RU2791572C1 - Sonodynamic therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to methods for sonodynamic therapy, including combined administration of a microbubble-chemotherapeutical agent complex together with a microbubble-sonosensitizer complex. In this case, the specified chemotherapeutical agent is 5-fluorouracil, doxorubicin, gemcitabine, docetaxel, or paclitaxel, and the specified sonosensitizer is Bengal red dye. Pharmaceutical compositions containing the specified complexes and their use in methods for sonodynamic therapy in the treatment of cancer, such as, for example, pancreatic cancer, are also proposed.

EFFECT: combined use of a microbubble-chemotherapeutical agent complex with a microbubble-sonosensitizer complex in a method for sonodynamic therapy leads to improvement of the efficiency of treatment of deeply located tumors.

33 cl, 19 dwg, 1 tbl, 17 ex

Description

FIELD OF TECHNOLOGY

This invention relates to improvements and relates to methods of sonodynamic therapy and, in particular, to the treatment of diseases characterized by hyperproliferative cells and/or idioplasts. More specifically, the invention relates to the targeted treatment of deep-seated tumors using combined sonodynamic and anti-cancer therapy.

The invention also relates to certain novel sensitizers, processes for their preparation and their use as sensitizers in photodynamic therapy (PDT) and/or sonodynamic therapy (SDT) methods. It also relates to the use of some of these agents as near infrared (NIR) imaging agents and their use in PDT diagnostic imaging techniques.

BACKGROUND OF THE INVENTION

The traditionally accepted treatment for deep-seated tumors usually involves extensive surgery, chemotherapy, radiation therapy, or a combination of all of these factors. All three interventions can lead to various complications, including sepsis. Thus, the search for more targeted and less invasive therapeutic approaches with improved treatment efficacy in these patients is in high demand. Pancreatic cancer is one example of a deep-seated tumor. It remains one of the most deadly types of cancer, with less than 20% of diagnosed patients eligible for curative surgery. It accounts for about 2% of all cancers with a five-year survival rate of 15-21% in patients with surgical resection followed by systemic chemotherapy.

Methods known for use in the treatment of cancer include photodynamic therapy (PDT). PDT involves the application of photosensitive agents to the affected area followed by exposure to photoactivating light to convert them into a cytotoxic form. This leads to the destruction of cells and the surrounding vasculature in the target tissue. The photosensitizers currently approved for use in PDT absorb light in the visible region (below 700 nm). However, light of this wavelength has a limited ability to penetrate the skin; it penetrates to a depth of only a few mm from the surface. While PDT can be used to treat deeper target cells, it usually involves using a device such as a fiber optic catheter system to activate the photosensitizer. This is not only a complex procedure, but also excludes access to certain areas of the body. It also compromises the non-invasive nature of the treatment. Thus, although suitable for the treatment of superficial tumors, the use of PDT in the treatment of deep-seated cells, such as tumor masses and anatomically less accessible lesions, is limited.

Sonodynamic therapy (SDT) is a newer concept and involves a combination of ultrasound and a sonosensitizer (also referred to herein as a "sonosensitizer"). Like PDT, activation of the sonosensitizer by acoustic energy results in the formation of reactive oxygen species (ROS), such as singlet oxygen, at the target site of interest. Such species are cytotoxic, thereby killing target cells or at least reducing their proliferative potential. Many known photosensitizing agents can be activated with acoustic energy and are thus suitable for use in SDT. Because ultrasound easily travels through several cm of tissue, SDT provides a way to treat tumors that are located deep within tissues. As with light, ultrasound energy can also be focused on the tumor mass to activate the sonosensitizer, thereby limiting its effect on the target site.

SDT offers some significant advantages over PDT: ultrasound is widely recognized as a cost-effective and safe clinical intravision method and, unlike light, can be highly focused with soft tissue penetration up to several tens of centimeters depending on the frequency of the ultrasound.

In WO 2012/143739, sonosensitizers are conjugated to a gas-filled microbubble (MB) to provide a microbubble-sonosensitizer complex for use in SDT. These complexes provide efficient delivery of the active sonosensitizer in a site-specific manner by controlled disruption of the vesicle using ultrasound. Subsequent or simultaneous sono-activation of the target sonosensitizer leads to cell destruction at the target site and regression of tumor tissues. The use of microbubbles also results in a reduction in toxic side effects due to screening of the sonosensitizer from potential light activation before reaching the desired target site.

Recently, the inventors have demonstrated the effectiveness of SDT using a microbubble-sleep sensitive agent complex for the treatment of pancreatic cancer in a preclinical model (McEwan et al. J Control Release. 2015; 203, 51-6). These studies showed that injection of ultrasonic sensitive (MB) microbubbles filled with oxygen gas and carrying Rose Bengal sensitizer provided a statistically significant SDT-mediated reduction in tumor growth in mice bearing a BxPC-3 human tumor xenograft compared to tumors treated with the same MW conjugate containing SF ₆ as core gas. The rationale for incorporating oxygen into the MB core was to increase the amount of ROS generated in the tumor microenvironment during the sonodynamic event, since oxygen is a substrate for ROS production in SDT. Pancreatic tumors are known to be highly hypoxic, and this further negatively impacts the effectiveness of approaches such as PDT/SDT that depend on the use of oxygen to generate cytotoxic ROS.

It has also been demonstrated that the combination of standard 5-fluorouracil (5-FUU) and gemcitabine therapy for pancreatic cancer with anti-mitabolites with adjunctive chemotherapy such as irinotecan and oxaliplatin can improve median survival in patients with pancreatic cancer (Lee et al. , Chemotherapy 2013; 59, 273-9). However, this combination, known as FOLFIRINOX, results in significant side effects and is only given to patients who are otherwise fit and healthy.

Thus, there is a need for alternative treatments for deep-seated, inaccessible tumors such as pancreatic cancer, in particular non-invasive or minimally invasive methods and which do not have adverse side effects. Such methods would have clear socio-economic benefits, for example in terms of reducing patient trauma, reducing treatment costs and reducing hospital costs. The present invention solves this need.

SUMMARY OF THE INVENTION

Because antimetabolite therapy and SDT exert their cytotoxic effects through different mechanisms (the first through inhibition of thymidylate synthase and the second through the oxidation of cellular substrates), the inventors now propose that their combination in a single therapeutic regimen may provide significant benefit to patients.

In particular, the inventors have now found that the use of microbubbles to deliver both a sonosensitizer and an antimetabolite confers a number of advantages when used in sonodynamic therapies. In particular, what they found is that delivery of both the sonosensitizer and the antimetabolite as a complex (or complexes) with a microbubble allows for efficient delivery of both agents in a site-specific manner (e.g., to an internal tumor) in a controlled manner. destruction of bubbles using ultrasound. Sono-activation of the target sonosensitizer results in the generation of ROS that destroy tumor cells at the target site. This action is complemented by the action of an antimetabolite, which exerts its cytotoxic effect directly at the intended target site. By using a microbubble as a carrier for both agents, their non-specific uptake by non-target tissues is reduced, which offers a significant advantage over systemic delivery. This therapy is expected to reduce side effects and, in turn, provide significant benefits to patients.

In addition, by using an oxygen-laden MT platform in combination with externally acquired ultrasound to deliver not only oxygen but also the antimetabolite and sensitizer to the tumor microenvironment, the inventors propose to realize highly precise therapy, in particular as a result of increasing the therapeutic performance of the sensitizer and antimetabolite of the chemotherapeutic drug. . The ability of the microbubble to deliver oxygen to the tumor is expected to further enhance such oxygen-dependent therapies to mediate their therapeutic effects.

This document describes the preparation of oxygenated lipid-stabilized MBs (O ₂ MB) with either Rose Bengal (O ₂ MB-RB) or 5-FU (O ₂ MB-5FU) attached to their surface. The resulting conjugates are characterized in terms of MP stability and ultrasound-mediated oxygen evolution and demonstrate the ultrasound-mediated cytotoxicity of combined antimetabolite/SDT treatment in an in vitro pancreatic cancer cell line panel. The therapeutic efficacy of the combined approach was demonstrated using a preclinical xenograft model of an ectopic human pancreatic tumor in mice and compared to conventionally accepted therapeutic approaches using only 5-FU or gemcitabine treatment. There is also evidence that SDT has a significant impact on the signaling processes that mediate the immune response and cell proliferation.

The results presented herein illustrate not only the potential of SDT/antimetabolite combination therapy as a stand-alone treatment option for pancreatic cancer, but also the ability of _O2- filled MBs to deliver _O2 to the tumor microenvironment to enhance the efficacy of therapies that rely on _O2 for mediating their therapeutic effect, the use of MB to facilitate O ₂ delivery as well as the sensitizer/antimetabolite, combined with the ability to activate the sensitizer with external sonication, provides a more targeted approach with improved efficacy and reduced side effects compared to conventional systemic routes of administration alone. antimetabolites..

This new approach to the treatment of pancreatic cancer extends to the treatment of other diseases and pathological conditions characterized by hyperproliferative and/or abnormal cells, in particular to the treatment of other deep-seated tumors. As will be described herein, this approach thus has a broader application that extends to the treatment of other such diseases and pathological conditions using other chemotherapeutic drugs.

DETAILED DESCRIPTION OF THE INVENTION

In its broadest aspect, the invention provides a microbubble-chemotherapeutic agent complex for use in a sonodynamic therapy method. In the context of this document, the term "sonodynamic therapy" is intended to refer to a method involving the combination of ultrasound and a sonosensitizer (also referred to herein as a "sonosensitizer drug"), in which activation of the sonosensitizer by acoustic energy results in the generation of reactive oxygen species such as singlet oxygen. .

The microbubble-chemotherapeutic agent complex comprises a microbubble attached to or otherwise associated with at least one chemotherapeutic agent. Where the microbubble is attached to more than one chemotherapeutic agent, these agents may be the same or different. However, in general, the chemotherapeutic agents attached to a particular microbubble will be identical. To the extent that such a complex is intended for use in SDT methods, it will be sensitive to ultrasound. In particular, it is contemplated that the microbubble component of the complex can be disrupted by the application of ultrasound, thereby releasing the chemotherapeutic agent at the desired target site.

The chemotherapeutic agent (or agents) can be associated with the microbubble in a covalent or non-covalent manner, for example, by electrostatic interaction, van der Waals forces and/or hydrogen bonding. In one embodiment, the microbubble is electrostatically bonded to the chemotherapeutic agent. In another embodiment, it may be covalently linked, ie the chemotherapeutic agent will be attached to the microbubble by one or more covalent bonds.

Preferably, the interaction between the chemotherapeutic agent (or agents) and the microbubble will include strong non-covalent binding, such as the interaction of biotin with avidin. In this embodiment, one component of the binding pair (eg, chemotherapeutic agent) is functionalized with biotin and the other (eg, microbubble) with avidin. Because avidin contains multiple binding sites for biotin, it will typically also be associated with the microbubble through the biotin-avidin interaction. For example, a microbubble can be functionalized with biotin to form a biotinylated microbubble which is then incubated with avidin. When avidin is bound to the vesicle, it allows any other biotinylated moieties to be bound, such as a chemotherapeutic agent. Thus, the resulting bond between the microbubble and the chemotherapeutic agent can take the form of a biotin-avidin-biotin interaction.

In the context of this document, the term "chemotherapeutic agent" is intended to broadly cover any chemical or biological compound useful in the treatment of cancer. It includes growth inhibitory agents and other cytotoxic agents. The term "growth inhibitory agent" refers to a compound that inhibits the growth of a cell, especially a cancer cell, either in vitro or in vivo.

Suitable classes of chemotherapeutic agents for use in this invention, and examples within those classes, include the following: antifolates (eg, methotrexate); 5-fluoropyrimidines (eg 5-fluorouracil or 5-FU); cytidine analogs (eg, gemcitabine); purine antimetabolites (eg mercaptopurine); alkylating agents (eg cyclophosphamide); non-classical alkylating agents (eg dacarbazine); platinum analogues (eg, cisplatin); anticancer antibiotics (eg actinomycin D, bleomycin, mitomycin C); bioreductive drugs (eg mitomycin C, banoxantrone (AQ4N)); anthracyclines (eg doxorubicin, mitoxantrone); topoisomerase I inhibitors (eg irinotecan); topoisomasase II inhibitors (eg etoposide); anti-microtubulin agents such as vinca alkaloids (eg vincristine), taxols (eg paclitaxel) and epothilones (eg ixabepilone); antiestrogen (eg, tamoxifen); antiandrogens (eg, biclutamide, cyproterone acetate); aromatase inhibitors (eg anastrozole, formestane); anti-angiogenic or hypoxia targeting drugs (naturally occurring, eg endostatin or synthetic, eg gefitinib, lenalidomide); antivascular agents (eg, cambrestatin); tyrosine kinase inhibitors (eg, gefitinib, erlotinib, vandetanim, sunitinib); agents targeting an oncogenic or signaling pathway (eg, tipifarnib, lonafarnib, naltrindol, rapamycin); stress protein targeting agents (eg geldanamycin and analogues thereof); autophagy targeting agents (eg, chloroquine); proteasome targeting agents (eg, bortezomib); telomerase inhibitors (targeted oligonucleotides or nucleotides); histone deacetylase inhibitors (eg, trichostatin A, valproic acid); DNA methyltransferase inhibitors (eg decitabine); alkylsulfonates (eg busulfan, improsulfan and piposulfan); aziridines (eg, benzodopa, carbokwon, meturedopa, and uredopa); ethyleneimines and methylamelamines (eg, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine); nitrogen mustards (eg, chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard); nitrosoureas (eg carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine); purine analogs (eg, fludarabine, 6-mercaptopurine, thiamiprin, thioguanine); pyrimidine analogs (eg ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, fluxuridine); androgens (eg calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone); and anti-adrenal agents (eg, aminoglutethimide, mitotane, trilostane). Pharmaceutically acceptable salts, derivatives or analogues of any of these compounds may also be used.

Examples of growth inhibitory agents for use in the present invention include agents that block cell cycle progression (at a location other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classic M-phase blockers include vinca alkaloids (vincristine and vinblastine); members of the taxane family, including paclitaxel, docetaxel and analogues thereof; topoisomerase inhibitors such as irinotecan, topotecan, camptothecin, lamellarin D, doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. G1 arresting agents include, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-FU, and ara-C.

The choice of chemotherapeutic agent will depend on various factors, including the nature of the tumor, the patient being treated, etc., but can be easily selected by those skilled in the art.

In one specific embodiment, the chemotherapeutic agent is an antimetabolite. Antimetabolites that are particularly suitable for use in the present invention include anti-folates, purine and pyrimidine antimetabolites, and antibiotics. One example of an antimetabolite that can be used in the treatment of pancreatic cancer is 5-fluorouracil (5-FU).

For use in SDT, the microbubble-chemotherapeutic agent complex is used in combination with at least one sonosensitizer (eg, a plurality of sonosensitizers) that is also associated with the microbubble (herein "microbubble-sonosensitiser complex"). The sonosensitizer may be associated with the same microbubble as the chemotherapeutic agent, or alternatively, it may be associated with a separate microbubble. Typically, the two agents will be associated with separate microbubbles.

The microbubble-sonosensitizer complex comprises a microbubble attached to or otherwise associated with at least one sonosensitizer, preferably a plurality of sonosensitizers. Where the microbubbles are attached to more than one sonosensitiser, they may be the same or different. However, in general, the sonosensitizers will be the same. To the extent that such a complex is intended for use in SDT methods, it will be sensitive to ultrasound. In particular, it is contemplated that the microbubble component of the complex can be disrupted by the application of ultrasound, thereby releasing the sonosensitizer at the desired target site. As described herein, activation of a sonosensitizer by acoustic energy also results in the formation of reactive oxygen species such as singlet oxygen, which are cytotoxic.

