Pancreatic Cancer Treatment

Abstract

The present invention relates to ultrasound-mediated delivery of therapeutic agents to the pancreas, and in particular to the treatment of pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). More particularly, the present invention provides cluster compositions and pharmaceutical compositions for use in the delivery of therapeutic agents and for the treatment of pancreatic cancer, including PDAC.

Description

[Technical field]

[0001] The present invention relates to ultrasound-mediated delivery of therapeutic agents to the pancreas, and in particular for the treatment of pancreatic cancer, such as pancreatic ductal adenocarcinoma (PDAC). Thus, the present invention provides cluster compositions and pharmaceutical compositions for use in the delivery of therapeutic agents and for use in the treatment of pancreatic cancer, such as PDAC. [Background technology]

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of malignant tumors with a 5-year survival rate of less than 9%. Current clinical treatment regimens include chemotherapy and / or surgery, with or without radiation therapy. Surgery remains the only possibility for cure, but is often not an option due to the late stage of diagnosis and the invasive nature of PDAC. If the cancer is downgraded after treatment allowing the possibility of surgery, the overall survival rate increases significantly, indicating the importance of tumor volume. PDAC is known to be highly resistant to conventional and targeted drugs. The reasons for such chemoresistance are thought to be multifactorial, but one of the major mechanisms is believed to be the low rate of drug penetration into the tumor bed. One of the main reasons for such extreme drug resistance is the desmoplastic and poorly vascularized stroma that forms a protective barrier around the PDAC cells. Thus, the major limitations to effectively treating such solid tumors with therapeutic agents, such as chemotherapeutic agents, are inadequate delivery to the target location due to tumor interstitial fluid pressure, lack of vascularization or perfusion, or the presence of a dense interstitial matrix. The stromal microenvironment is a complex structure composed of extracellular matrix (ECM), activated fibroblasts and myofibroblasts, inflammatory cells, and blood and lymphatic vessels that distort the normal architecture of pancreatic tissue. The complex interrelationships between tumor cells and stroma promote cancer cell motility, resistance to hypoxia, and stromal neovascularization. These physical barriers make the tumor inaccessible to most chemotherapy drugs. As a result, chemotherapy treatments suffer a similar fate in vivo, and compensatory dose increases exacerbate systemic side effects. Therefore, mechanical destruction of the tumor or normalization of tumor vascularization to allow improved delivery of drugs may offer better clinical prospects. Improved drug delivery may therefore have a significant impact on the therapeutic efficacy of chemotherapy in patients with PDAC.

[0003] Treatment options specifically focused on targeted drug delivery in solid tumors are currently under trial and include nanoparticles, molecular targeting, and ultrasound or microbubble mediated therapy, i.e. sonoporation. Sonoporation is a method in which gaseous microbubbles are injected into the vasculature and stimulated by ultrasound (US) to induce biomechanical effects that increase the permeability and extravasation of drugs into the vascular barrier at specific locations. Microbubbles are stabilized gas bubbles (2-3 μm in diameter) that are injected into blood vessels and are typically stable in vivo for up to 1-2 minutes without known side effects. Upon application of ultrasound, these microbubbles are believed to vibrate and interact with the nearby endothelium / vessel wall to form perforations through various biomechanical effects. The interactions may allow extravasation of drugs from the vascular compartment, increased intracellular drug uptake, and allow therapeutics to penetrate deeper into tissues as well as the vascular barrier. While the technique has shown promise, the inherent potential of sonoporation is limited due to the use of commercially available microbubbles that are designed and optimized for ultrasound imaging, not for therapeutic enhancement. As a result, substantial research has been focused on developing “next-generation” microbubbles optimized for ultrasound-mediated, targeted, and localized therapy. A major limitation is the size of the microbubbles. Being small, they are limited in the level of biomechanical effects they can induce within the vascular compartment. Furthermore, they are limited in their efficiency in causing perforation due to limited physical contact with the endothelial wall, and biomechanical effects typically decrease exponentially with distance from the vessel wall.

[0004] Recently, in US Patent Publication 1, the concept of ultrasound-mediated targeted delivery was proposed, where a microbubble / microdroplet cluster composition is administered with a therapeutic agent, and ultrasound irradiation of the targeted pathology can lead to an increased therapeutic effect compared to the therapeutic agent alone. This concept, called acoustic cluster therapy (ACT sonoporation or ACT), has since been investigated in a series of preclinical proof-of-principle and proof-of-concept studies. For example, S. Kotopoulis et al., Acoustic Cluster Therapy (ACT®) induces transient tumour volume reduction in a subcutaneous xenograft model of pancreatic ductal adenocarcinoma. J Control Release, 245 (2016) 70-80 (Non-Patent Document 1) describes a preclinical study in which ACT is combined with paclitaxel for the treatment of PDAC in mice. Unfortunately, due to several limitations in this study, the increase in pharmacological effect compared to the agent alone was only modest, with a small decrease in tumor growth rate, and no improvement in survival and no complete response.

[0005] In view of the above, there exists a significant unmet need for new and alternative compositions and methods for the treatment of subjects suffering from pancreatic cancer, such as PDAC. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2015 / 047103 [Non-patent literature]

[0007] [Non-Patent Document 1] J Control Release, 245 (2016) 70-80 Summary of the Invention

[0008] It is an object of the present invention to provide compositions and methods for use in the treatment of subjects suffering from pancreatic cancer, and in particular in the treatment of pancreatic ductal adenocarcinoma (PDAC). Based on conducted and newly designed studies, the inventors have demonstrated that acoustic cluster therapy is effective in the treatment of PDAC, and provides improved therapeutic efficacy of co-administered therapeutic agents compared to treating a subject with the therapeutic agent alone.

[0009] A pharmaceutical composition comprising a cluster composition that improves the delivery of therapeutic agents to the pancreas and the treatment of pancreatic cancer has been identified in the present invention. It uses ACT technology to generate large phase shift bubbles in vivo from an administered pharmaceutical composition comprising microbubbles / microdroplet clusters, which allows the delivery of separate, pre-, and / or co-, and / or post-administered therapeutic agents. This method provides a significant improvement in therapeutic efficacy over the use of therapeutic agents alone.

[0010] In one aspect, the present invention provides a pharmaceutical composition for use in a method for the treatment of pancreatic cancer, wherein the pharmaceutical composition comprises: (a) a cluster composition comprising a suspension of clusters in an aqueous biocompatible solvent, the clusters having an average diameter in the range of 1 to 10 μm and a circularity of less than 0.9; and (i) a first component comprising gas microbubbles and a first stabilizing agent for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizing agent for stabilizing the microdroplets, the second component comprising a diffusible component capable of diffusing into the gas microbubbles and thereby increasing their size at least temporarily; wherein the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via electrostatic interactions; and (b) a therapeutic agent selected from a chemotherapeutic agent and an immunotherapeutic agent, or a combination thereof, provided as a separate composition from (a). Includes.

[0011] The present invention further comprises: (a) a cluster composition comprising a suspension of clusters in an aqueous biocompatible solvent, the clusters having an average diameter in the range of 1 to 10 μm and a circularity of less than 0.9; and (i) a first component comprising gas microbubbles and a first stabilizing agent for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizing agent for stabilizing the microdroplets, the second component comprising a diffusible component capable of diffusing into the gas microbubbles and thereby increasing their size at least temporarily; wherein the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via electrostatic interactions; and (b) a therapeutic agent selected from a chemotherapeutic agent and an immunotherapeutic agent, or a combination thereof, provided as a separate composition from (a). The present invention provides a method for treating a subject suffering from pancreatic cancer, comprising administering to said subject a pharmaceutical composition comprising: [Brief description of the drawings]

[0012] [Figure 1] Figure 1 provides a visualization of cluster size versus product efficacy in vivo: the Y-axis shows the calculated correlation coefficient of Grey Scale enhancement from US imaging (i.e., the amount of bubbles deposited after activation) and the X-axis shows the cluster diameter in μm. [Figure 2A] FIG. 2A provides a photograph of the setup of the equipment used in the studies of Examples 2 and 3 for the application of the ACT sonoporation procedure including ultrasound activation and enhancement. [Figure 2B]FIG. 2B provides a setup sketch of the apparatus used in the studies of Examples 2 and 3 for the application of the ACT sonoporation procedure including ultrasound activation and enhancement. [Figure 2C] FIG. 2C provides an overview of the treatment schedule for the study of Example 2, in which therapeutic efficacy in treating a patient-derived xenograft mouse model of PDAC was evaluated. [Diagram 3] Figure 3 provides results from the study of Example 2. The Y-axis shows tumor volume as a function of time for treatment of pancreatic ductal adenocarcinoma in mice with liposomal irinotecan (closed squares), saline control (open squares), and liposomal irinotecan in combination with ACT (closed circles). The X-axis shows time in days from the start of treatment. Treatment was administered on days 0, 7, and 14. [Figure 4] Figure 4 provides results from the study of Example 2. The Y-axis shows tumor volume as a function of time for treatment of pancreatic ductal adenocarcinoma in mice with nab-paclitaxel / gemcitabine (closed squares), saline control (open squares), ACT before nab-paclitaxel / gemcitabine administration (open circles), and ACT after nab-paclitaxel (closed circles). The X-axis shows time in days from the start of treatment. Treatment was administered on days 0, 7, and 14. [Diagram 5] FIG. 5 provides graphs from an example of an ACT treatment performed during administration of the chemotherapy regimen FOLFOX (a 2-hour infusion of Recovorin and Oxaliplatin followed by a bolus injection of fluorouracil (5-FU) at 2 hours), followed by a 48-hour infusion of fluorouracil. Panel A: Each ACT treatment includes a.: injection of the cluster composition, then b.: activation of the cluster with conventional medical imaging US ultrasound exposure, and c.: enhancement with low-frequency US ultrasound exposure. Panel B: The Y-axis shows the plasma concentration relative to the peak. The X-axis shows the time in minutes. Three ACT procedures are performed at approximately 50, 80, and 110 minutes to cover all three drugs. [Figure 6]FIG. 6 is a graph from an example of a treatment schedule with a combination chemotherapy regimen of gemcitabine and nab-paclitaxel: nab-paclitaxel infusion at 30 minutes followed by gemcitabine infusion at 30 minutes. ACT procedures are applied three times during chemotherapy administration, as indicated by the gray ACT® sonoporation bar. Panel A: Each ACT procedure consists of a.: injection of the cluster composition, b.: activation of the cluster using 60 seconds of conventional medical imaging ultrasound, c.: an enhancement step using 5 minutes of 400-600 kHz ultrasound. Panel B: The Y-axis shows the plasma concentration of the administered chemotherapy in percent of peak, and the X-axis shows time in minutes. [Figure 7] 7 provides results from the study of Example 3, where the Y-axis shows tumor volume as a function of time for treatment of hepatocellular carcinoma (HCC) in mice with oncolytic reovirus (closed triangles), saline control (closed squares), and oncolytic reovirus in combination with ACT (closed circles). The X-axis shows time from start of treatment. Grey triangles below the X-axis indicate days of treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] definition As used herein, Acoustic Cluster Therapy (ACT), as defined in more detail below, involves the administration of a cluster composition (see definition below) in combination with at least one therapeutic agent (chemotherapeutic or immunotherapeutic agent), followed by application of ultrasound to a targeted pathological area (e.g., a tumor, etc.).

[0014] As used herein, "subject" means any human or non-human animal individual selected for treatment or therapy, and may include, but is not limited to, patients, particularly human patients diagnosed with pancreatic cancer, such as PDAC.

[0015] As used herein, the term "therapeutically effective amount" means an amount of a therapeutic agent that is effective to produce a desired therapeutic effect in a subject, at a reasonable benefit / risk ratio applicable to any treatment.

