-1beta; cumene hydroperoxide; t-butyl with the hydrogen peroxide. hydroperoxide, hydrogen peroxide, toxohormone, and a 0.025 A new Sonodynamic drug is presented where a combination of copper, hydrogen peroxide and ophenan quinone or a quinone containing Species is added to the throline. US 2003/0082101 A1 May 1, 2003

0034. Other compounds that can be used in combination cinic acid (EDDS), iminodisuccinate (IDSA), methylgly with the ultrasound therapy of the present invention include cinediacetic acid (MGDA), glutamate, N,N-bis (carboxym compounds that alter cell membrane permeability So that the ethyl) (GLUDA), diethylenetetraaminepentaacetic acid cell is more Susceptible to lysis or rupture during ultrasound (DTMPA), ethylenediaminediacetic acid (EDDA), 1,2- treatment. These compounds also enhance free radical pro bis(3,5-dioxopiperazine-1-yl)propane (ICRF-187), and duction. N,N'-dicarbozamidomethyl-N,N'-dicarboxymethyl-1,2-di 0035) Other compounds that can be used in the present aminopropane (ICRF-198). This list is representative of invention to enhance free radical production are those with chelants based on the aminocarboxylate Structure and is not demonstrated prooxidant activity. Examples include but are all inclusive. not limited to hydrazine derivatives, diamide, t-butylhydro 0039 Chelants that have available a coordination site that peroxide, hydrogen peroxide, Oxygen, and prooxidant drugs is free or occupied by an easily displaceable ligand Such as Such as primacquine. Additionally, compounds traditionally water are preferred; however this is not a strict requirement considered to be antioxidants may behave as prooxidants for activity. under certain conditions and at certain concentrations. Examples of these compounds are gallic acid, cumene 0040. In general, a 0.5:1 to 10:1 ratio of chelant to metal hydroperoxide, endotoxins (e.g., LPS), baiclain, Vitamins is preferred (Graf, (1984); Thomas, (1993); Inoue (1987)). (K, D and E), melatonin, bilirubin, N-(4-hydroxyphenyl 0041. The following chelants increase free radical pro )retinamide, beta-hematin, flavone, chalcone, chalconarige duction when exposed to ultrasound and a metal: hydroxy nin, naringenin, bleomycin, platinum derivatives (e.g., cis carboxylate chelants and related compounds including platin), nitrogen and Sulfur mustards, primaquine, organic alpha and beta hydroxycarboxylic acids, alpha and manadione, a-tocopherol, B-carotene, Trolox C, estrogen, beta ketocarboxylic acids and Salts thereof, their derivative, androgens (e.g., 5-alhpa-DHT), 1,4-naphthoguinone-2-me isomers, metal coordination compounds, and polymers. We thyl-3-Sulfonate, ascorbic acid gallic acid, captopril, enala demonstrated this using citrate. The chelant should be pril, buthionine, Sulfoximine, N-ethylmaleimide, and diaz present in a 0.5:1 to 100:1 ratio of chelant to metal. More enedicarboxylic acid bis (N,N'-dimethylamide), heme and preferably a ratio of 0.5:1 to 30:1 (chelant:iron) should be its degradation products (bile pigments) and heme precur used. Addition of a reducing agent Such as ascorbic acid, SOS. 1,4-naphthoguinone derivatives, 1,4 benzoquinone deriva 0.036 Compounds that exhibit increased thiobarbituric tives, and 1,4-anthraquinone derivatives and/or thiols further acid reactive substances (TBARS) in the presence of a metal increaseS radical production. and hydrogen peroxide are known to promote radical pro 0.042 Chelants that have available a coordination site that duction, usually via a Fenton and Haber-Weiss reaction is free or occupied by an easily displaceable ligand Such as mechanism. These compounds are therefore Suitable candi water are preferred; however this is not a strict requirement dates for use in Sonodynamic and photodynamic therapy. for activity. More preferably, compounds that exhibit increased thiobar 0043. Examples of other compounds are tartaric acid, bituric acid reactive substances (TBARS) in the presence of glucoheptonic acid, glycolic acid, 2-hydroxyacetic acid; a metal, hydrogen peroxide, and a radical generating Source 2-hydroxypropanoic acid, 2-methyl 2-hydroxypropanoic Such as an enzymatic Source or a radiolytic Source are acid; 2-hydroxybutanoic acid; phenyl 2-hydroxyacetic acid; excellent candidates for use as Sonodynamic agents, since phenyl 2-methyl 2-hydroxyacetic acid; 3-phenyl 2-hydrox ultrasound can be Substituted as the radical generating ypropanoic acid; 2,3-dihydroxypropanoic acid; 2,3,4-trihy SOCC. droxybutanoic acid; 2,3,4,5-tetrahydroxypentanoic acid; 0037. The following chelants increase free radical pro 2,3,4,5,6-pentahydroxyhexanoic acid; 2-hydroxydode duction when exposed to ultrasound and a metal: aminocar canoic acid; 2,3,4,5,6,7-hexahydroxyheptanoic acid; diphe boxylates and their Salts, derivatives, isomers, polymers, and nyl 2-hydroxyacetic acid, 4-hydroxymandelic acid, 4-chlo iron coordination compounds. Addition of a reducing agent romandelic acid; 3-hydroxybutanoic acid; Such as ascorbic acid, 1,4-naphthoguinone derivatives, 1,4 4-hydroxybutanoic acid; 2-hydroxyhexanoic acid; 5-hy benzoquinone derivatives, and 1,4-anthraquinone deriva droxydodecanoic acid; 12-hydroxydodecanoic acid; 10-hy tives and/or thiols further increaseS radical production. This droxydecanoic acid; 16-hydroxyhexadecanoic acid; 2-hy was demonstrated using the following aminocarboxylate droxy-3-methylbutanoic acid; 2-hydroxy-4- chelants: methylpentanoic acid; 3-hydroxy-4-methoxymandelic acid; 4-hydroxy-3-methoxymandelic acid; 2-hydroxy-2-meth ylbutanoic acid; 3-(2-hydroxyphenyl) lactic acid; 3-(4-hy droxyphenyl) lactic acid; hexahydromandelic acid; 3-hy Ethylenediaminetetraacetic acid Ethylene glycol-bis(2-aminoethyl)-N,N,N',N'- droxy-3-methylpentanoic acid, 4-hydroxy decanoic acid; tetraacetic acid 5-hydroxy decanoic acid; aleuritic acid, 2-hydroxypro Diaminocyclohexane-N,N,N',N'-tetraacetic acid panedioic acid, 2-hydroxybutanedioic acid; erythraric acid; Nitriloacetic acid N-(2-Hydroxyethyl)ethylenediamine-N,N',N'- threaric acid, arabiraric acid; ribaric acid; Xylaric acid; triacetic acid ly Xaric acid, glucaric acid, galactaric acid; mannaric acid; Diethylenetriaminepentaacetic acid gularic acid; allaric acid; altraric acid; idaric acid; talaric Picolinic acid acid; 2-hydroxy-2-methylbutanedioic acid; citric acid; isoc itric acid, agaricic acid, quinic acid; glucuronic acid; glu curonolactone; galacturonic acid; galacturonolactone; 0.038 Examples of other aminocarboxylate chelants are uronic acids; uronolactones, dihydroascorbic acid, dihy diethylenediamine pentaacetic acid, ethylenediaminedisuc droxytartaric acid; tropic acid, ribonolactone, gluconolac US 2003/0082101 A1 May 1, 2003 tone; galactonolactone; gulonolactone; mannonolactone; naphthoguinone derivatives, 1,4 benzoquinone derivatives, ribonic acid; gluconic acid; citramalic acid; pyruvic acid; and 1,4-anthraquinone derivatives and/or thiols further hydroxypyruvic acid; hydroxypyruvic acid phosphate; increases radical production (Quinlan, (1998)). methylpyruvate, ethyl pyruvate; propyl pyruvate, isopropyl pyruvate; phenyl pyruvic acid; methyl phenyl pyruvate; 0049. The following compounds increase free radical ethyl phenyl pyruvate, propyl phenyl pyruvate, formyl for production when exposed to ultrasound and a metal: mic acid; methyl formyl formate; ethyl formyl formate; hydroxy-1,4-naphthoguinones, their derivatives, isomers, propyl formyl formate; benzoyl formic acid; methylbenzoyl metal coordination compounds, Salts, and polymers. These formate; ethyl benzoyl formate; propyl benzoyl formate; compounds can act as chelants and/or reducing agents. We 4-hydroxybenzoyl formic acid, 4-hydroxyphenyl pyruvic demonstrated their effectiveness using the following com acid; and 2-hydroxyphenyl pyruvic acid. This list is repre pounds: Sentative of chelants based on the hydroxycarboxylic acid and ketocarboxylic acid structure but is not all inclusive (Toyokuni, (1993)). 10 uM 5-hydroxy-1,4-naphthoquinone (juglone) 0044) The following chelants increase free radical pro 15 uM 2-hydroxy-3-(3-methyl-2-butenyl)- duction when exposed to ultrasound and a metal: adenosine 1,4-naphthoquinone (lapachol) diphosphate (ADP), adenosine triphosphate (ATP) and gua 71 uM 5-hydroxy-2-methyl-1,4- nosine triphosphate (GTP). In general, a 0.5:1 to 10:1 ratio naphthoquinone (plumbagin) of chelant to metal is preferred. Addition of a reducing agent 106 uM 5.8 dihydroxy -1,4-naphthoquinone Such as ascorbic acid, 1,4-naphthoguinone derivatives, 1,4 benzoquinone derivatives, and 1,4-anthraquinone deriva 0050 Addition of a chelant such as aminocarboxylates, tives and/or thiols further increases radical production. We hydroxycarboxylates, or biologically relevant chelants Such demonstrated this using ADP. as ADP, ATP, or GTP further increases radical production. 004.5 The following compounds increase free radical Addition of a reducing agent Such as ascorbic acid, 1,4 production when exposed to ultrasound and a metal: benzoquinone derivatives, and 1,4-anthraquinone deriva phosphonoformic acid, phosphonoacetic acid, and pyro tives and/or thiols further increaseS radical production. phosphate. In general, a 0.5:1 to 30:1 ratio of compound to 0051. Other examples of hydroxylated 1,4-naphtho metal is preferred. These compounds can act as chelants quinones include the following compounds and their deriva and/or reducing agents. We demonstrated the activity of tives: 1,4-naphthalenedione, 2,3-dihydroxy; 1,4-naphtha these compounds when phosphonoformic acid was added to lenedione, 2.5,8-trihydroxy; 1,4-naphthalenedione, an iron/EDTA System and radical production was increased. 2-hydroxy; 1,4-naphthalenedione, 2-hydroxy-3-(3-methyl 0.046 Addition of a chelant such as aminocarboxylates, butyl); 1,4-naphthalenedione, 2-hydroxy-3-methyl, 1,4- hydroxycarboxylates, or biologically relevant chelants Such naphthalenedione, 5,8-dihydroxy-2-methyl, alkannin; as ADP, ATP, or GTP further increases radical production. alkannin dimethylacrylate; aristolindiguinone, chleone A, Addition of a reducing agent Such as ascorbic acid, 1,4- droSerone, isodiospyrin, naphthazarin; tricroZarin A, acti naphthoguinone derivatives, 1,4 benzoquinone derivatives, norhodine, euclein, and atovaquone. This list is representa and 1,4-anthraquinone derivatives and/or thiols further tive of hydroxy-1,4-naphthoguinones and is not all inclu increases radical production (Lindqvist, (2001)). SVC. 0047 The following compounds increase free radical 0052 The following compounds increase free radical production when exposed to ultrasound and a metal: tetra production when exposed to ultrasound and a metal: cycline antibiotics and their derivatives, Salts, and polymers. hydroxylated 1,4-benzoquinones, their derivatives, isomers, These compounds can act as chelants and/or reducing metal coordination compounds, Salts, and polymers. These agents. We demonstrated the activity of these compounds compounds can act as chelants and/or reducing agents. We when tetracycline was added to iron and radical production demonstrated their effectiveness using the following com was increased. Examples include but are not limited to pound: methacycline, doxycycline, Oxytetracycline, demeclocyline, meclocycline, chlortetracycline, bromotetracycline, dauno mycin, dihydrodaunomycin, adriamycin, Steffimycin, Steffi mycin B, 10-dihydrosteffimycin, 10-dihydrosteffimycin B, Tetrahydroxy 1,4-benzoquinone 13213 RP, tetracycline ref. 7680, baumycin A2, baumycin A1, baumycin B1, baumycin B2, antibiotic MA 144S1, 0053 Addition of a chelant such as aminocarboxylates, rhodomycin antibiotic complex, musettamycin, antibiotic hydroxycarboxylates, or biologically relevant chelants Such MA 144L1, aclacinomycin B, antibiotic MA 144 Y, aclaci as ADP, ATP, or GTP further increases radical production. nomycin A, antibiotic MA 144G1, antibiotic MA 144M1, Addition of a reducing agent Such as ascorbic acid, 1,4- antibiotic MA 144N1, rhodirubin B, antibiotic MA 144U1, naphthoguinone derivatives, and 1,4-anthraquinone deriva antibiotic MA 144G2, rhodirubin A, antibiotic MA 144M2, marcellomycin, Serirubicin, oxytetracycline, demeclocy tives and/or thiols further increaseS radical production. cline and minocycline. 0054 Embelin, methylembelin, and rapanone are 0.048. Addition of a chelant such as aminocarboxylates, examples of other hydroxylated 1,4-benzoquinones. hydroxycarboxylates, or biologically relevant chelants Such 0055. The following compounds increase free radical as ADP, ATP, or GTP further increases radical production production when exposed to ultrasound and a metal: Addition of a reducing agent Such as ascorbic acid, 1,4- hydroxylated anthraquinones, their derivatives, isomers, US 2003/0082101 A1 May 1, 2003 metal coordination compounds, Salts, and polymers. These thiol drugs are preferred. Examples of biological thiols compounds can act as chelants and/or reducing agents. include, but are not limited to cysteinylglycine, cysteamine, 0056. Addition of a chelant such as aminocarboxylates, thioglycollate and glutathione. Other thiol containing drugs hydroxycarboxylates, or biologically relevant chelants Such include but are not limited to Captopril, Pyritinol (pyridox as ADP, ATP, or GTP further increases radical production. ine disulfide), Thiopronine, Piroxicam, Thiamazole, 5-Thi Addition of a reducing agent Such as ascorbic acid, 1,4- opyridoxine, Gold Sodium thiomalate, and bucillamine. In naphthoguinone derivatives, 1,4 benzoquinone derivatives, addition, drugs classified as penicillins, cephalosporins, and and/or thiols further increaseS radical production. piroXicam may undergo hydrolytic breakdown in Vivo to 0057 Examples of hydroxylated anthraquinones include form thiols; therefore, they are thiol prodrugs. Addition of a but are not limited to the following compounds and their chelant Such as aminocarboxylates, hydroxycarboxylates, or derivatives: alizarin, aloe-emodin, anthragallol, aurantio biologically relevant chelants such as ADP, ATP, or GTP obtusin, barbaloin, cascaroside A, cassiamin C, 7-chloro further increaseS radical production. Addition of a reducing emodin, chrysazin, chrySo-obtusin, chrysophanic acid 9-an agent Such as ascorbic acid, 1,4-naphthoguinone derivatives, throne, digiferrugineol, 1,4-dihydroxy-2- 1,4 benzoquinone derivatives, and/or 1,4-anthraquinone methylanthraquinone, frangulin A, frangulin B, lucidin, derivatives. morindone, norobtusifolin, obtusifolin, phyScion, 0063 A comprehensive list of thiol compounds include pseudopurpurin, purpurin, danthron, and rubiadin. Prodrugs 1-(mercaptomethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2- Such as diacerein that are converted to hydroxylated one; 1,2,3-benzotriazine-4(3H)-thione; 1,2-benzisothiazole anthraquinones in the body are also relevant (Kagedal, 3(2H)-thione-1,1-dioxide;1,2-dihydro-3H-1,2,4-triazole-3- (1999); Lee, (2001); Lee, et al. (2001); Gutteridge, (1986); thione, 1,2-dihydro-3H-1,2,4-triazole-3-thione and Muller, (1993)). derivatives, 1,2-dihydro-4,5-dimethyl-2H-imidazole-2- 0.058 Flavonoids such as kaempferol, quercetin, and thione; 1,3-dihydro-1-methyl-2H-imidazole-2-thione; 1,3- myricetin and Sesquiterpenes Such as gossypol and feralin dihydro-2H-naphth 2,3-dimidazole-2-thione; 1,3-dihydro are reducing agents and/or chelants that increase free radical 4,5-diphenyl-2H-imidazole-2-thione, 1,4-benzoxazepine production when exposed to ultrasound and a metal. Addi 5(4H)-thione; 1,4-dihydro-5H-tetrazole -5-thione and tion of a chelant Such as aminocarboxylates, hydroxycar derivatives; 1,5-dihydro-4H-pyrazolo 3,4-dpyrimidine-4- boxylates, or biologically relevant chelants Such as ADP, thione; 1,5-dihydro-6H-imidazo[4,5-cpyridazine-6-thione; ATP, or GTP further increases radical production. Other 1,7-dihydro-6H-purine-6-thione, 1-adamantanethiol; 2(1H)- examples of flavonoids include, but are not limited to benzimidazolinethione; 2,4-diamino-6-mercapto-1,3,5-tri acacetin, apigenin, biochanin-A, daidZein, equiol, flavanone, azine; 2,4-dimethylbenzenethiol; 2,5-dimethylbenzenethiol; flavone, formononetin, genistin, glabranin, liquiritigenin, 2,6-dimethylbenzenethiol, 2-adamantanethiol, 2-amino-1,7- luteolin, miroestrol, naringenin, naringin, phaseollin, phlo dihydro-6H-purine-6-thione; 2H-1,4-benzothiazine-3(4H)- retin, prunetin, robinin, and Sophoricoside. Derivatives, thione; 2-imidazolidinethione; 2-Isopropyl-3-methylben polymers, and glycosylated forms of these compounds are Zenethiol, 2-isopropyl-4-methylbenzenethiol, 2-isopropyl also relevant. B-dihydroxy and B-trihydroxy flavonoids are 5-methylbenzenethiol; 2-mercapto-4H-1-benzopyran-4- preferred (Canada, (1990); Laughton, (1989)). thione, 2-mercapto-5-methyl-1,3,4-thiadiazole, 2-mercapto 0059. The following compounds increase free radical 5-nitrobenzimidazole, 2-mercaptothiazoline, 2-methyl-1- production when exposed to ultrasound and a metal: anti propenethiol, 2-methylene-1,3-propanedithiol, 2-propene-1- tumor antibiotic quinoid agents Such as benzoquinones, thiol: 3,4-dihydro-4,4,6-trimethyl-1-(4-phenyl-2-thiazolyl)- mitomycin, Streptonigrins, actinomycins, anthracyclines, 2(1H)-pyrimidinethione; 3,4-dihydro-4,4,6-trimethyl and Substituted anthraquinones. These compounds can act as 2(1H)-pyrimidinethione; 3-amino-5-mercapto-1H-1,2,4- triazole, 3-bromo-1-adamantanethiol, 3-mercapto-5-methyl chelants and/or reducing agents. 1,2,4-triazole and derivatives; 3-mercaptocyclohexanone 0060 Addition of a chelant such as aminocarboxylates, and derivatives; 3-quinuclidinethiol, 3-thio-9,10-secoc hydroxycarboxylates, or biologically relevant chelants Such holesta-5,7,10(19)-triene, 4-amino-2,4-dihydro-5-phenyl as ADP, ATP, or GTP further increases radical production. 3H-1,2,4-triazole-3-thione; 4-amino-3-hydrazino-5-mer Addition of a reducing agent Such as ascorbic acid or thiols capto-1,2,4-triazole; 4-benzocyclobutenethiol; further increases radical production (Gutteridge, (1985); 4-biphenylthiol, 4-Isopropyl-2-methylbenzenethiol; 5,6- Gutteridge, et al. (1984); Morier-Teissier, et al. (1990)). dichloro-2-mercapto-1H-indole, 5'-amino-2',3,3,4-tetrahy dro-4,4,6-trimethyl-2,2'-dithioXo1(2H),4'-bipyrimidin 0061 The following compounds increase free radical 6'(1H)-one; 5-isopropyl-2-methylbenzenethiol; production when exposed to ultrasound and a metal: ascor 5-mercapto-3-methyl-1,2,4-thiadiazole, 6-amino-2-mercap bic acid, its derivatives, Salts and polymers act as ultrasound topurine, 6-thioinosine; 7-(mercaptomethyl)-1,7-dimethyl enhanced reducing agents and/or chelants. Addition of a bicyclo2.2.1]heptan-2-one; 7-mercapto-3H-1,2,3-triazolo chelant Such as aminocarboxylates, hydroxycarboxylates, or 4,5-dpyrimidine, Azothiopyrine; benzocthiophene biologically relevant chelants such as ADP, ATP, or GTP 1(3H)-thione; bis(1-methylethyl)carbamothioic acid S-(2,3, further increases radical production (Schneider, (1988); 3-trichloro-2-propenyl) ester; Caesium 2,6-bis(2,4,6- Dognin, (1975)). triisopropylphenyl)phenylthiolate; (3,3)-cholest-5-ene-3- 0062) Thiol compounds, their derivatives, and polymers thiol; Cyclohexanethione; Lithium 2,6-bis(2,4,6- increase free radical production when exposed to ultrasound triisopropylphenyl)phenylthiolate, naphtho1,2-dthiazole and a metal. We demonstrated their effectiveness using -2(1H)-thione; naphtho2,1-dithiazole-2(3H)-thione; phe cysteine as an example of a biological thiol and pennicil nylmethanethiol; Potassium 2,6-bis(2,4,6-triisopropylphe lamine as an example of a thiol drug. Biological thiols and nyl)phenylthiolate; Rubidium 2,6-bis(2,4,6-triisopropy US 2003/0082101 A1 May 1, 2003