The sonosensitizer (or sonosensitizers) can be associated with the microbubble through covalent or non-covalent, for example, through electrostatic interaction, van der Waals forces and/or hydrogen bonding. In one embodiment, the microbubble is electrostatically coupled to the sonosensitiser. In another embodiment, it may be covalently bonded, ie the sonosensitiser will be attached to the microbubble by one or more covalent bonds. Preferably, however, the interaction will involve strong non-covalent binding, such as the interaction of biotin with avidin, as described above.

When a biotin-avidin interaction is used to bind the sonosensitizer(s) to a microbubble, one component of the binding pair (eg, the sonosensitizer) is functionalized with biotin and the other (eg, the microbubble) with avidin. Typically, the avidin molecule is also bound to the microbubble through the biotin-avidin interaction. For example, a microbubble can be functionalized with biotin to form a biotinylated microbubble which is then incubated with avidin. Once avidin is bound to the vesicle, this allows any other biotinylated moieties to be bound, such as the sonosensitizer. The resulting bond between the microbubble and the sonosensitizer may thus take the form of a "biotin-avidin-biotin" interaction.

In the context of this document, the term "microbubble" is intended to mean a microsphere containing a shell having an approximately spherical shape and which surrounds an internal void containing a gas or mixture of gases. "Shell" refers to the membrane that surrounds the internal void of a microbubble.

Microbubbles are well known in the art, for example as ultrasonic contrast agents. Thus, their composition and methods for their preparation are well known to those skilled in the art. Examples of methods for producing microbubbles are described, for example, in Christiansen et al., Ultrasound Med. Biol., 29: 1759-1767, 2003; Farook et al, J. R. Soc. Interface, 6: 271-277, 2009; and Stride & Edirisinghe, Med. Biol. Eng. Comput., 47: 883-892, 2009, the contents of which are hereby incorporated herein by reference.

Microbubbles contain a shell that surrounds an internal void containing gas. They are generally approximately spherical in shape, although the shape of the microbubble is not essential to the practice of the invention and is therefore not considered limiting. The size of the microbubble should be such that it can pass through the pulmonary system after administration, for example by intravenous administration. The microbubbles typically have a diameter of less than about 200 microns, preferably in the range of about 0.5 to about 100 microns. Particularly suitable for use in the present invention are microbubbles having a diameter of less than about 10 µm, more preferably from 1 to 8 µm, particularly preferably up to 5 µm, for example about 2 µm. The microbubble shell will vary in thickness and will typically be from about 10 to about 200 nm. The exact thickness is not essential as long as the sheath performs the desired function of containing the gas core.

Materials that can be used to form microbubbles must be biocompatible, and suitable materials are well known in the art. Typically, the microbubble shell will contain a surfactant or polymer. Surfactants that can be used include any material that is capable of forming and maintaining a microbubble by forming a layer at the interface between the gas inside the core and the outside, for example an aqueous solution that contains a microbubble. A surfactant or combination of surfactants may be used. Suitable include lipids, in particular phospholipids. Lipids that can be used include lecithins (ie phosphatidylcholine), for example natural lecithins such as egg yolk lecithin or soybean lecithin and synthetic lecithins such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine; phosphatidic acids; phosphatidylethanolamines; phosphatidylserines; phosphatidylglycerols; phosphatidylinositols; and their mixtures. The use of phospholipids having an overall net charge (eg, a negative charge), such as, for example, those derived from soybeans or egg yolk; phosphatidylserines; phosphatidylglycerols; phosphatidylinositols; and phosphatidic acids, is advantageous for binding the microbubble to the sonosensitiser via ionic bonding. In one embodiment, a long chain lipid such as dibegenoylphosphatidylcholine (DBPC) can be used to form the microbubble shell.

Suitable lipids can be selected based on their ability to improve the stability of the microbubbles with respect to oxygen retention. Suitable for use in this respect is dibegenoylphosphatidylcholine (DBPC).

Polymeric materials that are suitable for use in forming the microbubble shell include proteins, in particular albumin, in particular human serum albumin. Other biocompatible polymers that can be used include polyvinyl alcohol (PVA), poly(D,L-lactide-co-glycolide) (PLGA), cyanoacrylate, poloxamers (Pluronics), or combinations thereof.

Microbubble shells may contain one or more layers of the same or different materials. Multiple layers may, for example, be formed when the main shell material (eg lipid) carries one or more polymers or polysaccharides. Examples of such polymers include polyethylene glycol and polyvinylpyrrolidone.

The microbubble shell can also be coated with polymers such as poly-L-lysine and PLGA and/or polysaccharides such as alginate, dextran, diethylaminoethyldextran hydrochloride (DEAE) or chitosan. Methods for attaching these coating materials may include electrostatic or covalent interaction. Various coating materials (polymers, polysaccharides, proteins, etc.) can be used to improve microbubble properties, for example, by increasing the stiffness, circulation stability and/or tissue permeability of microbubble-based reagents by manipulating the net surface charge of the microbubble and perhaps most importantly, by increasing its payload. One way to achieve an increase in payload is to use a staged assembly method (LBL). This includes attaching multiple layers of sonosensitizer to preformed microbubbles to increase the capacity of the sonosensitizer. The LBL method is described by Borden et al. in DNA and polylysine adsorption and multilayer construction onto cationic lipid-coated microbubbles DNA and, Langmuir 23(18): 9401-8, 2007.

In addition, microbubble coating can improve payload stability, especially when the coating material serves as an immobilization matrix for a sonosensitizer or chemotherapeutic agent (eg, by crosslinking).

Lipids forming a monolayer, bilayer or multilayer structure can also be used. Examples of such lipids are unilamellar or multilamellar liposomes and micelles.

Any of the microbubble shells described herein may contain additional components that aid in the delivery of the bubble to the target site. For example, they may be functionalized such that they include or bind to it a ligand or targeting agent that is capable of binding to a target cell or tissue. Examples of suitable targeting agents include antibodies and antibody fragments, cell adhesion molecules and their receptors, cytokines, growth factors and receptor ligands. Such agents can be attached to microbubbles using methods known in the art, for example, by covalent coupling using molecular spacers (eg, PEG) and/or avidin-biotin complex formation. For example, incorporation of a lipid-PEG-biotin conjugate into lipid-based microvesicles followed by the addition of avidin allows the surface of the microvesicles to be functionalized with a biotinylated targeting ligand. Herceptin is an example of an antibody that can be conjugated to a microbubble coat for targeting.

The gas at the core of the microbubbles must be biocompatible. The term "gas" covers not only substances that are gaseous at room temperature and pressure, but also those that are in liquid form under given conditions. When the "gas" is liquid at ambient temperature, it usually undergoes a phase transition to a gas at a temperature of 30°C or higher, more preferably 35°C or higher. For any gas that is liquid at ambient temperature, it is generally preferred that the compound undergo a phase transition to a gas at a temperature between about 30 and 37°C, preferably at normal body temperature. Thus, any reference to "gas" should be considered to cover not only gases and liquids, but also liquid vapors and any combination thereof, such as a mixture of liquid vapor in a gas.

Gases that are suitable for inclusion in microbubbles for use in this invention include air, nitrogen, oxygen, carbon dioxide, hydrogen; inert gases such as helium, argon, xenon or krypton; sulfur fluorides such as sulfur hexafluoride, disulfur decafluoride; low molecular weight hydrocarbons such as alkanes (eg methane, ethane, propane, butane), cycloalkanes (eg cyclopropane, cyclobutane, cyclopentane), alkenes (eg ethylene, propene); and alkynes (eg acetylene or propyne); ethers; esters; halogenated low molecular weight hydrocarbons; and their mixtures.

Examples of suitable halogenated hydrocarbons are those containing one or more fluorine atoms and include, for example, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons.

Examples of suitable fluorocarbon compounds include perfluorocarbons. Perfluorocarbons include perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes, perfluoropentanes, perfluorohexanes and perfluoroheptanes; perfluoroalkenes such as perfluoropropene, perfluorobutenes; and perfluorocycloalkanes such as perfluorocyclobutane.

Microbubbles containing perfluorinated gases, in particular perfluorocarbons such as perfluoropropanes, perfluorobutanes, perfluoropentanes and perfluorohexanes, are suitable for use in this invention due to their stability in the bloodstream.

In the present invention, microbubbles containing a perfluorocarbon, in particular a perfluoroalkane, and a shell containing a phospholipid can be used, and are described, for example, Nomikou & McHale, Cancer Lett., 296: 133-143, 2010. One example of such a microbubble is Sonidel SDM202 ( available from Sonidel Ltd.). The perfluorocarbon may either be present as a gas or in liquid form. Those bubbles that contain a liquid core can be obtained from nanoemulsions, which can then be turned into a gas microbubble after exposure to ultrasound, for example, as described in Rapoport et al., Bubble Sci. Eng. Technol. 1:31-39, 2009.

Particularly preferred for use in the present invention are microbubbles that carry oxygen. Because oxygen is a key substrate for SDT and many cancers are hypoxic, filling the bladder core with oxygen gas enhances the sonodynamic effect and the amount of singlet oxygen produced.

When the microbubble is loaded with both a chemotherapeutic agent and a sonosensitiser, it will preferably contain oxygen (eg, it will contain oxygen gas). In the case of using different microbubbles in the combination therapy described herein, it is preferred that at least one type of microbubbles includes oxygen. For example, the microbubble conjugated to the sonosensitizer may include oxygen and/or the microbubble carrying the chemotherapeutic agent may include oxygen. In a preferred embodiment, all microbubbles used will contain oxygen (eg O ₂ gas), i. e. said bubbles will be "filled with O ₂ ".

Sonosensitizers that can be used in this invention include compounds that make target cells or tissues hypersensitive to ultrasound. In some cases, the sonosensitizer may be able to convert acoustic energy (eg, ultrasound) into ROS, resulting in cell toxicity. Others may cause hypersensitivity of the target cell or tissues to ultrasound, violating the integrity of the cell membrane. It is well known that many known sonosensitizers can facilitate photodynamic activation and can also be used to image cells or tissues that are sensitive to light.

In one embodiment of the present invention, the sonosensitizer may simultaneously function as an imaging agent, for example, as an NIR agent. Such sensitizers offer advantages in terms of their imaging potential for in vivo tracking of conjugates.

Examples of compounds suitable for use as sonosensitizers in this invention include phenothiazine dyes (e.g. methylene blue, toluidine blue), rose bengal, porphyrins (e.g. Photofrin®), chlorins, benzochlorins, phthalocyanines, naphthalocyanines, porphycenes, cyanines (e.g. , Merocyanine 540 and indocyanine green), azodipyromethines (eg BODIPY and their halogenated derivatives), acridine dyes, purpurins, pheophorbides, verdins, psoralens, hematoporphyrins, protoporphyrins and curcumins. Any known analogues or derivatives of these agents may also be used. Suitable derivatives include pharmaceutically acceptable salts.

Preferred for use as sonosensitizers in this invention are methylene blue, rose bengal, indocyanine green (ICG, also known as Cardio Green) and any analogues and derivatives thereof. The ICG has the following structure:

Known analogues of any of the sonosensitizers described herein can also be used in this invention. Particularly suitable are structural analogs of cyanine-based dyes, for example structural analogs of ICG and their pharmaceutically acceptable salts. Examples of such compounds include cyanine dyes IR820 and IR783, both of which are commercially available:

An FDA-approved near-infrared (NIR) absorbing fluorescent dye for use in medical imaging. It absorbs strongly in the NIR region (750-900 nm) and has the advantage that it can be activated by light at greater depths in human tissue (light penetration at 800 nm is four times that at 600 nm). However, the singlet oxygen generation (SOG) efficiency of cyanine dyes such as ICG, IR820 and IR783 is relatively low compared to other known sensitizers such as rose bengal. This can be overcome by concentrating more cyanine molecules on the microbubble.

Other attempts have been made to improve the ability of cyanine dyes to generate ROS by incorporating halogen atoms (eg, iodine and bromine) into their structure. For example, US 2013/0231604 (the entire content of which is incorporated herein by reference) suggests that cyanine-based dyes and dye analogs can be modified by including three iodine atoms in the benzene or naphthalene moiety of each benzazole or naphthazole. Any of the polymethine dyes (particularly cyanines) described herein can be used as the ROS sonosensitizer of this invention.

In developing the work documented in US 2013/0231604, the authors of this invention have obtained structural analogues of certain cyanine dyes (for example, IR783) bearing either one or two halogen atoms (for example, iodine or bromine, preferably iodine) on each of the benzazole rings, and have an increased ability to generate ROS and thus are more cytotoxic to cancer cells (eg, pancreatic cancer cells) when activated by ultrasound compared to ICG. Without wishing to be bound by theory, we believe that the presence of halogen atoms increases the intercombination conversion (ISC) from an excited singlet to an excited triplet state, which is known as the "heavy atom effect". The excited triplet state can then interact with molecular oxygen or other substrates to generate ROS. That this level of increased ability to generate ROS could be achieved by replacing fewer (i.e., 2 or 4 in total) hydrogen atoms in IR783 with halogen (i.e., iodine) atoms cannot be predicted in light of this fact of US 2013/0231604.

In addition, as will be discussed in more detail below, we surprisingly find that when IR783 is substituted with a total of two halogens (i.e., only one halogen, e.g., iodine, on each of the benzazole rings), the compound remains highly fluorescent and, thus, can also be used as an NIR imaging agent. Since any increase in ISC usually reduces the ability of a compound to emit fluorescence, this finding is unexpected. When combined, the NIR imaging potential and the sensitizer potential of these specific IR783 analogs means that these compounds have "therapeutic-diagnostic" potential, ie the ability to function as a therapeutic and diagnostic agent.

Halogenated (eg, iodinated) analogues of IR783, which are disclosed here, are new chemical objects and represent an additional aspect of the invention. According to a further aspect, the invention thus provides a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof:

(wherein in Formula I and Formula II, each X is independently selected from bromine and iodine, where preferably each X is iodine).

Suitable salts of such compounds and methods for their preparation can be readily selected by those skilled in the art. The compounds may, for example, be converted into a suitable pharmaceutically acceptable salt thereof with an inorganic or organic base. Bases that may be suitable for this purpose include alkali and alkaline earth metal hydroxides, for example sodium hydroxide, potassium hydroxide or cesium hydroxide, ammonia and organic amines such as diethylamine, triethylamine, ethanolamine, diethanolamine, cyclohexylamine and dicyclohexylamine. Methods of salt formation are generally accepted in the art.

Preferred compounds of formula I and II include the following and their pharmaceutically acceptable salts:

Pharmaceutical compositions containing any of the compounds of formula I, II, Ia or IIa, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or excipient, constitute a further aspect of the invention.

These new compounds can be obtained by methods known to experts in this field of technology and are illustrated using the examples given in this document. Methods for preparing such compounds are also part of this invention.

According to a further aspect of the invention, the invention thus provides a process for the preparation of a compound of formula I or II comprising the steps of:

(a) reacting a compound of formula III:

with a compound of formula IV or V:

(wherein, in formula IV, X represents a bromine or iodine atom, and in formula V, each X is independently selected from a bromine and iodine atom); and

(b) optionally converting the resulting compound into a pharmaceutically acceptable salt thereof.