[0016] The terms "microbubbles" or "regular, contrast microbubbles" are used herein to describe microbubbles having diameters in the range of 0.2-10 microns, typically with an average diameter between 2-3 μm. "Regular, contrast microbubbles" include commercially available agents such as Sonazoid® (GE Healthcare), Optison (GE Healthcare), Sonovue (Bracco Spa), Definity® (Lantheus Medical Imagin), Micromarker (VisualSonics Inc.) and Polyson L (Miltenyi Biotec GmbH).

[0017] The term HEPS / RFB is used herein to describe the microbubbles formed by reconstituting the first component (provided in Example 1) with 2 mL of water.

[0018] The terms "phase shift bubbles," "large, phase shift bubbles," "large, activated bubbles," and "activated bubbles" are used herein to describe large (greater than 10 μm) bubbles that are formed following ultrasound (US)-induced activation of the cluster composition.

[0019] The term "microdroplet" is used herein to describe microdroplets of emulsion having diameters in the range of 0.2 to 10 microns.

[0020] "Insonation" or "US insonation" is used to describe exposure to or treatment with ultrasound.

[0021] The term "regular medical imaging ultrasound" is used to describe ultrasound from off-the-shelf US scanners and probes for medical imaging, i.e., frequencies between 1-10 MHz and MI less than 1.9, preferably less than 0.7, and more preferably less than 0.4.

[0022] The term "deposit tracer" is used herein in reference to activated phase-shift bubbles in the sense that the temporary mechanical trapping of large bubbles in the microcirculation implies that the local deposition of phase-shift bubbles in tissue will reflect the amount of blood flowing through the tissue microcirculation at the time of deposition of the activated bubbles. Thus, the number of trapped "deposited" phase-shift bubbles will be linearly proportional to the tissue perfusion at the time of deposition.

[0023] The term "phase shift (process)" is used herein to describe the phase transition of a substance from a liquid to a gaseous state. In particular, the phase shift (process) is the change of the state of the oil component of the microdroplets of the cluster composition from liquid to gaseous upon US ultrasonic irradiation.

[0024] The term "bi-phasic" refers to a system that includes two phase states, particularly liquid and gas states, such as the microbubble (gas) and microdroplet (liquid) components of a cluster composition.

[0025] As used herein, the terms "therapy delivery / therapeutic agent(s)" and "drug delivery / drug(s)" are both understood to include drug molecules, nanoparticles, nanoparticle delivery systems, and liposomal delivery systems that contain at least one therapeutically active agent.

[0026] The term "1st component" (or first component, or C1) is used herein to describe the dispersed gas (microbubble) component. The term "2nd component" (or second component, or C2) is used herein to describe the dispersed oil phase (microdroplet) component that contains the diffusible component.

[0027] The term "cluster composition" is used herein to describe a composition resulting from the combination, e.g., mixing, of a first component (microbubbles) and a second component (microdroplets). Thus, a cluster composition having the characteristics further described herein is ready for administration to a subject and represents a formulated composition for use in acoustic cluster therapy.

[0028] The term "diffusible component" is used herein to describe a chemical moiety of the oil phase of a second component that is capable of diffusing in vivo into microbubbles in a first component, temporarily increasing their size.

[0029] The term "pharmaceutical composition" as used herein has its conventional meaning, and in particular is in a form suitable for administration to a mammal. The composition preferably comprises two separate compositions, a cluster composition (a) and a therapeutic agent (b), both of which are suitable for administration to a mammal, either by the same or different routes of administration, such as via parenteral, intraperitoneal, or intramuscular injection. The phrase "in a form suitable for mammalian administration" refers to a composition that is sterile, pyrogen-free, free of compounds that produce undue deleterious or adverse effects, and formulated at a biocompatible pH (about pH 4.0 to 10.5). Such compositions are formulated such that precipitation does not occur on contact with body fluids (such as blood), contain only biologically compatible excipients, and are preferably isotonic.

[0030] As used herein, the term "sonometry (system)" refers to an in vitro measurement system for dynamically classifying and measuring activated phase-shifting bubbles using acoustic techniques.

[0031] The term "reactivity" is used herein to describe the ability to form microbubbles / microdroplets by mixing of microbubbles in a first component and microdroplets in a second component. Coulter counting is suitable for quantifying the concentration and size distribution of microbubbles and microdroplets in C1 and C2, as well as for evaluating the properties of particles in cluster compositions (pharmaceutical products, DP). The reactivity (R) of a cluster composition is: R = (C c1 +C c2 -C DP )·100 / (C c1 +C c2 ) (In the formula, C c1 , C c2 and C DPare the concentration figures observed in C1, C2 and DP, respectively) Reactivity is therefore a measure of how many microbubbles and microdroplets in C1 and C2, respectively, form clusters in the DP. Reactivity is also related to how large these clusters are (i.e., how many individual microbubbles and microdroplets comprise a single cluster). From the Coulter analysis of C1, C2 and DP, reactivity can be easily calculated:

[0032] The term "microbubble / microdroplet cluster" or "cluster" as used herein refers to a group of microbubbles and microdroplets that are aggregated as an entity by electrostatic attraction and are permanently held together in a single particle. The term "clustering" as used herein refers to the process by which microbubbles in a first component and microdroplets in a second component form clusters.

[0033] In medical ultrasound, the acoustic power is usually described by the "Mechanical index" (MI). This parameter is expressed in MHz as the central frequency of the ultrasound field (F c It is defined as the maximum negative sound pressure in an ultrasound field (PNP) divided by 0.3 dB / cm / MHz, which is the square root of the power-negative noise (PNP) [American Institute of Ultrasound in Medicine. Acoustic Output Measurement Standard for Diagnostic Ultrasound Equipment. 1st ed. 2nd ed. Laurel, MD: American Institute of Ultrasound in Medicine; 1998, 2003].

[0034]

number

[0035] The regulatory requirement during medical US imaging is to use an MI less than 1.9. During US imaging with microbubble contrast agents, an MI less than 0.7 is recommended to avoid adverse biological effects such as microbleeds and irreversible vascular damage, and using an MI less than 0.4 is considered a "best practice."

[0036] In the context of the ACT procedure herein, the term "activation" or "activation step" refers to the induction of a phase shift in microbubbles / microdroplet clusters by ultrasound irradiation, i.e., the generation of large, activated bubbles.

[0037] The term "frequency" is defined as the number of (ultrasonic) cycles per second (Hz). As used herein, the term refers to the central frequency of the applied ultrasonic field.

[0038] In the context of the ACT procedure herein, the term "enhancement" or "enhancement step" refers to the induction of volume oscillations of large activated bubbles, the biomechanical effect of which is confirmed by US irradiation.

[0039] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention provides a cluster composition for use in a method of treating subjects suffering from pancreatic cancer, and in particular pancreatic ductal adenocarcinoma (PDAC). The present invention uses ACT technology to generate large phase-shifting bubbles in vivo from an administered pharmaceutical composition containing microbubbles / microdroplet clusters, which allows for the delivery of separate, pre- and / or co-administered and / or post-administered therapeutic agents. The therapeutic effect of the therapeutic agent is significantly increased compared to administration of the agent alone due to biomechanical mechanisms in the microvasculature, as further described below. The activated phase-shifting bubbles are approximately 10 times larger in diameter than typical, regular contrast agent microbubbles.

[0040] Pancreatic cancer includes any type of cancer of the pancreas, including PDAC, non-adenocarcinoma, and neuroendocrine tumors, and preferably the pancreatic cancer is PDAC. PDAC is a form of cancer in which epithelial tumors arise from the cells of the pancreatic duct, or pancreatic ductules, from which the pancreatic ducts act as conduits through which digestive enzymes and bicarbonate ions produced in the acinar cells reach the small intestine. Together, the pancreatic ductal cells and acinar cells constitute the "exocrine" pancreas, from which the vast majority of pancreatic neoplasms arise. Adenocarcinoma is defined as neoplasia, i.e., abnormal and excessive proliferation of epithelial tissue. Once a diagnosis of PDAC is confirmed or strongly suspected, staging of the tumor is typically attempted. This is primarily accomplished through triphasic CT scans of the abdomen. The American Joint Committee on Cancer (AJCC) 7th edition pancreatic cancer staging follows a standard "TNM" (tumor size, lymph node status, metastasis) format and is commonly used to stage tumors and determine prognosis. In stage 0, abnormal cells are found in the lining of the pancreas. These abnormal cells can become cancerous and spread into nearby normal tissue. Stage 0 is also called carcinoma in situ. In stage I, cancer has formed but is only found in the pancreas. Stage I is divided into stages IA and IB, depending on the size of the tumor. Stage IA: The tumor is 2 cm or smaller. Stage IB: The tumor is larger than 2 cm but not larger than 4 cm. Stage II is divided into stages IIA and IIB, depending on the size of the tumor and where the cancer has spread. Stage IIA: The tumor is larger than 4 cm. Stage IIB: The tumor is any size and the cancer has spread to 1-3 nearby lymph nodes. In stage III, the tumor is any size and the cancer has spread to four or more nearby lymph nodes or to major blood vessels near the pancreas. In stage IV, the tumor is any size and the cancer has spread to other parts of the body, such as the liver, lungs, or peritoneal cavity (the body cavity that contains most of the abdominal organs). Recurrent pancreatic cancer is cancer that has recurred after it has been treated.The cancer may come back in the pancreas or in other parts of the body.

[0041] The compositions for use and methods of treatment of the present invention may be useful in all stages of PDAC, and may be used in the treatment of one or more of stages 0, I, II, III, or IV. In certain embodiments, the pharmaceutical compositions of the present invention are for the treatment of PDAC at any of stages 0, I, II, III, or IV. In certain embodiments, the pharmaceutical compositions are for the treatment of PDAC at an earlier stage, such as the treatment of PDAC at stages 0, I, or II.

[0042] The present invention provides pharmaceutical compositions comprising cluster compositions for use in methods of delivery of therapeutic agents to the pancreas and for use in the treatment of pancreatic cancer, the methods including phase shifting technology to generate large phase shift bubbles in vivo from an administered cluster composition, allowing for the delivery and uptake of a separately administered therapeutic agent. The compositions and methods for use of the present invention enhance the therapeutic effect of a separately co-administered therapeutic agent and provide improved therapeutic outcomes compared to treatment without the use of a composition of the present invention.

[0043] Acoustic Cluster Therapy (ACT) is a novel technique that utilizes microbubbles, and more particularly microbubble / microdroplet clusters, activated by application of ultrasound to create local openings or perforations in tumor vasculature, leading to a temporary increase in vascular permeability, thereby allowing better penetration of drugs into the tumor bed. The present invention is based in part on findings from preclinical studies, where applicants have investigated the activity of ACT to enhance the therapeutic efficacy of clinically used chemotherapy regimens in patient-derived xenograft (PDX) mouse models for PDAC. Applicants have found that the ACT concept is an effective way to overcome biological barriers for improved therapeutic drug uptake. This has been found to be particularly beneficial for the treatment of pancreatic cancer, and in particular PDAC, due to the generally low drug penetration rate into the tumor bed, due to the scarce, adhesive and poorly vascularized stroma that forms a protective barrier around PDAC cells. Kotopoulis et al. briefly mention the possible use of ACT for the treatment of stage III PDAC, but do not point to the very specific therapeutic benefit that may entail in this disease, the use of ACT to overcome stromal transition formation, much less suggest the treatment of earlier or later stages of the disease. Furthermore, Kotopoulis et al. only cite one specific small molecule drug, and do not teach the use of the ACT concept with alternative larger molecule / composite therapeutics, such as liposomal formulations of drugs, or the use of immunotherapeutic drugs, such as oncolytic viruses or checkpoint inhibitors.