lphenyl)phenylthiolate; Sodium 2,6-bis(2,4,6- erate free radicals and reactive oxygen species. The addition triisopropylphenyl)phenylthiolate (Diez, (2001)). of ascorbic acid or thiols or sulfate or hydroxylated 1,4- naphthoguinones (either Systemically or encapsulated) 0.064 Sodium sulfide and sodium sulfite are reducing enhances the production of free radical and reactive oxygen agents that increase free radical production when exposed to Species. ultrasound and a metal. We demonstrated this using Sodium Sulfite. Addition of a chelant Such as aminocarboxylates, 0072 Quinones are well suited reductants in this inven hydroxycarboxylates, or biologically relevant chelants Such tion, Since they are only active in their Semiquinone form as ADP, ATP, or GTP further increases radical production which can be generated by the application of ultrasound. The (Cassanelli, (2001)). By Screening compounds using the Source of the quinone compounds can be azo dyes, which are TBARS assay in combination with ultrasound exposure, one treated by ultrasound to form quinones. These azo dyes can skilled in the art can readily identify compounds that are be thought of as prodrugs for quinone compounds under the particularly active during ultrasound exposure. influence of ultrasound. 0065. More preferably, compounds that exhibit increased 0073 For purposes of the present invention, an “activa thiobarbituric acid reactive substances (TBARS) in the tor” means at least one of a transition metal Such as iron, a presence of a metal, hydrogen peroxide, and a radical reductant, or a chelant, in any combination. Thus, one could generating Source Such as an enzymatic Source or a radi use a transition metal, a reductant, or a chelant alone, or a olytic Source, are excellent compounds for use as Sonody transition metal plus a reductant or a chelant, or a combi namic agents activated by ultrasound. Thus, using the nation of a transition metal, a reductant, and a chelant. TBARS assay with ultrasound exposure as the radical gen erating Source, one skilled in the art can readily identify 0074 The present invention also provides a method for useful compounds. The TBARS assay can be used in aque preventing development or metastasis of cancer by deliver ous, lipid, and biological Systems. ing a combination of a Sonodynamic or photodynamic agent and at least one activator which is a transition metal, a 0.066 Other compounds that can be used in the present reductant, or a chelant to precancerous or cancerous cells to invention are those that exhibit iron release from biological affected tissueS or organs of an animal, and then exposing compounds containing iron, Such as ferritin, hemoglobin, those tissueS or organs to irradiation which results in transferrin, etc., in the presence of ultrasound. For example, destruction of the cells. For purposes of the present inven anthraquinones are known to release iron from ferritin tion, irradiation refers to delivering light or Sound waves, or during exposure to a free radical generating Source Such as alpha, beta, or gamma emmissions. This enhanced form of a radiolytic or enzymatic Source. By Screening quinone Sonodynamic therapy or photodynamic therapy can be used compounds using an assay for the release of iron from in combination with conventional therapeutic regiments ferritin with ultrasound exposure as the free radical gener including radiation therapy, hormonal therapy, or one or ating Source, it is possible to identify Suitable quinone more chemotherapeutic agents. Sonondynamic agents. 0075. In another embodiment of the present invention, 0067 Copper and iron are the best metals for enhancing diseases or conditions which can be treated by destroying the Fenton and Haber-Weiss activity in the body, and thus tissue, e.g., cardiovascular disease, are treated by adminis are the preferred metals for use in the present invention. tering to the Site a combination of a photodynamic agent Platinum and chromium are also preferred metals. and/or Sonodynamic agent with at least one activator which 0068 The compounds described above for use as Sono is a transition metal, a reductant, or a chelant and exposing dynamic agents can be modified to increase their Solubility. the tissue to irradiation. Glycolysed or cyclodextrin modified compounds are Some 0076. In another aspect of the present invention, infec examples. tious diseases are treated by administering a Sonodynamic and/or photodynamic compound along with an activator to 0069. In one embodiment, high levels of ascorbic acid are enhance the formation of free radicals to a patient Suffering administered to a diseased body, followed by administration of liposomally or polymerically encapsulated Fe(II). Ultra from an infectious disease in order to destroy the microor Sound is used to rupture the liposome or polymer capsule to ganisms causing the disease. release iron at the target tissue. AScorbic acid acts as the 0077. The present invention can be used to enhance the reductant. Alternatively, ascorbic acid can be encapsulated Sterilizing effect of irradiation Such as light, ultrasound, alone or as part of the iron capsule and administered along microwave, etc., to destroy unwanted microorganisms by with the iron. administering to the desired Site a combination of a photo dynamic agent and/or Sonodynamic agent with at least one 0070 Another embodiment is treatment with EDTA, activator which is a transition metal, a reductant, or a chelant either Systemically or encapsulated in a bead. The bead is and exposing the Site to irradiation to destroy the pathogens. ruptured at the treatment Site with ultrasound or other exogenous energy Sufficient to rupture the material of which 0078. The present invention can also be used to arrest the capsule is made. Treatment is guided with ultrasound bleeding by delivering a combination of a photodynamic imaging. agent or Sonodynamic agent with an activator to the Site of 0071. Ultrasound mobilizes iron either reductively from bleeding and exposing the Site to irradiation. biological Storage or by degradation of heme compounds. 0079 According to the present invention, at least one Alternatively, iron is added to the EDTA prior to treatment Sonodynamic and/or photodynamic agent and an activator or delivered Separately. The iron, regardless of its Source, are combined prior to treating a Site, Such as a patient, chelates with EDTA and remains soluble and able to gen Surface, or reaction medium, with light or Sound energy. For US 2003/0082101 A1 May 1, 2003

treating diseases and conditions, at least one Sonodynamic Substance Such as a heavy metal compound, an insoluble and/or photodynamic agent, in combination with at least one iodinated organic compound, or a vesicle enclosing an activator, are administered either together or Separately as an iodinated organic compound or a heavy metal compound. injection or infusion, or applied directly, to a site. The Site is 0084. For MRI, the reporter is preferably a paramagnetic, then Subjected to the appropriate irradiation, at with which Superparamagnetic, ferromagnetic, or ferrimagnetic material time free radicals are formed which are capable of destroy Such as a chelated transition metal or lanthamide ion (Such ing the tissue or pathogen intended to be destroyed. as Gd, Dy, Mn, or Fe), or a Superparamagnetic metal oxide particle. BRIEF DESCRIPTION OF THE DRAWINGS 0085 For ultrasound imaging, in which case the imaging 0080 FIG. 1 illustrates the use of ultrasound to convert and therapy may be effected by the same or Similar appa methyl orange, O- and p-methyl red, and azobenzene to a ratus, the reported is preferably a particular Substance bound quinone. to the rest of the photodynamic or Sonodynamic compound, Such as a vesicle (liposome, micelle, or microballoon) DETAILED DESCRIPTION OF THE enclosing an echogenic contrast agent Such as a gas or a gas INVENTION precursor (a material which is gaseous at 37 Celcius), or a mixture thereof. Particularly useful echogenic materials are 0081. According to the present invention, production of perfluoroalkanes Such as perfluoropentane and perfluorobu free radicals is enhanced using the combination of at least one Sonodynamic agent and/or at least one photodynamic tane. agent in combination with at least one activator. The acti 0086 For Scintigraphy, the reporter is generally a Vator is any combination of a transition metal, a reductant or covalently bound non-metal radionuclide Such as an iodine a chelant. This combination is then treated at the desired site, isotope. e.g., reaction medium, with the appropriate light and/or 0087. For light imaging, the reporter is a chromophore Sonic energy to generate free radicals. In another embodi i.e., a compound which absorbs light at 300-1300 nm, ment of the present invention, a human or animal body is preferably 600 to 1300 nm, and includes fluorophores and treated by Sonodynamic and/or photodynamic therapy phosphorescent materials, and/or light ScattererS Such as wherein a photodynamic and/or Sonodynamic agent plus at particulates with or without associated chromophores. least one activator is administered to Said body and the body Reporters for magnetotomography include materials useful is exposed to light rays and/or ultrasound to achieve a as magnetic resonance reporters, particular chelated lantha cytopathogenic effect at a site therein. It has been found that combining at least one activator with a photodynamic and/or mides or Superparamagnetic metal oxides. Sonodynamic agent results in greatly increased rate of pro 0088 A more detailed list of reporters that can be used in duction of free radicals, thus greatly enhancing the effects of the present invention is given in Alfheim et al., PCT appli the photodynamic or Sonodynamic therapy. cation WO 98/52609, the entire contents of which are hereby incorporated by reference. 0082 In another aspect of the present invention, the photodynamic or Sonodynamic compound contains a 0089 For electrical impedance tomography, the reporter reporter moiety which is detectable by an in Vivo diagnostic is preferably a polyelectrolyte. imaging modality, and optionally a vector moiety which 0090. Imaging may be affected in a conventional fashion modifies the biodistribution of the photodynamic or Sono and using conventional imaging apparatus for the Selected dynamic compound, e.g., by prolonging the blood residence imaging modality. The reporter-containing photodynamic or time of the compound or by actively targeting the compound Sonodynamic compound plus metal is administered in a to particular body Sites Such as disease sites or other pro contrast-enhancing dose, e.g., a dose conventional for the posed sites for PDT or SDT. According to this aspect of the Selected imaging procedure, or at lower than conventional invention, a human or animal body can be treated by dose where the agent is administered near the target Site for photodynamic or Sonodynamic therapy wherein a photody SDT or PDT or where it is actively targeted to the target site namic or Sonodynamic compound which includes a reporter by a vector moiety. moiety is administered to the body in conjunction with an activator, the body is exposed to light or ultrasound to 0091. The photodynamic or Sonodynamic compound achieve a cytopathogenic effect at a Site therein. In this way, may optionally include a vector moiety which modifies the an image of the body to which the photodynamic or Sono biodistribution of the compound. Example of Suitable vec dynamic compound is distributed makes it possible to locate tors include antibodies, antibody fragments, proteins and sites for treatment by light or ultrasound, or to follow the oligopeptides which have affinity for cell Surface receptors, progreSS of the therapy at a Site within the body. Any Suitable especially receptors associated with Surfaces of diseased or imaging methods may be used, including X-ray, MRI, rapidly proliferating cells, and peptidic and non-peptidic ultrasound, light imaging, Scintigraphy, in Vivo microscopy, drugs which are preferentially taken up by diseased or Such as confocal, photoacoustic imaging, and acousto-Opti rapidly proliferating cells. cal imaging and Visual observation and photographic imag ing, magnetotomography, positron emission tomography or 0092) Definitions electrical impedance tomography. 0093. Unless indicated otherwise, the following defini 0.083. The choice of reporter moiety used depends on the tions obtain for the present invention. All percentages are by choice of imaging modality. For X-ray imaging, the reporter weight unless otherwise indicated. is preferably a heavy atom (atomic number greater than 37), 0094 Ultrasound comprises sound waves that occur at a a chelated heavy metal ion or complex ion, or a particular frequency above the audible frequency of the human ear (16 US 2003/0082101 A1 May 1, 2003 kHz). Ultrasound is generally associated with frequencies of 0102 Ligands are classified by the number of coordina about 20 kHz to about 500 MHZ. tion Sites available: 0.095 Cavitation is the formation of vapor bubbles during the negative pressure cycle of ultrasound waves. The bubbles can collapse, resulting in localized high tempera 1 site = monodentate tures and preSSures. Free radicals, Such as the hydroxyl 2 sites = bidentate 3 sites = tridentate radical hydrogen radical, Singlet oxygen, and Solvated elec 4 sites = tetradentate trons are typically generated form bubble collapse in aque 6 sites = hexadentate ous media. 0.096 Medical imagining involves the use of electromag netic radiation to produce images of internal Structures of the 0.103 Monodentate ligands are Cl-, NH, CN-, and human body for purposes of accurate diagnosis. Four imag F-. Examples of bidentate ligands are 1,10-phenanthroline ing modalities are most commonly used in medical practice and ethylene diamine. for diagnosis and therapy: ultrasound, MRI, X-rays, and 0104 Chelates are complex ions that involve ligands with nuclear medicine. two or more bonding sites. 0097 Contrast agents are pharmaceutical agents that are used in many medical imaging examinations to aid in 0105 Chelants or chelating agents are ligands with two Visualizing tumors, blood vessels, and other Structures. For or more bonding sites. example, gas filled microSpheres are used as a contrast agent 0106 Diagnostic or therapeutic ultrasound elements can for ultrasound imaging. Paramagnetic compounds can be be based on any method for focusing ultrasound, including used as MRI contrast agents. geometric, annular, or phase array, and the probe can include 0.098 Irradiation for purposes of the present invention both therapeutic and imaging capabilities. Focused or direct refers to any type of irradiation which is biologically com ultrasound refers to the application of ultrasound energy to patible. This includes visible light, infrared light, ultraviolet a particular region of the body, Such that the energy is light, ultrasound, microwaves, radio waves, laser light, concentrated to a Selected area or target Zone. Devices that magnetic files, or X-rayS. Irradiation can be applied Singly as are designed for administering ultrasound hyperthermia are a continuous wave or can be pulsed. Each type of irradiation also Suitable, as are ultrasound devices used in Surgery, Such can be applied in combination and/or Sequentially with one as high intensity focused ultrasound devices. or more additional types of irradiation. 0107 Transition metals which are preferred for use in the 0099 Photodynamic therapy involves the combined use present invention are those that can produce and/or react of photosensitizable compounds plus an appropriate light with molecular oxygen or molecular oxygen derived reac Source to generate a cytotoxic effect. In the present inven tive Species, Such as hydrogen peroxide and Superoxide. tion, a metal is present to enhance this effect. The photo This interaction is preferably via a Fenton and/or Haber Sensitizable compound is capable of absorbing or interacting Weiss mechanisms, or mechanisms related to the Fenton and with at least one specific wavelength of light. This wave Haber-Weiss reactions, Such as radical-driven Fenton reac length defines the type of irradiation used in photodynamic tions. Iron, copper, manganese, molybdenum, cobalt, Vana therapy. Generally, a visible wavelength of light provided by dium, chromium, nickel and Zinc are of particular pharma laser is used. cological importance. The (I), (II), (III), (IV), and/or (V) oxidation States or higher, and combinations thereof, 0100 Sonodynamic therapy involves the combined use depending upon the choice of metal(s), may be used. Water of SonoSensitizable compounds plus an appropriate ultra soluble or lipid-soluble forms of the transition metals can be Sound Source to generate a cytotoxic effect. The SonoSensi used. The metal can be administered in the form of free tizable compound is capable of absorbing or interacting with metal, or chelated or bound entities. The chelators may be the ultrasound irradiation. For purposes of the present inven free molecular entities or prosthetic groups in larger mol tion, the SonoSensitizable compound is used in combination ecules (e.g., porphyrin in hemoproteins). with a metal. Ultrasound within a frequency range of about 1 kHz to about 100 MHZ is generally used, with intensities 0108) Ferritin is a preferred vehicle for iron delivery in of about 0.1 W/cm to about 10,000 W/cm'. High intensity vivo. This protein contains up to 4500 atoms of Fe(III) focused ultrasound (HIFU) can deliver intensities of up to which can be released as Fe(II) by the application of 10,000 W/cm, with values typically in the range of 500-2, ultrasound. Furthermore, ferritin can be modified to include 000 W/cm. Ultrasound irradiation is generally applied from Surface moieties which enhance the release of iron or Fenton about 0.5 sec to about five hours, depending on the fre reactions. For example, reducing agents which are only quency, intensity, material treated, etc., as is well appreci active upon exposure to ultrasound will both aid the release ated by one skilled in the art. The ultrasound can be pulsed, of iron from ferritin but will also engage in radical driven Second harmonic, or continuous wave. Custom built Systems Fenton reactions. Non-enzymatically loaded ferritin may be can be used, or commercial diagnostic or therapeutic devices used, which has shown a greater ability to release iron. can be used in practicing the present invention. The particu While ferritin is the preferred biological source of iron, other lar type of apparatus used is not critical. biological Sources of iron can be used in the present inven 0101 Ligands are negatively charged chemicals that tion. combine with a positively charged metal. Monoatomic 0109) Other biological sources of iron or other metals examples are F-, Cl-, etc. Polyatomic examples are NH, Such as transferrin, lactoferrin, conalbumin, ovotransferrin, CNS-, HOH, etc. cytochrome C, heme compounds, myoglobin, porphyrin and US 2003/0082101 A1 May 1, 2003 porphyrin containing macromolecules, and metal containing (p-benzoquinone)bis(triphenylphosphine)palladium; 1,2- co-factors can be utilized. Synthetic versions, modifications naphthalenedione and amino, bromo, butyl, chloro, ethyl, or complexes of these compounds are also Suitable. ethynyl, fluoro, hydro, hydroxy, iodo, isopropyl, mercapto, 0110 Particulate forms of transition metals or combina methyl, methoxy, nitro, phenyl, phenylthio derivatives, 1,2- tions of metals in particulate form can be used. phenanthrenedione and hydroxy, derivatives, 1,4-an thracenedione and derivatives; 1,4-bis(2-(diethylamino)et 0111. The metal chelator can be chosen to enhance the hoxyanthraquinone; 1,4-naphthalenedione and amino, Fenton chemistry by maintaining the transition metal in a redox-active form and/or by lowering the redox potential of bromo, butyl, chloro, ethyl, ethynyl, fluoro, hydro, hydroxy, the metal. This enables the transition metal to act as a iodo, isopropyl, mercapto, methyl, methoxy, nitro, phenyl, prooxidant. A classic example is EDTA, which chelates iron phenylthio derivatives, 1,4-phenanthrenedione and deriva and lowers the redox potential of Fe(III)/Fe(II) by 0.65V. tives; 1,8-diphenyl-1,7-octadiyne-3,6-dione; 11,12,13 This greatly favors the reaction of iron with hydrogen Trinor-4-amorphene-3,8-dione, 2-(3-methyl-2-butenyl)-1,4- peroxide to form the toxic hydroxyl radical Species. Other benzenediol;2-(beta-D-glucopyranosyloxy)-1-hydroxy-9, Such chelators typically used with iron include nitrilotriace 10-anthracenedione; 2,5-cyclohexadiene-1,4-dione and tic acid (NTA), penicillamine (PCM), and triethylene tetra amine, bromo, carboxyl, chloro, ethoxy, ethyl, fluoro, mine (TTM). Additional chelants can also be used, including hydroxyl, methoxy, methyl, nitorSo, and phenyl derivatives, hydroxyethyleniminodiacetate (HEIDA), gallate (GAL), 2,5-dichloro-3,6-bis(p-nitroanilino)-p-benzoquinone; 2,6- heXaketocyclohexane, tetrahydroxy-1,4-quinone, gallic dimethylbenzoquinone; 2-demethylmultiorthoquinone; acid, rhodizonic acid, dipicolinic acid, alizarin, ascorbic 2-ethoxy -2a,3,4,5,5a,6,10b,10c-octahydro-5-hydroxy-8- acid, and picolinic acids. Other examples are given in U.S. methoxy-5a-methyl -2H-anthra 9.1-bcfuran-7,10-dione; Pat. Nos. 6,160,194 and 5,741,427, the entire contents of 2-Geranylemodin 005; 2-Hydroxygarveatin B; 2-methoxy which are hereby incorporated by reference. Flavonoids can 5-(1-phenyl-1H-tetrazol-5-yl)thio-p-benzoquinone, 2-me also be used as metal ion chelators which reduce the redox thylconospermone, 2-tetradecyl-1,4-benzenediol, 3,4-dihy dro-6(2H)-quinolinone; 3,4-phenanthrenedione and potential of metal ions. derivatives; 3,5-cyclohexadiene-1,2-dione; 3-(6-deoxy-al 0112 The choice of reductant can be guided by its redox pha-L-mannopyranosyl)oxyl-1,8-dihydroxy-6-methyl-9,10 potential, Such that the reduction of the transition metal back Anthracenedione; 3-tert-butyl-5,8-dimethyl-1,10-an to the active form after it has participated in the radical thraquinone; 4,5-dichloro-3,6-dioxo-1,4-cyclohexadiene-1, producing reaction is thermodynamically favorable. For 2-dicarbonitrile, 4.5-phenanthrenedione and derivatives, example, ascorbic acid has a Standard reduction potential of 5,10-dihydro-5,10-dioxo-naphtho2,3-b-1,4-dithiin-2,3-di -0.127V, and is therefore able to reduce Fe(III) to Fe(II), carbonitrile, 5,12-naphthacenedione, 5,6-dihydroxy-naph where the Fe(III)/Fe(II) standard reduction potential is tho2,3-fcuinoline-7,12-dione; 5-Methylaltersolanol A;6, 0.77V. The Fe(II) form is then able to react with species such 13-pentacenedione; 6, 15-dihydro-5,9,14, 18 as hydrogen peroxide, with the production of radical Species anthrazinetetrone; 6,6'-biembelin; 6-2-(4.9-dihydro-8- Such as hydroxyl radical ion. hydroxy-5,7-dimethoxy-4,9-dioxonaphtho2,3-blfuran-2- 0113 Reducing agents are often metal chelators. For yl-1H-2-benzopyran-3-carboxylic acid;7-beta-D- example, oxalate can chelate iron and reduce it from Fe(III) glucopyranosyl-9,10-dihydro-3,5,6,8-tetrahydroxy-1- to Fe(II). methyl-9,10-dioxo-2-Anthracenecarboxylic acid; 9,10 anthracenedione and amine, azido, benzoyl, bromo, chloro, 0114. In preferred modes utilizing ultrasound, such as ethyl, ethenyl, fluoro, hydroxyl, methoxy, methyl, nitroSo, Sonodynamic therapy, the preferred reducing agent is a and phenyl derivatives, 9,10-phenanthrenedione and amino, Species which is activated by ultrasound. Such species bromo, chloro, fluoro, hydroxy, methyl, and nitro deriva readily becomes a radical upon exposure to ultrasound, and tives, Acequinocyl, Aclacinomycin A; Actinorhodine, exhibits no cytotoxic behavior in the absence of ultrasound. Alizarin Cyanin Green F: Alkannin; AloeSaponol I, Aloetic Compounds containing a quinone Structure are preferred acid, AlterSolanol G, Ametantrone; Aminoanthraquinones compounds, and the most preferred quinones are hydroxy and carboxylic acid derivatives, Anthraflavone; Anthrimide; lated 1,4-naphthoguinones, which are activated by ultra Antibiotic BE 69785A; Antibiotic JTNC; Antibiotic Q Sound and remain inactive without ultrasound. Upon acti 6916Z; ASterriquinone, Atovaquone, Aurantiogliocladin; Vation by ultrasound they form a semiquinone radical which Austrocortilutein, Austrocortirubin; AVerantin; Averythrin; can then reduce metals. 1-4 benzoquinone and 1-2 benzo AZanzone A, BenZaanthracene-7,12-dione; Benzo quinone, which are also preferred quinones, are the Simplest quinoniom Cl; Betulachrysoquinone; Bis-(4-amino-1-an quinones which can be used. Higher molecular weight thraquinonyl)amine; Bis(phenanthrenequinone)bis(py compounds which contain 1-4 benzoquinone or 1-2 benzo ridine) nickel; Bostrycin; Bostrycoidin; Buparvaquone; C.I. quinone moieties can be used. Such structures include Vat Yellow; C.I. Violet 43; Canaliculatin; Carboquone; napthoguinones, anthraquinones, and mitomycins. Carubicin; Cassumunacquinone 1; Conospermone; Cord Examples include, but are not limited to, acamelin, alizarin, eauxione; Cordiachrome A, B, and C, Cycloleucomelone; alkannin, arisianone, arStolindicquinone, barbaloin, cas Daunorubicin; Decylplastoquinone; Decylubiquinone; Der Siamin, cypripedin, 2,6-dimethoxybenzoquinone, dioSpyrin, moquinone; Diacerein; Diaziquone; Dibenza,hanthracene embelin, echinone, lapachone, juglone, isodiospyrin, hyperi 7,14-dione; Didyronic acid; Dihydodioxoanthracene cin, lawsone, primin, ubiquinones, rapanone, ramentaceone, Sulfonic acid derivatives, Sennoside, Vitamin K, coenzyme Q, and anthracycline anti Dihydrodioxoanthracenedicarboxylic acid and derivatives, biotics. Doxorubicin; Echinochrome A, Epirubicin; Frangulin A and 0115 Additional examples of quinones, both hydroxy B; Fredericamycin A; Frenolicin; Fusarubin; Geldanamycin; lated and non-hydroxylated, include, but are not limited to, Gossyrubilone; Granaticin; Granatomycin D; Herbimycin US 2003/0082101 A1 May 1, 2003