The invention also provides for the use of any of the compounds of formulas I, II, Ia and IIa and their pharmaceutically acceptable salts as a drug, for example, as a therapeutic, diagnostic or therapeutic agent (i.e. having both therapeutic and diagnostic functionality). In particular, they can be applied in a PDT and/or SDT technique or in an in vivo imaging technique (eg NIR imaging), especially in such methods for treatment and/or diagnostic imaging of deeply located cells such as tumor masses. When used in PDT methods, activating light having a wavelength in the near IR spectrum, for example, from 700 to 900 nm, more specifically from 750 to 850 nm, can be used. When activated by NIR light or ultrasonic activation, these compounds have an increased ability to generate ROS and are more toxic to cancer cells (eg, pancreas and cervical cancer cells) than ICG.

The use of compounds of formula I or Ia or their pharmaceutically acceptable salts as NIR imaging agents, preferably as combined sensitizers and NIR imaging agents in PDT and/or SDT, is a preferred embodiment of the invention.

Microbubbles carrying any of the halogenated sensitizers described herein, in particular compounds of formula I, II, Ia, IIa and their pharmaceutically acceptable salts, and methods for preparing such microbubbles are also part of the invention. Such microbubble sensitizer complexes can be prepared using any of the methods described herein with respect to attaching a sonosensitizer to a microbubble. Preferably, such methods will include the step of biotinylating the halogenated sensitizer and coupling it to a microbubble with functionalized biotin-avidin.

Methods for the formation of microbubbles are known in the art. Such methods include forming a gas suspension in an aqueous medium in the presence of a selected sheath material. Methods used to form microbubbles include sonication, high speed agitation (mechanical agitation), coaxial electrohydrodynamic atomization, and T-junction microfluidic processing (see, for example, Stride & Edirisinghe, Med. Biol. Eng. Comput., 47: 883-892, 2009). Ultrasonic processing is widely used and generally preferred. This method can be performed using an ultrasonic emitting transducer. More specifically, an aqueous suspension of microbubble shell components is sonicated in the presence of an appropriate gaseous microbubble component, eg oxygen.

Other methods that can be used to form microbubbles include evaporation of the core of nanodroplets in a nanoemulsion (see, for example, Rapoport et al., supra). The core of such nanodroplets, as a rule, is formed by an organic perfluorine-containing compound, which is coated with the walls of a biodegradable amphiphilic block copolymer, such as poly(ethylene oxide)-copoly(L-lactide) or poly(ethylene oxide)-co-caprolactone. Alternatively, nanoemulsions can be obtained by extrusion through sizing membranes, for example using albumin as the shell material. The transition from drop to bubble can be induced by physical and/or mechanical means, which include heat, ultrasound, and injection through a fine gauge needle. Such microbubbles can be formed at the time of administration to a patient (eg, during the injection step using a fine gauge needle) or in vivo in target cells or tissues (eg, by sonicating the nanoemulsion).

Administration of a nanoemulsion capable of forming the desired microbubble complex(s) as defined herein, either at the stage of administration to a patient or after administration (ie, in vivo), is within the scope of this invention. When it is desired that the resulting microbubble contains oxygen gas, this can be provided in dissolved form in the liquid core of the perfluorocarbon phase shift nanoemulsion.

The microbubble complexes provided herein can be prepared using methods and techniques known in the art. Methods that can be used to covalently attach a chemotherapeutic agent and/or a sonosensitiser to a microbubble include known chemical bonding techniques. The particular method used will depend on the precise nature of the microbubbles, the chemotherapeutic agent and the sonosensitizer, in particular the nature of any pendant functional groups. If necessary, one or both components of the complex (eg, microbubble and/or sonosensitivity or microbubble and/or chemotherapeutic agent) can be functionalized, for example, to include reactive functional groups that can be used to connect molecules. Suitable reactive groups include an acid group, hydroxy, carbonyl, acid halide, thiol, and/or a primary amine. Methods for introducing such functional groups are well known in the art.

Examples of methods that can be used to covalently link microbubbles to one or more chemotherapeutic agents and/or sonosensitizers include, but are not limited to, the following: a) carbodiimide-based linking methods. They can be used to link microbubbles containing an amino or carboxyl functional group to a moiety having either a carboxyl or amino functional group. Such methods lead to the formation of ester or amide bonds; b) "CLICK" reaction (i.e. 1,3-dipolar cycloaddition reaction). This reaction can be used to interact with azide- or acetylene-functionalized microbubbles with a fragment having either an acetylene or azide functional group; c) the formation of a Schiff base (ie the formation of an imino bond). This reaction can be used to couple aldehyde or amine functionalized microbubbles to an amino or aldehyde functional moiety; and d) Michael addition reaction.

Linking the microbubble to one or more chemotherapeutic agents and/or sonosensitizers via a biotin-avidin bond can be accomplished by methods known to those skilled in the art. In such methods, both parts will typically be biotinylated and avidin is then used to form a bond between them. An example of a method for preparing a microbubble-chemotherapeutic agent conjugate bound by the interaction of biotin with avidin is shown in Scheme 1 in Example 2.

As an alternative to combining a chemotherapeutic agent and/or a sonosensitizer with a preformed microbubble, these fragments can alternatively be linked to a lipid (e.g., using any of the methods described above) and that the lipid can subsequently be incorporated into the lipid envelope of the microbubble during its preparation.

Charged sonosensitizers and/or chemotherapeutic agents may be electrostatically coupled to the charged microbubble. For example, an anion bubble may be associated with a cationic sonosensitiser or a cationic chemotherapeutic agent and vice versa. One example of a charged sonosensitizer is methylene blue, which can be electrostatically attached to an anionic microbubble.

Examples of methods for preparing a microbubble-sonosensitizer complex are described in WO 2012/143739, the entire contents of which are incorporated herein by reference. As an example, in FIG. 2 is (a) a schematic illustration of the preparation of a rose bengal derivative (labeled "RB1") and (b) a schematic illustration of the covalent bonding of RB1 to a microbubble. Any of the methods described in WO 2012/143739 can be used similarly to the method for preparing a microbubble-chemotherapeutic agent complex described herein.

The microbubble-chemotherapeutic agent complexes described herein are novel in themselves and form a further aspect of the invention. In one embodiment, these complexes may also be associated with one or more of the sonosensitizers described herein. Methods for preparing microbubble-chemotherapeutic agent complexes comprising the step of coupling at least one chemotherapeutic agent to the microbubble, for example using any of the methods described herein, form another aspect of the invention.

The microbubble complexes described herein have properties that make them useful in sonodynamic therapy techniques.

The complexes are useful in the treatment of disorders or abnormalities of cells or tissues in the body that are responsive to sonodynamic therapy. These include malignant and precancerous conditions such as cancerous tumors or tumors and their metastases; tumors such as sarcomas and carcinomas, in particular solid tumors. The invention is particularly useful in the treatment of tumors, especially those below the surface of the skin.

Examples of tumors that can be treated using this invention are sarcomas, including osteogenic and soft tissue sarcomas; carcinomas, eg breast, lung, brain, bladder, thyroid, prostate, colon, rectum, pancreas, stomach, liver, uterus, liver, kidney, prostate, cervical and ovarian carcinomas; lymphomas including Hodgkin's lymphoma and non-Hodgkin's lymphoma; neuroblastoma, melanoma, myeloma, Wilms tumor; leukemias, including acute lymphoblastic leukemia and acute myeloid leukemia; astrocytomas, gliomas and retinoblastomas. The treatment of pancreatic cancer is a preferred aspect of the present invention.

In one aspect, the complexes described herein can be used in a sonodynamic therapy method and simultaneously in an in vivo imaging method (eg, a diagnostic imaging method). In such methods, imaging can be used to monitor payload deposition and/or accumulation of the complex (or complexes) at the object of interest. As described above, this aspect of the invention can be implemented by selecting a sensitizer that has imaging potential, such as a sensitizer that simultaneously functions as an NIR imaging agent. Alternatively, a known imaging agent, such as an NIR imaging agent, may also be conjugated to at least one of the microbubbles for use in the present invention. Where a single microbubble is used, it can thus carry the chemotherapeutic agent, sensitizer and NIR imaging agent. When different microbubbles are used to carry a chemotherapeutic agent and a sensitizer, the NIR imaging agent can be conjugated (eg, via a non-covalent bond such as a biotin-avidin interaction) to one or both types of microbubbles. Each of these types of microbubbles is new and they form additional aspects of the invention.

In addition to providing a means to target a chemotherapeutic agent and a sonosensitizer to a specific site in vivo, the methods described herein can be further applied ex vivo. For example, in autologous bone marrow transplantation for the treatment of leukemia, the patient's bone marrow can be treated ex vivo by molecularly targeting the microbubble complex (or complexes) to cancer cells. These mixtures can then be sonicated to kill the cancer cells, and the treated bone marrow can then be used to restore hematopoiesis in a patient after radiation therapy. Alternatively, the methods of this invention can be performed ex vivo to remove unwanted tissue from organs harvested for conventional transplantation. Surgically removed tissues can be targeted and lesions destroyed prior to re-grafting of treated tissue.

For use in any of the methods described herein, microbubble complexes will typically be provided in a pharmaceutical composition along with at least one pharmaceutically acceptable carrier or excipient. Such compositions form a further aspect of the present invention.

Pharmaceutical compositions for use in the present invention may be formulated using methods well known in the art. The route of administration will depend on the intended use. Typically, they will be administered systemically and thus may be provided in a form suitable for parenteral administration, for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Suitable pharmaceutical forms include suspensions and solutions which contain active microbubble complexes together with one or more inert carriers or excipients. Suitable carriers include saline, sterile water, phosphate buffered saline, and mixtures thereof.

The compositions may further include other agents such as emulsifiers, suspending agents, dispersing agents, solubilizers, stabilizers, buffering agents, preservatives, and the like. The compositions can be sterilized by conventional sterilization methods.

Solutions containing complexes can be stabilized, for example, by adding agents such as viscosity modifiers, emulsifiers, solubilizing agents, etc.

Preferably, the compositions for use in the present invention will be in the form of an aqueous suspension of the complex (or complexes) in water or saline, for example phosphate buffered saline. The complexes can be presented as a lyophilized powder for re-dissolution at the site of application, for example, for re-dissolution in water, saline or PBS.

The methods described herein include administering a therapeutically effective amount of a composition that contains loaded microbubbles. The microbubble complexes can then be distributed to the desired part or target area of the body prior to activation. After introduction into the body, the target area is exposed to ultrasound at a frequency and intensity to achieve the desired therapeutic effect. With respect to a microbubble loaded with sensitizer, a typical activation procedure is schematically depicted in the attached FIG. 1. This figure depicts a two-step process in which microbubbles (MB) are first destroyed by focused ultrasound, thereby releasing the sonosensitizer (SS), which is then able to penetrate into the desired target tissue (eg, tumor). Subsequent sonoactivation of the sonosensitizer in target cells results in the production of singlet oxygen, which can oxidize various cellular components such as proteins, lipids, amino acids, and nucleotides, thereby destroying the target cells. While it is contemplated that activation of the sonosensitizer typically occurs post-delivery (ie, after the microbubbles break to release the sonosensitizer), delivery of the complex and activation of the sonosensitizer may nonetheless be simultaneous.

The effective dosage of the compositions described herein will depend on the nature of the complex, the route of administration, the condition being treated, the patient, and the like. And can be adjusted accordingly.

The frequency and intensity of the ultrasound that may be used may be selected based on the need to achieve selective disruption of the microbubbles in the target area and may, for example, correspond to the resonant frequency of the microbubbles. Ultrasonic frequencies are typically in the range of 20 kHz to 10 MHz, preferably 0.1 to 2 MHz. Ultrasound can be delivered as a single frequency or as a combination of different frequencies. The intensity (ie, power density) of the ultrasound may be from about 0.1 W/cm ² to about 1 kW/cm ² , preferably from about 1 to about 50 W/cm ² . The processing time is usually in the range of 1 ms to 20 minutes and this will depend on the chosen intensity, ie. for low ultrasound intensity, the treatment time will be longer, and for higher ultrasound intensity, the treatment time will be shorter. Ultrasound can be applied in continuous or pulsed mode and can be either focused or delivered as a columnar beam.

Any radiation source capable of producing acoustic energy (eg, ultrasound) can be used in the methods described herein. The source must be capable of directing energy to the target site and may include, for example, a transducer or device capable of directing energy to the target tissue from a body surface.

In cases where the frequencies and/or intensities of ultrasound needed to achieve cavitation (or destroy microbubbles) and those needed to activate the activation of the sonosensitizer are different, these different sets of ultrasound parameters (frequency/intensity) can be applied simultaneously or in two (or several) staged methodology.

Another aspect of this invention relates to a method of sonodynamic therapy of cells or tissues of a patient, which includes:

(a) introducing into the affected cells or tissues an effective amount of the composition described herein; and

(b) exposing said cells or tissues to ultrasound.

In the case where a sonosensitizer that is also responsive to light is used, ultrasonic activation may be accompanied by light activation. Photothermal activation can also be used, for example by using NIR dye as a sonosensitiser.

Where different microbubbles are used to carry the chemotherapeutic agent and the sonosensitizer, it is expected that they will typically be co-administered in the same pharmaceutical formulation, eg, as an aqueous solution. However, in another embodiment, they can be administered separately (eg, simultaneously or sequentially) in separate preparations.

In a further aspect, the invention thus provides a product comprising a microbubble-chemotherapeutic agent complex as described herein and a microbubble-sonosensitizer complex as described herein for simultaneous or sequential use in a sonodynamic therapy and/or diagnostic imaging technique.

In yet another aspect, the invention provides a kit comprising: (i) a microbubble-chemotherapeutic agent complex as described herein; and separately (ii) the microbubble-sonosensitizer complex described herein; optionally together with instructions for use (i) and (ii) in a sonodynamic therapy and/or diagnostic imaging method. When used, the active components of the kit (ie (i) and (ii)) may be administered simultaneously, separately or sequentially. In one embodiment of the kit, component (i) and/or (ii) may be provided in dry form, such as lyophilized powders. In this case, the kit may also contain a container containing a sterile physiologically acceptable liquid for re-dissolving the powdered forms of the active substances, for example saline or PBS.

While the various methods and uses of the invention are generally described herein in the context of administering a "off-the-shelf" microbubble-sonosensitizer complex, in an alternative embodiment, a complex precursor may be administered. As used herein, the term "precursor" is intended to refer to a microbubble-sonosensitizer complex precursor that is converted in vivo to it and thus is substantially equivalent to it. Thus, for example, the term "precursor" encompasses nanoemulsion or nanodroplet formulations that are capable of converting to the desired microbubble-sonosensitizer complex, either in vivo or during administration. In one embodiment, such precursors are capable of being converted into the desired complex upon accumulation in a target tissue (eg, tumor tissue). Subsequent distribution to tissue or target cells, the transition from droplet to bubble can be induced by methods that include ultrasound. Alternatively, the step of introducing the complex precursor may itself cause the formation of the microbubble-sonosensitizer complex of the present invention. For example, when the precursor takes the form of a nanoemulsion, the transition from droplet to bubble can be induced by injection through a fine gauge needle. Direct administration of suitable nanoemulsions into target cells or tissues, such as tumors, is a preferred aspect of the present invention.

As will be appreciated, in any of the compositions of the methods or uses described herein, any reference to the microbubble-sonosensitizer complex of this invention may be replaced by a suitable "precursor" as defined herein.