[0044] When referring to the ACT technique of the present invention, in addition to separate administration of therapeutic agents, it includes administration of premixed cluster compositions, which are described in more detail below. The therapeutic agents are administered according to the standard of care, i.e. according to their respective Summary of Product Characteristics (SmPC). In one embodiment of the present invention, this includes the use of cluster formulations in which negatively charged microbubbles are mixed with positively charged microdroplets before administration to the subject. The administered clusters can be activated by ultrasound. These microbubble and microdroplet mixtures are held together by electrostatic forces resulting in small microbubble-microdroplet clusters. The microdroplets typically have a boiling point below 50° C. and contain oil components with low solubility in blood. The cluster composition, i.e. dispersion, is intended for administration together with a drug, i.e. with a therapeutic agent. When the clusters of the cluster composition are sonicated, the vibrating microbubbles initiate a momentary vaporization (phase shift) of the attached microdroplets. The resulting enlarged bubbles have been shown to form in capillary-sized blood vessels in vivo and can be excited by low-frequency US to induce biomechanical effects that allow extravasation and increase drug permeability in the insonified tissue.

[0045] Thus, drug delivery to the pancreas and treatment of pancreatic cancer according to the present invention is achieved by the use of a two-component, biphasic microbubble / microdroplet formulation system (i.e., cluster composition) in which the microbubbles in the first component are physically attached by electrostatic attraction to micron-sized emulsion microdroplets in the second component prior to administration. The compositions for use in the method of treating PDAC according to the present invention provide improved uptake of therapeutic agents, resulting in beneficial treatments including, for example, reduced tumor volume.

[0046] Mixing the first component with the second component prior to administration is a prerequisite for efficient formation of microbubble / microdroplet clusters, and the cluster composition can be stable at ambient conditions. The clusters are easily activated in vivo using low-power conventional medical imaging ultrasound, i.e., MI less than 1.9, preferably less than 0.7, and most preferably less than 0.4, which induces a liquid-to-gas transition (phase shift) of the diffusible components. The therapeutic agents, i.e., chemotherapeutic or immunotherapeutic agents, or combinations thereof, are administered separately according to their approved SmPC. The large activated bubbles are transiently incorporated within the microvasculature of the insonified tissue, and further application of low-power, low-frequency ultrasound facilitates the uptake of the agent into the target tissue of the pancreas. The activated phase-shift bubbles are approximately 10 times larger in diameter than typical microbubbles, resulting in Transient deposition / trapping of activated bubbles within the microvasculature of the targeted (i.e., insonified) lesion; Close contact of the activated bubble with the endothelium; Orders of magnitude greater biomechanical effects during post-activation US treatment, avoiding the inertial cavitation mechanism compared to conventional contrast microbubbles; results.

[0047] The cluster composition, i.e., the combination of the first and second components, is a suspension or dispersion of individual microbubbles and microdroplets in the form of clusters of gas microbubbles and oil microdroplets, i.e., stable microbubble / microdroplet clusters. The analytical procedures for quantitative detection and characterization of the clusters are described in Example 1. As used herein, the term "clusters" refers to groups of microbubbles and microdroplets that are permanently held together in a single particle, aggregated as an entity by electrostatic attraction. The content and size of the clusters in the cluster composition are substantially stable for a period of time (e.g., more than 1 hour) after mixing of the first and second components in vitro, i.e., they do not spontaneously collapse, form larger aggregates, or spontaneously activate (phase shift), and are substantially stable for a period of time even during continued stirring after dilution. Thus, it is possible to detect and characterize the clusters in the cluster composition using various analytical techniques that require dilution and / or stirring. Furthermore, the stability of the cluster composition allows for necessary clinical procedures to be performed (e.g., reconstitution, withdrawal and administration of single doses, etc.) The first and second components, as well as the cluster composition, are prepared in accordance with Good Manufacturing Practice (GMP).

[0048] After mixing of the two components (in vitro), such as by reconstituting a lyophilized microbubble component with a microdroplet component in the form of an emulsion, a cluster composition prepared according to the present invention exhibits in-use stability suitable for its intended use, and exhibits stable properties during a time window suitable for administration, such as greater than 1 hour, preferably greater than 3 hours, after mixing of the components, within which the cluster composition may be administered to a subject.

[0049] Each cluster in the cluster composition comprises at least one microbubble and at least one microdroplet, typically 2-20 individual microbubbles / microdroplets, and the clusters typically have an average diameter in the range of 1-10 μm, and thus can flow freely within the vasculature. They are further characterized and separated from the individual microbubbles and microdroplets by a circularity parameter. The circularity of a two-dimensional shape (e.g., a microbubble, microdroplet, or a projection of a microbubble / microdroplet cluster) is the ratio of the perimeter of a circle having the same area as the shape divided by the actual perimeter of the shape. Thus, a perfect circle (i.e., a two-dimensional projection of a spherical microbubble or microdroplet) has a theoretical circularity value of 1, while any other geometric shape (e.g., a projection of a cluster) has a circularity less than 1. The clusters of the present invention have a circularity less than 0.9. The definition of the circularity parameter is further described in WO 2015 / 047103.

[0050] According to the present invention, compositions comprising clusters having an average size in the range of 1-10 μm, and especially 3-10 μm, and defined by a circularity smaller than 0.9, are considered to be particularly useful, as shown in the examples. In one embodiment, the average cluster diameter is in the range of 3-10 μm, and preferably 4-9 μm, more preferably 5-7 μm. Clusters in this size range are free-flowing in the vasculature before activation, they are easily activated by US irradiation, and they generate activated bubbles large enough to temporarily deposit and remain in the microvasculature, for example in pancreatic tissue or pancreatic cancer tissue. The microbubbles in the clusters allow efficient energy transfer of ultrasound energy in the diagnostic frequency range (1-10 MHz), i.e. upon activation, and allow evaporation (phase shift) of the emulsion microdroplets at low MI (less than 1.9, and preferably less than 0.7, and more preferably less than 0.4, but greater than 0.1), as well as diffusion of the vaporized liquid into the microbubbles and / or fusion between the vapor bubbles and the microbubbles. The activated bubbles then expand further due to the inward diffusion of matrix gas (eg, blood gases) to reach a volume-weighted median diameter of greater than 10 μm but less than 40 μm.

[0051] It has been discovered that the formation of these clusters, i.e. by preparing a cluster composition from the first and second components prior to administration, is a prerequisite for an efficient phase shifting event, and that their number and size characteristics are strongly related to the efficacy of the composition, i.e. its ability to form large, activated (i.e. phase shifted) bubbles in vivo, and are a prerequisite for the intended functionality in vivo. The number and size characteristics can be controlled by various formulation parameters, such as, but not limited to, the strength of the attraction between the microbubbles in the first component and the microdroplets in the second component (e.g., the difference in surface charge between the microbubbles and the microdroplets); the size distribution of the microbubbles and the microdroplets; the ratio of the microbubbles and the microdroplets; and the composition of the aqueous matrix (e.g., pH, buffer concentration, ionic strength, etc.). When the cluster composition is prepared and administered, the average equivalent circular diameter of the clusters formed is preferably greater than 3 μm, more preferably between 5-7 μm, but preferably less than 10 μm. Also, the concentration of clusters between 3 and 10 μm in the mixed preparation (cluster composition) should preferably be greater than 10 million / mL, more preferably greater than 20 million / mL. As shown in Example 1, the cluster composition for use according to the invention had a cluster concentration of 40 to 44 million / mL, measured 0 to 3 hours after mixing of the first and second components, with an average diameter of 5.8 to 6.2 μm. In one embodiment, based on the results shown in Tables 5 and 6 of Applicant's WO 2015 / 047103, the composition for administration should contain at least 600,000 clusters / mL with a diameter between 5 and 10 μm. In another embodiment, the cluster concentration of clusters with a size in the range of 1 to 10 μm should be at least about 25 million / mL.

[0052] The size of the activated bubbles (in vivo) can be designed by varying various formulation parameters of the first and second components and the size characteristics of the prepared clusters (see Example 1). After administration, the clusters are activated to generate large bubbles under imaging control by application of external ultrasound energy, such as from a clinical ultrasound imaging system. The generated large phase-shifted bubbles are typically 10 μm or larger in diameter. Low MI energy levels that are well within the exposure limits of diagnostic imaging (MI<1.9) are sufficient to activate the clusters, making this technique significantly different from other available phase transition techniques, such as acoustic microdroplet vaporization (ADV). Due to the large size of the activated bubbles, they temporarily remain in the microvasculature and can be spatially localized to a tissue or organ of interest, such as the pancreas, and more preferably to cancerous tissue (such as a tumor) within the pancreas, by spatially localized application of ultrasound energy to activate the clusters. Thus, following administration of the cluster composition, the clusters can be activated in, at, or near the pancreas by directed application of ultrasound energy to the abdomen and pancreatic area. The large activated bubbles generated (diameter 10 μm or greater) acoustically resonate at low ultrasound frequencies (1 MHz or less).

[0053] It will be appreciated that the further ultrasonic irradiation of the large activated bubbles by application of low frequency ultrasound for the compositions and methods for use of the present invention further enhances the uptake of the therapeutic agent, and thus it has been found that application of low frequency ultrasound close to the resonant frequency of the large activated bubbles can be used to produce a mechanism of mechanical and / or thermal biological effects to increase the permeability and / or sonoporation and / or endocytosis of the vasculature of the pancreatic cancer tissue, thus increasing the delivery and retention of the therapeutic agent in the targeted tissue. Thus, frequency components in the range of 0.05-2 MHz, preferably in the range of 0.1-1.5 MHz, more preferably in the range of 0.1-1.0 MHz, even more preferably in the range of 0.2-1 MHz, most preferably in the range of 0.4-0.6 MHz, such as in particular in the range of 0.33-0.65 MHz, are used in the further ultrasonic irradiation to increase the uptake. Surprisingly, a greater therapeutic effect has been found when the activated bubbles are ultrasonically irradiated to induce increased uptake by applying ultrasound, such as in the range of 0.4-0.6 MHz, such as 500 Hz, as used in the examples. And thus, the problem caused by fibrosis of the interstitium is overcome.It has been found that ultrasound guidance allows the therapeutic agent to penetrate better into the tumor bed of the pancreas.The MI of this expansion step is preferably less than 0.5, more preferably less than 0.4, and most preferably less than 0.3, but greater than 0.15, preferably greater than 0.2.If the MI applied during the expansion step is less than 0.2, it is expected that the biomechanical effect produced will be insufficient, and therefore the therapeutic efficacy will be significantly reduced.

[0054] While the direct mechanism of action, i.e., the mechanical and / or thermal biological effects generated, increases the delivery of the therapeutic agent and improves its distribution, it should be understood that the nature of these biomechanical effects is a direct result of the chemical attributes of the cluster composition, i.e., the chemical composition and properties of the cluster. For example, the lifetime of a gas bubble in an aqueous matrix is ​​inversely proportional to the solubility and diffusion coefficient of the gas in the matrix and proportional to the density of the gas. Thus, bubbles made from heavier gases with low solubility and diffusivity will grow larger and persist longer than bubbles made from lighter gases with high solubility and diffusivity. For example, 5 μm perfluorobutane microbubbles can persist 500 times longer in water than 5 μm air microbubbles. Thus, the chemical composition of the microdroplet components will govern the lifetime of the activated bubbles in vivo and, therefore, the level of biomechanical forces that can be induced and the level of therapeutic effect that can be achieved using the ACT method. For this reason, perfluorinated oils would be particularly useful for use in microdroplets of the second component since gases therefrom have very low solubility and diffusivity in water, and high density.