A, Idarubicin; Ilimaquinone; ISOcordeauxione; ISOfusarubin; present invention by using them in a Fenton reaction. These Javanicin; Juglomycin F, Kermesic acid; Laccaic acid A, B, chelators include phenolate derivatives, glycopeptides, and C, and D, Lagopodin A, Lapinone; Latinone; Leucoqui hydroxamic acid derivatives. nizarin; Mansonone A.C., and G. Menaquinone 4, 6, and 7; Menatetrenone; Menoctone; Menogaril; Miltirone; Mimo 0120 Catechols and other phenolic compounds are also cin; Mimosamycin; Mitomycin A, B, and C; Mitoxantrone; low molecular weight chelants that can be used in the Mollisin; Morindin; Murayaquinone; Murrapanine; present invention. Several of these compounds also lower Mycenone; Mycochrysone; N-(4-amino-3-methyl-1-an the redox potential of the metals with which they interact. It thraquinonyl)-benzamide; N-(4-amino-9,10-dihydro-3- is believed that wood rot fungu use hydroquinone-driven methoxy-9,10-dioxo-1-anthracenyl)-4-methyl-benzene Fenton reactions. For example, 4,5-dimethoxy-1,2-benzene Sulfonamide; N-(4-amino-9,10-dihydro-9,10-dioxo-1- diol and 2,5-dimethoxy-1,4-benezenediol have been isolated anthracenyl)-benzamide, N-(4-chloro-9,10-dihydro-9,10 from one Such fungus and these compounds are believed to dioxo-1-anthracenyl)-benzamide; N-(5-amino-9,10 chelate iron is a manner that facilitates free radical produc dihydro-9,10-dioxo-1-anthracenyl)-benzamide; N,N'-(9,10 tion by the Fenton reaction. Is was recently discovered that dihydro-9,10-dioxo-1,4-anthracenediyl)bis-benzamide; Fenton chemistry is involved in Wood rot mechanisms, So Naphthoherniarin; Naphthomevalin; Naphthyridinomycin other low molecular weight compounds that enhance the A; Nogalamycin; Noravanicin; Novarubin; Oncocalyxone Fenton reaction are likely to be isolated from wood rot in the A., Oosporein; Paeciloquinone A, Parvaquone; Perezone; future. These compounds are of interest because their activ Phenicin; Piloquinone; Pirarubicin; Pleurotin(e); Porfiromy ity will be enhanced when exposed to ultrasound due to the cin; Resistomycin; Rhacodione B; Rhodocomatulin; Rhodo availability of iron during ultrasound exposure as well as the mycin A and B; Rhodoquinone; Ruberytheric acid; Rubia ability of ultrasound to accelerate the Fenton reaction. nin; Seratrodast, Sodium B-naphthoguinone-4-Sulfonate; Quinolines can also be used to enhance the Fenton reaction Sodium alizarineSulfonate; Solaniol, Spiranthocquinone; via chelation. Streptonigrin; Sudan blue GA; Tabebuin; Tectoleafcuinone; 0121 Oxalate can be used in conjunction with Fenton Triaziquone; Triptone; Ubiquinone 30 and 50; Versiconol; therapies to increase the rate of production of hydroxyl Vitamin K, Xanthoviridicatin D; Zorubicin. radicals by preventing ferric iron from reacting with oxygen 0116 Bipyridyl herbicides, Such as paraquat and diquat, to form hydro(oxide) complexes. and compounds containing the bipyridyl Structure, are also 0.122 The chelant can be chosen to modify the hydro good candidates for ultrasound activated reductants. philicity of the metal compound Such that it has a longer residence time in the blood. These chelators are commonly 0117 Chemical compounds which undergo chemical used in MRI contrast agents, as disclosed in EP 187947 and transformation upon application of ultrasound to form WO 89/06979, the entire contents of which patents are quinone compounds can also be used. Such compounds can hereby incorporated by reference. Using these patents as be considered quinone pro-drugs. These include azobenzene guides, one skilled in the art can create Similar chelated and related azo-dyes, dinitrobenzene and compounds con metal compounds which react via Fenton-like mechanisms. taining a dinitrobeZene Structure, nitrophenol and com Binding the chelant to a macromolecule Such as a polysac pounds containing a nitrophenol Structure, phenol, com charide (e.g., dextran or derivatives thereof) to produce a pounds containing a phenolic structure, flavanols, catechol Soluble macromolecular chelant having a molecular weight and structures containing a catechol moiety. above the kidney threshold, about 40 kD, ensures relatively 0118. The reductant, if administered alone, can be an long term retention of the contrast agent within the Systemic activator Since biological Sources of metals, Such as iron, vasculature. Other examples can be found in U.S. Pat. Nos. exist in the body. Additionally, Several reductants are known 4,687,658; 4,687,659, and EP 299975 and EP 130934, the to mobilize iron from biological Stores, Such as ferritin, entire contents of which are hereby incorporated by refer therefore increasing the amount of metal present at the CCC. treatment Site. Iron is also released during ultrasound expo 0123 Vanadium metallocene complexes, such as Sure by cell lysis during mechanical Shearing from ultrasonic described in U.S. Pat. No. 6,051,603, can also produce cavitation and by degradation of heme compounds during reactive oxygen Species via Fenton-type reactions. This cavitation. The reductant can therefore Substantially increase patent is hereby incorporated in its entirety by reference. the formation of cytotoxic species. This increase can be further improved by the addition of metals, free bound or 0.124 Fullerene derivatives can be used as metal delivery chelated, to the body. vehicle when a chelating moiety is attached to the carbon Surface. These modified Fullerene compounds can carry up 0119 For in vivo use, low molecular weight chelators are to 30 or more metal atoms. The metal atoms can also be favored, since they allow easier diffusion of iron into cell incorporated inside of Fullerenes and Fullerene compounds. walls, where the hydroxyl radical will be generated in close proximity to the polyunsaturated fatty acids and lipids of the 0.125 Other metal chelators which can be used in the cell wall. The hydroxyl radical can therefore initiate and present invention include but are not limited to citrates, engage in the chain reactions which ultimately lead to gluconates, Succinctness, Sulfates, phosphates, tartrates, alu hydroperoxide formation. These chelators include classes of minates, Saccharide, lactates, oxalates, formats, fumigates, compounds recently isolated from Wood decay fungi, and glycerophosphates, chlorides, ammonium compounds, have been termed “redox cycling chelators' because of their nitrates, pentonates, Sugars, ADP, ATP, PDTA, thiosulfates role in the Fenton mediated degradation of wood by certain and thiosulfates, and polymer chelants Such as polyvinylpy fungi. One can readily determine without undue experimen rollidone and other polyamines. Of these compounds, ami tation, if a “wood rot compound is applicable for use in the nocarboxylates, hydroxcarboxylates, and the biological US 2003/0082101 A1 May 1, 2003