Nanoemulsion or nanodroplet formulations for use as precursors to the microbubble sonosensitiser of this invention can be prepared by appropriate modification of methods and techniques known in the art, such as those described in Rapoport et al. (supra). In such formulations, nanoemulsion droplet cores, which may be formed by liquid perfluorocarbon (e.g., perfluoroalkane), are wall-coated with suitable polymeric, proteinaceous, or lipid-forming shell materials (e.g., any of the polymers described herein with respect to the microbubble-sonosensitizer complex). Associating the nanodroplet shells with the sonosensitizer can be achieved using conventionally accepted methods and includes any of the methods described above for attaching the sonosensitizer to a preformed microbubble. The exact method will depend on the exact nature of the shell material and the sonosensitiser, in particular the nature of any pendant functional groups. Optionally functionalized, both the shell and/or the sonosensitizer may, for example, include reactive functional groups that can be used to link the fragments. Suitable reactive groups include acid, hydroxy, carbonyl, acid halide, thiol and/or primary amine. In one embodiment, the shell may be functionalized with biotin and then bound to avidin to subsequently facilitate binding of the biotinylated sonosensitizer. When it is desired that the resulting microbubble contain oxygen gas, the perfluorocarbon could act as a carrier for the oxygen in liquid form. After complex formation, the perfluorocarbon liquid is saturated with oxygen, which then evaporates to form oxygen gas.

In a similar manner as described above with respect to microbubble-sonosensitizer complexes, precursors of the microbubble-chemotherapeutic agent complex can also be used in the present invention. Likewise, they may be in the form of a nanoemulsion that is capable of forming the desired complex, either during administration or at the intended target site.

Hereinafter the invention will be further described with reference to the following non-limiting examples and accompanying drawings, in which:

Fig. 1 is a schematic representation of an ultrasonic-activated microbubble-sonosensitizer sonosensitizer complex.

On FIG. 2 depicts (a) a schematic illustration of the preparation of a rose bengal derivative (designated "RB1") and (b) a schematic illustration of the covalent bonding of RB1 to a microbubble.

On FIG. 3 shows photomicrographs taken with a 40 × objective lens of O ₂ MB after dilution (1:10) in PBS. The scale scale is 20 µm; (b) Size distribution of O ₂ MB after centrifugation obtained from analysis of 30 images with an optical microscope (unfilled boxes on the left side of the graph represent MB that were detected by the image analysis software, but less than 450 nm, the optical resolution of the system).

On FIG. 4 is a plot of % MB remaining after incubation of MB dispersions in PBS prepared from DBPC or DSPC at 37°C. The error rates are ± standard error, where n=4. *p<0.05 and **p<0.01

On FIG. 5 is a graph of the % increase in dissolved oxygen for degassed PBS solutions containing either O ₂ MB or PFBMB. The arrow indicates the time of application of ultrasound.

On FIG. 6 is a graph of cell viability for cells (a) BxPc-3, (b) MIA PaCa-2 and (c) PANC-1 treated (from left to right) (i) without treatment (ii) gemcitabine (iii) 5-FU ( iv) O ₂ MB-5FU+US (v) O ₂ MB-RB+US (vi) O ₂ MB-RB / O ₂ MB-5FU - US mixture and (vii) O ₂ MB-RB / O ₂ MB mixture -5FU+US. [RB], [5-FU] and [gemcitabine] were kept constant at 5 μM, 100 μM and 100 μM, respectively. The sonication was carried out for 30 seconds at a frequency of 1 MHz, the power density of ultrasound equal to 3.0 W.cm ^-2 and duty cycle equal to 50%, pulse rate =100 Hz. Error values are ± standard error, where n=4. *p<0.05, **p<0.01 and ***p<0.001.

On FIG. 7 is a plot of cell viability for BxPc-3 (black), MIA PaCa-2 (grey) and PANC-1 (white) cells treated with PFBMB-RB/PFBMB-5FU+US mixture (left) or (ii) O ₂ mixture MB-RB / O ₂ MB-5FU+US (right). US concentrations and parameters as in FIG. 6. Error rates are ± standard error, where n=4 **p<0.01

On FIG. 8 shows a plot of change in (a) % tumor volume and (b) mean body weight for mice treated with (i) no treatment (blank diamonds) (ii) ultrasound alone (solid diamonds) (iii) gemcitabine (open triangles) (b) (iv) O ₂ MB-RB / O ₂ MB-5FU - US mixture (open circles) (v) O ₂ MB-RB+US (filled squares) (vi) O ₂ MB-RB / O ₂ MB-5FU mixture +US (filled squares). Not shown for ease of illustration, 5-FU treatment only, O ₂ MB-RB - US, O ₂ MB-5FU+US, O ₂ MB-5FU - US. The concentrations of RB, 5-FU, and gemcitabine were held constant at 0.184 mg/kg (90.8 μM), 0.115 mg/kg (440 μM), and 0.264 mg/kg (440 μM), respectively, and sonication was performed for 30 seconds at a frequency equal to 1 MHz, ultrasonic power density equal to 3.0 W⋅cm ^-2 and duty cycle equal to 50%, pulse rate =100 Hz. Error values are ± standard error, where n=4. *p<0.05, **p<0.01 and ***p<0.001 for (vi) vs. (i) and ^Δp <0.05, ^ΔΔp <0.01 and ^ΔΔΔp <0.001 for (vi) versus (v).

On FIG. 9 is a plot of % tumor volume change in mice treated with gemcitabine IP (filled diamonds). The error rates are ± standard error, where n=4.

Figure 10 depicts (a) Bcl3 and Bcl2 protein expression using immunohistochemistry. The inner image is the entire section and the main image is the selected area at ×20 magnification. (b) Histology score for Bcl3 and Bcl2 expression.

On FIG. 11 depicts (a) RT-PCR quantitative expression of Bcl3 mRNA. (b) Plot of Bcl3 gene expression profiles for (i) untreated (black), (ii) O ₂ MB-5FU+US (grey), and (iii) O ₂ MB-RB/O ₂ MB-5FU+US mixture ( white). Error values are ± standard error, where n=3. ***p<0.001.

On FIG. 12 depicts (a) typical fluorescent images of nude mice bearing BxPC-3 ectopic tumors before (t=0), 5 min after (t=5), and 30 min after (t=30) intravenous administration of MB-9 conjugate. with with (+US) or without (-US) ultrasound applied to the tumor during IV injection. (b) The plot of % increase in tumor fluorescence was recorded 5 and 30 min after intravenous administration of MB-9 conjugates with (US) or without (control) ultrasound applied to the tumor during IV injection. Increase in intensity measured relative to tumors before treatment. Error values are ± SEM, where n=3. (c) Densitometry data (compared to GAPDH loading control) depicting Hif1α tumor protein expression for mice treated with IV O ₂ MB suspension or PFBMB. The inset is a typical Western blot image of HIF1α protein expression in tumors treated with IV O ₂ MB suspension or PPMMB. Error values are ± SEM, where n=3. *p<0.05, **p<0.01 and ***p<0.001.

On FIG. 13 is a plot of tumor growth versus time for mice bearing ectopic human pancreatic BxPC3 tumors treated with (i) vehicle alone (open circles) or (ii) intratumoral injection of I ₂ -IR783 (100 μl, 1 mg/kg) in carrier medium PBS : DMSO (98:2) when irradiated with light of 780 nm for 3×3 min with a 1-minute delay between treatments (open squares). Mice in the treatment group received a second treatment on day 8, which included 100 μl of O ₂ MBs (1×10 ⁸ MP/ml).

On FIG. 14 shows (a) UV visible spectra and (b) Fluorescence spectra of ICG (……), I2-IRCYDYE (solid line) and I4-IRCYDYE (-----).

On FIG. 15 is a graph of the increase in SOSG intensity at 410 nm for ICG, I2-IRCYDYE and I4-IRCYDYE. An increased SOSG intensity indicates the formation of singlet oxygen.

On FIG. 16 is a plot of cell viability for Mia Raca cells (upper plots) and for BxPC3 cells (lower plots) treated with (a) ICG, (b) I2-IRCYDYE and (c) I4-IRCYDYE with (white bars) and without (black bars). strips) 780 nm (200 mW) light for 1 min.

On FIG. 17 shows treatment of MiaPaCa2 cells with rose bengal1 (RB), 5-fluorouracil (5FU) and RB/5FU ± ultrasound combination therapy to determine if there is any synergy between SDT and 5-FU treatment. Cells were incubated with 3 μM RB and 50 μM 5FU (or both) for 3 hours as these represent sublethal doses and allow identification of synergy if evident. Ultrasonic exposure was 30 sec, 3 W/cm ² , 1 MHz, 50% duty cycle; the pulse repetition rate is 100 MHz. Cell viability was determined 24 hours after treatment using the MTT assay.

On FIG. 18 is a schematic representation of oxygenated microbubbles with doxorubicin (Dox-O ₂ MB) and rose bengal (RBO ₂ MB) attached to the surface.

On FIG. 19 is a plot of % tumor volume versus time for MDA-MB-231 xenograft of human breast tumors treated with (i) vehicle alone (ii) DoxO ₂ MB+US (iii) RBO ₂ MB+US or (iv) mixed DoxO ₂ MB / RBO ₂ MB+US. An intrauterine injection of 100 μl was administered on days 0 and 14, reflecting the dose of MB containing 300 μM and 475 μM RB and DOX, respectively, for groups (ii) and (iii) and 150 μM and 237.5 μM RB and DOX, respectively, for group (iv ). Ultrasonic exposure was equal to 3.5 min, 3 W/cm ² , 1 MHz, 50% duty cycle; pulse repetition rate 100 MHz.

EXAMPLES

Reagents and equipment:

Rose bengal sodium, 2-bromoethylamine, NHS-biotin, MTT, avidin, FITC-avidin, chloroacetic acid, 4-dimethylaminopyridine (DMAP), hydroxybenzotriazole (HOBt), N,N'-dicyclohexylcarbodiimide (DCC), anhydrous dimethylformamide (DMF) ) and ethanol were purchased from Sigma Aldrich (UK) of the highest degree of stonecrop. Biotin, 5-fluorouracil, di-(N-succinimidyl) carbonate and 2-aminoethanol were purchased from Tokyo Chemical Industry UK Ltd. 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DSPC), dibegenoylphosphatidylcholine (DBPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 (DSPE-PEG ( 2000)) and DSPE-PEG(2000)-biotin were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). Doxorubicin was purchased from HABC (China). Oxygen gas was purchased from BOC Industrial Gases United Kingdom while perfluorobutane gas (PFB) was purchased from Apollo Scientific Ltd. Phosphate buffered saline (PBS) was purchased from Gibco, Life Technologies, United Kingdom.

NMR spectra were recorded on a Varian 500 MHz spectrometer. ESI-MS characterization was achieved using an LCQTM quadrupole ion trap mass spectrometer (Finnigan MAT, San Jose, CA, USA) using electrospray ionization (ESI). Optical images were obtained using an optical microscope of an optical microscope (optical microscope Leica DM500). Dissolved oxygen was measured using a Thermo Scientific Orion Star A216 tabletop dissolved oxygen meter. Error was expressed as ± SEM (standard deviation of the mean) while statistical comparisons were made using unpaired Student's t-test data.

Example 1 - Obtaining microbubbles loaded with O ₂ (O ₂ MBs)

DSPC MB was prepared as described in McEwan et al. (J Control Release. 2015; 203, 51-6). However, to improve both the physical stability of MBs and their resistance to O ₂ retention, the long chain lipid dibegenoylphosphatidylcholine (DBPC) was used instead of distearoylphosphatidylcholine (DSPC) as shown in previous work to reduce the diffusivity of the MB surface and therefore improve stability.

For MB DBPC, DBPC (4.0 mg, 4.43 µmol), DSPE-PEG (2000) (1.35 mg, 0.481 µmol) and DSPE-PEG (2000)-biotin (1.45 mg, 0.481 µmol ) in a molar ratio of 82:9:9 was dissolved in chloroform and placed in a glass tube. The solution was heated to 40° C. until all the chloroform had evaporated. Added PBS (pH 7.4±0.1) (5 ml) to the dried lipid film, and the contents were heated above the phase transition temperature of the lipid (>70°C) with constant stirring on a magnetic stirrer for 30 minutes. The slurry was then sonicated with a Microson ultrasonic disruptor for 1.5 min (100 Watts, 22.5 kHz at power setting 4), the headspace was filled with perfluorobutane (PFB) gas, and the gas/liquid surface was sonicated (power 19) at flow20 to form PFBMB. The MP suspension was cooled in an ice bath for about 10 minutes. Then, an aqueous solution of avidin (50 μl, 10 mg/ml) was added to the cooled MB suspension and stirred for an additional 10 minutes. The suspension was then centrifuged (300 rpm, 10 min) and the resulting MB cake was concentrated in 1 ml of PBS (pH 7.4±0.1). This solution was divided into two tubes for freeze drying. For PFBMB, the tubes were then crimped (sealed with a metal cap). To create oxygen-filled MBs, the headspace of the tube and the MB suspension were purged under a positive pressure of oxygen gas for 2 min, after which the tube was crimped. After preparation as described above, the MB samples were visualized using conventional optical microscopy to determine their size distribution and concentration. 10 µl samples were removed from each suspension and diluted in 90 µl PBS (pH 7.4±0.1) followed by hemocytometer examination (Bright-Line, Hausser Scientific, Horsham, PA, USA). Images were taken with 40x objective lenses on a Leica DM500 optical microscope. The MB size distribution and concentration was then obtained using custom image analysis software Matlab (2010B, The MathWorks, Natick, Massachusetts, USA).

The MB data had an average diameter of 1-2 µm with a concentration of about 1x109 MB/mL as determined by optical microscopy image analysis (FIG. 3). To determine the effect of DBPC incorporation on MB stability, we incubated PBS dispersions of MB prepared with DBPC or DSPC at 37°C and counted the number of viable MBs remaining at various time intervals. The results are depicted in Fig. 4 and indicate a significant improvement in stability for MB derived from DBPC compared to preparations derived using DSPC. Indeed, after a three-hour incubation, 80% of DBPC MB remained, while the amount of DSPC MB was reduced to 54%. These results are consistent with the results of previous studies, which showed that increasing the length of the lipid acyl chain reduces both the mechanical flexibility of microbubbles and surface diffusion.

Example 2 - Preparation of Biotinylated Rose Bengal and Biotinylated 5-FU

Scheme 1 (a) Synthetic scheme for obtaining biotin-5-FU (5). (b) Schematic representation of the structure of O ₂ MB-PvB and O ₂ MB-5FU conjugates.

Biotin functionalized Rose Bengal (6) was prepared as described in McEwan et al. (J Control Release. 2015; 203, 51-6). Biotin-functionalized 5-FU (5) was synthesized according to scheme 1a according to the procedures outlined below.

Preparation of N-(2-hydroxyethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (2):

To an ice-bath cooled solution of biotin-N-hydroxysuccinimide ester (1) (obtained by the reaction between biotin and di(N-succinimidyl) carbonate) described in Kang et al., Jr. Rapid Commun Mass Spectrom. 2009, 23(11), 1719-1726) (3.75 g, 11 mmol) in anhydrous DMF (40 ml), 2-aminoethanol (1.0 ml, 16.4 mmol) was added and the mixture was stirred at 25°C within 30 min. The progress of the reaction was monitored by thin layer chromatography (TLC) (Merck Silica 60, HF 254, 20:80 methanol-dichloromethane, v/v). Biotin-N-hydroxysuccinimide ester (R _f =0.76) was absorbed within 15 min with the concomitant formation of an alcohol product (R _f =0.47). The reaction mixture was concentrated under reduced pressure and the residue co-evaporated with DMF to remove excess 2-aminoethanol. The white residue was recrystallized from water to give 2 as a light yellow solid (1.7 g, 38%). An analytical sample was obtained from the second recrystallization, M.p. 192-195°C. ¹ HNMR (500 MHz, D ₂ O) 4.49-4.47 (m, 1H, -CH), 4.31-4.30 (m, 1H, -CH), 3.53-3.51 ( m, 2H, CH ₂ ), 3.23-3.18 (m, 3H, CH and CH ₂ ), 2.85-2.64 (m, 2H, CH ₂ ), 2.15 (t, 2H, -CH ₂ ), 1.62-1.46 (m, 4H, CH ₂ X 2 ), 1.32-1.26 (m, 2H, CH ₂ ).