[0055] When the compositions and methods of the present invention are compared with those in which free-flowing regular contrast agent microbubbles are used, the large phase-shifted microbubbles generated in vivo from the administered clusters of the present invention are incorporated into the blood vessel segments, and the activated bubble surface is in close contact with the endothelium. In addition, the volume of the activated bubbles is typically 1000 times that of regular microbubbles. At the same mechanical index (MI), when insonified at frequencies close to the resonant frequency in both bubble types (0.5 MHz for phase-shifted microbubbles and 3 MHz for regular contrast agent microbubbles), the absolute volume displacement during vibration (i.e., the exerted biomechanical force) was shown to be three orders of magnitude larger for phase-shifted bubbles than for regular contrast agent microbubbles. Thus, insonification of phase-shifted bubbles will create a biomechanical effect with significantly larger size and deeper penetration at a completely different level than that of regular contrast agent microbubbles. The biological effects observed with free-flowing regular contrast microbubbles are likely dependent on cavitation mechanisms, with safety concerns such as microbleeding and irreversible vascular damage. However, larger phase-shift bubbles from clusters can be oscillated in a gentler manner (lower MI, e.g., less than 0.4), avoiding cavitation mechanisms, while still inducing sufficient mechanical forces to increase drug uptake from the vasculature and into the target tissue of the pancreas. The capture of large phase-shift bubbles would also act as a deposition tracer. This would further allow quantification of the number of activated clusters and tissue perfusion, as well as contrast imaging of tissue vasculature to identify the spatial extent of the lesion being treated.

[0056] The chemical composition of the administered clusters and the processes that occur during their activation are crucial for the efficacy of the clusters. For example, the chemical nature of the encapsulated oil droplets influences the amount of activated bubbles that deposit upon US irradiation and their lifetime in vivo. The physicochemical properties of the oil, such as vapor pressure, boiling point, and water solubility, are all correlated with the amount of activated bubbles that deposit and the time they have been deposited. For C4-C6 homologs of perfluorinated hydrocarbon chains, the amount of activated bubbles and their lifetime increase depending on the chain length as the water solubility and vapor pressure decrease and the boiling point increase. Furthermore, it should be noted that the activated large bubbles of the clusters can act mechanically on cells of the vasculature and generate biochemical signals, which leads to increased uptake of therapeutic agents.

[0057] The cluster composition is designed to cluster and phase shift in a controlled manner. Upon exposure to ultrasound, e.g., standard medical imaging frequencies and intensities, at the targeted pancreatic lesion, the microbubbles of the administered cluster composition transfer acoustic energy to the attached oil microdroplets and can either act as evaporative seeds or bind with the microbubbles such that the oil undergoes a phase shift (vaporization) from liquid to gas. The resulting bubbles undergo an initial rapid expansion due to oil vaporization, followed by a slower expansion due to inward diffusion of blood gases, and temporarily block the microcirculation (aorta and capillary network) for about 1 minute or more, preferably 2-3 minutes or more, most preferably 3-6 minutes or more. In the methods of the invention, or in pharmaceutical compositions for use, a therapeutic agent is further administered to the subject, e.g., co-administered or pre- or post-administered with the cluster composition. The clusters are activated by application of external ultrasound energy to generate larger bubbles, which are then trapped within the microvasculature of the tumor (e.g., in the pancreas). Furthermore, application of low frequency ultrasound after capture promotes infiltration of therapeutic agents into target tissues of the pancreas. Thus, the major limitations of existing techniques in effectively treating pancreatic solid tumors due to insufficient delivery to the target location, whether due to tumor interstitial fluid pressure, lack of vascularization or perfusion, or the presence of dense interstitial matrix, may be overcome by the techniques of the present invention, as it has been found that the accessibility of therapeutic agents to the tumor is significantly increased.

[0058] First component of the cluster composition The first component includes gas microbubbles and a first stabilizing agent for stabilizing the microbubbles. Thus, the first component is an injectable aqueous medium that includes dispersed gas and a material for stabilizing the gas. The microbubbles may resemble conventional ultrasound contrast agents that are commercially available and approved for use in several clinical applications, such as Sonazoid®, Optison, Definity®, or Sonovue, or similar agents used in preclinical applications, such as Micromarker and Polyson L. Any biocompatible gas may be present in the gas dispersion, where the term "gas" as used herein includes any substance (including mixtures) that is at least partially, e.g., substantially or completely, in gaseous (including vapor) form at normal human body temperature of 37°C. Thus, the gas may be, for example, air; nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas, such as helium, argon, xenon or krypton; a fluoride, such as sulfur hexafluoride, disulfur decafluoride or trifluoromethyl sulfur pentafluoride; selenium hexafluoride; an optionally halogenated silane, such as methylsilane or dimethylsilane; a low molecular weight hydrocarbon (e.g., containing up to 7 carbon atoms), such as an alkane, such as methane, ethane, propane, butane or pentane, a cycloalkane, such as cyclopropane, cyclobutane or cyclopentane, an alkene, such as ethylene, propene, propadiene or butene, or an alkyne, such as acetylene or propylene, an ether, such as dimethyl ether; a ketone; an ester; a halogenated low molecular weight hydrocarbon (e.g., containing up to 7 carbon atoms); or a mixture of any of the above.Preferably, the gas is a halogenated gas, and more preferably a perfluorinated gas.Advantageously, at least some of the halogen atoms in the halogenated gas are fluorine atoms: thus, the biocompatible halogenated hydrocarbon gas may be selected, for example, from bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons. Representative perfluorocarbons include perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (e.g., perfluoro-n-butane, optionally mixed with other isomers such as perfluoro-iso-butane), perfluoropentane, perfluorohexane, or perfluoroheptane; perfluoroalkenes such as perfluoropropene, perfluorobutene (e.g., perfluorobut-2-ene), perfluorobutadiene, perfluoropentene (e.g., perfluoropent-1-ene), or perfluoro-4-methylpent-2-ene; perfluoroalkynes such as perfluorobut-2-yne; and perfluoroalkynes such as perfluorocyclobutane; and perfluorocycloalkanes such as perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluorocyclohexane, perfluoromethylcyclohexane, or perfluorocycloheptane. Other halogenated gases can include methyl chloride, fluorinated (eg, perfluorinated) ketones, such as perfluoroacetone, and fluorinated (eg, perfluorinated) ethers, such as perfluorodiethyl ether.

[0059] The use of perfluorinated gases, such as sulfur hexafluoride, and perfluorocarbons, such as perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane, is particularly advantageous in view of the high stability of microbubbles containing such gases in the bloodstream. In one embodiment, the first component gas is selected from the group of sulfur fluorides and halogenated low molecular weight hydrocarbons (e.g., containing up to 7 carbon atoms). Other gases having physicochemical properties that lead to the formation of highly stable microbubbles in the bloodstream may be useful as well. Most preferably, the dispersed gas comprises sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane (i.e., C3-C6 perfluorocarbons), nitrogen, air, or mixtures thereof. Even more preferably, the dispersed gas comprises sulfur hexafluoride, perfluoropropane, or perfluorobutane, or mixtures thereof. And even more preferably, the dispersed gas is perfluorobutane.

[0060] The dispersed gas may be in any convenient form, for example any suitable gas-containing ultrasound contrast agent formulation may be used as the gas-containing component, such as, for example, Sonazoid®, Optison, Sonovue or Definity®, or a preclinical agent, such as, for example, Micromarker or PolySon L. The first component will also contain a material to stabilize the microbubble dispersion, referred to herein as a "first stabilizer." Representative examples of such formulations include gas microbubbles stabilized (e.g., at least partially encapsulated) by, for example, a first stabilizing agent, such as a clump-resistant surface membrane (e.g., gelatin), a membrane-forming protein (e.g., albumin, such as human serum albumin), a polymeric material (e.g., synthetic biodegradable polymers, elastic interfacial synthetic polymeric membranes, particulate biodegradable polyaldehydes, particulate N-dicarboxylic acid derivatives of polyamino acid-polycyclic imides, etc.), non-polymeric and non-polymeric wall-forming materials, or surfactants (e.g., polyoxyethylene-polyoxypropylene block copolymer surfactants, such as Pluronic, polymeric surfactants, or membrane-forming surfactants, such as phospholipids). Preferably, the dispersed gas is in the form of gas microbubbles stabilized with phospholipids, proteins, or polymers. Thus, in one embodiment, the first stabilizing agent is selected from the group of phospholipids, proteins, and polymers. Particularly useful first stabilizers are selected from the group of surfactants including phospholipids, including molecules with a net overall negative charge, such as naturally derived (e.g., from soybean or egg yolk), semi-synthetic (e.g., partially or fully hydrogenated) and synthetic phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid and / or cardiolysin.Alternatively, the phospholipids applied for stabilization may have an overall neutral charge and may be added with a negative surfactant, such as a fatty acid, such as phosphatidylcholine-added palmitic acid, or may be a mixture of differently charged phospholipids, such as phosphatidylethanolamine and / or phosphatidylcholine and / or phosphatidic acid. For the first stabilizing agent, i.e. stabilizing the microbubbles of the first component, various examples are shown in WO2015 / 047103, Example 5 and Tables 9 and 10, where various microbubble formulations with various excipients are tested. This result demonstrates that the ACT concept used in the present invention is applicable to a wide variety of microbubble formulations and also with regard to the composition of the stabilizing membrane.

[0061] The size of the microbubbles of the dispersed gaseous components should preferably be less than 7 μm, more preferably less than 5 μm, and most preferably less than 3 μm to allow unhindered transport through the pulmonary system even when in microbubble / microliquid clusters.

[0062] Second component of the cluster composition The second component comprises microdroplets comprising an oil phase and a second stabilising agent for stabilising the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles of the first component and at least temporarily increasing their size. With respect to the second component, the "diffusible component" is preferably a gas / vapor, a volatile liquid, a volatile solid or a precursor thereof capable of generating gas, e.g. upon administration, the main requirement being that the component: The main requirement is that the dispersed gas should have or be capable of producing sufficient gas or vapor pressure in vivo (e.g., at least 50 torr, and preferably greater than 100 torr) to facilitate inward diffusion of gas or vapor molecules into the dispersed gas. The "diffusible component" is preferably formulated as a microdroplet emulsion (i.e., a stabilized suspension) in a suitable aqueous medium, since in such a system the vapor pressure in the aqueous phase of the diffusible component will be substantially equal to the vapor pressure of the pure component material, even in a very dilute emulsion.

[0063] The diffusible component in such microdroplets is advantageously liquid at the processing and storage temperatures, which may be, for example, as low as -10°C if the aqueous phase contains a suitable antifreeze material, while being gaseous or exhibiting a substantial vapor pressure at body temperature. Suitable compounds may be selected, for example, from the various lists of emulsifiable low-boiling liquids given in patent applications WO 94 / 16379 or WO 2015 / 047103, the contents of which are incorporated herein by reference. Specific examples of emulsifiable diffusible components include aliphatic ethers such as diethyl ether; polycyclic oils or alcohols such as menthol, camphor or eucalyptol; heterocyclic compounds such as furan or dioxane; aliphatic hydrocarbons, which may be saturated or unsaturated, linear or branched, such as n-butane, n-pentane, 2-methylpropane, 2-methylbutane, 2,2-dimethylpropane, 2,2-dimethylbutane, 2,3-dimethylbutane, 1-butene, 2-butene. , 2-methylpropene, 1,2-butadiene, 1,3-butadiene, 2-methyl-1-butene, 2-methyl-2-butene, isoprene, 1-pentene, 1,3-pentadiene, 1,4-pentadiene, butene-in, 1-butyne, 2-butyne or 1,3-butanediene, cycloaliphatic hydrocarbons such as cyclobutane, cyclobutene, methylcyclopropane or cyclopentane, and halogenated low molecular weight hydrocarbons containing up to 7 carbon atoms. Representative halogenated hydrocarbons include dichloromethane, methyl bromide, 1,2-dichloroethylene, 1,1-dichloroethane, 1-bromoethylene, 1-chloroethylene, ethyl bromide, ethyl chloride, 1-chloropropene, 3-chloropropene, 1-chloropropane, 2-chloropropane, and t-butyl chloride.Advantageously, at least some of the halogen atoms are fluorine atoms, such as dichlorofluoromethane, trichlorofluoromethane, 1,2-dichloro-1,2-difluoroethane, 1,2-dichloro-1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane, 2-bromo-2-chloro-1,1,1-trifluoroethane, 2-chloro-1,1,2-trifluoroethyl difluoromethyl ether, 1-chloro-2,2,2-trifluoroethyl difluoromethyl ether, partially fluorinated alkanes, such as pentafluoropropane, for example 1H,1H,3H-pentafluoropropane. propane, hexafluorobutane, nonafluorobutanes such as 2H-nonafluoro t-butane, decafluoropentanes such as 2H,3H-decafluoropentane, etc.), partially fluorinated alkenes (e.g. heptafluoropentenes such as 1H,1H,2H-heptafluoropent-1-ene, and nonafluorohexenes such as 1H,1H,2H-nonafluorohex-1-ene, etc.), fluorinated ethers (e.g. 2,2,3,3-pentafluoropropyl methyl ether or 2,2,3,3-pentafluoropropyl difluoromethyl ether, etc.), and more preferably perfluorocarbons.Examples of perfluorocarbons include perfluoroalkanes such as perfluorobutane, perfluoropentane, perfluorohexane (e.g., perfluoro-2-methylpentane), perfluoroheptane, perfluorooctane, perfluorononane, and perfluorodecane; perfluorocycloalkanes such as perfluorocyclobutane, perfluorodimethylcyclobutane, perfluorocyclopentane, and perfluoromethylcyclopentane; perfluoroalkenes such as perfluorobutene (e.g., perfluorobut-2-ene or perfluorobuta-1,3-diene), perfluoropentene (e.g., perfluoropent-1-ene), and perfluorohexene (e.g., perfluoro-2-methylpent-2-ene or perfluoro-4-methylpent-2-ene); perfluorocycloalkenes such as perfluorocyclopentene or perfluorocyclopentadiene; and perfluorinated alcohols such as perfluoro-t-butanol. Thus, the oil of the second component (the diffusible component) may be selected from the group of aliphatic ethers, heterocyclic compounds, aliphatic hydrocarbons, halogenated low molecular weight hydrocarbons, and perfluorocarbons. In one embodiment, the oil phase of the second component comprises a perfluorocarbon.