chelants ADP, ATP, and GTP are preferred because their acid; 2-hydroxybutanoic acid; phenyl 2-hydroxyacetic acid; involvement in radical production is greatly accelerated by phenyl 2-methyl 2-hydroxyacetic acid; 3-phenyl 2-hydrox ultrasound. ypropanoic acid; 2,3-dihydroxypropanoic acid; 2,3,4-trihy droxybutanoic acid; 2,3,4,5-tetrahydroxypentanoic acid; 0.126 Chelants that increase free radical production when 2,3,4,5,6-pentahydroxyhexanoic acid; 2-hydroxydode exposed to ultrasound and a metal include aminocarboxy canoic acid; 2,3,4,5,6,7-hexahydroxyheptanoic acid; diphe lates and their Salts, derivatives, isomers, polymers, and iron nyl 2-hydroxyacetic acid, 4-hydroxymandelic acid, 4-chlo coordination compounds. Addition of a reducing agent Such romandelic acid; 3-hydroxybutanoic acid; as ascorbic acid, 1,4-naphthoguinone derivatives, 1,4-ben 4-hydroxybutanoic acid; 2-hydroxyhexanoic acid; 5-hy Zoquinone derivatives and 1,4-anthraquinone derivatives droxydodecanoic acid; 12-hydroxydodecanoic acid; 10-hy and/or thiols further increases free radical production. This droxydecanoic acid; 16-hydroxyhexadecanoic acid; 2-hy was demonstrated using the following aminocarboxylate droxy-3-methylbutanoic acid; 2-hydroxy-4- chelants: methylpentanoic acid; 3-hydroxy-4-methoxymandelic acid; 0127 Ethylenediaminetetraacetic acid 4-hydroxy-3-methoxymandelic acid; 2-hydroxy-2-meth ylbutanoic acid; 3-(2-hydroxyphenyl) lactic acid; 3-(4-hy 0128. Ethylene glycol-bis-(2-aminoethyl)-N,N,N', droxyphenyl) lactic acid; hexahydromandelic acid; 3-hy N'-tetraacetic acid droxy -3-methylpentanoic acid, 4-hydroxy decanoic acid; 0129. Diaminocyclohexane-N,N,N',N'-tetraacetic 5-hydroxy decanoic acid; aleuritic acid, 2-hydroxypro acid panedioic acid, 2-hydroxybutanedioic acid; erythraric acid; threaric acid, arabiraric acid; ribaric acid; Xylaric acid; 0130 Nitriloacetic acid ly Xaric acid, glucaric acid, galactaric acid; mannaric acid; 0131) N-2-(hydroxyethyl)ethylenediamine-N,N',N'- gularic acid; allaric acid; altraric acid; idaric acid; talaric triacetic acid acid; 2-hydroxy-2-methylbutanedioic acid; citric acid; isoc itric acid, agaricic acid, quinic acid; glucuronic acid; glu 0132) Diethylenetriaminepentaacetic acid curonolactone; galacturonic acid; galacturonolactone; uronic acids; uronolactones, dihydroascorbic acid, dihy 0133 Picolinic acid droxytartaric acid; tropic acid, ribonolactone, gluconolac 0134 Examples of other aminocarboxylate chelants are tone, galactonolactone; gulonolactone; mannonolactone; diethylenediamine pentaacetic acid, ethylenediaminedisuc ribonic acid; gluconic acid; citramalic acid; pyruvic acid; cinic acid (EDDS), iminodisuccinate (IDSA), methylgly hydroxypyruvic acid; hydroxypyruvic acid phosphate; cinediacetic acid (MGDA), glutamate, N,N-bis-(carboxym methylpyruvate; ethyl pyruvate; propyl pyruvate; isopropyl ethyl) (GLUDA), diethylenetetraaminepentaacetic acid pyruvate; phenyl pyruvic acid; methyl phenyl pyruvate; (DTMPA), ethylenediaminediacetic acid (EDDA), 1,2-bis ethyl phenyl pyruvate, propyl phenyl pyruvate; formyl for (3,5-dioxopiperazine-1-yl)propane (ICRF-187), and N,N'- mic acid; methyl formyl formate; ethyl formyl formate; dicarboxamidomethyl-N,N'-dicarboxylmethyl-1,2-diamino propyl formyl formate; benzoyl formic acid; methylbenzoyl propane (ICR198). This is not an exclusive list of formate; ethyl benzoyl formate; propyl benzoyl formate; aminocarboxylate chelants, but is merely presented to illus 4-hydroxybenzoyl formic acid, 4-hydroxyphenyl pyruvic trate Some of the aminocarboxylate chelants that can be used acid; and 2-hydroxyphenyl pyruvic acid. This list is repre in the present invention. Sentative of chelants based on the hydroxycarboxylic acid and ketocarboxylic acid structure but is not all inclusive 0135 Chelants that have available a coordination site that is free or occupied by an easily displaceable ligand Such as (Toyokuni (1993)). water are preferred. However, this is not a strict requirement 0140. The following chelants increase free radical pro for activity. duction when exposed to ultrasound and a metal: adenosine diphosphate (ADP), adenosine triphosphate (ATP) and gua 0.136 While any ratio of chelant to metal can be used, nosine triphosphate (GTP). In general, a 0.5:1 to 10:1 ratio generally a ratio of about 0.5:1 to about 10:1 of chelant to of chelant to metal is preferred. Addition of a reducing agent metal is preferred. Such as ascorbic acid, 1,4-naphthoguinone derivatives, 1,4 0.137. A number of chelants have been found to increase benzoquinone derivatives, and 1,4-anthraquinone deriva free radical production when exposed to ultrasound and a tives and/or thiols further increases radical production. We metal: hydroxycarboxylate chelants and related compounds, demonstrated this using ADP. including organic alpha and beta hydroxycarboxylic acid, 0.141. The following compounds increase free radical alpha and beta ketocarboxylic acids and Salts thereof, their production when exposed to ultrasound and a metal: derivatives, isomers, metal coordination compounds, and phosphonoformic acid, phosphonoacetic acid, and pyro polymers. phosphate. In general, a 0.5:1 to 30:1 ratio of compound to 0138 While a preferred ratio of chelant to metal is about metal is preferred. These compounds can act as chelants 0.5:1 to about 100:1, a preferred ratio is about 0.5:1 to about and/or reducing agents. We demonstrated the activity of 30:1. Addition of a reducing agent Such as ascorbic acid, these compounds when phosphonoformic acid was added to 1,4-naphthoguinone derivatives, 1,4-benzoquinone deriva an iron/EDTA System and radical production was increased. tives, or 1,4-anthraquinone derivatives, and/or thiols used 0142. Addition of a chelant such as aminocarboxylates, increase radical productions. hydroxycarboxylates, or biologically relevant chelants Such 0139 Examples of other compounds are tartaric acid, as ADP, ATP, or GTP further increases radical production, glucoheptonic acid, glycolic acid, 2-hydroxyacetic acid; particularly when added to the compounds listed in para 2-hydroxypropanoic acid, 2-methyl 2-hydroxypropanoic graph 0107. Addition of a reducing agent Such as ascorbic US 2003/0082101 A1 May 1, 2003 acid, 1,4-naphthoquinone derivatives, 1,4 benzoquinone 2-hydroxy; 1,4-naphthalenedione, 2-hydroxy-3-(3-methyl derivatives, and 1,4-anthraquinone derivatives and/or thiols butyl); 1,4-naphthalenedione, 2-hydroxy-3-methyl, 1,4- further increaseS radical production particularly when added naphthalenedione, 5,8-dihydroxy-2-methyl, alkannin; to the compounds listed in paragraph 01.07 (Lindqvist alkannin dimethylacrylate; aristolindiguinone, chleone A, (2001)). droSerone, isodiospyrin, naphthazarin; tricroZarin A, acti 0143. The following compounds increase free radical norhodine, euclein, and atovaquone. This list is representa production when exposed to ultrasound and a metal: tetra tive of hydroxy-1,4-naphthoguinones and is not all inclu cycline antibiotics and their derivatives, Salts, and polymers. SVC. These compounds can act as chelants and/or reducing 0.148. The following compounds increase free radical agents. We demonstrated the activity of these compounds production when exposed to ultrasound and a metal: when tetracycline was added to iron and radical production hydroxylated 1,4-benzoquinones, their derivatives, isomers, was increased. Examples include but are not limited to metal coordination compounds, Salts, and polymers. These methacycline, doxycycline, Oxytetracycline, demeclocyline, compounds can act as chelants and/or reducing agents. We meclocycline, chlortetracycline, bromotetracycline, dauno demonstrated their effectiveness using the following com mycin, dihydrodaunomycin, adriamycin, Steffimycin, Steffi pound: mycin B, 10-dihydrosteffimycin, 10-dihydrosteffimycin B, 13213 RP, tetracycline ref. 7680, baumycin A2, baumycin A1, baumycin B1, baumycin B2, antibiotic MA 144S1, rhodomycin antibiotic complex, musettamycin, antibiotic Tetrahydroxy 1,4-benzoquinone MA 144L1, aclacinomycin B antibiotic MA 144 Y, aclaci nomycin A, antibiotic MA 144G1, antibiotic MA 144M1, 0149 Addition of a chelant such as aminocarboxylates, antibiotic MA 144N1, rhodirubin B, antibiotic MA 144U1, hydroxycarboxylates, or biologically relevant chelants Such antibiotic MA 144G2, rhodirubin A, antibiotic MA 144M2, as ADP, ATP, or GTP further increases radical production, marcellomycin, Serirubicin, oxytetracycline, demeclocy particularly when added with a compound as described cline and minocycline. above. Addition of a reducing agent Such as ascorbic acid, 0144. Addition of a chelant such as aminocarboxylates, 1,4-naphthoguinone derivatives, and 1,4-anthraquinone hydroxycarboxylates, or biologically relevant chelants Such derivatives and/or thiols further increaseS radical production as ADP, ATP, or GTP further increases radical production, especially in combination with a compound as described particularly when added with a compound described in above. paragraph 0109. Addition of a reducing agent Such as 0150 Embelin, methylembelin, and rapanone are ascorbic acid, 1,4-naphthoguinone derivatives, 1,4 benzo examples of other hydroxylated 1,4-benzoquinones. quinone derivatives, and 1,4-anthraquinone derivatives and/ or thiols further increaseS radical production, particularly in 0151. The following compounds increase free radical combination with a compound from paragraph 0109 (Quin production when exposed to ultrasound and a metal: lan (1998)). hydroxylated anthraquinones, their derivatives, isomers, metal coordination compounds, Salts, and polymers. These 0145 The following compounds increase free radical compounds can act as chelants and/or reducing agents. production when exposed to ultrasound and a metal: hydroxy-1,4-naphthoguinones, their derivatives, isomers, 0152. Addition of a chelant such as aminocarboxylates, metal coordination compounds, Salts, and polymers. These hydroxycarboxylates, or biologically relevant chelants Such compounds can act as chelants and/or reducing agents. We as ADP, ATP, or GTP further increases radical production demonstrated their effectiveness using the following com especially in combination with a compound as described pounds: above. Addition of a reducing agent Such as ascorbic acid, 1,4-naphthoguinone derivatives, 1,4 benzoquinone deriva tives, and/or thiols further increaseS radical production more particularly, when and in combination with a compound as 10 uM 5-hydroxy-1,4-naphthoquinone described above. (juglone) 15 uM 2-hydroxy-3-(3-methyl-2-butenyl)- 1,4-naphthoquinone (lapachol) 0153. Examples of hydroxylated anthraquinones include 71 uM 5-hydroxy-2-methyl-1,4- but are not limited to the following compounds and their naphthoquinone (plumbagin) derivatives: alizarin, aloe-emodin, anthragallol, aurantio 106 uM 5.8 dihydroxy -1,4-naphthoquinone obtusin, barbaloin, cascaroside A, cassiamin C, 7-chloro emodin, chrysazin, chrySo-obtusin, chrysophanic acid 9-an throne, digiferrugineol, 1,4-dihydroxy-2- 0146 Addition of a chelant such as aminocarboxylates, methylanthraquinone, frangulin A, frangulin B, lucidin, hydroxycarboxylates, or biologically relevant chelants Such morindone, norobtusifolin, obtusifolin, phyScion, as ADP, ATP, or GTP further increases radical production. pseudopurpurin, purpurin, danthron, and rubiadin. Prodrugs Addition of a reducing agent Such as ascorbic acid, 1,4 Such as diacerein that are converted to hydroxylated benzoquinone derivatives, and 1,4-anthraquinone deriva anthraquinones in the body are also relevant (Gutteridge, et tives and/or thiols further increaseS radical production. al. (1986); Kagedal, et al., (1999); Lee (1999); Lee, et al. 0.147. Other examples of hydroxylated 1,4-naphtho (2001); Muller, et al. (1993)). quinones include the following compounds and their deriva 0154 Flavonoids such as kaempferol, quercetin, and tives: 1,4-naphthalenedione, 2,3-dihydroxy; 1,4-naphtha myricetin and Sesquiterpenes Such as gossypol and feralin lenedione, 2.5,8-trihydroxy; 1,4-naphthalenedione, are reducing agents and/or chelants that increase free radical US 2003/0082101 A1 May 1, 2003

production when exposed to ultrasound and a metal. Addi derivatives; 1,5-dihydro-4H-pyrazolo 3,4-dpyrimidine-4- tion of a chelant Such as aminocarboxylates, hydroxycar thione; 1,5-dihydro-6H-imidazo[4,5-cpyridazine-6-thione; boxylates, or biologically relevant chelants Such as ADP, 1,7-dihydro-6H-purine-6-thione, 1-adamantanethiol; 2(1H)- ATP, or GTP further increases radical production. Other benzimidazolinethione; 2,4-diamino-6-mercapto-1,3,5-tri examples of flavonoids include, but are not limited to azine; 2,4-dimethylbenzenethiol; 2,5-dimethylbenzenethiol; acacetin, apigenin, biochanin-A, daidZein, equiol, flavanone, 2,6-dimethylbenzenethiol, 2-adamantanethiol, 2-amino-1,7- flavone, formononetin, genistin, glabranin, liquiritigenin, dihydro-6H-purine-6-thione; 2H-1,4-benzothiazine-3(4H)- luteolin, miroestrol, naringenin, naringin, phaseollin, phlo thione; 2-imidazolidinethione; 2-Isopropyl-3-methylben retin, prunetin, robinin, and Sophoricoside. Derivatives, Zenethiol, 2-isopropyl-4-methylbenzenethiol, 2-isopropyl polymers, and glycosylated forms of these compounds are 5-methylbenzenethiol; 2-mercapto-4H-1-benzopyran-4- also relevant. B-dihydroxy and B-trihydroxy flavonoids are thione, 2-mercapto-5-methyl-1,3,4-thiadiazole, 2-mercapto preferred (Canada (1990); Laughton. (1989)). 5-nitrobenzimidazole, 2-mercaptothiazoline, 2-methyl-1- O155 The following compounds increase free radical propenethiol, 2-methylene-1,3-propanedithiol, 2-propene-1- production when exposed to ultrasound and a metal: anti thiol: 3,4-dihydro-4,4,6-trimethyl-1-(4-phenyl-2-thiazolyl)- tumor antibiotic quinoid agents Such as benzoquinones, 2(1H)-pyrimidinethione; 3,4-dihydro-4,4,6-trimethyl mitimycins, Streptonigrins, actinomycins, anthracyclines, 2(1H)-pyrimidinethione; 3-amino -5-mercapto-1H-1,2,4- triazole, 3-bromo-1-adamantanethiol, 3-mercapto-5-methyl and Substituted anthraquinones. These compounds can act as 1,2,4-triazole and derivatives; 3-mercaptocyclohexanone chelants and/or reducing agents. and derivatives; 3-quinuclidinethiol, 3-thio-9,10-secoc 0156 Free radical production by compounds as described holesta-5,7,10(19)-triene, 4-amino-2,4-dihydro -5-phenyl above is enhanced by adding chelants Such as aminocar 3H-1,2,4-triazole-3-thione; 4-amino-3-hydrazino-5-mer boxylates, hydroxycarboxylates, or biologically relevant capto-1,2,4-triazole; 4-benzocyclobutenethiol; chelants such as ADP, ATP, or GTP, or reducing agents such 4-biphenylthiol, 4-Isopropyl-2-methylbenzenethiol; 5,6- as ascorbic acid or thiols (Gutteridge, et al. (1985); Gut dichloro-2-mercapto-1H-indole, 5'-amino-2',3,3,4-tetrahy teridge, et al. (1984); Morier-Teissier, et al. (1990)). dro-4,4,6-trimethyl-2,2'-dithioXo1(2H),4'-bipyrimidin 6'(1H)-one; 5-isopropyl-2-methylbenzenethiol; O157 The following compounds increase free radical 5-mercapto-3-methyl-1,2,4-thiadiazole, 6-amino-2-mercap production when exposed to ultrasound and a metal: ascor topurine; 6-thioinosine; 7-(nercaptomethyl)-1,7-dimethylbi bic acid, its derivatives, Salts and polymers act as ultrasound cyclo2.2.1]heptan-2-one, 7-mercapto-3H-1,2,3-triazolo.4, enhanced reducing agents and/or chelants. Addition of a 5-dipyrimidine; Azothiopyrine; benzocthiophene-1(3H)- chelant Such as aminocarboxylates, hydroxycarboxylates, or thione; bis(1-methylethyl)carbamothioic acid S-(2,3,3- biologically relevant chelants such as ADP, ATP, or GTP trichloro-2-propenyl) ester; Caesium 2,6-bis(2,4,6- further increases radical production (Dognin (1975); triisopropylphenyl)phenylthiolate; (3,3)-cholest-5-ene-3- Schneider (1988)). thiol; Cyclohexanethione; Lithium 2,6-bis(2,4,6- 0158. Thiol compounds, their derivatives, and polymers triisopropylphenyl)phenylthiolate, naphtho1,2-dthiazole increase free radical production when exposed to ultrasound -2(1H)-thione; naphtho2,1-dithiazole-2(3H)-thione; phe and a metal. We demonstrated their effectiveness using nylmethanethiol; Potassium 2,6-bis(2,4,6-triisopropylphe cysteine as an example of a biological thiol and pennicil nyl)phenylthiolate; Rubidium 2,6-bis(2,4,6-triisopropy lamine as an example of a thiol drug. Biological thiols and lphenyl)phenylthiolate; Sodium 2,6-bis(2,4,6- thiol drugs are preferred. Examples of biological thiols triisopropylphenyl)phenylthiolate (Diez (2001)). include, but are not limited to cysteinylglycine, cysteamine, 0160 Sodium sulfide and sodium sulfite are reducing thioglycollate and glutathione. Other thiol containing drugs agents that increase free radical production when exposed to include but are not limited to Captopril, Pyritinol (pyridox ultrasound and a metal. We demonstrated this using Sodium ine disulfide), Thiopronine, Piroxicam, Thiamazole, 5-Thi Sulfite. Addition of a chelant Such as aminocarboxylates, opyridoxine, Gold Sodium thiomalate, and bucillamine. In hydroxycarboxylates, or biologically relevant chelants Such addition, drugs classified as penicillins, cephalosporins, and as ADP, ATP, or GTP further increases radical production. piroXicam may undergo hydrolytic breakdown in Vivo to form thiols; therefore, they are thiol prodrugs. Addition of a 0.161 The terms “a receptor” and “an antigen” refer to a chelant Such as aminocarboxylates, hydroxycarboxylates, or chemical group in a molecule which comprises an active site biologically relevant chelants such as ADP, ATP, or GTP in Said molecule, or to an array of chemical groups in a further increaseS radical production. Addition of a reducing molecule which comprise one or more active sites in the agent Such as ascorbic acid, 1,4-naphthoguinone derivatives, molecule, or to a molecule comprised of one or more 1,4 benzoquinone derivatives, and/or 1,4-anthraquinone chemical groups or one or more arrays of chemical groups, derivatives. which group or groups or array of groups comprise one or more active sites in the molecule. An “active site of a 0159. A comprehensive list of thiol compounds include receptor' has a specific capacity to bind to or has an affinity 1-(mercaptomethyl)-7,7-dimethylbicyclo[2.2.1]heptan-2- for binding to a vector. With respect to use with the term “a one; 1,2,3-benzotriazine-4(3H)-thione; 1,2-benzisothiazole receptor' or with the term “active site in a receptor', the 3(2H)-thione -1,1-dioxide;1,2-dihydro-3H-1,2,4-triazole-3- term "vector” as used herein refers to a molecule comprised thione; 1,2-dihydro-3H-1,2,4-triazole-3-thione and of a specific chemical group or a Specific array of chemical derivatives, 1,2-dihydro-4,5-dimethyl-2H-imidazole-2- groups receptor recognizing group, which molecule, group, thione; 1,3-dihydro-1-methyl-2H-imidazole-2-thione; 1,3- or array of groups is complementary to or has a specific dihydro-2H-naphth 2,3-dimidazole-2-thione; 1,3-dihydro affinity for binding to a receptor, especially to an active Site 4,5-diphenyl-2H-imidazole-2-thione, 1,4-benzoxazepine in a receptor, to which otherwise modifies the biodistribution 5(4H)-thione; 1,4-dihydro-5H-tetrazole -5-thione and of the overall composition of matter in a desired manner. US 2003/0082101 A1 May 1, 2003