¹³ SNMR (125 MHz, D ₂ O) 177.09 (C=O), 61.98 (CH ₂ ), 60.19 (CH), 59.91 (CH), 55.24 (CH), 41, 29 (CH ₂ ), 39.61 (CH ₂ ), 35.42 (CH ₂ ), 27.77 (CH ₂ ), 27.56 (CH ₂ ), 25.02 (CH ₂ ).

ESI-MS (M+H ⁺ ): found 288.70, calculated for C ₁₂ H ₂₁ N ₃ O ₃ S=287.13.

Preparation of 5-fluorouracil-1-carboxylic acid (4):

A mixture of 5-fluorouracil (3) (5 g, 38.4 mmol), potassium hydroxide (9.07 g, 161.6 mmol) and chloroacetic acid (3.63 g, 38.4 mmol) in 100 ml of water was heated with reflux for 2 hours at 70°C. After cooling to room temperature, the pH of the solution was adjusted to 5.5 by adding concentrated hydrochloric acid. The reaction mixture was then kept in a refrigerator (5° C.) for 18 h and the resulting white crystals were isolated by filtration and washed with cold water to obtain 4 with a yield of 52.5%.

mp>200°C.

¹ HNMR (500 MHz, D ₂ O) 7.76 (d, 1H, J=6 Hz, CH), 4.29 (s, 2H, CH ₂ ).

¹³ C NMR (D ₂ O): 173.58 (C=O), 159.97 (C=O), 150.80 (C=O), 141.20 (C), 131.74 (CH), 51.48 (CH ₂ ).

ESI-MS (MH ⁺ ): found 187.10, calculated for C ₆ H ₅ O ₄ N ₂ F=188.11.

Preparation of 2-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethyl-2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidine- 1(2H)-yl)acetate (5):

N-(2-Hydroxyethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (2) (0.5 g, 1.7 mmol), 5-fluorouracil -1-carboxylic acid (4) (0.4 g, 2.1 mmol), DMAP (0.023 g, 0.17 mmol) and HOBT (0.023 g, 0.17 mmol) were added to 20 ml of anhydrous DMF in 100 ml two-necked round bottom flask under N ₂ atmosphere. The mixture was heated at 40°C and stirred until a homogeneous solution was obtained. DCC (0.4 g, 1.9 mmol) was then added to the reaction mixture and allowed to stir at room temperature for 12 hours. DMF was removed under reduced pressure, diethyl ether (50 ml) was added and the contents were stirred for 20 minutes. The resulting white semi-solid was removed by filtration, and after removal of excess diethyl ether under reduced pressure, the crude product was purified by preparative HPLC (C-18 column) using acetonitrile/water (80:20 by volume) as the mobile phase. Product 5 was obtained after lyophilization of the desired fractions as a white semi-solid (0.24 g, 30% pure yield).

¹ HNMR (500 MHz, D ₂ O): 7.67 (d, 1H, J=6.0 Hz, CH), 4.50-4.47 (m, 1H, CH), 4.31-4, 29 (m, 1H, CH), 4.19 (s, 2H, CH ₂ ), 3.54 (t, 2H, CH ₂ ), 3.22-3.19 (m, 2H, CH ₂ ), 2 .89-2.86 (m, 1H, CH), 2.67-2.64 (m, 2H, CH ₂ ), 2.17-2.14 (m, 2H, CH ₂ ), 1.61- 1.47 (m, 4H, CH ₂ X 2 ), 1.47-1.28 (m, 2H, CH ₂ ).

¹³ SNMR 125 MHz, D ₂ O): 177.12 (C=O), 173.74 (C=O), 165.33 (C=O), 160.01 (C=O), 159.81 ( C=O), 141.14 (C), 131.71 (CH), 62.00 (CH ₂ ), 60.22 (CH), 59.94 (CH), 55.26 (CH), 51, 53 (CH ₂ ), 41.31 (CH ₂ ), 39.64 (CH ₂ ), 35.45 (CH ₂ ), 27.79 (CH ₂ ), 27.58 (CH ₂ ), 25.14 ( _CH2 ).

ESI-MS (M - H ⁺ ) found 456.20, calculated for C ₁₈ H ₂₄ FN ₅ O ₆ S=457.48.

Example 3 - Preparation of O ₂ MB-Rose Bengal and O ₂ MB-5FU Conjugates

Saturated solutions 5 (91.2 mM) and 6 (0.61 mM) were prepared in 0.5% DMSO in PBS (pH 7.4 ± 0.1). A 0.3 ml aliquot of these stock solutions was then added separately to 1 μl of two suspensions of PFBMB-functionalized avidin (1×10 ⁹ MP/ml) and the contents were mixed by shaking for 15 minutes. The suspensions were then centrifuged (900 rpm) for 5 min and the MB conjugates were isolated as a milky suspension floating on top of the solution. The solution was removed and replaced with another 0.3 ml stock solution containing either 5 or 6 and the mixing/centrifugation steps repeated. The MB suspensions were then washed with PBS (5 ml), centrifuged (900 rpm) for 5 minutes, and the MB was transferred to a clean centrifuge tube. This washing procedure was repeated again and PFBMB-RB and isolated PFBMB-5FU conjugates were placed in a glass vial. The PFBMB-RB and PFBMB-5FU conjugates were then sparged with oxygen gas for 2 min, and the resulting O ₂ MB-RB and O ₂ MB-5FU conjugates were mixed together at a ratio of 1:3.25 to obtain a final suspension containing 6, 8×10 ⁷ MP/ml with 90.8 μM RB and 440 μM 5-FU.

This O ₂ MB-RB/O ₂ MB-5FU mixture was used directly in the in vitro and in vivo experiments described herein.

Example 4 - Preparation of MB-IR820 O ₂ conjugates

Scheme 2 (a) Synthesis of biotin-functionalized dye absorbing in the NIR region (9). (b) Schematic representation of the MB-9 conjugate used in the imaging experiments.

Synthesis of 2-((E)-2-((E)-2-((4-aminophenyl)thio)-3-((E)-2-(1,1-dimethyl-3-(4-sulfobutyl)- 1H-benzo[e]indole-2(3H)-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e] indole-3-ia (8):

Compound 7 was prepared using the following literature procedure (James et al., Evaluation of Polymethine Dyes as Potential Probes for Near Infrared Fluorescence Imaging of Tumors: Part - 1. Theranostics. 2013, 3(9), 692-702). 4-Aminothiophenol (0.63 g, 5 mmol) was dissolved in anhydrous DMF (50 ml) under N ₂ atmosphere. To this solution was added 7 (0.6 g, 0.7 mmol) and the mixture was stirred for 18 hours at room temperature. The progress of the reaction was monitored by TLC (Merck Silica 60, HF 254 using 25% MeOH/DCM as mobile phase). DMF was removed under reduced pressure and the residue was redissolved in DMF (5 ml) and precipitated with Et ₂ O (15 ml). The solid was filtered off, washed with Et ₂ O (30 ml) and purified by column chromatography (silica gel, 60-120 mesh) using MeOH-DCM (1:3) as eluting agent. The product (230 mg, 4.8%) was isolated as a reddish brown semi-solid. This compound was not stable and was directly used in the next step.

¹ H NMR (500 MHz, MeOH-d ₄ ): 8.96-8.93 (m, 2H, Ar-CH), 8.81-8.78 (m, 2H, Ar-CH), 8.09 -8.07 (m, 2H, Ar-CH), 7.90-7.89 (m, 6H, Ar-CH), 7.57-7.51 (m, 4H, Ar-CH), 7, 38 (brs, 2H, NH ₂ ), 7.38-7.28 (m, 2H, Ar-CH), 6.34-6.31 (m, 2H, CH X 2 ), 4.23 (brs, 4H, CH X 2, CH ₂ ), 2.87-2.80 (m, 8H, CH ₂ X 4), 1.98-1.91 (m, 10H, CH ₂ X 5), 1.70 ( s, 12H, CH ₃ X 4).

¹³ C NMR (125 MHz, dmso-d ₆ ): 173.4, 170.2, 150.1, 148.4, 143.7, 144.6, 142.7, 134.3, 133.9, 132 .4, 128.0, 126.1, 126.2, 125.5, 125.7, 117.5, 115.4, 104.7, 61.8, 59.3, 49.4, 48.9 , 46.8, 30.2, 28.6, 26.8, 26.9, 25.2, 21.0.

ESI-MS calculated for C ₅₂ H ₅₈ N ₃ O ₆ S ₃ Na ₂ ⁺ = 961.1, found 960.3.

Synthesis of 2-((E)-2-((E)-3-((E)-2-(1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indole-2(3H )-ylidene)ethylidene)-2-((4-(5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)phenyl)thio)cyclohex-1-en-1 -yl)vinyl)-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indol-3-ium (9):

Compound 8 (100 mg, 0.1 mmol) was added to a stirred solution of 1 (40.9 mg, 0.12 mmol) in anhydrous DMF (50 ml) to which was added a catalytic amount of triethylamine. The solution was stirred at room temperature for 5 hours. The reaction mixture was added to ether (100 ml) and the contents were stirred for 30 minutes. The precipitate was collected by filtration and purified by preparative TLC using MeOH:DCM (1:4) as eluting agent and the product was isolated as a green powder. Yield = 21 mg, 18.4%.

¹ H NMR (500 MHz, MeOH-d ₄ ): 8.77 (d, J=7.8 Hz, 2H, Ar-CH), 8.2l (d, J=7.5 Hz, 2H, Ar- CH), 8.03-7.99 (m, 2H, Ar-CH), 7.73 (d, J=7.5 Hz, 2H, Ar-CH), 7.60-7.57 (m, 2H, Ar-CH), 7.47-7.44 (m, 2H, Ar-CH), 7.20-7.17 (m, 2H, Ar-CH), 7.16 (d, J=12 Hz, 1H, CH), 6.89-6.83 (m, 2H, Ar-CH), 6.58 (d, J=12 Hz, 1H, CH), 6.42 (brs, 1H, NH) , 6.36 (brs, 2H, NH X 2 ), 4.29-4.27 (m, 6H, CH X 2, NCH ₂ ), 4.10 (brs, 2H, -CH ₂ ), 3.14 -3.06 (m, 3H, CH, CH ₂ ), 2.80-2.74 (m, 4H, CH ₂ X 2), 2.57-2.48 (m, 4H, CH ₂ X 2) , 2.19-2.16 (m, 2H, CH ₂ ), 1.88-1.59 (m, 2H, CH ₂ ), 1.76 (s, 12H, CH ₃ X 4), 1.59 -1.57 (m, 2H, CH ₂ ), 1.48-1.28 (m, 12H, CH ₂ X 6).

¹³ C NMR (125 MHz, dmso-d ₆ ): 177.5, 174.3, 169.9, 166.2, 152.5, 150.2, 148.0, 145.3, 144.8, 140 .7, 134.8, 132.6, 131.3, 130.0, 128.5, 126.3, 124.7, 120.1, 116.8, 114.8, 102.5, 64.0 , 62.3, 60.1, 54.9, 50.1, 48.6, 48.1, 42.2, 36.7, 32.8, 30.2, 28.4, 28.3, 26 .9, 26.0, 24.5, 22.8.

ESI-MS calculated for C ₆₂ H ₇₂ N ₅ O ₈ S ₄ ⁺ = 1142.4 (protonated form, M ⁺ ), found 1143.4.

Obtaining conjugates O ₂ MB-IR820:

Biotin-functionalized IR-820 (9) was attached to the O ₂ MB surface following the procedure described above for 5-FU and Rose Bengal. [MB]=2.6×10 ⁸ ; [9]=280 μM.

Example 5 - Ultrasound mediated release of O ₂ from O ₂ MB

0.5 ml of the suspension O ₂ MB (1×10 ⁸ ) obtained in Example 1 was added to degassed PBS (pH 7.4±0.1) (4.5 ml). The level of dissolved oxygen in this solution was measured over a period of 20 min at 2 min intervals using a dissolved oxygen meter. Ultrasound was applied after 4.5 minutes for 1 minute using a frequency of 1 MHz, an ultrasound power density of 3.0 Wcm ^-2 and a duty cycle of 50% (pulse frequency = 100 Hz). Control experiments using PFBMB were also performed according to the same procedure.

If MB O ₂ is to be successful as a carrier for in vivo oxygen delivery, it is important that gas exchange between the MB core and blood be minimized until the MB is sonicated at the target site. The half-life of commercially available MBs ranges from 0.97 minutes in men to 1.23 minutes in women. Therefore, it is important that the O ₂ MB be able to retain their oxygen for at least the specified time period in situations where an oxygen diffusion gradient may exist. In an attempt to mimic such a scenario, O ₂ MB (0.5 ml, 1×10 ⁸ ) was added to 4.5 ml of degassed PBS (pH 7.4±0.1) in a glass vial and the contents were stirred intermittently at 37°C. Because O ₂ MB floated on top of the PBS solution, they were in direct contact with the air in the headspace of the open vial. The amount of dissolved O ₂ in the PBS solution was determined using a dissolved oxygen meter and measured 4.5 minutes before and 14.5 minutes after sonication. As a control, experiments were also carried out using PFBMB. The results are shown in Fig. 5 and illustrate that O ₂ MB effectively retain their O ₂ until destroyed by external ultrasound, in which the level of dissolved oxygen increases by more than 40% five minutes after irradiation. In contrast, the level of dissolved oxygen in the control PFBMB experiment increased by about 20% 1 min after sonication and then decreased to 5% 5 minutes after sonication. We believe that this initial increase in dissolved O ₂ in the control preparation was caused by ultrasonic-mediated agitation of the liquid in the measurement chamber. However, the results show that O ₂ MB is effective at retaining oxygen and sonication leads to an increase in dissolved oxygen, which is maintained for a relatively long period of time in this system. We believe that the specified time frames for both retention and release mediated by ultrasound would allow sufficient time to allow targeting of microbubbles and their gas load to a specific anatomical site and provide an increase in the concentration of dissolved oxygen in the tissue microenvironment that would be sufficient. to maintain elevated ROS levels during SDT.