[0064] Particularly useful in the present invention are 1×10 -4 M or less, more preferably 1×10 -5 The diffusible component has a water solubility of less than M. However, it should be noted that when a mixture of diffusible components and / or cosolvents is used, a substantial fraction of the mixture may contain compounds with higher water solubility. Examples of suitable oils (diffusible components) based on water solubility are perfluorodimethylcyclobutane, perfluoromethylcyclopentane, 2-(trifluoromethyl)perfluoropentane, and perfluorohexane.

[0065] It will be appreciated that mixtures of two or more diffusible components may be employed in accordance with the present invention if desired, and references herein to "the diffusible component" may be interpreted as such mixtures.

[0066] The second component may also include a material for stabilizing the microdroplet dispersion, herein referred to as a "second stabilizer." The second stabilizer may be the same as or different from any material used to stabilize the gas dispersion, e.g., a surfactant, e.g., a phospholipid, polymer, or protein. The nature of any such material may greatly affect factors such as the growth rate of the dispersed gas phase. In general, a wide range of surfactants are useful as stabilizers and may be selected from the extensive list set forth in, for example, EP-A-0727225, the contents of which are incorporated herein by reference. Representative examples of useful surfactants include fatty acids (e.g., linear, saturated or unsaturated fatty acids, e.g., containing 10-20 carbon atoms) and carbohydrate and triglyceride esters, phospholipids (e.g., lecithin), fluorine-containing phospholipids, proteins (e.g., albumin, e.g., human serum albumin), polyethylene glycols, and polymers such as block copolymer surfactants (e.g., polyoxyethylene-polyoxypropylene block copolymers, e.g., Pluronics, and other extended polymers, e.g., acyloxyacyl polyethylene glycols, e.g., polyethylene glycol methyl ether 16-hexadecanoyloxy-hexadecanoate, e.g., where the polyethylene glycol moiety has a molecular weight of 2300, 5000 or 10000), and fluorine-containing surfactants (e.g., those sold under the product names Zonyl and Fluorad, or those described in WO 96 / 39197, the contents of which are incorporated herein by reference). Particularly useful surfactants include phospholipids, and in particular phospholipids that contain molecules with an overall neutral charge, such as distearoyl-sn-glycerol-phosphocholine (DSPC). In the second component, a range of stabilizing agents can be used to stabilize the microdroplets. In addition, a wide range of ionic, preferably cationic, materials may be used to promote the appropriate surface charge.

[0067] It will be appreciated that to promote favorable electrostatic interactions to achieve clustering between the microbubbles in the first component and the emulsion microdroplets in the second component, they should have opposite surface charges. Thus, if the microbubbles of the first component are negatively charged, the microdroplets of the second component should be positively charged, or vice versa. In a preferred embodiment, the surface charge of the microbubbles of the first component is negative, and the surface charge of the microdroplets of the second component is positive. To promote the appropriate surface charge of the oil microdroplet structure, a cationic surfactant may be added to stabilize the structure. A wide range of cationic substances may be used, such as at least some hydrophobic and / or substantially water insoluble compounds with a basic nitrogen atom, such as primary, secondary or tertiary amines and alkaloids. A particularly useful cationic surfactant is stearylamine. In one embodiment, the second stabilizer is a neutral phospholipid-added cationic surfactant, such as DSPC-membrane with stearylamine.

[0068] In one embodiment, the first stabilizing agent and the second stabilizing agent each independently comprise a phospholipid, a protein, a polymer, a polyethylene glycol, a fatty acid, a positively charged surfactant, a negatively charged surfactant, or a mixture thereof.

[0069] In one embodiment, the first component comprises a dispersed gas selected from the group of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, nitrogen and air or mixtures thereof, stabilized by a first stabilizing agent selected from the group of phospholipids, proteins and polymers, and the second component comprises a diffusible component selected from the group of perfluorocarbons, such as perfluorocycloalkanes, stabilized with a second stabilizing agent selected from the group of surfactants, including phospholipids, polymers and proteins. More particularly, both stabilizing agents are selected from phospholipids.

[0070] The first and second components are mixed for use in an appropriate time frame immediately prior to intended use to prepare the cluster composition. It will also be understood that mixing of the first and second components can be accomplished in various ways depending on the form of the components; for example, mixing two fluid components, reconstituting one component in dry powder form with one component in fluid form, mixing two components in dry form before reconstitution with a fluid (e.g., water for injection or buffer), etc. It will also be understood that other components, such as but not limited to, the level of surface charge of the microbubbles / microdroplets, the concentration of the microbubbles / microdroplets in the two components, the size of the microbubbles / microdroplets, the composition and concentration of ions in the liquid matrix, pH, the composition and concentration of excipients (e.g., buffer or isotonic solution), etc. (see WO2015 / 047103, Example 1), can affect the ability of the microbubbles and microdroplets to form clusters when mixed. Such properties of the components and compositions also affect the size and stability (both in vitro and in vivo) of the generated clusters and may be important factors influencing the biological attributes (e.g., efficacy and safety profile). It will also be understood that not all microbubbles / microdroplets in a cluster composition may be present in a clustered form, and a significant portion of the microbubbles and / or microdroplets may be present in a free (unclustered) form along with a population of microbubbles / microdroplet clusters. In addition, the manner in which the two components are mixed may affect these aspects, such as, but not limited to; the shear stress applied during homogenization (e.g., gentle manual homogenization or strong mechanical homogenization), and the time range for homogenization. The cluster composition should be administered to a subject within a time frame in which the properties of the clusters have not substantially changed, such as within 5 hours, e.g., within 3 hours, of mixing the two components. Applicant's in-use stability studies indicate that the clusters exhibit stable properties for at least 3 hours (see Example 1).

[0071] The microdroplet size of the dispersed diffusible components in emulsions intended for intravenous infusion should be less than 7 μm, more preferably less than 5 μm, even more preferably less than 3 μm, but greater than 0.5 μm, more preferably greater than 1 μm, to allow unhindered transport through the pulmonary system while still maintaining a volume sufficient for retention of the activated bubbles in the microvasculature.

[0072] The growth of the dispersed gas phase in vivo may involve, for example, the expansion of any encapsulating material (if it is sufficiently flexible) and / or the extraction of excess surfactant from the administered material to the growing gas-liquid interface. However, it is also possible that the stretching of the encapsulating material and / or the interaction of the material with ultrasound may substantially increase its porosity. Whereas such disruption of the encapsulating material has previously often been found to lead to a rapid loss of echogenicity via outward diffusion and dissolution of the gas exposed thereby, the applicant has found that when using the composition according to the invention, the exposed gas exhibits substantial stability. Without wishing to be bound by theoretical calculations, the applicant believes that the exposed gas, for example in the form of released microbubbles, is stabilized, for example against the collapse of the microbubbles, by the supersaturated environment created by the diffusible components, which provides an inward pressure gradient that opposes the tendency of the microbubble gas to diffuse outward. By virtue of the substantial absence of encapsulating material, the exposed gas surface may be activated such that the activated bubbles exhibit particularly favorable acoustic properties, evidenced by high backscatter and low energy absorption (e.g., as represented by a high backscatter:attenuation ratio) at typical diagnostic imaging frequencies, and this reverberation effect may continue for significant periods of time, even during sustained ultrasound exposure.

[0073] In vivo activation and enhancement The present invention relates to a method for producing a method for manufacturing a semiconductor device comprising the steps of: (i) administering to a mammalian subject suffering from pancreatic cancer a pharmaceutical composition, wherein at least one therapeutic agent is administered separately, prior to, and / or together with, and / or subsequent to the cluster composition; (ii) optionally imaging the clusters of pharmaceutical composition using ultrasound imaging to identify target areas for treatment within the subject; (iii) activating a phase shift of the diffusible component of the second component of the cluster composition from step (i) by ultrasonic irradiation of a target area within the subject; and (iv) promoting extravasation of the therapeutic agent administered in step (i) by further exposure to ultrasound. The present invention provides a pharmaceutical composition as described above for use in Acoustic Cluster Therapy (ACT) treatment, comprising: In the method of the present invention, for treatment or delivery, the method comprises the steps described above.

[0074] The acoustic resonance of the microbubble component of the clusters is within the diagnostic frequency range (1-10 MHz). When the cluster composition is administered to a subject, activation of the clusters is easily obtained with mid-to-low mechanical indices, i.e., MIs less than 1.9, and preferably less than 0.7, and more preferably less than 0.4, but greater than 0.1, using standard diagnostic ultrasound imaging pulses, for example, as used in conventional medical ultrasound abdominal and cardiac applications. Activation of the clusters to phase shift to create larger (10 μm or more in diameter) phase shift bubbles can be achieved using clinical imaging systems within the millimeter spatial resolution range by employing imaging pulses. Upon activation, the oil within the microdroplets vaporizes and the resulting large activated bubbles are temporarily deposited within the microvasculature. Further application of low frequency ultrasound after activation and deposition allows for an enhancement of the delivery mechanism by effectively overcoming biological barriers, improving the efficiency of drug delivery to cancer pancreatic tissue. These mechanisms may include the process of sonoporation, i.e., ultrasonic irradiation of microbubbles within vascular compartments and subsequent volumetric vibration increases the permeability of the vascular barrier. In other words, the ACT approach increases the permeability of the endothelial wall and therefore promotes the extravasation, distribution, and cellular uptake of therapeutic agents. Other mechanisms, such as the generation of cellular signals to enhance therapeutic effects, mechanical disruption of interstitial structures to increase drug penetration, may also be induced.