Examples include cell Surface antigens, cell Surface and 0.167 The ultrasound transducer used in Sonodynamic intracellular receptors which bind hormones, and cell Sur therapy may be applied externally or may be implanted. It face and intracellular receptors which bind drugs. Sites of can be introduced into the body via endoscopy or catheter. Specific association of Specific hormone binding to cellular receptors and Specific binding of drugs or cellular receptors 0168 Focused ultrasound can be guided by imaging are examples of active sites of the receptors, and the modalities, Such as MRI. The applied ultrasound can act as hormones or the drugs are examples of vectors for the both the irradiation Source and as an imaging modality. respective receptors. 0169. The exact operating parameters for photodynamic 0162 The vector group can be selected from a wide therapy and Sonodynamic therapy are determined depending variety of naturally occurring or Synthetically prepared upon the Specific irradiation System being used, as well as on materials, including but not limited to enzymes, amino the target tissue or other application. acids, peptides, polypeptides, proteins, lipoproteins, glyco 0170 For purposes of the present invention, “a metal” proteins, lipids, phospholipids, hormones, growth factors, means an element that forms positive ions when its com Steroids, Vitamins, polysaccharides, lectins, toxins, nucleic pounds are in Solution and whose oxides form hydroxides acids (including oligonucleotides), haptens, avidin and rather than acids with water. Metals occur in every group of derivatives thereof, biotin and derivatives thereof, antibod the periodic table except VIIA and the noble gas group. ies (monoclonal and polyclonal), anti-antibodies, antibody 0171 The preferred metals for use in the present inven fragments and antigenic materials (including proteins and tion are transition metals, lanthamides, and actinides. The carbohydrates). The vector group can also be components or metal, can be in the form of free metal ions, metal Salts products of Viruses, bacteria, protozoa, fungi, parasites, (inorganic or organic), metal oxides, metal hydroxide, metal rickettsia, molds, as well as animal and human blood, tissue Sulfides, coordinate compounds, or clathrates. The metal can and organ compositions. The vector group can also be a be present in one or more oxidations States. A combination pharmaceutical drug or Synthetic analog of any of the of different metals can be used in combination or Sequen materials mentioned above, as well as others known to one tially. These metals may be bound, covalently or nonco skilled in the art. Additional Specific vector groups are Valently, to complexing or chelating agents, including lipo described in WO 96/40285, the entire contents of which are philic derivatives thereof, or to proteinaceous hereby incorporated by reference. macromolecules. The metals can be incorporated into lipo 0163 Preferred vectors are antibodies and various immu Somes or vesicles. Polymerized and particulate forms of noreactive fragments thereof, proteins and peptides, as long metals can also be used. Biological Sources of iron, Such as as they contain at least one reactive site for reacting with a ferritin and transferrin, can also be used. Metals and metal vector reactive group or with linking groups. The Site can be compounds that are used as MRI contrast agents can also be inherent to the vector or it can be introduced though appro used. Typical MRI contrast agent compositions are priate chemical modification of the vector. The antibodies described in U.S. Pat. Nos. 6,088,613; 5,861,140; 5,820, and fragments thereof can be produced by any conventional 851; 5,534,241; 5,460,700; 5,411,730; 5,409,689; 5,407, means, including molecular biology, phage display, and 657; 5,336,762; 5,314,679; 5,242,681; 5,236,915; 5,336, genetic engineering. 695; 5,213,788; 5,155,215; 5,120,527; 5,055,288; and SO 0164. The term “antibody fragments” refers to a vector 30688A2, the entire contents of with which are hereby which comprises a residue of an antibody, which character incorporated by reference. istically has an affinity for binding to an antigen. Antibody 0172 Sonotherapeutic delivery systems, with which gen fragments exhibit at least a percentage of affinity for binding erally involve rupturing drug filed microSpheres at the to an antigen, this percentage being in the range of 0.001 desired site by application of ultrasound energy, are Suitable percent to about 1000 percent, preferably about 0.1 percent delivery vehicles for the Sonotherapeutic agents and/or met to about 1000 percent, of the relative affinity of the antibody als. These delivery systems are described in detail in U.S. for binding to the antigen. Pat. Nos. 6,028,066; 5,997.898; 6,039,967; PCT applica 0.165 Additional preferred vectors are peptides, oli tions 991391A1, 98.51284A1, 9842384A1, 0012062A1, gopeptides, or peptoids, which vectors are composed of one 993.9697A1; European applications 988061A1, 98.1333A1, or more amino acids whose Sequence and composition 959908A1, 831932A1, 0097907A1; and Japanese applica comprise a molecule, Specific chemical group or a specific tion 101.301.69A. array of chemical groups, which are complementary to or 0173 Sonodynamic or photodynamic delivery systems have a specific affinity for binding to a receptor, especially can be in the form of a microSphere containing the Sonody to an active site of a receptor. Especially preferred vectors namic agent in which the activator metal is covalently or are peptidomimetic molecule, which are fully Synthetic non-covalently attached to the Surface or components of the organic materials that are the Structural or functional equiva microSphere. Two types of microSpheres, one containing the lent of receptor groups derived or identified form antibodies, antibody fragments, proteins, fusion proteins, peptides, or Sonodynamic agent and one containing the activator, can be peptoids, and that have affinity for the same receptor. Other used in combination or Sequentially. peptidometric vectors include chemical entities Such as 0.174. The Sonodynamic or photodynamic agent and drugs, for example, which show affinity for the receptor, and metal activator can be combined via covalent or non especially for the active site of the receptor of interest. covalent bonds. In a preferred embodiment, this is achieved 0166 Peptidometric vectors can be identified using mol by attaching the Sonodynamic agent to the Surface of ferritin ecule biological techniques Such as protein mutation, phage or modified ferritin through ionic or covalent attachments. display, genetic engineering, and other Such techniques 0.175. In one embodiment of the invention, the activator know to those skilled in the art. may include a molecule with which is detectable via an in US 2003/0082101 A1 May 1, 2003

Vivo diagnostic imaging modality, Such as X-ray, MRI, ESR, bility issue. However, it is also known that alteration of the NMR, ultrasound, light imaging Scintigraphy, in vivo molecular structure to control the relative hydrophobicity of microScopy Such as confocal microscopy, photoacoustic the molecule within a preferred range can increase the oral imaging and acousto-optical imaging, Visual observation, availability of the agent. photographic imaging, magnetotomography, or electrical impedance tomography. The metal activator itself is Suitable 0181 Any known route of administration of drugs or for MRI imaging, permitting Simultaneous treatment and agents to mammals are envisaged by the present invention. imaging. 0182. The photodynamic or Sonodynamic compounds 0176) The metal activator can include a moiety to modify can be formulated with conventional pharmaceutical or its biodistribution, thus targeting the desired location with Veterinary aids, Such as emulsifiers, fatty acid esters, gelling greater Specificity. Examples of these moieties include anti agents, Stabilizers, antioxidants, OSmolality adjusting agents, bodies, antibody fragments, proteins, and oligopeptides buffers, pH adjusting agents, etc., and may be in a form which have an affinity for cell Surface receptors, particularly Suitable for parenteral or enteral administration. Thus, the receptors associated with Surfaces of diseased or rapidly photodynamic or Sonodynamic compounds of the present proliferating cells, and peptides and non-peptide drugs with invention, which may be formulated with the metal activator which are preferentially taken up by diseased or rapidly or administered Separately from the metal activator, can be proliferating cells. These targeting moieties also include in conventional pharmaceutical administration forms Such tumor-targeting drug compound, blood residence prolonging as tablets, capsules, powders, Solutions, Suspensions, disper compounds, folic acid and derivatives thereof. Activators Sions, Syrups, Suppositories, etc. with which contain Sulfonic acid groups of derivatives 0183) To treat patients according to the present invention, thereof promote retention at tumor Sites. the Sonodynamic therapy may be effected by exposing the 0177. The metal activator can be administered prior to patient to an effective amount of ultrasound acoustic energy administering the Sonodynamic or photodynamic agent, or as described in the literature. Generally, frequency and power levels that produce ultraSonic cavitation or mechani in combination with the Sonodynamic or photodynamic cal shearing in the body are preferred. Generally, this will agent. Different routes may be used for administering the involve exposure to focused ultrasound, e.g., at a power metal activator and the Sonodynamic or photodynamic level of about 0.1 to about 20 Wem', preferably about 4 to agent. DOSage about 12 Wem', a frequency of about 0.01 to about 10.0 0.178 For photodynamic therapy or Sonodynamic MHZ, preferably about 0.1 to about 5.0 MHZ, particularly therapy, the photodynamic and/or Sonodynamic compound about 0.001 to about 2.2 MHz, for periods of 10 millisec is administered in conjunction with at least one activator. onds to 60 minutes, preferably for about one second to about The dosage used will depend on the mode of administration, five minutes. AS one skilled in the art can readily appreciate, the nature of the condition being treated, the patient's size these values depend on the transducer frequency, type of and Species. Where a reporter is used, the dosage also tissue irradiated, and Sonodynamic agent used, and these depends on the nature of the imaging modality and the values are merely illustrative. The important characteristic is nature of the reporter. Where the reporter is a non-radioac that mechanical Shearing and/or cavitation are required for tive metalion, generally dosages of about 0.001 to about 5.0 treatment moles of chelated imaging metalion per kilogram of patient 0.184 Particularly preferably, the patient is exposed to body weight are effective to achieve adequate contrast ultrasound at an acoustic power of about 5 mW to 10W with enhancements. a fundamental frequency of about 0.01 to about 1.2 MHZ 0179 The photodynamic or Sonodynamic compounds and a corresponding Second harmonic frequency, as this plus activator according to the present invention may be produces the exposure necessary to achieve a cytopatho administered by any convenient route, Such as by injection genic effect. or infusion into muscle, tumor tissue, or the vasculature, Subcutaneously, or interstitially, by administration into an 0185. “Treatment” or “treating” means any treatment of eternally voiding body cavity Such as into the digestive tract a disease in a mammal, including: (orally or rectally), vagina, uterus, bladder, ears, nose or 0186 preventing the disease, i.e., preventing the lung, by transdermal administration by iontophoresis or by clinical Symptoms of the disease from developing; topical application, or by topical application to a Surgically 0187 inhibiting the disease, i.e., arresting the devel exposed site. Direct injection into a tumor is one preferred opment of clinical Symptoms, and/or administration route. 0188 relieving the disease, i.e., causing the regres 0180. The administration forms used may be any con Sion or disappearance of clinical Symptoms. ventional form for administration of pharmaceuticals, Such as Solutions, Suspensions, dispersions, Syrups, powders, tab 0189 Photodynamic and Sonodynamic Therapy lets, capsules, Sprays, creams, gels, and the like. Oral 0190. For photodynamic therapy, the parameters of the administration of photodynamic or Sonodynamic com pulse of light required for activation of the photoSensitizable pounds plus metal activators is often preferred because of compound may be determined empirically, for example, by enhanced patient compliance and ease of administration. direct measurement of the fluorescence activity of the Sen While not every agent is bioavailable by this route, since not Sitizer plus activator under different irradiation regimes, or all molecules are chemically stable in the environs of the by measuring the slope of effect evoked on final Subtract of gut, transportable acroSS alimentary membranes for absorp the Sensitizer activity under different radiation regimes tion into the blood/lymphatics, or active even if accessible which change can be easily determined by a fluorescence or due to metabolic processes within the gut or possible Solu activity effect on a Substrate. US 2003/0082101 A1 May 1, 2003

0191 It should be noted that there exists an inverse expansion of immune cells challenged by pathogens or other relationship between the intensity of irradiation and the Stimuli, and as a defense mechanism to remove Self-reactive duration, i.e., the lower the intensity above the threshold of lymphocytes. In aging cells and/or tissues that exhibit func activation, the longer the duration should be. Therefore, for tional deficiencies, apoptosis is a useful approach for each specific photosensitizable compound, there exist Sev increasing the turnover of Senescent cells and thus trigger eral pulses which can be used for treatment purposes. the renewal of cellular function and Structure. 0.192 For Sonodynamic therapy, ultrasound or any other 0199 Accordingly, Sonodynamic or photodynamic externally controllable Sonic energy Source is administered, therapy according to the present invention is effective in the toxicity of which is Selectively enhanced by a Sensitizer. treating conditions characterized by neoplastic tissue, 0193 The preferred Sonodynamic agent employed in the including the cancerS Sarcoma, lymphoma, leukemia, carci present invention is ultrasound, particularly low intensity, noma and melanoma, cardiovascular diseases Such as arte non-thermal ultrasound, i.e., ultrasound generated within the riosclerosis, atherosclerosis, intimal hyperplasia and rest wavelengths of about 0.1 MH and about 5.0 MHZ and at enosis, and other activated macrophage-related disorders intensities between about 3.0 and about 5.0 W/cm. Ultra including autoimmune diseases Such as rheumatoid arthritis, Sound can be generated by a focused array transducer, driven Sjogrens Scleroderma, Systemic lupus erthematosis, non by a power amplifier. The diameter of the focused array Specific vasculitis, Kawasaki's disease, psoriasis, Type I transducer varies in size and Spherical curvature to allow for diabetes, and pemphiguS Vulgaris. Other diseases and con variation of the focus of the ultrasonic output. Commercially ditions that can be treated by the process of the present available therapeutic ultrasound devices can be used. Fre invention include granulomatous diseases Such as tubercu quency and power levels that produce ultraSonic cavitation losis, Sarcoidosis, lymphomatoid granulomatosis, and Weg or mechanical shearing in the body are preferred. ner's granulomatosis, inflammatory diseases Such as inflam matory lung diseases Such as interstitial penumonitis and 0194 The photodynamic or Sonodynamic compounds asthma, inflammatory bowel disease Such as Crohn's dis may be used alone or in any desired combination of photo ease, inflammatory arthritis, and in transplant rejection, Such dynamic or Sonodynamic compounds. Where there is a as in heart/lung transplants. Additional treatment options plurality of photodynamic or Sonodynamic compounds, they include cervical dysplasia and cervical ablation, endometrio may be administered Separately, Sequentially, or Simulta sis and endometrial ablation, fibroids, treatment of diseased neously. The metal activator can be administered Separately, tissues after Surgery (e.g., treating tissue Surrounding a Sequentially, or Simultaneously with the photodynamic or tumor after its Surgical removal), bone marrow purging to Sonodynamic compounds. remove tumor cells that may contaminate bond marrow 0.195 Sonodynamic or photodynamic therapy using a during autologous bone marrow transplants, prostate cancer Sonodynamic or photodynamic agent along with a metal and benign prostate hyperplasia (BPH), age-related macular enhancer can be used for all types of therapy for which degeneration (AMD), and for immunomodulation (e.g., to Sonodynamic and/or photodynamic therapy can be used. For SuppreSS development of contact hyperSensitivity, abrogate example, patients can be treated according to the present development of acute adjuvant enhanced arthritis, and pro invention to induce apoptosis or programmed cell death long Survival of skin allografts). Cosmetic treatments are thereby to prevent and/or treat a variety of diseases or also included, Such as removal of skin discoloration, moles, conditions and provide a variety of benefits. Cancer can be birthmarks, Spider and varicose veins, and unwanted hair. prevented by applying ultrasound energy or light energy The parameters of the pulse (light, ultrasound, microwave, along with an enhancer and a metal to induce apoptosis or etc.) required for activation of the photosensitizable or programmed cell death of precancerous cells in different SonoSensitizable compound in the presence of at least one tissues and organs of a mammal. metal can be determined empirically, for example by direct measurement of the fluorescence activity of the Sensitizer 0196. Additionally, cancer cells can be exposed to ultra under different irradiation regimes, or by measuring the Sonic or light energy along with an enhancer and a metal in slope of effect on the effect of sensitizer activity under an amount effective to induce apoptosis of cancer cells. The different radiation regimes, which change may be easily present invention can be used to induce apoptosis undergo determined by a fluorescence or activity effect on a Sub ing abnormal proliferation in target cells having one or more Strate. The parameters of energy irradiation which are Suf growth factors including, but not limited to, EGF, TGF, ficient to terminate or significantly reduce the change in NGF, FGF, IFG, and PDGF. fluorescence can be used in accordance with the present 0197) The present invention can also be used to affect invention. cells undergoing other types of abnormal proliferation, Such 0200. It is also possible to combine photodynamic as, for example, in conditions including arteriosclerosis, therapy with Sonodynamic therapy for enhanced effect of vascular and fibrotic proliferative diseases, retinopathies, each therapy. In this case, a patient is treated with a eczema or psoriasis, by applying Sound and/or light energy Sonodynamic compound and exposed to Sound waves, as along with an enhancer and a metal. well as with a photodynamic compound and exposed to light 0198 Apoptosis is a general property of most cells, being waves. Because the activator enhances both the Sonody fundamental for the organization and life span of any namic compound and the photodynamic compound, only organism to control homeostasis and cell populations. It is one activator need be administered for both forms of treat necessary to achieve an adequate balance between the ment. However, if one activator is more effective than sufficient survival of cells and overwhelming proliferation another activator in photodynamic therapy as opposed to and expansion. This is of particular importance in preventing Sonodynamic therapy, then a combination of activators may and treating malignant growth, but is also necessary to limit be administered. US 2003/0082101 A1 May 1, 2003