Example 6 In Vitro Cytotoxicity Experiments

Primary human pancreatic adenocarcinoma cell lines MIA PaCa-2 and PANC-1 were stored in Dulbecco's Modified Eagle's medium, while BxPC-3 cells were stored in RPMI-1640 medium, all supplemented with 10% (v/v .) fetal bovine serum in a humidified atmosphere of 5% CO ₂ at 37°C. These cell lines were seeded in the wells of a 96-well plate at a concentration of 5×10 ³ cells per well and incubated for 21 h at 37°C in a humidified 5% CO ₂ atmosphere before being transferred to a hypoxic chamber at 37°C ( O ₂ /CO ₂ /N ₂ , 0.1:5:94.9 v/v/v) for 3 hours (this is intended to mimic the hypoxic conditions found in the tumor site). Media was then removed from each well and replaced with O ₂ MB-RB (50 μl, 5 μM RB) and O ₂ MB-5FU (50 μl, 100 μM 5FU) conjugates. Individual wells were then sonicated with a Sonorel SP100 sonoporator (30 sec, frequency = 1 MHz, sonic power density = 3.0 Wcm ^-2 , duty cycle = 50% with pulse repetition rate = 100 Hz). The cells were kept in a hypoxic environment for an additional 3 hours before the treatment solution was removed, the cells were washed with PBS and fresh medium (200 μl per well) was added. The plates were then incubated in a normal oxygen environment (i.e. a humidified atmosphere of 5% CO ₂ at 37° C.) for an additional 21 hours before cell viability was determined using the MTT assay (McHale et al., Cancer Lett 1988; 41,315-21). A similar procedure was repeated only for vehicle, gemcitabine (a drug approved for use in the treatment of pancreatic cancer), 5-FU, O ₂ MB-5FU+US, O ₂ MB-RB+US, and O ₂ MB-RB/O mixture. _2MB -5FU - US. Bo all experiments used the amount of RB, 5-FU and gemcitabine equal to 5 μm, 100 μm and 100 μm, respectively. All groups were also repeated using PFBMB conjugates with the same amount of attached RB or 5-FU.

The results depicted in Fig. 6 show that a statistically significant decrease in viability was observed in all three cell lines for cells treated with SDT/antimetabolite combination therapy (i.e. O ₂ MB-RB/O ₂ MB-5FU mixture + US) compared to cells treated with either antimetabolite therapy (i.e. 5-FU or gemcitabine). Indeed, a statistically significant reduction in viability was also observed for cells treated with combination therapy compared to cells treated with SDT alone (ie, O ₂ MB-RB+US). That the effect of SDT observed under such hypoxic conditions is significantly enhanced by the application of O ₂ MBs was confirmed by comparing the difference in cytotoxicity between the sonicated O ₂ MB-RB/O ₂ MB-5FU mixture and the otherwise identical conjugate mixture. PFBMB with ultrasonic treatment (Fig. 7). Indeed, a statistically significant (p<0.01) decrease in cell viability of more than 20% was observed for all three cell lines treated with O ₂ MB conjugates compared to PFBMB conjugates. Taken together, the results depicted in FIG. 6 and 7 clearly show the benefits of combining SDT with antimetabolite therapy, especially in hypoxic environments where O ₂ MBs can provide additional O ₂ to improve the effect of SDT.

Example 7 In Vivo Cytotoxicity Experiments

BxPc-3 cells were stored in RPI-1640 medium supplemented with 10% fetal serum albumin as above. Cells (1×10 ⁶ ) were resuspended in 100 μl of ^Matrigel® and implanted in the dorsum of female Balb/c SCID (C.B-17/IcrHan®Hsd-Prkdcscid) mice. Tumor formation occurred about 2 weeks after implantation, and tumor measurements were taken every other day using calipers. Once the tumors reached a mean volume of 218 mm ³ , calculated from the geometric mean diameter using the equation for tumor volume = 4πR ³ /3, animals were randomly assigned to 10 groups (n=4). After administration of anesthesia (intraperitoneal injection of Hypnorm/Hypnovel), 100 µl of PBS mixture containing O ₂ MB-RB (MB=1.6×10 ⁷ , [RB]=90.8 µM) and O ₂ MB-5FU (MB= 5.2×10 ⁷ , [5FU]=440 μM) was injected directly into each tumor. Intratumoral injection was chosen as the route of administration to exclude experimental changes caused by the pharmacokinetic behavior of the platform. If necessary, the tumors were then sonicated for 3.5 min at an ultrasound frequency of 1 MHz, an ultrasound power density of 3.5 W⋅cm ^-2 (I _SATP ; spatial mean temporal peak) and using a 30% duty cycle while repeating the pulse rate equal to 100 Hz. Additional treatment groups included (i) no drug; (ii) MB-RB O ₂ conjugate only ± sonication; and (iii) MB-5FU O ₂ conjugate only ± sonicated. Treatment with only gemcitabine (440 μM) and 5-FU (440 μM) was also performed. After treatment, the animals were allowed to emerge from anesthesia, and tumor volume and body weight were recorded daily for nine days. The % tumor volume increase was calculated using pretreatment measurements for each group.

Tumor volume was measured daily for 9 days and % change in tumor volume for each group as a function of time. For ease of interpretation, only the results of six of the ten groups are depicted in FIG. 8a. These results demonstrate a dramatic reduction in tumor volume in mice treated with SDT/antimetabolite combination therapy compared to gemcitabine or 5-FU therapy alone. Indeed, 9 days post-treatment, tumors in mice treated with gemcitabine or 5-FU alone grew by 125.1% and 123.3%, respectively, while tumors treated with O ₂ MB-RB/O ₂ MB-5FU+US increased by only 29.1% compared to the original initial volume over the same time period. In addition, there was also a statistically significant decrease in tumor volume for tumors treated with SDT/5-FU combination therapy (i.e., MB-RB O ₂ /MB-5FU+US O 2 mixture) relative to _SDT treatment alone (t .e O ₂ MB-RB+US) with tumors on average 30.2% less than 9 days after treatment. Analysis of mean body weight (FIG. 8b) for animals in each of the groups showed no significant reductions over the course of the experiment, suggesting that the treatment did not cause any acute side effects.

In these experiments, gemcitabine was administered as an intratumoral injection at a concentration of 0.264 mg/kg to allow a direct molar comparison with the amount of 5-FU used (440 μM). Although this amount was delivered directly to the tumor, it is significantly less than the normal systemic dose of gemcitabine (120 mg/kg) used in mice.

To compare the efficacy of SDT/5-FU combination therapy versus systemic gemcitabine therapy, we treated mice bearing BxPC-3 ectopic tumors with gemcitabine (120 mg/mg/kg) injected intraperitoneally (IP) at 0.3 and Day 8 Tumor volume was measured daily as before and compared with untreated control animals. These results (FIG. 9) show that although the tumor volume in the control group increased by about 100%, the tumor volume increased by 38% in the gemcitabine-treated group, and under no circumstances did the therapy reduce the tumor volume below the initial tumor volume. In contrast, with a single treatment for SDT/5FU combination therapy (Figure 8), tumor volume decreased below the initial treatment volume and remained so until 6 days post-treatment, while tumors in the gemcitabine group showed a 20% increase in tumor volume at day 6. day. That such a dramatic response can be achieved with relatively small amounts of sensitizer/5-FU and after a single treatment is extremely promising and suggests that targeted delivery of such agents may provide increased therapeutic benefit with reduced side effects.

Example 8 - In Vivo Near-IR Fluorescence (NIR) Imaging of O ₂ MB-9 Conjugates Followed by IV Administration to Tumor Bearing Mice

Nude athymic mice were anesthetized (intraperitoneal injection of Hypnorm/Hypnovel) and MB-9 O ₂ conjugate (100 ml) was administered via tail vein injection. In the treatment group, ultrasound (conditions described above in 2.10) were applied to the tumors during and for 3 minutes after injection, while no ultrasound was applied to the tumors in the control group (n=3 in each group). Following administration (at t=5 min and t=10 min), animals were placed in the Xenogen IVIS® Lumina fluorescence imaging system chamber using an ICG filter set (excitation: 705-780 nm; emission: 810-885 nm). Data was collected and analyzed using the Living Image® software package version 2.60. Quantitative data were obtained by drawing the region of interest around the tumor and comparing the fluorescent signal (photons/sec) at t=5 and t=10 min after injection of O ₂ MB-9 with the fluorescent signal obtained before injection.

Example 9 Immunohistochemistry and qRT-PCR Analysis

We were also interested in studying the effects of SDT/5-FU combination therapy at the molecular level compared to 5-FU therapy. For this tumor in the control (i.e., untreated) group, the O ₂ MB-5FU+US group (i.e., 5-FU), and the O ₂ MB-RB/O ₂ MB-5FU mixture + US group ( ie combination treatment) were collected at the end of the monitoring period and subjected to immunohistochemistry and qRT-PCR analysis.

Expression of HIF1α in tumors after administration of IV O ₂ MB:

Nude athymic mice were anesthetized (intraperitoneal injection of Hypnorm/Hypnovel) and both PFBMB and O ₂ MB (100 μl) were injected into the tail vein (n=3 in each group). Ultrasound (conditions as in 2.10 above) was applied to the tumor during and within 3 minutes after the IV injection and the tumors were excised 30 minutes later. For Western blot analysis of HIF-1α protein expression, total protein was extracted using urea buffer. The primary mouse antibodies used in these studies are HIF1α (Millipore, 1:500) and anti-GAPDH (Sigma, 1:1000). Protein samples were electrophoresed on a 4-12% ^TruPAGE® gel and transferred to nitrocellulose membranes. Non-specific binding blocking was performed in 5% (w/v) bovine serum albumin diluted in 1x Tris buffered saline containing 0.05% (v/v) Tween 20. The membranes were then incubated in the appropriate secondary goat anti-mouse IgG-HRP antibody (1:10,000 stock solution). Secondary antibodies were purchased from Santa Cruz Biotechnology, Heidelberg, Germany. Densitometry was performed to quantify HIF1α protein expression using GAPDH as a standard.

Characterization of the immune response:

To characterize the immune response in treated tissues, Bcl3 and Bcl2 protein expression was examined using immunohistochemistry in tissue samples collected at the end of the monitoring period. Immunohistochemical (IHC) evaluation of Bcl2 and Bcl3 proteins was performed on paraffin sections. Paraffin-embedded tissue samples were cut to a thickness of 4 µm using a Leica RM2235 microtome (Leica Biosystems Ltd., Newcastle) and examined on a coated slide. IHC analysis for Bcl2 (clone: BCL-2/100/D5) and Bcl3 (clone: 1E8) were diluted 1:200 and 1:150, respectively. Both antibodies were mouse antibodies obtained from Leica Biosystems. Immunostaining was performed using an automated Bond-Max system (Leica Biosystems Ltd., Newcastle) using adapted high temperature antigen demasking with antigen demasking binding solution 2 (based on EDTA pH 9.0) for 30 min. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide for 5 minutes. Histological samples were incubated with primary antibody for 15 min at room temperature, and slides were incubated with rabbit anti-mouse antibody for 8 min at room temperature. The slides were then incubated with goat anti-rabbit polymer reagent for 8 minutes at room temperature. Reactions were developed using a purified polymer binding kit followed by color development with 3,3'-diaminobenzidine tetrahydrochloride as chromogen for 10 minutes. Immunohistochemistry intensity and scores were performed according to Allred et al. (Prognostic and predictive factors in breast cancer by immunohistochemical analysis. 1998, 11 (2): 155-68). To confirm immunohistochemical studies, Bcl3 expression was also studied at the transcriptional level, Bcl3 mRNA expression was measured by gene-specific qRT-PCR using the primers listed in Table 1:

qRT-PCR and analysis was performed according to previously published protocols (Hamoudi et al., Leukemia, 2010, vol. 24, no. 8, pp. 1487-1497; and Bi et al., Haematologica, 2012, 97, 926-930 ). Briefly, RNA was extracted from microdissected slides using the RecoverAll Kit (Life Technologies, Paisley, UK). cDNA synthesis was performed using the Superscript III First Strand cDNA Synthesis Kit (Life Technologies, Paisley, UK) using the reverse primer of each of the genes, including the two constitutive genes; 18S rRNA and β-actin. qRT-PCR was performed using the SYBR Green kit on a CFX96 instrument (BioRad, UK). The qRT-PCR cycle was as follows: 95°C for 3 min, 95°C for 10 sec, 60°C for 45 sec for 40 cycles. For analysis, the geometric mean of 18S rRNA and β-actin was taken as a single constitutive value. Statistical comparison between groups was performed using a two-way ANOVA with Bonferroni post-hoc analysis.

Results:

Immunohistochemistry results showed that at the protein level, deregulation of Bcl3 and Bcl2 was observed between both treatment groups and the control group. At this level of analysis, the intensity and proportion of Bcl3 were higher in the control and 5FU groups, but decreased in the combined treatment group. Similarly, Bcl2 protein expression was highest in the control group, decreased in the 5FU group, and was not detectable in the combination treatment group (FIG. 10). At the mRNA level, a similar pattern was observed for Bcl3 (FIGS. 11a and 11b) with ΔΔCt showing a significant decrease of about 5 and 7 times for 5FU and combination treatment groups, respectively, relative to the control group (p<0.001). Bcl3 is a key element of the NF-κB pathway and is involved in the regulation of many cellular pathways, including survival, proliferation, inflammation, and immune response. Expression and activation of Bcl3 have been associated with increased cell proliferation or survival, depending on the tissue and type of stimuli. Its transcriptional repressor function has been shown to be involved in the regulation of immune responses and in the development and activation of immune cells (Wessells et al., J Biol Chem 2004; 279: 49995-50003, and Kuwata et al., Blood 2003; 102: 4123-4129). The fact that Bcl3 expression was deregulated suggests an alteration in the immune response as well as signaling cell survival and proliferation. This was confirmed by the fact that Bcl2, which is an important anti-apoptotic gene, was higher in the control, but its expression was significantly reduced after the combined treatment. Indeed, Bcl2 expression is known to be upregulated in most primary pancreatic tumors (Campani et al., Pathol. 2001, 194(4), 444-450) and the use of Bcl2-specific siRNA to reduce its expression has been shown to have an antiproliferative effect. and a pro-apoptotic effect on pancreatic tumor growth in vitro and in vivo (Ocker et al., Gut, 2005, 54(9), 1298-1308). Recently, a G-quadruplex-binding compound (MM41), which exhibits antitumor activity using the MIA PaCa-2 pancreatic cancer xenograft model, has been shown to reduce Bcl2 levels by 40% after analysis at the protein level (Ohnmacht et al., Sci Rep 2015, 16(5): 11385). Taken together, these results indicate a marked effect on cellular signaling pathways from SDT/5-FU combination therapy and suggest that SDT may provide a significant therapeutic benefit for patients with pancreatic cancer when used in conjunction with established chemotherapy-based regimens.

Example 10 - NIR Imaging

To be suitable for transition to clinical use, MP suspension must be administered intravenously and MB destroyed at the tumor site using appropriate ultrasound conditions. Such a strategy would enhance localization of the sensitizer/chemotherapeutic agent and increase the pO ₂ of the tumor at the tumor site. To test the feasibility of this approach, a biotin-functionalized cyanine dye absorbing near infrared (9) was used as a surrogate for RB and 5-FU (Scheme 2) - see Example 4. UV visible spectra and fluorescence spectra of 9 detected absorption (750 nm) and emission maxima (818 nm) in the NIR region make this compound ideal for in vivo imaging.

As described in Example 4, dye (9) was loaded onto the surface of MB following the same procedure as for RB and 5-FU. Nude nude mice carrying ectopic Bx-PC3 tumors were then intravenously injected with MB-9 O ₂ conjugate via the tail vein. Ultrasound was applied directly to the tumor during and for 3 minutes after IV administration. Control experiments in the absence of ultrasound were used for comparative purposes.

Mice were imaged before, 5 and 30 minutes after administration using the IVIS whole body imaging system. Representative images (Fig. 12a) show strong tumor fluorescence 30 min after treatment of mice in the sonicated group, while mice in the control group showed negligible tumor fluorescence, with most of the radiation observed from the liver region.