[0075] The clusters are not activated at low MI (below the cluster activation threshold, which is about 0.1), which allows standard medical ultrasound contrast imaging to be performed, such as to identify tumor microvascular lesions without cluster activation. Thus, in one embodiment, the method includes using a low MI contrast imaging mode (MI<0.15) to image the microbubble components, i.e., dispersed gas, without activating the clusters, to identify lesion locations for treatment. Thus, since the clusters are not activated at low MI (below the activation threshold), standard medical ultrasound contrast imaging can be performed prior to the activation step, such as to identify tumor microvascular lesions. Activation under medical ultrasound imaging control with imaging pulses allows spatially targeted activation of the clusters in tissue regions examined by the ultrasound field. After activation, the large phase-shifted bubbles generated are temporarily trapped within the pancreatic microvasculature due to their size. The resulting large phase-shifted bubbles are approximately 1000 times the volume of the evaporated emulsion microdroplets (20 μm diameter bubbles from 2 μm diameter oil microdroplets). The scattering cross-section of these large phase-shifted bubbles is orders of magnitude larger than that of the micron-sized microbubbles in the clusters before activation. As a result, the large phase-shifted bubbles generate a large amount of backscattered signal and are easily imaged in basic imaging modes with diagnostic imaging systems. The mechanical resonance frequency of the large phase-shifted bubbles is also an order of magnitude lower (below 1 MHz) than that of the microbubbles in the clusters before activation. Application of an acoustic field commensurate with the resonance frequency of the larger phase-shifted bubbles creates relatively large radial vibrations in the MI within the range for medical diagnostics.Thus, low frequency ultrasound in the range of 0.05-2 MHz, preferably 0.1-1.5 MHz, more preferably 0.1-1.0 MHz, even more preferably 0.2-1 MHz, most preferably 0.4-0.6 MHz, and especially 0.33-0.65 MHz, can be applied to create the mechanism of biological effects that promote the uptake of administered drugs and thus extravasation. After in vivo activation, the majority of activated bubbles were found to be within 10-20 μm in diameter. The Minnaert resonance frequency of free microbubbles of this size is 0.65-0.33 MHz. The most preferred frequency range for the low frequency enhancement step is therefore 0.33-0.65 MHz. The exploitation of the resonance effect of activated bubbles allows better control of the onset of these biological effects at lower acoustic intensities and lower frequencies than is possible with other techniques. Combined with the fact that large phase-shifting bubbles are activated under imaging control and deposited within the tissue microvasculature (allowing for spatial targeting of large activated bubbles in tissue) and their extended residence time allows for a more efficient and controlled implementation of the drug delivery mechanism.

[0076] It is envisaged that the ACT concept for drug delivery to the pancreas and treatment of PDAC, i.e. compositions for use in the present invention, is a concept that applies to a wide range of combinations of components and (first and second) ingredients, and also to an even wider range of therapeutic agents.

[0077] Treatment The therapeutic agent, also referred to as "the drug", that is delivered to the subject is selected from the group of chemotherapeutic and immunotherapeutic agents. It is administered as a separate composition from the cluster composition. Examples of classes of therapeutic agents and specific agents useful in the claimed invention include, but are not limited to, the following:

[0078] Alkylating Agents Nitrogen Mustard: Mechlorethamine Hydrochloride (Mustargen) Nitrosoureas: Carmustine (BiCNU), Streptozocin (Zanosar), Lomustine (CeeNU) Tetrazines: Dacarbazine (DITC-Dome), Temozolomide (Temodar) Aziridines: Thiotepa (Thioplex), Mitomycin (Mutamycin), Aziridinyl Benzoquinone (AZQ) Cisplatin: Cisplatin (Platinal), Carboplatin (Paraplatin), Oxaliplatin (Eloxatin)

[0079] Antimetabolites Antifolates: Methotrexate (Otrexup, Rasuvo, Trexall), Pemetrexed (Altima) Fluoropyrimidines: Fluorouracil (Adrucil), Capecitabine (Xeloda) Deoxynucleotides Analogs: cytarabine (Cytosar-U), decitabine (Dacogen), azacitidine (Vidaza), gemcitabine (Gemzar), fludarabine (Fludara), nelarabine (Arranon), pentostatin (Nipent) Thiopurines: Thioguanine (Tabloid), Mercaptopurine (Purinethol, Purixan)

[0080] Microtubule Inhibitors Vinca alkaloids: Vinorelbine (Navelbine), Vinicristine (Oncovin, Vincasar Pfs), Vindesine (Eldisine), Vinflunine (Javlor) Taxanes: Paclitaxel or nab-paclitaxel (Onxol, Abraxane), Cabazitaxel (Jevtana), Docetaxel (Docetaxel, Docefrez) Podophyllotoxin: Etoposide (Eposin, Etoposide), Teniposide (Vumon)

[0081] Topoisomerase inhibitors Topoisomerase I: Irinotecan (Onivyde), Topotecan (Act Topotecan, Hycamtin) Topoisomerase II: doxorubicin (Adriamycin, Caelyx), mitoxantrone (Novantrone), teniposide (Vumon), novobiocin (Novobiocin Sodium), mervalone, aclarubicin

[0082] Cytotoxic Antibiotics Anthracyclines: doxorubicin (Adriamycin, Caelyx), daunorubicin (Cerubidine, DaunoXome), epirubicin (Ellence), idarubicin (Idamycin), bleomycin (Blenoxane), mitomycin (Mitosol, Mutamycin)

[0083] immunotherapy CAR-T cell therapy: Sipuleucel-T (Provenge), Tisangenlecleucel (Kymriah), Axicabtadine ciloleucel (Yescarta) Antibody therapy: alemtuzumab (Campath CD52), atezolizumab (Tecentriq PD-L1), ipilimumab (Yervoy CTLA4), pembrolizumab (Keytruda PD-1), durvalumab (Imfinizi IgG1k) Oncolytic viruses: talimogene laherparepvec (OncoVEX GM-CSF / T-vecIMLYGIC), Ad2 / 5 dl1520 (Onyx-015), GLV-1h68 (GL-ONC1), CV706 Cancer vaccines: Oncophage, Sipuleucel-T (Provenge)

[0084] Cytokine therapy Interferon: IFNα (Infergen), IFNβ (Actimmune) Interleukin: Not commercially available, in clinical trials.

[0085] In one embodiment, the therapeutic agent is selected from the group of alkylating agents, antimetabolites, microtubule inhibitors, topoisomerase inhibitors, cytotoxic antibiotics, immunotherapeutic agents, and cytokine therapeutic agents.

[0086] In one embodiment, the therapeutic agent is formulated in a vehicle that is included, for example, in the form of a liposome, conjugate, nanoparticle, or microsphere, which is used as a vehicle for the therapeutic agent. Thus, the therapeutic agent may be part of a larger drug construct, for example, a nanodrug, for example, in a liposomal or microparticle formulation, or as a monoclonal antibody. Thus, in one embodiment, the therapeutic agent is formulated in a closed lipid sphere, such as including the therapeutic agent in a liposomal formulation.

[0087] As shown in the examples, we evaluated the synergistic effects of combining ACT with larger constructs, the nanodrug nab-paclitaxel (paclitaxel bound to albumin protein in nanoparticles) and the nanodrug liposomal irinotecan. As mentioned above, the ACT concept may be particularly useful in combination with such larger drug molecules or constructs, as also shown in Example 3.

[0088] Examples of suitable approved therapeutic agents that may be formulated in a vehicle include the following: the liposomal chemotherapy drug Doxil, in which doxorubicin is encapsulated in liposomes; The liposomal chemotherapy drug Onivide® (irinotecan encapsulated in liposomes); and Paclitaxel Albumin-bound Particle Suspension Abraxane® It is.

[0089] In addition, combination therapy between one or more chemotherapeutic agents and one or more immunotherapeutic agents is preferred. Furthermore, new generation liposomes containing two anticancer drugs using a single liposome, and immunoliposomes containing antibodies bound to liposomes are encompassed by the present invention.

[0090] In a preferred embodiment, the therapeutic agent is selected from the group of large molecules (such as proteins or antibodies) or nanostructured drugs, including, but not limited to, nab-paclitaxel / gemcitabine, liposomal irinotecan, liposomal doxorubicin, and monoclonal antibody combinations.

[0091] In a second preferred embodiment, the therapeutic agent is selected from the group of immunotherapeutic agents, such as oncolytic viruses, including but not limited to adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus, and vaccinia. The virus can cause the cancer cells to "burst", killing the cancer cells and releasing cancer antigens. These antigens can stimulate an immune response that can seek out and eliminate any tumor cells that remain nearby and may be elsewhere in the body.

[0092] In one embodiment, the therapeutic agent comprises nab-paclitaxel (Abraxane) in combination with gemcitabine. In another embodiment, the therapeutic agent is liposomal irinotecan (Onivyde®).

[0093] In yet another embodiment, multiple therapeutic agents are administered as a combination regimen.Examples of suitable combination regimens include combination regimens that include leucovorin, fluorouracil, irinotecan and oxaliplatin (FOLFIRINOX), combination regimens that include leucovorin, fluorouracil and irinotecan (FOLFIRI), and combination regimens that include leucovorin, fluorouracil and oxaliplatin (FOLFOX).

[0094] In yet another embodiment, the therapeutic agent is an immunotherapeutic agent, such as, for example, a checkpoint inhibitor, for example, an anti-PD1, anti-CTLA4, or anti-PDL1 checkpoint inhibitor.

[0095] In one embodiment of the invention, the method comprises treatment with at least one chemotherapeutic agent in combination with treating the subject with immunotherapy.

[0096] Disclaimer: In one embodiment, the therapeutic agent is not paclitaxel, i.e., as a free non-albumin bound form. In one embodiment, the therapeutic agent is not a combination of gemcitabine with nab-paclitaxel.

[0097] In addition to a separately administered therapeutic agent, in one embodiment, the second component of the cluster composition includes an additional therapeutic agent contained in the microdroplets.

[0098] Route of administration The cluster composition is administered parenterally, preferably intravenously, to the mammalian subject. The route of administration may also be selected from intra-arterial, intramuscular, intraperitoneal, intratumoral or subcutaneous administration. For administration to the subject, the therapeutic agent is administered separately, prior to, and / or together with, and / or after the cluster composition as a separate composition. The therapeutic agent is administered according to its respective approved Summary of Product Characteristics. Typically, the route is selected from the group consisting of, but not limited to, intravenous, intraperitoneal, intratumoral and intramuscular administration. Thus, the two compositions, i.e., cluster composition (a) and therapeutic agent composition (b), may be administered via the same route of administration or via different routes of administration.

[0099] Treatment schedule It will be understood that the compositions for use, methods of treatment, and / or methods of drug delivery of the present invention can be carried out, for example, as part of a multi-drug combination treatment regimen. In one embodiment, the pharmaceutical composition for use according to the present invention includes the use of more than one therapeutic agent. Such a combination chemotherapy regimen can include, for example, leucovorin, fluorouracil, irinotecan (FOLFIRI).

[0100] Furthermore, in one embodiment, several ACT treatments may be performed during the period of administering the therapeutic agent, for example, as illustrated in Figures 5 and 6. In one embodiment, the treatment method includes 1-5, for example 2-4 treatments. An "ACT treatment" or "ACT procedure" includes at least the administration of a cluster composition, activation of the clusters by conventional medical imaging US ultrasound exposure, and subsequent low frequency US ultrasound exposure to induce enhanced uptake. Figures 5 and 6 provide an example of a treatment schedule.

[0101] Figure 5 shows a graph of ACT treatments administered during treatment with the following chemotherapy combination regimen: FOLFOX. A 2-hour infusion of oxaliplatin and recovorin is followed by a bolus injection of fluorouracil (5-FU) and a 48-hour infusion of 5-FU. The ACT procedure is applied three times between chemotherapy drug administrations, as indicated by the gray ACT® sonoporation bar. Panel A of Figure 5: Each ACT procedure is as follows: a. Injection of cluster composition b. Activation of the clusters using 60 seconds of conventional medical imaging ultrasound; and c.: Enhancement step using 400-600 kHz ultrasound irradiation for 5 minutes It consists of: Panel B of Figure 5: The Y-axis shows the plasma concentration of the administered chemotherapy drug in percent of peak, and the X-axis shows time in minutes. In this example, three ACT procedures are performed at approximately 50, 80, and 110 minutes to cover all three drugs. A similar overview is shown in Figure 6 for the standard therapeutic combination regimen for the treatment of PDAC; gemcitabine and nab-paclitaxel.