0201 Ultrasound according to the present invention can 0208 Free-radical polymerization can be conducted in a also be used to induce hemostasis, particularly following an variety of ways, including bulk polymerization, Solution automobile accident which internal organs are damaged and polymerization, Suspension polymerization, and emulsion endoscopic fibers or catheters cannot be used to treat rup polymerization. tured organs or intra-liver bleeding. Moreover, bleeding 0209 For generation of free radicals, a photoinitiator gastric ulcers or ruptured eSophageal varices can be treated and/or Sononinitiator plus a metal is Subjected to the appro by the method of the present invention. In this embodiment, priate wavelength of light or Sound for an appropriate a Sonodynamic agent is introduced to the body along with a amount of time. The free radicals thus generated are used for metal activator. Ultrasound energy is applied at a Selected initiating and accelerating a variety of reactions, as Site in the body at a frequency Sufficient to create hemostasis. described above. This embodiment is particularly useful immediately after bleeding has begun, So that bleeding can be halted while the 0210 Any conventional Sonodynamic or photodynamic patient is being transported in an ambulance to an urgent agents can be used in the present invention along with a care center. Death rates from trauma are lowered by tem metal to enhance their Sonodynamic or photodynamic effect. porarily Stopping the bleeding until major Surgical interven 0211 Conventional Sonodynamic agents include the fol tion can be performed in a hospital. lowing classes of compounds: 0202 Deep locations of the body including but not lim 0212 1. Porphyrins, comprising four pyrrole rings ited to the liver, abdominal aorta, and their bleeding organs together with four nitrogen atoms and two replaceable can be treated to halt bleeding without Surgical intervention. hydrogen atoms, for which various metal atoms can be Because ultrasound energy is used, body organs and struc readily substituted. Porphyrins include hemins, chloro ture are not damaged. phylls, and cytochromes. Specific porphyrins used include gallium porphyrin, porphyrin analogs and derivatives, meso 0203. In the present invention, photodynamic and Sono porphyrin, proptoprohyrin, and hematoporphyrin, dynamic agents are combined with an activator, followed by 0213 2. Texaphyrins, aromatic pentadentate macrocyclic irradiation of the activator-agent combination. In one expanded porphyrins, also described as an aromatic benzan embodiment of this invention where the activator is a nulene containing both 18 pi and 22 pi electron delocaliza transition metal, the photodynamic and Sonodynamic agents tion pathways, are preferably capable of chelation with a metal, i.e., the metal ion is attached by coordinate links to two or more 0214 3. Cyanines and phthalocyanines, dyes consisting non-metal atoms in the same molecule. Generation of Free of two heterocyclic groups connected by a chain of conju Radicals for Chemical Reactions gated double bonds containing an odd number of carbon atoms. Cyanines include isocyanines, merocyanines, cryp 0204 Free radicals are reactive chemical species possess tocyanines, and dicyanines. Phthalocyanines are any group ing a free (unbonded or unpaired) electron. Radicals may of benzoporphyrins which comprise four isolindole groups also be positively or negatively charged species carrying a joined by four nitrogen atoms, free electron (ion radicals). Free radicals are very reactive chemical intermediates and generally have a short lifetime, 0215 4. Chromophores, compounds which absorb and/or emit light, particularly those with delocalized electron SyS generally a half-life of less than 10-3 seconds. tems. Chromophores can alternatively contain a complexed 0205 Once they are formed, radicals undergo two types metal ion. The term includes fluorophores as well as phos of reactions: propagation reactions and termination reac phorescent compounds. A more complete listing of chro tions. In propagation, a radical reacts to form a covalent mophores can be found in WO9852609, the entire contents bond and to generate a new radical. Three of the most of which are hereby incorporated by reference; common propagating reactions are atom abstractions, beta Scission, and addition to carbon-carbon double bonds or 0216) 5. Water soluble polymers (hexamers and higher aromatic rings. In a termination reaction, two radicals inter polymers), particularly polyalkyleneoxide compounds Such act in a mutually destructive reaction in which both radicals as those described in WO9852609, the entire contents of form covalent bonds and the reaction terminates. The two which are hereby incorporated by reference. The sensitizer most common termination reactions are coupling and dis agent is Selected form the group consisting of water Soluble proportionation. polymers and derivatives thereof, Surfactants, oil-in-water emulsions, Stabilized particles, and chromophoric groups 0206 Radical chain reactions are involved in many com Such as Sulfonated dyes. Preferably the Sensitizer agent is a mercial processes, including polymerization and copolymer water Soluble polymer Such as a polyalkylene oxide or a ization, polymer crosslinking, and polymer degradation. derivative thereof; Other radical-initiated polymer processes include curing of 0217 6. DMSO (dimethylsulfoxide) and DMF (dimeth resins or rubber, grafting of Vinyl monomers onto polymer ylformamide); backbones, and telomerizations. 0218 7. Chemotherapeutic compounds such as adriamy 0207 Radical reaction initiation with ultraviolet radiation is widely used in industrial processes. This proceSS generally cin and derivatives thereof, mitomycin and derivatives requires the presence of a photoinitiator. According to the thereof, diazaquinone, and amphotercin; present invention, however, visible light as well as ultravio 0219 8. Chlorines, pheophorbide, acridine orange and let or other types of light can be used in connection with a acridine derivatives, methylene blue, fluorescein, neutral photoinitiator and a metal to generate free radicals. red, rhodamins, Rose-Bengal, tetracycline, and purpurins, US 2003/0082101 A1 May 1, 2003

0220 9. Antioxidants, such as vitamin E, N-acetylcys bands extended toward longer wavelengths. Substituted teine, glutathione, Vitamin C, cysteine, methionine, 2-mer perylenequinones as described in WO 98/33470 include: captoethanol, and/or photoSensitizing molecules. A com plete listing is provided in U.S. Pat. No. 5,984,882, the entire contents of which are hereby incorporated by reference. OH O 0221 10. Xanthene dyes. OMe 0222 11. Hypericine, hypocrellins, and perylenequino nes. Examples can be found in WO 02/34708 and WO MeO RNH2 98/33470, the entire contents of which are hereby incorpo He rated by reference. MeO Me 0223) The hypocrellin derivates of WO 02/34708 consist of amino-Substitued demethoxylated hypocrellins A and B, O COMe whose structures are shown as V and VI: OMe

OH O Hypocrellin B RNH O

V OMe O OH R MeO -- MeO Me R OH R2 CH O COMe OMe

COCH RNH O R4 Isomer A O HNR O OH OMe

MeO

MeO Me VI O OH O COMe R3 OMe

O HNR Isomer B R CH R2 0226 Conventional photodynamic agents include texa COCH phyrins, porphyrins, phthalocyanines, chlorine, rhodamine R4 derivatives as described above for Sonodynamic agents.

O OH 0227 Additional photodynamic agents include precur Sors to porphyrin Such as 5-aminolevulinic acid; benzophe noxazine analogs, chlorophyll and conjugates of chlorophyll and bacteriochlorophyll derivatives with amino acids, pep 0224 where R, R, R, R are OCH or NHCHAr (Ar tides and proteins, porphycenes, pyrylium compounds; thi are phenyl or pyridyl group), NHCH(CH), where opyrylium compounds, Selenopyrylium compounds, telluro -CH(CH), are alicyclic group and N=3, 4, 5, 6). 2-BA-2- pyrylium compounds; fullerene derivatives, phylloerythrins, DMHB is where R, R, R are OCH, and R is pyropheophorbides; boron difluoride compounds, ethylene NH(CH2)CH. Alternatively, R,R,R, R may be OCH glycol esterS Substituted perylenequinones; 1, 3, 4, 6-tet or NHCH (CH), Ar, wherein Ar is a phenyl, naphthyl, rahydroxyhelianthrone and its derivatives, quinolines; thi polycyclic aromatic or a heterocyclic moiety, and n is 0-12. azine dyes, polycyclic quinines, and other biocompatible chromophores capable of cytotoxic effects upon irradiation 0225. These hypocrellin derivatives also include 2-buty with light waves. lamino-2-demethoxy-hypocrellin B (2-BA-2-DMNB), which exhibits Strong absorption in the red spectral region. 0228. The following nonlimiting examples will further Compared with its parent compound HB its absorption describe the present invention. US 2003/0082101 A1 May 1, 2003

EXAMPLE 1. tion. After 10 minutes of treatment, 1 mL test Solution was 0229. The following example uses the thiobarbituric placed in a test tube followed by 2 mL of 1%. 2-thiobarbituric acid-reactive substances (TBARS) assay to identify chelants acid and 2 mL of 2.8% trichloroacetic acid. The test tube was that enhance radical production during ultrasound exposure. sealed and heated to 90 degrees Celsius for 30 minutes and All solutions were prepared in pH 7.5 phosphate buffer allowed to cool to room temperature for 20 minutes. The containing approximately 2 mM deoxyribose, 0.01% hydro absorbance at 532 nm was measured. The enhanced radical gen peroxide, 0.025 mM ferrous iron, and 0.03–0.04 mM production during ultrasound exposure was determined by chelant. Solutions were Sonicated at 30 W, 2 MHz, 32-34 comparing the amount of deoxyribose degradation that degrees Celsius for ten minutes using a PZT-8 1.8 cm occurs in the Sonicated Solution verSuS the control Solution diameter custom transducer. The Sonicated Solution was using the following equation: placed on an orbit shaker rotating at 25 RPM to ensure even Sonication of the Solution while the transducer was held Stationary. Control Solutions were placed in a controlled temperature bath at 32-34 degrees Celsius without Sonica % activity = AbS532 sonicated solution - Abss32 control solution x 100 tion. After 10 minutes of treatment, 1 mL test Solution was AbS532 control solution placed in a test tube followed by 2 mL of 1%. 2-thiobarbituric acid and 2 mL of 2.8% trichloroacetic acid. The test tube was sealed and heated to 90 degrees Celsius for 30 minutes and allowed to cool to room temperature for 20 minutes. The absorbance at 532 nm was measured. The enhanced radical Results: production during ultrasound exposure is determined by % Ultrasound comparing the amount of deoxyribose degradation that Mediated occurs in the Sonicated Solution verSuS the control Solution Activity vs using the following equation: Chelant Control No chelant O% Ethylenediaminetetraacetic acid 575% % activity = AbS532 sonicated solution- AbS532 control solution x 100 Ethylene glycol-bis(2- 5.20% AbS532 control solution aminoethyl)-N,N,N',N'- tetraacetic acid Diaminocyclohexane-N,N,N',N'- 446% tetraacetic acid Nitriloacetic acid 23.8% N-(2- 224% Results: Hydroxyethyl)ethylenediamine N,N',N'-triacetic acid % Ultrasound Diethylenetriaminepentaacetic 177% Mediated acid Activity vs Chelant Control Desferrioxamine mesylate 81% No chelant 19% Desferrioxamine mesylate 92% Nitriloacetic acid 69% EXAMPLE 3 Ethylenediaminetetraacetic acid 64% Diaminocyclohexane-N,N,N',N'- 61% tetraacetic acid 0231. The following example uses the thiobarbituric N-(2- 34% acid-reactive substances (TBARS) assay to identify chelants Hydroxyethyl)ethylenediamine that enhance radical production during ultrasound exposure. N,N',N'-triacetic acid Ethylene glycol-bis(2- 29% All solutions were prepared in pH 7.5 phosphate buffer aminoethyl)-N,N,N',N'- containing approximately 2 mM deoxyribose, 0.01% hydro tetraacetic acid gen peroxide, 0.025 mM ferrous iron, and 0.07-0.11 mM chelant. Solutions were Sonicated at 30 W, 2 MHz, 32-34 degrees Celsius for ten minutes using a PZT-8 1.8 cm EXAMPLE 2 diameter custom transducer. The Sonicated Solution was 0230. The following example uses the thiobarbituric placed on an orbit shaker rotating at 25 RPM to ensure even acid-reactive substances (TBARS) assay to identify chelants Sonication of the Solution while the transducer was held that enhance radical production during ultrasound exposure. Stationary. Control Solutions were placed in a controlled All solutions were prepared in pH 7.5 phosphate buffer temperature bath at 32-34 degrees Celsius without Sonica containing approximately 2 mM deoxyribose, 0.01% hydro tion. After 10 minutes of treatment, 1 mL test Solution was gen peroxide, 0.02-0.03 mM ferric iron, and 0.04-0.05 mM placed in a test tube followed by 2 mL of 1%. 2-thiobarbituric chelant. Solutions were Sonicated at 30 W, 2 MHz, 32-34 degrees Celsius for ten minutes using a PZT-8 1.8 cm acid and 2 mL of 2.8% trichloroacetic acid. The test tube was diameter custom transducer. The Sonicated Solution was sealed and heated to 90 degrees Celsius for 30 minutes and placed on an orbit shaker rotating at 25 RPM to ensure even allowed to cool to room temperature for 20 minutes. The Sonication of the Solution while the transducer was held absorbance at 532 nm was measured. The enhanced radical Stationary. Control Solutions were placed in a controlled production during ultrasound exposure was determined by temperature bath at 32-34 degrees Celsius without Sonica comparing the amount of deoxyribose degradation that US 2003/0082101 A1 May 1, 2003 20 occurs in the Sonicated Solution verSuS the control Solution using the following equation:

Results:

AbS532 sonicated solution- Abss32 control solution 100 % Ultrasound % activity = X Mediated y AbS532 control solution Activity vs Chelant Control No chelant O% Adenosine diphosphate (ADP) 280% 3-(2-Pyridyl)-5,6-bis(5-sulfo- 2O7% 2-furyl)-1,2,4-triazine Results: (ferene) Picolinic Acid 175%, % Ultrasound Citrate 16.1% Mediated 3-(2-Pyridyl)-5,6-diphenyl- 13.6% 1,2,4-triazine-4,4-disulfonic Activity vs acid (ferrozine) Chelant Control

No chelant 19% Picolinic Acid 53% EXAMPLE 5 3-(2-Pyridyl)-5,6-bis(5-sulfo- 50% 2-furyl)-1,2,4-triazine 0233. The following example uses the thiobarbituric (ferene) acid-reactive substances (TBARS) assay to identify com 3-(2-Pyridyl)-5,6-diphenyl- 45% pounds that enhance radical production during ultrasound 1,2,4-triazine-4,4g exposure of Solutions containing iron or iron plus a chelant. disulfonic acid (ferrozine) All solutions were prepared in pH 7.5 phosphate buffer 110 Phenanthroline 33% containing approximately 2 mM deoxyribose, 0.01% hydro Citrate 31% gen peroxide, 0.08-0.1 mM ferric iron, and the additives Adenosine diphosphate (ADP) 26% indicated in the table below. Solutions were Sonicated at 30 W, 2 MHz, 32-34 degrees Celsius for ten minutes using a PZT-8 1.8 cm diameter custom transducer. The Sonicated solution was placed on an orbit shaker rotating at 25 RPM EXAMPLE 4 to ensure even Sonication of the Solution while the trans ducer was held Stationary. Control Solutions were placed in 0232 The following example uses the thiobarbituric a controlled temperature bath at 32-34 degrees Celsius acid-reactive substances (TBARS) assay to identify chelants without Sonication. After 10 minutes of treatment, 1 mL test that enhance radical production during ultrasound exposure. solution was placed in a test tube followed by 2 mL of 1% All solutions were prepared in pH 7.5 phosphate buffer 2-thiobarbituric acid and 2 mL of 2.8% trichloroacetic acid. containing approximately 2 mM deoxyribose, 0.01% hydro The test tube was sealed and heated to 90 degrees Celsius for gen peroxide, 0.02-0.03 mM ferric iron, and 0.07-0.11 mM 30 minutes and allowed to cool to room temperature for 20 chelant. Solutions were Sonicated at 30 W, 2 MHz, 32-34 minutes. The absorbance at 532 nm was measured. The degrees Celsius for ten minutes using a PZT-8 1.8 cm enhanced radical production during ultrasound exposure diameter custom transducer. The Sonicated Solution was was determined by comparing the amount of deoxyribose placed on an orbit shaker rotating at 25 RPM to ensure even degradation that occurs in the Sonicated Solution versus the Sonication of the Solution while the transducer was held control Solution using the following equation: Stationary. Control Solutions were placed in a controlled temperature bath at 32-34 degrees Celsius without Sonica % activity = AbS532 sonicated solution - Abss32 control solution x 100 tion. After 10 minutes of treatment, 1 mL test Solution was AbS532 control solution placed in a test tube followed by 2 mL of 1%. 2-thiobarbituric acid and 2 mL of 2.8% trichloroacetic acid. The test tube was sealed and heated to 90 degrees Celsius for 30 minutes and allowed to cool to room temperature for 20 minutes. The absorbance at 532 nm was measured. The enhanced radical Results: production during ultrasound exposure was determined by % Ultrasound comparing the amount of deoxyribose degradation that Mediated occurs in the Sonicated Solution verSuS the control Solution Activity vs using the following equation: Additive Control No additive 76% EDTA (0.15 mM) 277% Foscarnet (phosphonoformic 380% % activity = AbS532 sonicated solution- Abss32 control solution x 100 acid) (0.15 mM) + EDTA (0.15 AbS532 control solution mM) US 2003/0082101 A1 May 1, 2003

EXAMPLE 6 degrees Celsius for ten minutes using a PZT-8 1.8 cm 0234. The following example uses the thiobarbituric diameter custom transducer. The Sonicated Solution is placed acid-reactive substances (TBARS) assay to identify metals on an orbit shaker rotating at 25 RPM to ensure even that enhance radical production during ultrasound. All Solu Sonication of the Solution while the transducer is held tions were prepared in pH 7.5 phosphate buffer containing Stationary. Control Solutions are placed in a controlled approximately 2 mM deoxyribose, 0.01% hydrogen perox temperature bath at 32-34 degrees Celsius without Sonica tion. After 10 minutes of treatment, 1 mL test Solution is ide, and the additives indicated in the table below. Solutions placed in a test tube followed by 2 mL of 1%. 2-thiobarbituric were Sonicated at 30W, 2 MHz, 32-34 degrees Celsius for acid and 2 mL of 2.8% trichloroacetic acid. The test tube is ten minutes using a PZT-8 1.8 cm diameter custom trans sealed and heated to 90 degrees Celsius for 30 minutes and ducer. The Sonicated Solution was placed on an orbit shaker allowed to cool to room temperature for 20 minutes. The rotating at 25 RPM to ensure even Sonication of the solution absorbance at 532 nm is measured. The enhanced radical while the transducer was held Stationary. Control Solutions production during ultrasound exposure is determined by were placed in a controlled temperature bath at 32-34 comparing the amount of deoxyribose degradation that degrees Celsius without Sonication. After 10 minutes of occurs in the Sonicated Solution verSuS the control Solution treatment, 1 mL test Solution was placed in a test tube using the following equation: followed by 2 mL of 1%. 2-thiobarbituric acid and 2 mL of 2.8% trichloroacetic acid. The test tube was sealed and heated to 90 degrees Celsius for 30 minutes and allowed to AbS532 sonicated solution - Abss32 control solution cool to room temperature for 20 minutes. The absorbance at %o activitytivitv = AbS532 control solution x 100 532 nm was measured. The enhanced radical production during ultrasound exposure was determined by comparing the amount of deoxyribose degradation that occurs in the Sonicated Solution versus the control Solution using the 0236 Results: following equation:

Approximate Chelant:Iron Ratio % activity = AbS532 sonicated solution- AbS532 control solution x 100 AbS532 control solution for Optimum Ultrasound Mediated Chelant Activity vs Control Desferrioxamine mesylate 1:1 to 1:10 Nitriloacetic acid 1:1 to 1:10 Ethylenediaminetetraacetic 1:1 to 1:10 Results: acid Diaminocyclohexane-N,N,N',N'- 1:1 to 1:10 % Ultrasound tetraacetic acid Mediated N-(2- 1:1 to 1:10 Activity vs Hydroxyethyl)ethylenediamine Additive Control N,N',N'-triacetic acid Ethylene glycol-bis(2- 1:1 to 1:10 No additive O% aminoethyl)-N,N,N',N'- Ferrous iron added as Fe(NH) (SO) (0.05 34% tetraacetic acid mM) + ferric iron added as FeCls (approx Diethylenetriaminepentaacetic 1:1 to 1:10 0.05 mM) acid Ferrous iron added as Fe(NH) (SO) (0.1 mM) 12% Adenosine diphosphate (ADP) 3:1 to 30:1 Ferric iron added as FeCls (approx 0.1 mM) 76% 3-(2-Pyridyl)-5,6-bis(5- 3:1 to 30:1 Ferritin (approx. 0.2 mg/mL) 6% sulfo-2-furyl)-1,2,4-triazine Ferrous iron added as Fe(NH) (SO) (0.05 25.3% (ferene) mM) + ferric iron added as FeCls (approx Picolinic Acid 3:1 to 30:1 0.05 mM) + 0.15 mM EDTA Citrate 3:1 to 30:1 Ferrous iron added as Fe(NH) (SO) (0.1 mM) + 106% 3-(2-Pyridyl)-5,6-diphenyl- 3:1 to 30:1 O.15 nM EDTA 1,2,4-triazine-4,4-disulfonic Ferric iron added as FeCls (approx 0.1 mM) + 277% acid (ferrozine) O.15 nM EDTA 110 Phenanthroline 3:1 to 30:1 Ferritin (approx. 0.2 mg/mL) + EDTA (0.15 18% mM) Cupric chloride (0.026 mM) 82% EXAMPLE 8 0237) The following example uses the release of iron EXAMPLE 7 from ferritin assay to show the effect of naphthoguinones on 0235. The following example uses the thiobarbituric the release of iron from ferritin during ultrasound exposure. acid-reactive substances (TBARS) assay to show the effect All Solutions were prepared in pH 7 acetic acid Solution of chelant concentration on the enhancement of radical containing approximately 0.2 mg/mL ferritin and 1 mM production during ultrasound exposure. All Solutions are ferrozine, and the concentration of naphthoguinone indi prepared in pH 7.5 phosphate buffer containing approxi cated in the table below. Solutions were Sonicated at 30W, mately 2 mM deoxyribose, 0.01% hydrogen peroxide, 0.025 2 MHz, 32-34 degrees Celsius for fifteen minutes using a mM ferrous iron, approximately 0.025 mM ferric iron, and PZT-8 1.8 cm diameter custom transducer. The Sonicated the ratio of chelant to combined iron indicated in the table solution was placed on an orbit shaker rotating at 25 RPM below. Solutions are Sonicated at 30 W, 2 MHz, 32-34 to ensure even Sonication of the Solution while the trans US 2003/0082101 A1 May 1, 2003 22 ducer was held Stationary. Control Solutions were placed in to a ferrozine-iron Standard curve to determine the amount a controlled temperature bath at 32-34 degrees Celsius of iron released. The enhanced iron release due to ultrasound without Sonication. After 15 minutes of treatment, an aliquot exposure in the presence of the anthraquinone is compared was tested for the presence of the iron-ferrozine chelate via to the amount of enhanced iron release due to ultrasound absorbance at 562 nm. The amount of iron released was determined using the control Solution corrected absorbance exposure in the absence of any additives as follows: (Subtract the absorbance of the control solution from the absorbance of the ultrasound solution). The corrected absor bance was compared to a ferrozine-iron Standard curve to % activity = determine the amount of iron released. The enhanced iron release due to ultrasound exposure in the presence of the iron release (with additive) - iron release (without additive) 100 X naphthoguinone was compared to the amount of enhanced iron release (with additive) iron release due to ultrasound exposure in the absence of any additives as follows: 0240 Results: % activity = iron release (with additive) - iron release (without additive) 100 X % Ultrasound iron release (with additive) Additive Mediated Activity Anthraquinone-2- <10% sulfonic acid 0238 Results: 0.05 mM Alizarin Red S; >50% 3,4-dihydroxy-9,10

anthracenesulfonic acid 0.05 mM Rhein: 9,10- >50% % Ultrasound dihydro-4,5-dihydroxy Additive Mediated Activity 9,10-dioxo-2- 18 uM 2-methyl-1,4- 5.3% anthracenecarboxylic naphthoquinone acid (menadione) 0.05 mM Chrysophanol: 50% 10 uM 5-hydroxy-1,4- 93% 1,8-dihydroxy-3- naphthoquinone methylanthraquinone (juglone) 0.05 mM Emodin; 6- >50% 15 uM 2-hydroxy-3-(3- 72% methyl-1,3,8- methyl-2-butenyl)-1,4- trihydroxyanthraquinone naphthoquinone (lapachol) 71 uM 5-hydroxy-2- 155% methyl-1,4- naphthoquinone EXAMPLE 10 (plumbagin) 106 uM 5.8 dihydroxy - 185% 0241 The following example uses the release of iron 1,4-naphthoquinone from ferritin assay to show the effect of additives on the release of iron from ferritin during ultrasound exposure. All Solutions were prepared in pH 7 acetic acid Solution con EXAMPLE 9 taining approximately 0.2 mg/mL ferritin and 1 mM fer rozine, and the concentration of 1,4-quinone indicated in the 0239). The following example uses the release of iron from ferritin assay to show the effect of anthraquinones on table below. Solutions were Sonicated at 30 W, 2 MHz, the release of iron from ferritin during ultrasound exposure. 32-34 degrees Celsius for fifteen minutes using a PZT-81.8 All solutions are prepared in pH 7.5 phosphate buffer cm diameter custom transducer. The Sonicated Solution was containing approximately 0.2 mg/mL ferritin and 1 mM placed on an orbit shaker rotating at 25 RPM to ensure even ferrozine, and the concentration of anthraquinone indicated Sonication of the Solution while the transducer was held in the table below. Solutions are Sonicated at 30W, 2 MHz, Stationary. Control Solutions were placed in a controlled 32-34 degrees Celsius for fifteen minutes using a PZT-81.8 temperature bath at 32-34 degrees Celsius without Sonica cm diameter custom transducer. The Sonicated Solution is tion. After 15 minutes of treatment, an aliquot was tested for placed on an orbit shaker rotating at 25 RPM to ensure even the presence of the iron-ferrozine chelate via absorbance at Sonication of the Solution while the transducer is held 562 nm. The amount of iron released was determined using Stationary. Control Solutions are placed in a controlled temperature bath at 32-34 degrees Celsius without Sonica the control Solution corrected absorbance (Subtract the tion. After 15 minutes of treatment, an aliquot is tested for absorbance of the control Solution from the absorbance of the presence of the iron-ferrozine chelate via absorbance at the ultrasound Solution). The corrected absorbance was 562 nm. The amount of iron released is determined using the compared to a ferrozine-iron Standard curve to determine the control Solution corrected absorbance (Subtract the absor enhanced iron release due to ultrasound exposure. The bance of the control Solution from the absorbance of the enhanced iron release due to ultrasound exposure in the ultrasound Solution). The corrected absorbance is compared presence of the additive was compared to the amount of US 2003/0082101 A1 May 1, 2003

enhanced iron release due to ultrasound exposure in the absence of any additives as follows: -continued % Ultrasound Mediated % activity = Activity vs Additive Control iron release (with additive) - iron release (without additive) 100 X Quercetin (0.075 mM) >100% iron release (with additive) Myricetin (0.075 mM) >100% Addition of 0.075 mM ascorbate or cysteine significantly increased radical 0242) Results: production in the sonicated versus control solution. EXAMPLE 12

% Ultrasound Mediated 0245. The following example uses the thiobarbituric Additive Activity acid-reactive substances (TBARS) assay to identify anti 1,4-benzoquinone O% tumor antibiotics that enhance radical production during Tetrahydroxy 1,4- 186% ultrasound. All Solutions are prepared in pH 7.5 phosphate benzoquinone buffer containing approximately 2 mM deoxyribose, 0.01% (0.11 mM) hydrogen peroxide, 0.005 mM ferrous iron, 0.005 mM ferric DIHYDROXYFUMARATE (0.01 mM) 72% iron, and the additives indicated in the table below. Solutions CYSTEINE (0.45 mM) 160% are sonicated at 30 W, 2 MHz, 32-34 degrees Celsius for PENICILLAMINE (0.11 mM) 1.17% fifteen minutes using a PZT-8 1.8 cm diameter custom transducer. The Sonicated Solution is placed on an orbit EXAMPLE 11 shaker rotating at 25 RPM to ensure even Sonication of the solution while the transducer is held stationary. Control 0243 The following example uses the thiobarbituric Solutions are placed in a controlled temperature bath at acid-reactive substances (TBARS) assay to identify metals 32-34 degrees Celsius without Sonication. After 15 minutes that enhance radical production during ultrasound. All Solu of treatment, 1 mL test Solution was placed in a test tube tions are prepared in pH 7.5 phosphate buffer containing followed by 2 mL of 1%. 2-thiobarbituric acid and 2 mL of approximately 2 mM deoxyribose, 0.01% hydrogen perox 2.8% trichloroacetic acid. The test tube is Sealed and heated ide, 0.05 mM ferrous iron added as FeSO hydrate, 0.075 to 90 degrees Celsius for 30 minutes and allowed to cool to mM EDTA, and the additives indicated in the table below. room temperature for 20 minutes. The absorbance at 532 nm Solutions are sonicated at 30 W, 2 MHz, 32-34 degrees is measured. The enhanced radical production during ultra Celsius for ten minutes using a PZT-8 1.8 cm diameter Sound exposure is determined by comparing the amount of custom transducer. The Sonicated Solution is placed on an deoxyribose degradation that occurs in the Sonicated Solu orbit shaker rotating at 25 RPM to ensure even Sonication of tion versus the control Solution using the following equation: the solution while the transducer is held stationary. Control Solutions are placed in a controlled temperature bath at 32-34 degrees Celsius without Sonication. After 10 minutes % activity = Abs532 sonicated solution- Abs532 control solution x 100 of treatment, 1 mL test Solution is placed in a test tube Abs532 control solution followed by 2 mL of 1%. 2-thiobarbituric acid and 2 mL of 2.8% trichloroacetic acid. The test tube is sealed and heated to 90 degrees Celsius for 30 minutes and allowed to cool to 0246 Results: room temperature for 20 minutes. The absorbance at 532 nm is measured. The enhanced radical production during ultra Sound exposure is determined by comparing the amount of deoxyribose degradation that occurs in the Sonicated Solu % Ultrasound tion versus the control Solution using the following equation: Mediated Activity vs Additive Control No additive <20% Mitomycin C, 0.025 mM >100% % activity = Abs532 sonicated solution- Abs532 control solution x 100 o activity Abs532 control solution Streptonigrin, 0.025 mM >100% Mithramycin, 0.025 mM >100% Olivomycin, 0.025 mM >100% Chromomycin, 0.025 mM >100% 0244 Results: Carminic acid, 0.025 mM >100% Daunomycin, 0.1 mM >100% Epirubicin, 0.1 mM >100%

% Ultrasound Mediated Activity vs EXAMPLE 13 Additive Control 0247 The following example uses the thiobarbituric No additive <20% acid-reactive Substances (TBARS) assay to identify existing Gossypol (0.075 mM) >100% Sonodynamic agents that exhibit enhanced radical produc tion during ultrasound exposure in the presence of a metal. US 2003/0082101 A1 May 1, 2003 24

All solutions were prepared in pH 7.5 phosphate buffer radation that occurs in the Sonicated Solution versus the containing approximately 2 mM deoxyribose, 0.01% hydro control Solution using the following equation: gen peroxide, 0.025 mM ferrous iron, 0.025 mM ferric iron, and the additives indicated in the table below. Solutions were Sonicated at 30W, 2 MHz, 32-34 degrees Celsius for % activity = Abs532 sonicated solution- Abs532 control solution x 100 ten minutes using a PZT-8 1.8 cm diameter custom trans o activity Abs532 control solution ducer. The Sonicated Solution was placed on an orbit shaker rotating at 25 RPM to ensure even Sonication of the solution while the transducer was held Stationary. Control Solutions 0250 Results: were placed in a controlled temperature bath at 32-34 degrees Celsius without Sonication. After 10 minutes of treatment, 1 mL test Solution was placed in a test tube % Ultrasound followed by 2 mL of 1%. 2-thiobarbituric acid and 2 mL of Mediated Activity 2.8% trichloroacetic acid. The test tube was sealed and Additive vs Control heated to 90 degrees Celsius for 30 minutes and allowed to No additive 20% cool to room temperature for 20 minutes. The absorbance at Hypocrellin A (0.025 mM) >30% 532 nm was measured. The enhanced radical production Hypericin (0.025 mM) >30% Iron(III) phthalocyanine- >30% during ultrasound exposure was determined by comparing 44'4"4"-tetrasulfonic the amount of deoxyribose degradation that occurs in the acid (0.025 uM) Sonicated Solution versus the control Solution using the following equation: 0251 Metal toxicity occurs by three mechanisms. First, metals propagate free radical chain reactions on which % activity = Abs532 sonicated solution- Abs532 control solution x 100 continued radical production depends. Second, traces of Abs532 control solution metals are required for Fenton type reactions. Third, metals provide for Site-specific production of active Species, as in binding to DNA to provide centers for repeated generation 0248 Results: of ferryl species or hydroxyl radicals. Therefore, it is believed that the compositions of the present invention may be effective because of one of these mechanisms or a combination of mechanisms. % Ultrasound Mediated Activity 0252) The foregoing description of the specific embodi Additive vs Control ments will So fully reveal the general nature of the invention that others can, by applying current knowledge, readily No additive 19% Hematoporphyrin (0.027 mM) 24% modify and/or adapt for various applications Such specific Rose Bengal (0.028 mM) 28% embodiments without undue experimentation and without Adriamycin (0.029 mM) 29% departing from the generic concept. Therefore, Such adap Tetracycline (0.030 mM) 51% tations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the EXAMPLE 1.4 purpose of description and not of limitation. The means and 0249. The following example uses the thiobarbituric materials for carrying our various disclosed functions may acid-reactive Substances (TBARS) assay to identify existing take a variety of alternative forms without departing from Sonodynamic agents that exhibit enhanced radical produc the invention. Thus, the expressions “means to” and “means tion during ultrasound exposure in the presence of a metal. for as may be found in the specification above and/or in the All solutions are prepared in pH 7.5 phosphate buffer claims below, followed by a functional Statement, are containing approximately 2 mM deoxyribose, 0.01% hydro intended to define and cover whatever Structural, physical, gen peroxide, 0.025 mM ferrous iron, 0.025 mM ferric iron, chemical, or electrical element or Structures which may now and the additives indicated in the table below. Solutions are or in the future exist for carrying out the recited function, Sonicated at 30 W, 2 MHz, 32-34 degrees Celsius for ten whether or not precisely equivalent to the embodiment or minutes using a PZT-81.8 cm diameter custom transducer. embodiments disclosed in the Specification above; and it is The Sonicated Solution is placed on an orbit Shaker rotating intended that Such expressions be given their broadest at 25 RPM to ensure even Sonication of the Solution while interpretation. the transducer is held Stationary. Control Solutions are placed 0253 All references cited herein are incorporated by in a controlled temperature bath at 32-34 degrees Celsius reference. without Sonication. After 10 minutes of treatment, 1 mL test solution is placed in a test tube followed by 2 mL of 1% References 2-thiobarbituric acid and 2 mL of 2.8% trichloroacetic acid. 0254 Babbs, C. F. Free radicals and the etiology of The test tube is sealed and heated to 90 degrees Celsius for colon cancer. Free Radicals Biology & Medicine. Vol. 30 minutes and allowed to cool to room temperature for 20 8 pp 191-200 (1990). minutes. The absorbance at 532 nm is measured. The 0255 Canada, A. The production of reactive oxygen enhanced radical production during ultrasound exposure is species by dietart flavonols. Free Radical Biology & determined by comparing the amount of deoxyribose deg Medicine. Vol 9. pp441-449 (1990). US 2003/0082101 A1 May 1, 2003

0256 Cassanelli, S. Sulfide is an efficient iron releas 0271 Lee H Z.Effects and mechanisms of emodin on ing agent for mammalian ferritins. Biochimica et Bio cell death in human lung Squamous cell carcinoma. Br physica Acta. Vol 1547 pp.174-182 (2001). J. Pharmacol. Vol. 134(1) pp 11-20 (2001). 0257 Chen, F. One-electron reduction of chromium 0272 Lee HZ, Hsu SL, Liu MC, Wu CH.Effects and (VI) by alpha-lipoic acid and related hydroxyl radical mechanisms of aloe-emodin on cell death in human generation, dG hydroxylation and nuclear transcription lung Squamous cell carcinoma. Eur J. Pharmacol. Vol factor kB activation. Archives of Biochemistry and 431(3) pp. 287-95 (2001). Biophysics. Vol 338 pp 165-172 (1997). 0273 Lawson, R. C., Sonochemistry of quinones in 0258 Diez, L. High performance liquid chromato argon-Saturated aqueous Solutions: enhanced cyto graphic assay of hydroxyl free radical using Salicylic chrome c reduction. Chem. Res. Toxicol. Vol 12. pp acid hydroxylation during in vitro experiment Sinvolv 850-854 (1999). ing thiols. Journal of Chromatography B, Vol 763 0274) Lindqvist, C. Generation of hydroxyl radicals by pp.185-193 (2001). the antiviral compound phosphoneformic acid (foscar 0259 Dognin, J. Mobilisation of iron from ferritin net). Pharmacology & Toxicology. Vol. 89 pp. 49-55 fractions of defined iron content by biological reduc (2001). tants. FEBS Letters. Vol 54. pp234-236 (1975). 0275 Mascio, P. D. DNA damage by 5-aminoevulinic 0260 Donlin, M. J. Analysis of iron in ferritin, the and 4.5 dioxovaleric acids in the presence of ferritin. iron-storage protein: a general chemistry experiment. J. Archives of Biochemistry and Biophysics. Vol 373 pp Chem. Edu. 75, 437-441 (1998). 368-374 (2000). 0261 Graf, E. Iron-catalyzed hydroxyl radical forma 0276 Morier-Teissier E. Free radical production and tion. The Journal of Biological Chemistry. Vol 259, No. DNA cleavage by copper chelating peptide-an 6, pp. 3620-3624 (1984). thraquinones. Anticancer Drug Des. Vol. 5(3) pp. 291 305 (1990). 0262 Gutteridge9. J. M. Free radical damage9. to deox yribose by anthracycline, aureolic acid and amino 0277 Muller K, Gurster D. Hydroxyl radical damage quinone antitumour antibiotics. An essential require to DNA sugar and model membranes induced by ment for iron, Semiquinones and hydrogen peroxide. anthralin (dithranol). Biochem Pharmacol. Vol 46(10) Biochem Pharmacol. Vol. 34(23) pp. 4099-103 (1985). pp. 1695-704 (1993). 0263 Gutteridge J M Mitomycin C-induced deoxyri 0278 Ou, Z. Metal ions affect on the photodynamic bose degradation inhibited by Superoxide dismutase. A actions of cyclodextrin-modified hypocrellin. Cancer reaction involving iron, hydroxyl and Semiquinone Letters. pp. 206-207 (2002). radicals. FEBS Lett. Vol. 167(1) pp. 37-41 (1984). 0279 Quinlan, G. Hydroxy radical generation by the tetracycline antibiotics with free radical damage to 0264 Gutteridge JM, Carminic acid-promoted oxygen DNA, lipids, and carbohydrates in the presence of iron radical damage to lipid and carbohydrate. Food Addit and copper salts. Free Radical Biology & Medicine. Vol Contam. Vol 3(4) pp 289-93 (1986). 5 pp341-348 (1998). 0265 Gutteridge, J. M. C. Damage to biological mol 0280 Ryter, S.W. The heme synthesis and degradation ecules by iron and copper complexes. Lipofuscin. pathways: role in oxidant Sensitivity. Free Radical Pp69-82 (1987). Biology & Medicine. Vol 28 pp289-309 (2000). 0266 Halliwell, B. The deoxyribose method: a simple 0281 Schneider, J. E. Ascorbate/iron mediation of test-tube assay for determination of rate constants for hydroxyl free radical damage to PBR322 plasmid reactions of hydroxyl radicals. Analytical Biochemis DNA. Free Radical Biology & Medicine. Vol 5 pp287 try. Vol 165 pp. 215-219 (1987). 295 (1988). 0267 Hristov, P. Lipid peroxidation induced by ultra 0282 Stadtman, E. R. Fenton chemistry. The Journal Sonication in Ehrlich asciitic tumor cells. Cancer Let of Biological Chemistry. Vol 266 pp 17201-17211 ters. Vol. 121 pp 7-10 (1997). (1991). 0268 Inoue, S. Hydroxyl radical production and O283 Th omas, C. The hvdrolvsihydrolySIS productod fICRF-187 human DNA damage induced by ferric nitrilotriacetate promotes iron-catalyzed hydroxyl radical production and hydrogen peroxide. Cancer Research. Vol. 47 pp via the Fenton reaction. Biochemical Pharmacology. 6522-6527 (1987). Vol. 45, No. 10, pp 1967-1972 (1993). 0269. Kagedal K, Bironaite D, Ollinger K. 0284 Thomas, C. E. Release of iron from ferritin by Anthraquinone cytotoxicity and apoptosis in primary cardiotoxic anthracycline antibiotics. Arch. Biochem. cultures of rat hepatocytes. Free Radic Res. Vol. 31(5) Biophys. 248, 684-689 (1986). pp. 419-28 (1999). 0285 Tonetti, M. Enhanced formation of reactive spe 0270. Laughton, M. Antioxidant and prooxidant cies from cis-diammine-(1,1-cyclobutanedicarboxy actions of the plant phenolics quercetin, gossypol, and lato)-platinum(II) (carboplatin) in the presence of oxy myricetin. Biochemical Pharmacology. Vol. 38. gen free radicals. Biochemical Pharmacology. Vol 46 pp.2859-2865 (1989). pp. 1377-1383 (1993). US 2003/0082101 A1 May 1, 2003 26