When the tumor fluorescence intensity was measured relative to the pretreatment value (FIG. 12b), a statistically significant 7-fold increase was observed for the sonicated group compared to the control group 30 minutes after treatment (p<0.01). In addition, when O ₂ MB or PFBMB were administered to tumor bearing animals by tail vein injection and subsequently sonicated, protein extracts from surgically excised tumors revealed a significant decrease in Hif-1α in O ₂ MB treated tumors (Fig. 12c). These results suggest that the application of ultrasound to the tumor during and immediately after administration of the O ₂ MB-9 conjugate promotes stimulus-dependent destruction of MB in the tumor vasculature, which in turn facilitates the release of both O ₂ and payload. purposefully. The end result is an increase in tumor pO ₂ as evidenced by decreased Hif1α protein expression and a higher drug concentration in the tumor as evidenced by enhanced fluorescence (9).

Example 11 - Synthesis of monoiodo ICG (I ₂ -IR783 or "I2-IRCYDYE")

Synthesis of (4-iodophenyl)hydrazine (1):

20 g (91.3 mmol) of 4-iodananiline was stirred with a solution of 15 ml of concentrated hydrochloric acid and 15 ml of water. The mixture was cooled to about -10°C and a solution of 12.6 g (182.6 mmol) NaNO ₂ in 45 ml of water was added dropwise with continuous stirring. The suspension was allowed to stir for another 30 minutes and then an ice-cold solution of SnCl ₂ ,2H ₂ O (67.99 g, 301.3 mmol in 40 ml concentrated HCl) was added dropwise while maintaining the temperature at -10°C, for example. The reaction mixture was stirred at this temperature for 1.5 h and at 5°C overnight. The resulting light brown precipitate was filtered off and washed three times with water. The solid mass thus obtained was then stirred with a saturated solution of NaOH in water (100 ml) and extracted with ether (200 ml). The ether layer was washed with an aqueous solution of NaOH, Na ₂ S ₂ O ₃ and water. After drying with MgSO ₄ (anhydrous), the ether layer was evaporated to dryness to give 17.94 g of (4-iodophenyl)hydrazine as a brown powder. mp=104-106°C.

¹ H NMR (CDCl ₃ ): 7.48 (d, J=8.0 Hz, 2H, Ar-CH), 6.62 (d, J=8.0 Hz, 2H, Ar-CH), 5, 18 (brs, 1H, NH), 3.55 (brs, 2H, NH ₂ ).

ESI-MS (M+H) found = 235.00, calculated for C ₆ H ₇ IN ₂ = 234.04.

Synthesis of 5-iodo-2,3,3-trimethyl-3H-indole (2):

12.68 g (54.1 mmol) of (4-iodophenyl)hydrazine (1) and 8 g (92.8 mmol) of 3-methyl-2-butanone were refluxed in 100 ml of glacial acetic acid for 20 hours. The acetic acid was evaporated and the residue was taken up in ether. The insoluble precipitate was filtered off and the ethereal solution was washed with an aqueous solution of NaOH, followed by washing with Na ₂ S ₂ O ₃ and water. The organic layer was dried over anhydrous Na ₂ SO ₄ and the ether was removed under reduced pressure to obtain 10.5 g of 5-iodo-2,3,3-trimethyl-3H-indole (2) as a sticky red liquid.

¹ H NMR (CDCl ₃ ): 7.60 (dd, J=4.5, 8.0 Hz, 2H, Ar-CH), 7.28 (d, J=8.0 Hz, 1H, Ar-CH ), 2.25 (s, 3H, CH ₃ ), 1.20 (s, 6H, CH ₃ X 2).

¹³ C NMR (CDCl ₃ ): 153.4 (C), 148.1 (C), 139.3 (C), 136.6 (CH), 130.6 (CH), 121.8 (CH), 89.9 (C), 54.0 (C), 23.0 (CH ₃ ), 22.9 (CH ₃ ), 15.3 (CH ₃ ).

ESI-MS (M+H) found = 286.1, calculated for C ₁₁ H ₁₂ IN=285.12.

Synthesis of 5-iodo-2,353-trimethyl-1-(4-sulfobutyl)-3Н-indol-1-ium (3):

Toluene (70 ml), 5-iodo-2,3,3-trimethyl-3H-indole (2) (12 ggg, 42.1 mmol) and 1,4-butanesultone (8.6 g, 63.1 mmol) heated under reflux for 18 hours. The reaction mixture was allowed to cool to room temperature. The resulting brown crystals were filtered and washed with acetone (3 X 10 ml). The filtered product was recrystallized from a solution of MeOH and diethyl ether. The crystals were collected and dried in vacuo to give 8 g of 5-iodo-2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indol-1-ium (3).

¹ H NMR (dmso-d ₆ ): 8.27 (s, 1H, Ar-CH), 7.95 (s, 1H, Ar-CH), 7.82 (s, 1H, Ar-CH), 4 .42 (brs, 2H, CH ₂ ), 2.79 (s, 3H, CH ₃ ), 2.47 (brs, 2H, CH ₂ ), 1.90 (brs, 2H, CH ₂ ), 1.69 (brs, 2H, CH ₂ ), 1.49 (s, 6H, CH ₃ X 2).

¹³ C NMR (DMSO-d ₆ ): 176.2, 148.4, 139.9, 136.7, 132.5, 126.8, 96.8, 49.8, 46.8, 42.6, 26.8, 25.6, 10.5.

ESI-MS (M+H) found = 422.10, calculated for C ₁₅ H ₂₁ INO ₃ S ⁺ = 422.30.

Synthesis of 2-((E)-2-((E)-2-chloro-3-((E)-2-(5-iodo-3,3-dimethyl-1-(4-sulfobutyl)indoline-2- ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-5-iodo-3,3-dimethyl-1-(4-sulfobutyl)-3H-indol-1-ium (5):

Solution 3 (0.2 g, 0.47 mmol), 4 (prepared according to the method described in Flanagan et al., Bioconjugate Chem, 1997, 8, 751-756) (0.153 g, 0.47 mmol) and anhydrous sodium acetate (0.077 g, 0.93 mmol) in absolute EtOH (10 ml) under N ₂ was heated under reflux for 4 hours. EtOH was removed under reduced pressure and the residue was purified by column chromatography (silica gel 60-120 mesh) using 25% MeOH-CHCl ₃ as eluting agent. The product (0.152 g, 33% yield) was isolated as a green powder.

¹ H NMR (MeOH-d ₄ ): 8.26 (d, J=7.8 Hz, 1H, Ar-CH), 8.03-7.98 (m, 2H, Ar-CH), 7.68 -7.63 (m, 2H, Ar-CH), 7.63-7.49 (m, 1H, Ar-CH), 6.39-6.36 (m, 2H, CH X 2), 4, 34-4.33 (m, 2H, CH X 2), 3.33-3.34 (m, 4H, CH ₂ X 2), 2.92-2.90 (m, 2H, CH ₂ ), 2 .89-2.80 (m, 2H, CH ₂ ), 2.08-l.96 (m, 26H, CH ₂ X 7, CH ₃ X 4).

¹³ C NMR (DMSO-d ₆ ): 174.7, 173.9, 150.1, 149.6, 148.0, 146.7, 145.9, 130.8, 134.8, 132.6, 130.1, 129.8, 128.3, 126.4, 124.7, 120.7, 116.1, 114.9, 104.6, 102.8, 98.6, 62.1, 60, 1, 50.4, 29.1, 48.7, 30.5, 28.4, 28.5, 26.3, 26.2, 24.6.

ESI-MS (MH ⁺ ) found = 977.2, calculated for C ₃₈ H ₄₆ ClI ₂ N ₂ O ₆ S ₂ ⁺ = 979.06.

Example 12 - Synthesis of diiodine-IR-820 (I ₄ -IR783 or "I4-IRCYDYE")

Synthesis of 3,5-dniodnitrobenzene (2):

To a concentrated solution of H ₂ SO ₄ (96%, 15 ml), cooled to 0°C, 2,6-diiodo-4-nitroaniline 1 (3.9 g, 10 mmol) was added in small portions. This solution was stirred for 20 minutes at the same temperature and NaNO ₂ (1.5 g, 22 mmol) was added. Stirring was continued at 0° C. for 2 hours. Then the viscous solution was poured onto ice (100 g) and any solid material was filtered off. The yellow filtrate was carefully poured into a refluxing solution of CuSO ₄ .5H ₂ O (160 mg, 1 mmol) in EtOH (200 ml) and stirred for 2 h to reduce the diazonium salt. After cooling to room temperature, solid 3,5-diiodonitrobenzene (2) was separated. The product was filtered and washed with water until neutral. The product was recrystallized from EtOH to give 2.48 g (66% yield) of fine brown needles.

¹ H NMR (CDCl3) δ=8.43 (t, J=1.4 Hz, 2H, Ar-CH X2), 8.29 (s, 1H, Ar-CH);

¹³ C NMR (CDCl ₃ )δ=94.1, 131.7, 148.4, 151.0.

ESI-MS[M+H ⁺ ]: calculated for C ₆ H ₃ I ₂ NO ₂ Na 397.8, found 398.9 m/z.

Synthesis of 3,5-diiodoaniline (3):

To a suspension of 2 (7.15 g, 19 mmol) in anhydrous EtOH (75 ml) under argon was added SnCl ₂ .2H ₂ O (21.6 g, 96 mmol). This mixture was brought to a boil and a solution of NaBH ₄ (361 mg, 9.5 mmol) in EtOH (40 ml) was added dropwise. The reaction mixture was stirred at reflux for 45 min. After the reaction mixture was cooled to 0°C, was added water (60 ml) and the mixture was neutralized with NaOH (2.5 M in H ₂ O). The aniline derivative was extracted with diethyl ether, dried over Na ₂ SO 4 and evaporated under reduced pressure to give aniline 3 (5.86 g, 89% crude yield).

¹ H NMR (CDCl3) δ=7.39 (s, 1H, Ar-CH), 6.97 (s, 2H, Ar-CH X 2 ), 3.66 (brs, 2H, NH ₂ ).

¹³ C NMR (CDCl3) δ=148.5, 134.8, 122.9, 95.1.

ESI-MS[M+H ⁺ ]: calculated for C ₆ H ₅ I ₂ N 344.8, found 345.5 m/z.

Synthesis of 3,5-diiodophenylhydrazine (4):

This compound was synthesized according to the procedure described in US 2013/0231604.

Synthesis of 4,6-diiodo-2,3,3-trimethyl-3H-indole (5):

This compound was synthesized according to the procedure described in US 2013/0231604.

Synthesis of 4,6-diiodo-2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indol-1-ium

Toluene (10 ml), 4,6-diiodo-2,3,3-trimethyl-3H-indole (5) (2.1 g, 5.1 mmol) and 1,4-butanesultone (3.5 g, 25 .7 mmol) was heated at reflux for 18 hours. The reaction mixture was allowed to cool to room temperature. The resulting brown crystals were filtered off and washed with acetone (3 X 10 ml). The filtered product was recrystallized from a solution of MeOH and diethyl ether. The crystals were collected and dried in vacuo to give 1.9 g of 4,6-diiodo-2,3,3-trimethyl-1-(4-sulfobutyl)-3H-indol-1-ium (6).

¹ H NMR (MeOH-d ₄ ): 8.42 (s, 1H, Ar-CH), 8.36 (s, 1H, Ar-CH), 4.51-4.48 (m, 2H, CH ₂ ), 2.88-2.85 (m, 2H, CH ₂ ), 2.09-2.00 (m, 2H, CH ₂ ), 1.99-1.82 (m, 2H, CH ₂ ), 1.73 (s, 6H, CH ₃ X 2), 1.16 (s, 3H, CH ₃ ).

ESI-MS[M-H ⁺ ]: calculated for C ₁₅ H ₂₀ I ₂ NO ₃ S ⁺ 547.9, found 546.1 m/z.

Synthesis of 2-((E)-2-((E)-2-chloro-3-((E)-2-(4,6-diiodo-3,3-dimethyl-1-(4-sulfobutyl)indoline- 2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-4,6-diiodo-3,3-dimethyl-1-(4-sulfobutyl)-3H-indol-1-ium (8):

Solution 8 (0.84 g, 1.5 mmol), 7 (prepared according to the method described in Flanagan et al., Bioconjugate Chem, 1997, 8, 751-756) (0.25 g, 0.7 mmol ) and anhydrous sodium acetate (0.13 g, 1.5 mmol) in absolute EtOH (10 ml) under N ₂ was heated under reflux for 4 hours. EtOH was removed under reduced pressure and the residue was purified by column chromatography (silica gel 60-120 mesh) using 25% MeOH-CHCl ₃ as eluting agent. The product (0.153 g, 8% yield) was isolated as a brown powder.

¹ H NMR (MeOH-d ₄ ): 8.59 (s, 2H, Ar-CH X 2 ), 8.29 (s, 2H, Ar-CH X 2 ), 6.77-6.75 (m, 2H, CH X 2), 5.30 (brs, 2H, CH X 2), 4.82-4.72 (m, 4H, CH ₂ X 2), 3.39 (brs, 4H, CH ₂ X 2 ), 2.60-2.47 (m, 14H, CH ₂ X 7), 2.23 (s, 12H, CH ₃ X 4).

¹³ C NMR (DMSO-d ₆ ): 170.2, 169.9, 158.9, 150.1, 149.7, 148.6, 146.8, 144.9, 140.8, 139.3, 134.2, 132.1, 126.7, 124.3, 104.0, 100.4, 96.7, 96.2, 94.5, 64.1, 59.5, 50.5, 48, 7, 48.1, 30.3, 28.7, 28.2, 26.3, 26.1, 24.3.

ESI-MS[M-H ⁺ ]: calculated for C ₃₈ H ₄₄ CI ₄ N ₂ O ₆ S ₂ Na ⁺ 1253.85, found 1252.81 m/z.

Example 13 - In vivo PDT effect of I ₂ -IR783 in mice bearing human xenografts of ectopic BxPc-3 human pancreatic cancer tumors

BxPc-3 cells were stored in RPMI-160 medium supplemented with 10% fetal bovine serum. Cells were cultured at 37°C in 5% CO2 in air. BxPc-3 cells (1×10 ⁶ ) were resuspended in 100 μl of Matrigel and implanted in the back of the back of male SCID mice. Tumor formation occurred about 2 weeks after implantation, and tumor measurements were taken every day using a caliper. After the tumors reached a mean volume of 267 mm ³ calculated from the geometric mean diameter using the equation for tumor volume = 4πR ³ /3, the animals were randomly assigned to 2 groups (n=2). After administration of anesthesia (IP injection of Hypnorm/Hypnovel), the treatment group received a 100 μl aliquot of I ₂ -IR783 (1 mg/kg) in OCB:DMSO (98:2) vehicle directly into each tumor and irradiated with 780 nm light (100 mW) for 3×3 min with 1 minute delay between treatments. The second group (control) received only the carrier medium. Following treatment, animals were allowed to recover from anesthesia and tumor volume was monitored at the indicated time points. The % increase in tumor volume was calculated using preliminary measurements for each group. On day 8, the treatment group received a second treatment as described above, but also received an intratumoral injection of 100 μl O ₂ MB (1×10 ⁸ MB/ml) prior to light exposure. The results are shown in FIG. 13.

Example 14 - Fluorescence of IR783 analogs 12 and 14 ("I2-IRCYDYE" and "I4-IRCYDYE")

On FIG. 14 shows the spectra of (a) UV visible and (b) fluorescent emission of I2-IRCYDYE and I4-IRCYDYE compared to Cardio green. The new compounds clearly show absorption profiles similar to Cardio Green. However, although the fluorescent emission of I2-IRCYDYE remains similar to that of Cardio green, the emission of I4-IRCYDYE is significantly quenched. This is explained by the increase in ISC due to the presence of additional iodine atoms.