[0102] The inventors found that it is beneficial to repeat the ACT procedure rather than applying a single ACT procedure for each administration of the therapeutic agent. Using US imaging during the activation of ACT, the inventors observed a significant effect in the deposition of ACT bubbles in the tumor. A strong variance in the deposition pattern from injection to injection in the same animal was observed; the density of the deposited ACT bubbles differed between various segments of the tumor, and this pattern changed between injections. Although not fully understood, it is hypothesized that these effects are due to temporary fluctuations in the perfusion to various tumor segments. Based on these observations, in the examples, the inventors applied the ACT procedure three times in succession, in quick succession, in order to reach as much of the tumor volume as possible. This also points out the advantage of applying several ACT treatments during clinical use, as described above for FOLFOX and NAB / GEM regimens.

[0103] Thus, in one embodiment, multiple therapeutic agents, e.g., 1-5 therapeutic agents, are administered simultaneously or sequentially over a time span, e.g., up to 3 hours, where at least one ACT treatment, e.g., 1-5, is administered during the same period.

[0104] In one embodiment, the following ACT procedure is provided: Following administration of the cluster composition, for example intravenously, local US ultrasound irradiation of the tumor using conventional medical imaging US (activation), followed by low-frequency US ultrasound irradiation to induce increased uptake (promoting extravasation), and these steps are performed 2-5 times in succession, for example 3 times in succession. Thus, steps (i)-(iv) of the ACT treatment are repeated 1-4 times. This is performed simultaneously with the administration of the therapeutic agent. The activation, i.e., the first US ultrasound irradiation, should be initiated immediately, for example within 1 minute, after each administration of the cluster composition and last for, for example, 30-120 seconds. Ultrasonic irradiation using low-frequency ultrasound follows the activation step, which should typically last for 3-10 minutes, for example about 5 minutes. Immediate initiation of step (iv) after step (iii) is preferred. Dual-frequency transducers can be beneficially used in the treatment for both the activation and enhancement steps. By using such, the switch from the activation ultrasound irradiation in step (iii) to the enhancement ultrasound irradiation in step (iv) can be performed without delay. Applying the enhancement field immediately after activation may be important for the resulting therapeutic effect. In this regard, it would be beneficial to apply both the activation and the enhanced ultrasound exposure using a broadband or dual-frequency US transducer; that is, a transducer that can deliver sufficient US pressure (i.e., MI) across all frequencies required for the described preferred ranges. For example, a transducer that can deliver an MI of up to 0.4 at both 1-10 MHz and 400-600 kHz.

[0105] In another embodiment, the polytherapy regimen includes anti-PD1, anti-PDL1 or CTLA4 monoclonal antibodies and chemotherapeutic drugs, such as pembrolizumab + cisplatin + oxaliplatin (5-FU) or capecitabine. Thus, several therapeutic drugs can be used, and several ACT procedures can be applied during the treatment regimen. In a preferred embodiment, the ACT treatment is performed when the active therapeutic molecule shows a maximum or near-maximum concentration in the blood after administration. Thus, the timing of the ACT treatment can vary depending on the pharmacokinetics of the therapeutic drug.

[0106] The therapeutic agent is administered separately, before, and / or together with, and / or after the cluster composition. In a preferred embodiment, the therapeutic agent is administered after administration of one of at least one cluster composition. Surprisingly, in Example 2, it was found that ACT treatment, i.e., administration of clusters and ultrasound irradiation, before administration of the therapeutic agent shows a similar effect size as ACT treatment starting after administration of the therapeutic agent (i.e., when the therapeutic agent is in the bloodstream). This result shows that the biomechanical effect induced by ACT, i.e., increased vascular barrier permeability, persists for some time after the US procedure has ended. This may be clinically beneficial since ACT treatment may be performed before therapeutic administration and treatment begins. Thus, in one embodiment, the cluster composition is administered and US ultrasound irradiation is performed in vivo, followed by administration of the therapeutic agent. In another embodiment, the cluster composition is administered either immediately before or immediately after administration of a therapeutic agent, such as chemotherapy.

[0107] result The inventors have found that applying the ACT concept in the treatment of PDAC, in combination with treatment with at least one chemotherapeutic and / or immunotherapeutic agent, i.e. as provided by the present invention, results in a significant increase in therapeutic effect compared to using the therapeutic agent alone and at the same dose, i.e. without using the claimed method or composition. The pharmaceutical composition for use and the method of treatment result in improved uptake of the therapeutic agent, for example as a result of enhanced extravasation of the therapeutic agent, and the therapeutic effect is significantly enhanced. The treatment provides a significant inhibition of tumor growth, induced tumor shrinkage, and therefore a reduction in tumor volume. As an overall result, the method can provide an improvement in survival time. Using the method of the present invention, a reduction in PDAC tumor volume of more than 50%, for example more than 75%, for example more than 90%, can be achieved. As shown in Examples 2 and 3, using the method of the present invention, a reduction in tumor volume of 85% or even 90% can be achieved 50 days after the first drug administration, compared to treatment with the drug alone. Moreover, as shown in Example 2, even complete remission can be achieved, thus significantly increasing the rate of complete response compared to treatment without ACT. Using the method of the present invention, the percentage of treated subjects who experience complete remission, for example, 120 days after the first drug administration, is at least 40%, for example at least 50%, or even at least 60%. It should be noted that these efficacy levels were observed in preclinical studies in mice, and efficacy levels in humans may be lower. A further advantage of the method is that the dosage of the therapeutic agent can be reduced compared to the doses normally used, resulting in reduced systemic side effects while maintaining or improving therapeutic efficacy. In Example 3, a similar synergistic effect using a combination with ACT was shown using the immunotherapeutic oncolytic reovirus.

[0108] In one embodiment, the pharmaceutical composition of the present invention is for use in the delivery of therapeutic agents to pancreas, particularly to subjects diagnosed with PDAC.The composition for use and the use of ACT technology provide site-specific delivery of therapeutic agents to reach effective local concentration of therapeutic agents, and further provide improved uptake in the area of ​​interest.

[0109] Thus, in one embodiment, the present invention provides a pharmaceutical composition for use in a method of delivering a therapeutic agent, wherein the method comprises the steps of: (i) administering to a mammalian subject suffering from PDAC a pharmaceutical composition as defined in the first aspect; wherein at least one therapeutic agent is administered prior to, and / or together with, and / or after the cluster composition, and prior to or after any of steps (ii)-(iii); (ii) optionally imaging the clusters of pharmaceutical composition using ultrasound imaging to identify target areas for treatment within the subject; (iii) insonifying a target area within the subject to induce a phase shift of a diffusible component of the second component of the cluster composition from step (i); (a) the microbubbles of the cluster are expanded by the diffusive component of step (iii) to result in an expanded bubble that is localized to the target region due to transient deposition in the microcirculation at the target region by the expanded bubble; and (b) promoting extravasation of the therapeutic agent administered in step (i); activating the compound; (iv) further promoting extravasation of the therapeutic agent administered in step (i) by further exposure to ultrasound. Includes.

[0110] Similarly, the present invention provides a method for delivering at least one therapeutic agent to a mammalian subject, comprising the steps of: (i) administering to a mammalian subject a pharmaceutical composition as defined in the first aspect; (ii) optionally imaging the microbubbles of the pharmaceutical composition using ultrasound imaging to identify target areas for treatment within the subject; (iii) insonifying a target area within the subject to induce a phase shift of a diffusible component of the second component of the cluster composition from step (i); (a) the microbubbles of the cluster are expanded by the diffusive component of step (iii) to result in an expanded bubble that is localized in the target area due to temporary blockage of microcirculation in the target area by the expanded bubble; and (b) said activating step (iii) promotes extravasation of the therapeutic agent administered in step (i). activating the compound; (iv) further promoting extravasation of the therapeutic agent administered in step (i) by further exposure to ultrasound. The present invention provides a method comprising:

[0111] The method and composition for use of the present invention may involve diagnostic imaging, for example, by performing ultrasound imaging, including using clustered microbubbles as a contrast agent, as disclosed above, but may also be combined with other types of imaging examinations, typically to diagnose and / or evaluate the outcome of treatment. Such imaging may include abdominal computed tomography (CT), for example, as an initial test to identify pancreatic masses, or abdominal magnetic resonance imaging (MRI) with or without cholangiopancreatography (MRCP), abdominal ultrasound (US), ultrasound (EUS) with or without endoscopic cholangiopancreatography (ERCP), etc. EUS and ERCP are the most invasive of the above-mentioned examinations, but are the only ones among those listed that allow biopsy, which can provide an accurate diagnosis.

[0112] Embodiments and features described in the context of one aspect, e.g. relating to a composition for use in treatment, also apply to other aspects of the invention relating to use in delivery or methods for treatment or delivery.

[0113] The following examples are provided to illustrate the invention in accordance with the principles of the invention, but should not be construed as limiting in any sense.

[0114] Working Example The applicant's patent application WO 2015 / 047103, in particular the examples therein which provide a description of analytical methodologies for characterizing the composition of clusters, results from the use of the clusters, etc., are incorporated herein by reference.

[0115] In the following examples, the first component is referred to as C1, the second component is referred to as C2, and the cluster composition, i.e. the composition resulting from the combination of the first and second components, is referred to as DP (drug product).

[0116] Example 1 provides a description of the analytical methodology for characterization and quantification of microbubble / microdroplet clusters in DPs, and describes the associated responses and attributes, including concentration, size, and circularity. Details of analytical techniques for characterization and quantification of activated bubble size and concentration are also provided. In addition, data on the stability of clusters after preparation is provided, as is a comparison of the properties of premixed and co-injected DPs. Engineering steps for controlled manipulation of cluster content and size in DPs are also detailed.

[0117] Example 1 further presents results from an in vivo study elucidating the effect of cluster characteristics on product efficacy as their ability to deposit large activated bubbles in the microcirculation. These data are further analyzed and it is concluded that clusters having an average size between 3-10 μm, defined by a circularity of less than 0.9, contribute to the efficacy of the cluster composition.

[0118] Example 1. Cluster preparation, analysis tools, and basic properties The microbubble / microdroplet clusters formed by the mixing of C1 and C2, i.e. present in the DP, are crucial for a key quality attribute of the composition, i.e. functionality with respect to drug delivery. Analytical techniques to characterize and control the formed clusters in terms of concentration and size are therefore essential tools for evaluating the invention and for the quality control (QC) of pharmaceutical products. The inventors have identified three different analytical tools that can be applied for this purpose: Coulter counting, flow particle image analysis (FPIA) and microscopy / image analysis.

[0119] In addition to these techniques applied to the characterization of clusters in cluster compositions, an analytical methodology has been developed to study the activation of clusters in vivo, i.e. the formation of large activated bubbles by ultrasound irradiation. This methodology, "sonometry", is detailed in WO 2015 / 047103, E1-6. The main reported responses from sonometry analysis are the number and volume of activated bubbles, as well as their size distribution, both versus time after activation. The activation response may also be examined by microscopy / image analysis, as detailed in WO 2015 / 047103, E1-5.

[0120] Ingredients and Compositions The first component (C1) in the compositions investigated in the disclosed examples consisted of perfluorobutane (PFB) microbubbles stabilized by a hydrogenated egg phosphatidylserine sodium (HEPS-Na) membrane and embedded in lyophilized sucrose. HEPS-Na has a negatively charged head group, resulting in a negatively charged surface for the microbubbles. Each vial of C1 contained approximately 16 μL or 2×10 9 The lyophilized formulation contains microbubbles having an average diameter of about 2.0 μm. This lyophilized formulation exhibits a long shelf life, more particularly a shelf life of 3 years, when stored at room temperature.