0286 Toyokuni, S. Induction of oxidative single and 18. The method according to claim 15 wherein the metal double strand breaks in DNA by ferric citrate. Free is administered after administration of the photodynamic Radical Biology & Medicine. Vol 15 pp 117-123 agent or the Sonodynamic agent. (1993). 19. The method according to claim 15 wherein the metal What is claimed is: is Selected from the group consisting of transition metals, 1. A Sonodynamic composition comprising a Sonody lanthamides, and actinides. namic agent and at least one metal. 20. The method according to claim 19 wherein the metal 2. The Sonodynamic composition according to claim 1 is in a form Selected from the group consisting of free metal wherein the metal is Selected from the group consisting of ions, inorganic metal Salts, organic metal Salts, metal oxides, transition metals, lanthamides, and actinides. metal hydroxides, metal Sulfides, coordination compounds, 3. The Sonodynamic composition according to claim 2 chelates and clathrates. wherein the metal is in a form Selected from the group 21. The method according to claim 15 wherein the mam consisting of free metal ions, inorganic metal Salts, organic mal is also administered an activator for a photodynamic metal Salts, metal oxides, metal hydroxides, metal Sulfides, agent or a Sonodynamic agent, Said activator Selected from coordination compounds, chelates, and clathrates. the group consisting of transition metals, chelants, a com 4. A photodynamic composition comprising a photody pound that exhibits increased thiobarbituric acid resistance namic agent and at least one metal. in the presence of a metal and hydrogen peroxide, a reduc 5. The photodynamic composition according to claim 4 tant, a macrophage/neutrophil Stimulator, and compounds wherein the metal is Selected from the group consisting of with prooxidant activity. transition metals, lanthamides, and actinides. 22. A method for enhancing the formation of free radicals 6. The photodynamic composition according to claim 5 comprising Subjecting the combination of a Sonodynamic wherein the metal is in a form Selected from the group agent and an activator for the Sonodynamic agent to Sound consisting of free metal ions, inorganic metal Salts, organic WWCS. metal Salts, metal oxides, metal hydroxides, metal Sulfides, 23. The method according to claim 22 wherein the acti coordination compounds, chelates, and clathrates. Vator is Selected from the group consisting of iron, reduc 7. A method for enhancing the formation of free radicals tants, chelants, and mixtures thereof. comprising Subjecting the combination of a photodynamic 24. The method according to claim 15 wherein the Sono agent and a metal to light waves. dynamic agent is a quinone compound. 8. The method according to claim 7 wherein the metal is 25. The method according to claim 24 wherein the Selected from the group consisting of transition metals, quinone compound is generated from an azo dye upon lanthamides, and actinide. exposure to ultrasound. 9. The method according to claim 8 wherein the metal is 26. The method according to claim 24 wherein the in a form Selected from the group consisting of free metal quinone compound is an anthraquinone. ions, inorganic metal Salts, organic metal Salts, metal oxides, 27. The method according to claim 23 wherein the acti metal hydroxide, metal Sulfides, coordination compounds, Vator comprises a mixture of iron, a reductant, and a chelant. chelates, and clathrates. 28. A method for generating free radicals comprising 10. The method according to claim 7 wherein the com Subjecting aqueous ferrous iron in the presence of a reducing bination of photodynamic agent and a metal further includes agent to ultrasound. a compound that produces a bicarbonate. 29. The method according to claim 27 wherein the reduc 11. A method for enhancing the formation of free radicals ing agent is oxidized ascorbic acid. comprising Subjecting the combination of a Sonodynamic 30. The method according to claim 29 wherein the iron is agent and a metal to Sound waves. in the form of ferritin. 12. The method according to claim 11 wherein the metal 31. The method according to claim 15 wherein the acti is Selected from the group consisting of transition metals, Vator is a combination of iron and ascorbic acid and at least lanthamides, and actinide. one of the activators is encapsulated in a material which is 13. The method according to claim 12 wherein the metal destroyed by contact with ultrasound. is in a form Selected from the group consisting of free metal 32. A Sonodynamic composition comprising a Sonody ions, inorganic metal Salts, organic metal Salts, metal oxides, namic agent, at least one metal, and at least one compound metal hydroxide, metal Sulfides, coordination compounds, that enhances free radical production. chelates, and clathrates. 33. The Sonodynamic composition according to claim 32 14. The method according to claim 12 wherein the com further including at least one compound that alters cell bination of Sonodynamic agent and a metal further includes membrane permeability. a compound that produces a bicarbonate. 34. The Sonodynamic composition according to claim 33 15. A method for treating a mammal by photodynamic further including a compound that exhibits iron release from therapy or Sonodynamic therapy comprising administering a biological compounds containing iron in the presence of photodynamic agent or a Sonodynamic agent and a metal to ultrasound. the mammal and exposing the mammal to light waves or to 35. A photodynamic composition comprising a photody Sound waves. namic agent, at least one metal, and at least one compound 16. The method according to claim 15 wherein the metal that enhances free radical production. is administered simultaneously with the photodynamic 36. The method according to claim 7 wherein the com agent. bination of a photodynamic agent and a metal further 17. The method according to claim 15 wherein the metal includes are least one member of the group consisting of is administered prior to administration of the photodynamic compounds that Show increased thiobarbituric acid reactive agent or the Sonodynamic agent. substances (TBARS) in the presence of a metal and hydro US 2003/0082101 A1 May 1, 2003 27 gen peroxide, compounds that exhibit iron release from diospyrin, naphthazarin, tricroZarin A, actinorhodine, biological compounds containing iron in the presence of euclein, and atovaquone, hydroxylated 1,4-benzoquinones, ultrasound, chelants which produce free radical production their derivatives, isomers, metal coordination compounds, when exposed to ultrasound including aminocarboxylates Salts, and polymers thereof, hydroxylated anthraquinones, and their Salts, derivatives, isomers, polymers, and iron their derivatives, isomers, metal coordination compounds, coordination compounds, reducing agents, chelants that Salts, and polymers, hydroxylated anthraquinones and their have available a coordination site that is free or occupied by derivatives, including alizarin, aloe-emodin, anthragallol, an easily displaceable ligand, tartaric acid, glucoheptonic aurantio-obtusin, barbaloin, cascaroside A, cassiamin C, acid, glycolic acid, 2-hydroxyacetic acid; 2-hydroxypro 7-chloroemodi, chrysazin, chryso-obtusin, chrysophanic panoic acid, 2-methyl 2-hydroxypropanoic acid; 2-hydrox acid 9-anthrone, digiferrugineol, 1,4-dihydroxy-2-methy ybutanoic acid; phenyl 2-hydroxyacetic acid; phenyl 2-me lanthraquinone, frangulin A, frangulin B, lucidin, morin thyl 2-hydroxyacetic acid; 3-phenyl 2-hydroxypropanoic done, norobtusifolin, obtusifolin, phyScion, pseudopurpurin, acid; 2,3-dihydroxypropanoic acid; 2,3,4-trihydroxybu purpurin, danthron, and rubiadin; flavonoids including tanoic acid; 2,3,4,5-tetrahydroxypentanoic acid; 2,3,4,5,6- kaempferol, quercetin, and myricetin and Sesquiterpenes pentahydroxyhexanoic acid; 2-hydroxydodecanoic acid; including gossypol and feralin, cacetin, apigenin, biochanin 2,3,4,5,6,7-hexahydroxyheptanoic acid; diphenyl 2-hy A, daidzein, equiol, flavanone, flavone, formononetin, genis droxyacetic acid, 4-hydroxymandelic acid, 4-chloroman tin, glabranin, liquiritigenin, luteolin, miroestrol, naringenin, delic acid; 3-hydroxybutanoic acid, 4-hydroxybutanoic acid; naringin, phaseollin, phloretin, prunetin, robinin, and 2-hydroxyhexanoic acid; 5-hydroxydodecanoic acid; 12-hy Sophoricoside, derivatives, polymers, and glycosylated droxydodecanoic acid, 10-hydroxy decanoic acid; 16-hy forms thereof, anti-tumor antibiotic quinoid agents includ droxyhexadecanoic acid; 2-hydroxy-3-methylbutanoic acid; ing benzoquinones, mitimycins, Streptonigrins, actinomy 2-hydroxy-4-methylpentanoic acid; 3-hydroxy-4-methoxy cins, anthracyclines, and Substituted anthraquinones; thiol mandelic acid, 4-hydroxy-3-methoxymandelic acid; 2-hy compounds, their derivatives, and polymers including cyS droxy-2-methylbutanoic acid; 3-(2-hydroxyphenyl) lactic teinylglycine, cysteamine, thioglycollate and glutathione, acid; 3-(4-hydroxyphenyl) lactic acid; hexahydromandelic Captopril, Pyritinol (pyridoxine disulfide), Thiopronine, acid; 3-hydroxy-3-methylpentanoic acid, 4-hydroxyde Piroxicam, Thiamazole, 5-Thiopyridoxine, Gold sodium thi canoic acid; 5-hydroxydecanoic acid; aleuritic acid; 2-hy omalate, bucillamine, 1-(mercaptomethyl)-7,7-dimethylbi droxypropanedioic acid; 2-hydroxybutanedioic acid; eryth cyclo2.2.1]heptan-2-one, 1,2,3-benzotriazine-4(3H)- raric acid; threaric acid, arabiraric acid, ribaric acid; Xylaric thione; 1,2-benzisothiazole-3(2H)-thione-1,1-dioxide;1,2- acid, lyxaric acid; glucaric acid, galactaric acid, mannaric dihydro-3H-1,2,4-triazole-3-thione; 1,2-dihydro-3H-1,2,4- acid; gularic acid; allaric acid; altraric acid, idaric acid; triazole-3-thione and derivatives; 1,2-dihydro-4,5-dimethyl talaric acid; 2-hydroxy-2-methylbutanedioic acid, citric 2H-imidazole-2-thione; 1,3-dihydro-1-methyl-2H acid, isocitric acid, agaricic acid, quinic acid; glucuronic imidazole-2-thione; 1,3-dihydro-2H-naphth 2,3-d acid; glucuronolactone; galacturonic acid, galacturonolac imidazole-2-thione; 1,3-dihydro-4,5-diphenyl-2H tone; uronic acids; uronolactones, dihydroascorbic acid; imidazole-2-thione, 1,4-benzoxazepine-5(4H)-thione, 1,4- dihydroxytartaric acid; tropic acid, ribonolactone; glucono dihydro-5H-tetrazole-5-thione and derivatives; 1,5-dihydro lactone, galactonolactone; gulonolactone, mannonolactone; 4H-pyrazolo 3,4-dpyrimidine-4-thione; 1,5-dihydro-6H ribonic acid; gluconic acid; citramalic acid; pyruvic acid; imidazo[4,5-cpyridazine-6-thione; 1,7-dihydro-6H-purine hydroxypyruvic acid; hydroxypyruvic acid phosphate; 6-thione, 1-adamantanethiol, 201H)-benzimidazolinethione; methylpyruvate, ethyl pyruvate; propyl pyruvate, isopropyl 2,4-diamino-6-mercapto-1,3,5-triazine; 2,4-dimethylben pyruvate; phenyl pyruvic acid; methyl phenyl pyruvate; Zenethiol; 2,5-dimethylbenzenethiol; 2,6-dimethylben ethyl phenyl pyruvate, propyl phenyl pyruvate, formyl for Zenethiol, 2-adamantanethiol, 2-amino-1,7-dihydro-6H-pu mic acid; methyl formyl formate; ethyl formyl formate; rine-6-thione, 2H-1,4-benzothiazine-3(4H)-thione; propyl formyl formate; benzoyl formic acid; methylbenzoyl 2-imidazolidinethione; 2-Isopropyl-3-methylbenzenethiol; formate; ethyl benzoyl formate; propyl benzoyl formate; 2-isopropyl-4-methylbenzenethiol, 2-isopropyl-5-methyl 4-hydroxybenzoyl formic acid, 4-hydroxyphenyl pyruvic benzenethiol; 2-mercapto-4H-1-benzopyran-4-thione; acid; 2-hydroxyphenylpyruvic acid, chelants which increase 2-mercapto-5-methyl-1,3,4-thiadiazole, 2-mercapto-5-ni free radical production when exposed to ultrasound and a trobenzimidazole, 2-mercaptothiazoline, 2-methyl-1-pro metal, including adenosine diphosphate (ADP), adenosine penethiol, 2-methylene-1,3-propanedithiol, 2-propene-1- triphosphate (ATP) and guanosine triphosphate (GTP), thiol: 3,4-dihydro-4,4,6-trimethyl-1-(4-phenyl-2-thiazolyl)- reducing agents including ascorbic acid, 1,4-naphtho 2(1H)-pyrimidinethione; 3,4-dihydro-4,4,6-trimethyl quinone derivatives, 1,4 benzoquinone derivatives, and 1,4- 2(1H)-pyrimidinethione; 3-amino-5-mercapto-1H-1,2,4- anthraquinone derivatives and/or thiols, phosphonoformic triazole, 3-bromo-1-adamantanethiol, 3-mercapto-5-methyl acid, phosphonoacetic acid, and pyrophosphate, biological 1,2,4-triazole and derivatives; 3-mercaptocyclohexanone chelants including ADP, ATP, and GTP, tetracycline antibi and derivatives; 3-quinuclidinethiol, 3-thio-9,10-secoc otics and their derivatives, Salts, and polymers thereof, holesta-5,7,10(19)-triene, 4-amino-2,4-dihydro-5-phenyl hydroxy-1,4-naphthoguinones, their derivatives, isomers, 3H-1,2,4-triazole-3-thione; 4-amino-3-hydrazino-5-mer metal coordination compounds, Salts, and polymers thereof, capto-1,2,4-triazole; 4-benzocyclobutenethiol; including 1,4-naphthalenedione, 2,3-dihydroxy; 1,4-naph 4-biphenylthiol, 4-Isopropyl-2-methylbenzenethiol; 5,6- thalenedione, 2.5,8-trihydroxy; 1,4-naphthalenedione, 2-hy dichloro-2-mercapto-1H-indole, 5'-amino-2',3,3,4-tetrahy droxy; 1,4-naphthalenedione, 2-hydroxy-3-(3-methylbutyl); dro-4,4,6-trimethyl-2,21-dithioXo1(2H),4'-bipyrimidin 1,4-naphthalenedione, 2-hydroxy-3-methyl, 1,4-naphtha 6'(1H)-one; 5-isopropyl-2-methylbenzenethiol; lenedione, 5,8-dihydroxy-2-methyl; alkannin; alkannin dim 5-mercapto-3-methyl-1,2,4-thiadiazole, 6-amino-2-mercap ethylacrylate; aristolindicquinone, chleone A, droSerone, iso topurine, 6-thioinosine; 7-(mercaptomethyl)-1,7-dimethyl US 2003/0082101 A1 May 1, 2003 28 bicyclo2.2.1]heptan-2-one; 7-mercapto-3H-1,2,3-triazolo phenylmethanethiol; Potassium 2,6-bis(2,4,6-triisopropy 4,5-dpyrimidine, Azothiopyrine; benzocthiophene lphenyl)phenylthiolate; Rubidium 2,6-bis(2,4,6-triisopro 1(3H)-thione; bis(1-methylethyl)carbamothioic acid S-(2,3, pylphenyl)phenylthiolate; Sodium 2,6-bis(2,4,6-triisopro 3-trichloro-2-propenyl) ester; Caesium 2,6-bis(2,4,6- pylphenyl)phenylthiolatedrugs classified as penicillins, triisopropylphenyl)phenylthiolate; (3,3)-cholest-5-ene-3- cephalosporins, and pirOXicam, reducing agents including thiol; Cyclohexanethione; Lithium 2,6-bis(2,4,6- Sodium Sulfide and Sodium Sulfite. triisopropylphenyl)phenylthiolate; naphtho1,2-dthiazole 2(1H)-thione; naphtho2,1-dthiazole-2(3H)-thione;