Example 15 - Singlet Oxygen Production and In Vitro Cytotoxicity of IR783 I2 and I4 Analogs ("I2-IRCYDYE" and "I4-IRCYDYE")

On FIG. 15 shows that both I2-IRCYDYE and I4-IRCYDYE produce more singlet oxygen than Cardio Green when excited at 780 nm.

On FIG. 16 shows that both I2-IRCYDYE and I4-IRCYDYE are significantly more cytotoxic to two different pancreatic cancer lines (Mia Race and BxPC-3) than Cardio Green when exposed to 780 nm irradiation. The compounds also appeared to be more toxic to the cervical cancer (HeLa) cell line than Cardio Green at 780 nm excitation. In vivo experiments in mice using ectopic BxPC-3 pancreatic cancer tumors also demonstrate that I2-IRCYDYE localizes to the tumor 18 hours after tail vein injection.

These results suggest that both I2-IRCYDYE and I4-IRCYDYE are effective NIR-activated sensitizers and that I2-IRCYDYE also has potential as an imaging agent given its high NIR fluorescence. This provides the potential to image PDT and/or SDT of solid tumors such as pancreatic tumors.

Example 16 - Combination Therapy Antimetabolite/Sonodynamic Therapy of Human Pancreatic Cancer Cells MiaPaCa-2 Using Rose Bengal and 5-FU.

Methodology:

Primary human pancreatic cancer adenocarcinoma cell lines MIA PaCa-2 were stored in Dulbecco's Modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum in a humidified 5% CO ₂ atmosphere at 37°C. Cells were seeded in the wells of a 96-well plate at a concentration of 4×10 ³ cells per well and incubated for 21 h at 37°C in a humidified 5% CO ₂ atmosphere. The medium was then removed and the wells were treated with either rose bengal, (3 μM), 5-fluorouracil (50 μM) or a combination of both RB (3 μM) and 5-FU (50 μM) for 3 h. Drug solutions were then removed, added fresh medium and sonicated harvested wells were delivered using a Sonidel SP100 sonoporator (30 sec, frequency = 1 MHz, sonic power density = 3.0 Wcm ^-2 , duty cycle = 50% with pulse repetition rate = 100 Hz) . Cells were then incubated for 24 hours until cell viability was determined using the MTT assay.

Results:

The results are shown in Fig. 17. The results show that SDT treatment (ie RB+US) reduces cell viability by 11.1% compared to RB alone (RB-US). Treatment with 5FU + ultrasound reduced cell viability by 5.9% more than 5FU. Mixed SDT/5FU + sonication (combo + US) resulted in a 21.9% reduction compared to RB/5FU - sonication. Surprisingly, this difference is larger than would be expected by adding the effects caused by both treatments (17%) and indicates synergy when both treatments are combined. (n=6).

Only active agents participated in this experiment. However, they were effectively released molecules when the microbubbles collapsed. Thus, it is expected that the results will be extended to the situation in which active agents are supplied using the microbubble technology described herein.

Example 17 Combination Anthracycline/Sonodynamic Therapy of MDA-MB-231 Human Breast Cancer Tumors Using Rose Bengal-Doxorubicin Conjugate Oxygenated Microbeads.

Synthesis of Biotin-Bengal Rose and Biotin-Doxorubicin:

Synthesis of Biotin-Bengal rose is described in detail in Example 2. Biotin-Doxorubicin (Biotin-Dox) was prepared according to Scheme 3:

Scheme 3: Synthesis scheme for obtaining Biotin-Dox

To an ice-bath cooled solution of biotin-N-hydroxysuccinimide ester (0.14 g, 0.41 mmol) in DMF (10 ml) was added doxorubicin (0.3 g, 0.41 mmol) under nitrogen atmosphere. After stirring for 30 minutes, triethylamine (0.5 ml, 2 mmol) was added to this reaction mixture and stirred for an additional 12 hours at room temperature. The progress of the reaction was monitored by TLC (Merck Silica 60, HF 254, 20:80 methanol-dichloromethane, v/v). After completion of the reaction, an excess of diethyl ether (100 ml) was added to the reaction mixture. The red solid thus obtained was filtered off and washed three times with diethyl ether (50 ml X 3). This red solid was then purified by PTLC using methanol-dichloromethane (20:80, v/v) as eluent to give 0.25 g (yield=78%) of biotinylated doxorubicin. An analytical sample was obtained by recrystallization of this product from ethanol.

¹ H NMR (MeOH-d ₄ ) δ: 8.54 (brs, 1H, NH), 7.82-7.76 (m, 2H, aromatic), 7.47 (d, J=7.5 Hz, 1H, aromatic), 5.39 (brs, 1H, NH), 5.05 (brs, 2H, NH, OH), 4.71 (s, 2H, -CH ₂ -OH), 4.67 (brs, 2H, OH X 2), 4.36-4.33 (m, 1H, CH), 4.25-4.22 (m, 1H, CH), 4.16-4.13 (m, 1H, CH ), 3.99 (s, 3H, OCH ₃ ), 3.60-3.58 (m, 1H, CH), 3.55 (brs, 2H, OH X2), 3.30-2.5 (m , 4H, CH ₂ X1, CH X 2), 2.18-2.14 (m, 3H, CH ₂ X 1, CH), 2.00-1.96 (m, 1H, CH), 1.63 -1.50 (m, 4H, CH ₂ X 2), 1.42-1.26 (m, 11 H, CH ₃ X 1, CH ₂ X4).

ESI-MS [MH]: calculated for C ₃₇ H ₄₃ I ₂ N ₃ O ₁₃ S=769.25, found = 767.9 m/z.

Preparation of oxygen-filled microbubbles of rose bengal (RBO ₂ MB) and doxorubicin (DoxO ₂ MB) conjugates:

Solutions containing Biotin-RB (2.5 mg/ml) and Biotin-Dox (2.5 mg/ml) were prepared in a solution of 0.5% DMSO in PBS (pH 7.4±0.1). A 2 ml aliquot of these stock solutions was then added separately to two 2 μl of PFBMB-functionalized avidin suspension (1×10 ⁹ MV/ml) and the contents were mixed with shaking for 15 minutes. The suspensions were then centrifuged (900 rpm) for 5 min and the MB conjugates were isolated as a milky suspension floating on top of the solution. The solution was removed and replaced with another 2 ml stock solution containing either Biotin-RB or Biotin-Dox and the mixing/centrifugation steps repeated. The MB suspensions were then washed with PBS (5 ml), centrifuged (900 rpm) for 5 min, and the MB was transferred to clean centrifuge tubes. This washing procedure was repeated again and the isolated PFBMB-RB and PFBMB-Dox conjugates were placed in a glass vial. The PFBMB-RB and PFBMB-Dox conjugates were then sparged with gaseous oxygen for 2 min, and as a result RBO ₂ MB and DoxO ₂ MB (see Fig. 18) were used directly in animal experiments.

Processing of human MDA-MB-231 xenograft using SCID mouse breast cancer tumors:

All animals used in this study were handled humanely and according to licensed procedures in accordance with the UK Animals (Scientific Procedures) Act 1986. MDA-MB-231 cells were stored in RPI-1640 medium supplemented with 10% fetal serum albumin as indicated higher. Cells (1×10 ⁶ ) were resuspended in 100 μl of ^Matrigel® and implanted in the dorsum of female Balb/c SCID (CB-17/IcrHan®Hsd-Prkdcscid) mice. Tumor formation occurred about 2 weeks after implantation, and tumor measurements were taken every other day with a caliper. Once the tumors reached an average volume of 100 mm ³ calculated from the geometric mean diameter using the equation for tumor volume = 4πR ³ /3, the animals were randomly assigned to 3 groups (n=3). After administration of anesthesia (intraperitoneal injection of Hypnorm/Hypnovel), group 1 received 100 μl RBO ₂ MB (300 μM RB); group 2 received 100 μl DoxO ₂ MB (475 μM) and group 3 received 100 μl containing RBO ₂ MB (150 μM RB) and DoxO ₂ MB (237.5 μM). Intratumoral injection was chosen as the route of administration in order to exclude experimental changes caused by the pharmacokinetic behavior of the platform. The tumors were then sonicated for 3.5 min at an ultrasound frequency of 1 MHz, an ultrasound power density of 3.5 W cm ^-2 (I _SATP ; spatial mean temporal peak) and using a duty cycle of 30% at a pulse rate of 100 Hz. Treatments were repeated on the 14th day. After treatment, the animals were allowed to recover from anesthesia, and tumor volume and body weight were recorded daily for nine days. The % tumor volume increase was calculated using pretreatment measurements for each group.

Results:

The results are shown in Fig. 19. The results show that the DoxO ₂ MB/RBO ₂ MB+US mixed treatment was more effective than RBO ₂ MB+US and as effective as DoxO ₂ MB+US using half the concentration of doxorubicin and rose bengal. The results show that the platform can be used to treat breast cancer.

Claims (33)

1. Use of a microbubble-chemotherapeutic agent complex for the manufacture of a medicament for the treatment of pancreatic, breast, or prostate cancer through sonodynamic therapy, said sonodynamic therapy method comprising simultaneous or sequential use of a microbubble-sonosensitizer complex; wherein said chemotherapeutic agent is 5-fluorouracil, doxorubicin, gemcitabine, docetaxel or paclitaxel, and said sonosensitizer is rose bengal dye. 2. Use of a microbubble-chemotherapeutic agent complex in the manufacture of a medicament for the treatment of pancreatic, breast, or prostate cancer through sonodynamic therapy, said complex being attached to or associated with at least one sonosensitizer; wherein said chemotherapeutic agent and said sonosensitizer are as defined in claim 1. 3. Use according to claim 2, characterized in that said complex is attached to or associated with at least one said sonosensitizer via a biotin-avidin interaction. 4. Use according to claim 1, characterized in that the microbubble-chemotherapeutic agent complex comprises a microbubble attached to or associated with at least one chemotherapeutic agent, which is 5-fluorouracil, doxorubicin, gemcitabine, docetaxel, or paclitaxel. 5. Use according to claim 4, characterized in that the microbubble-chemotherapeutic agent complex comprises a microbubble attached to or associated with said at least one chemotherapeutic agent via a biotin-avidin interaction. 6. Application according to any one of paragraphs. 1-5, characterized in that the microbubble-chemotherapeutic agent complex contains a microbubble having a shell that retains gas. 7. Use according to claim 6, characterized in that said gas is oxygen. 8. Application according to any one of paragraphs. 1 and 4-7, characterized in that the microbubble-sonosensitizer complex contains a microbubble having a shell that retains gas. 9. Use according to claim 8, characterized in that said gas is oxygen. 10. Application according to any one of paragraphs. 1-9, characterized in that said microbubble-chemotherapeutic agent complex and/or said microbubble-sonosensitizer complex contains a microbubble having a diameter in the range from 0.5 to 100 µm. 11. Application according to any one of paragraphs. 1-10, characterized in that said microbubble-chemotherapeutic agent complex and/or said microbubble-sonosensitizer complex contains a phospholipid monolayer shell with one or more polymers associated with it. 12. Pharmaceutical composition for the treatment of pancreatic, breast or prostate cancer through sonodynamic therapy, wherein said composition contains a microbubble-chemotherapeutic agent complex as defined in paragraph 2 or 3, together with at least one pharmaceutical carrier or excipient . 13. Pharmaceutical composition for the treatment of pancreatic, breast or prostate cancer through sonodynamic therapy, while the specified composition contains a microbubble-chemotherapeutic agent complex, as defined in any of paragraphs. 1, 4-7, 10 and 11, and the microbubble-sonosensitiser complex as defined in any one of paragraphs. 1 and 8-11, together with at least one pharmaceutical carrier or excipient. 14. The use of a pharmaceutical composition according to claim 12 or 13 in a method for the manufacture of a medicament for the treatment of pancreatic, breast or prostate cancer through sonodynamic therapy. 15. Use according to claim 14, characterized in that said drug is brought into contact with cells or tissues of the patient and, simultaneously or sequentially, said cells or tissues are exposed to ultrasound. 16. Use according to claim 14 or 15, characterized in that said cancer is breast or prostate carcinoma. 17. Use according to claim 14 or 15, characterized in that said cancer is pancreatic cancer. 18. The use of a product containing a microbubble-chemotherapeutic agent complex and a microbubble-sonosensitizer complex in the manufacture of a medicament for simultaneous or sequential use in a method for treating pancreatic, breast or prostate cancer through sonodynamic therapy, wherein said chemotherapeutic agent is a 5- fluorouracil, doxorubicin, gemcitabine, docetaxel or paclitaxel, and said sonosensitizer is a rose bengal dye. 19. A microbubble for use in the treatment of pancreatic, breast, or prostate cancer through sonodynamic therapy, wherein said microbubble contains associated at least one chemotherapeutic agent and at least one sonosensitizer, wherein said chemotherapeutic agent and said sonosensitizer are as defined in paragraph 1. 20. Microbubble according to claim 19, which contains gaseous oxygen. 21. A method for the treatment of pancreatic, breast or prostate cancer by means of sonodynamic therapy, which includes the simultaneous or sequential administration of a microbubble-sonosensitizer complex and a microbubble-chemotherapeutic agent complex into cells or tissues of a patient in need thereof, exposing said cells or tissues to ultrasonic exposure to break said microbubbles and activate said sonosensitizer; wherein said chemotherapeutic agent is 5-fluorouracil, doxorubicin, gemcitabine, docetaxel or paclitaxel, and said sonosensitizer is rose bengal dye. 22. A method for the treatment of pancreatic, breast or prostate cancer by means of sonodynamic therapy, which includes the introduction into the cells or tissues of a patient in need of it, a microbubble-chemotherapeutic agent complex, wherein said complex is attached to or associated with at least one sonosensitizer, and wherein said method includes exposing said cells or tissues to ultrasonic action to break said microbubbles and activate said sonosensitizer; and wherein said chemotherapeutic agent and said sonosensitizer are as defined in claim 21. 23. The method of claim 22 wherein said complex is attached to or associated with at least one said sonosensitizer via a biotin-avidin interaction. 24. The method of claim 21, wherein the microbubble-chemotherapeutic agent complex comprises a microbubble attached to or associated with at least one chemotherapeutic agent, which is 5-fluorouracil, doxorubicin, gemcitabine, docetaxel, or paclitaxel. 25. The method of claim 24 wherein the microbubble-chemotherapeutic agent complex comprises a microbubble attached to or associated with said at least one chemotherapeutic agent via a biotin-avidin interaction. 26. The method according to any one of paragraphs. 21-25, characterized in that the microbubble-chemotherapeutic agent complex contains a microbubble having a shell that retains gas. 27. The method of claim 26, wherein said gas is oxygen. 28. The method according to any one of paragraphs. 21 and 24-27, characterized in that the microbubble-sonosensitizer complex contains a microbubble having a shell that retains gas. 29. The method of claim 28, wherein said gas is oxygen. 30. The method according to any one of paragraphs. 21-29, characterized in that said microbubble-chemotherapeutic agent complex and/or said microbubble-sonosensitizer complex contains a microbubble having a diameter in the range of 0.5 to 100 µm. 31. The method according to any one of paragraphs. 21-30, characterized in that said microbubble-chemotherapeutic agent complex and/or said microbubble-sonosensitizer complex contains a phospholipid monolayer shell having one or more polymers associated with it. 32. The method according to any one of paragraphs. 21-31, characterized in that said cancer is a breast or prostate carcinoma. 33. The method according to any one of paragraphs. 21-31, characterized in that said cancer is pancreatic cancer.

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