[0121] The second component (C2) in the composition investigated in this example consisted of perfluoromethyl-cyclopentane (pFMCP) microdroplets stabilized by a 1,2-distearoyl-sn-glycerol-3-phospocholine (DSPC) membrane with 3% mol / mol of stearylamine (SA) added to provide a positive surface charge. The microdroplets in C2 were dispersed in 5 mM TRIS buffer. The standard formulation of C2 investigated in these studies was approximately 4 μL or 0.8 × 10 per mL. 9 The second component comprises microdroplets having an average diameter of about 1.8 μm. The second component exhibits a long shelf life, more particularly a shelf life of 18 months or more, when stored refrigerated.

[0122] In some cases, various formulation variables, such as SA content, microdroplet size, microdroplet concentration, TRIS concentration, and pH, were varied in a controlled manner to elucidate their effect on cluster properties. When such samples were used, these aspects are detailed herein.

[0123] Cluster Composition (DP) was aseptically prepared by reconstituting a vial of C1 with 2 mL of C2, followed by manual homogenization for 30 seconds. A sterile, single-use syringe and needle were used to remove 2 mL from the vial of C2. The contents of the syringe were added through the stopper of the vial of C1, and the resulting DP was homogenized to prepare the composition for administration.

[0124] As shown in WO 2015 / 047103, the first and second components, i.e., the microbubble and microdroplet formulations, may be varied. For example, as shown in Tables 9 and 10 of WO 2015 / 047103, both the gas and stabilizing membrane of the first component may be altered to prepare clusters with suitable properties expected to be useful in therapy according to the present invention.

[0125] Stability of clusters in the cluster composition under analysis Clusters in DP are formed and held together by electrostatic attraction between microbubbles and microdroplets. These forces are finite and clusters can break up after formation through various pathways / influences, e.g. mechanical stress or thermal (Brownian) motion. For accurate and precise characterization, it is important that the clusters remain stable over the analysis time. This stability was tested using all the methodologies mentioned above. To assess stability, 3-5 analyses were repeated for one DP sample for longer than 5 min. No significant changes in either concentration or size were observed between these replicates, indicating that microbubbles, microdroplets and clusters are stable under the described analysis conditions, i.e. after dilution in PBS or water and under continuous homogenization (agitation) for longer than 5 min.

[0126] Formulation Many different formulation aspects can be explored to control the cluster content and size in DP and target optimal properties. Parameters that can be used to engineer cluster content and size distribution include, but are not limited to, the difference in surface charge between microbubbles and microdroplets, such as SA%:microdroplet size in C2:pH:TRIS concentration in C2:and the concentration of microbubbles and microdroplets. In addition, chemical degradation of components, such as from extended storage at high temperature, may affect the cluster formation ability of C1 and C2 during the preparation of DP.

[0127] From the in vivo characterization of the 30 compositions, as reported in WO 2015 / 047103, several important correlations can be extracted that elucidate the nature and properties of the system. The size of the clusters formed was also found to be strongly related to the reactivity of the system. At relatively low levels of reactivity (e.g., below 20%), only small clusters (1-5 μm) and medium sized clusters (5-10 μm) are formed. As the reactivity increases, larger clusters begin to form, with 10-20 μm clusters beginning to form when R is greater than about 20%, and 20-40 μm clusters beginning to form when R is greater than about 50%. The formation of large clusters comes at the expense of smaller and medium sized clusters. At cluster concentrations of 1-5 μm and 5-10 μm, a clear optimum between content and reactivity was found. It was found that the formation of larger clusters (i.e., greater than 10 μm or greater than 20 μm) is detrimental to the efficacy of the composition, and that the ability to form clusters must be properly balanced.

[0128] Based on the applicant's experiments and the results shown in Tables 5 and 6 of WO 2015 / 047103, the efficacy of the cluster composition (linear enhancement in Grey Scale units (GS)) correlates with the cluster mean size and the concentration of clusters (million / mL). Grey Scale enhancement is the increase in brightness (contrast) observed by US imaging after administration of the cluster composition and activation in vivo, and is a measure of the amount of activated bubbles in the imaged tissue. The results reported therein are from a multivariate, principal component analysis (PCA) of the contribution of clusters of various size classes to the linear enhancement (in Grey Scale units) in ultrasound signals from canine myocardium upon iv administration of the cluster composition and activation in the left ventricle, see Example 2 of WO 2015 / 047103. The PCA was performed on the data of 30 samples detailed in Tables 5 and 6 therein. The results demonstrate that small and medium sized clusters (smaller than 10 μm) contribute significantly to the efficacy of the cluster composition, whereas larger clusters (larger than 10 μm) do not. These results and conclusions also apply to the present invention. The size distribution of the clusters is important, and the average size should be in the range of 3-10 μm, and preferably 4-9 μm, more preferably 5-7 μm.

[0129] The cluster concentration and average diameter of the cluster composition prepared according to Example 1 was analyzed and found to be maintained for several hours at a cluster concentration of about 40-44 million / mL and an average cluster diameter of about 5.8-6.2 μm. The results are shown in Table 1 below and are consistent with the results in Table 6 of WO 2015 / 047103. The data in Table 1 indicate that the prepared cluster composition has acceptable stability and that optimal cluster size and concentration can be achieved.

[0130] [Table 1]

[0131] Cluster size influences efficacy. Figure 1 visualizes cluster size and product efficacy, showing that clusters with a mean diameter in the range of 3-10 μm have optimal efficacy. Thus, in Figure 1, the relationship between product efficacy and cluster diameter is provided. The Y-axis shows the correlation coefficient for the Grey Scale enhancement from US images of canine myocardium after injection and activation of clusters into the left ventricle, and reflects the amount of activated bubbles deposited. The X-axis shows the cluster diameter in μm. The grey boxes indicate the different cluster size bins evaluated: 1-5 μm, 5-10 μm, 10-20 μm, and 20-40 μm. The solid line represents a continuous function of efficacy versus cluster diameter. The error bars represent the standard error. Figure 1 is an alternative visualization of Figure 12 (left side) of WO 2015 / 047103 and is based on the data provided in Table 2 below.

[0132] [Table 2]

[0133] In contrast to the co-injection of the two components taught in WO 99 / 53963, applying the concept of the present invention, i.e., by preparing the cluster composition from C1 and C2 prior to administration, thus forming the microbubble / microdroplet clusters, allows for a greater than 10-fold increase in efficacy. The formation of microbubble / microdroplet clusters by the combination of the first and second components, and the administration of these pre-made clusters, is a prerequisite for the intended functionality in vivo. The cluster composition can be administered to a subject during a time window during which the properties of the clusters do not substantially change, such as within 3 hours of mixing the two components.

[0134] Example 2 Therapeutic Efficacy in a Patient-Derived Xenograft Mouse Model of PDAC - Acoustic Cluster Therapy with Chemotherapy Study Design: Acoustic Cluster Therapy (ACT) and compositions for use according to the present invention (referred to as ACT) were investigated for therapeutic efficacy levels with two different chemotherapy regimens.

[0135] The following ACT procedure was used; intravenous administration of the cluster composition + localized ultrasound (US) irradiation of the tumor was performed three times consecutively either immediately before or after administration of chemotherapy drugs. The experimental setup (rig) and treatment schedule are shown in Figure 2, where Figure 2A) provides a set-up photograph of the equipment used in the study for ultrasound activation and enhancement, and Figure 2B) provides a set-up sketch. In Figure 2B, the symbols indicate the following: 1 is a dual frequency ultrasound transducer (2.7 MHz and 500 kHz output), 2 is an ultrasound waveguide, 3 is a water bath, 4 is ultrasound gel, 5 is an ultrasound absorbing pad, 6 is an injection syringe with the cluster composition, 7 is a VeVo imaging table, 8 is a catheter, and 9 is a tumor.

[0136] Additionally, Figure 2C outlines the treatment schedule, where the cluster composition was formulated by reconstituting one vial of Sonazoid (GE Healthcare) (Component 1 as outlined in Example 1) with 2 mL of perfluoromethylcyclopentane (PFMCP) emulsion containing 6.8 mg PFMCP / mL. The PFMCP droplets were stabilized with distearoylphosphatidylcholine containing 3 M stearylamine (Component 2 as outlined in Example 1).

[0137] In Figure 2C, the following designations are used: A. Anesthetized, catheterized mouse. b.: Injection of cluster composition; c.: Ultrasonic activation at 2.7 MHz for 60 seconds, and d.: Ultrasonic enhancement at 500 kHz for 5 min. Gemcitabine and nab-paclitaxel infusions as shown before or after ACT treatment. Or liposomal irinotecan infusions before ACT treatment as shown.

[0138] Each ACT treatment included an injection of the cluster composition at 2 mL / kg, followed by a US activation and enhancement step.

[0139] Tumor xenografts from PDAC patients were cultured with a tumor size of 163 ± 3 (mean ± SEM) mm 3 At the time of reaching 100% CI, tumors were subcutaneously implanted into Atomitic nude mice enrolled in the treatment study. Mice (7–10 / group, see Table 3) were treated with chemotherapy drugs alone or in combination with ACT. In addition, a saline + ACT control group was also performed. Two chemotherapy regimens were tested: - Nab-paclitaxel (NAB, 15mg / kg, iv, weekly x 3) and gemcitabine (GEM, 60mg / kg, ip, weekly x 3) combination (NAB / GEM), and - Liposomal irinotecan (ONI, 15 mg / kg, iv, weekly x 3).

[0140] Table 3 provides an overview of the treatment groups and the number of animals in each group.

[0141] Gemcitabine was administered intraperitoneally (Gemcitabine IP), nab-paclitaxel was administered intravenously (Nab-Paclitaxel IV), and liposomal irinotecan was administered intravenously (Liposomal irinotecan IV).

[0142] [Table 3]

[0143] Animals were monitored daily for 49 days after the start of the study, with body weight and tumor size measured with calipers. On day 49, animals showing progressive disease were culled, whereas animals showing significant remission (tumor size 60 mm or greater) were culled. 3Animals that showed a complete response (defined as less than 150 mm on the first treatment day (day 0)) were monitored for up to 120 days. At day 120, the proportion of complete remissions in each group, defined as animals with no measurable residual tumor burden, was determined. Tumor burden was determined by the mean of 150 mm on the first treatment day (day 0). 3 was normalized to.

[0144] result The results of the mean tumor volumes and standard error of the mean (SEM) are set forth in Tables 4 and 5 below.

[0145] [Table 4]

[0146] [Table 5]

[0147] The results shown in Tables 4 and 5 are illustrated in Figures 3 and 4, respectively.

[0148] FIG. 3 provides the therapeutic effect of ACT combined with liposomal irinotecan for the treatment of patient-derived pancreatic ductal adenocarcinoma. The Y-axis represents the mean (mm) 3 Figure 1 shows tumor volumes at 10-day post-treatment with ACT. The x-axis shows time in days since study initiation. Treatments were applied on days 0, 7, and 14. Treatment groups were as follows: saline control (open squares), liposomal irinotecan alone (closed squares), and liposomal irinotecan with ACT (closed circles).

[0149] Figure 4 provides the therapeutic effect of ACT combined with gemcitabine and nab-paclitaxel on pancreatic ductal adenocarcinoma. The Y-axis is mm 3Figure 1 shows tumor volumes at 0-day post-treatment. The x-axis shows time in days since study initiation. Treatments were applied on days 0, 7, and 14. Treatment groups were as follows: saline control (open squares), gemcitabine and nab-paclitaxel alone (closed squares), gemcitabine and nab-paclitaxel with ACT applied prior to drug administration (open circles), and gemcitabine and nab-paclitaxel with ACT applied after drug administration (closed circles).

[0150] In summary, the results show that the administration of nab-paclitaxel / gemcitabine (ABX / GEM) or liposomal irinotecan (ONI) alone showed a significant inhibition of tumor growth at the doses used. In addition, however, the application of ACT according to the invention before or after chemotherapy treatment dramatically improved the activity and therapeutic effect, and significantly induced tumor shrinkage, even more than 100 days after the last administration of treatment. The application of ACT after the administration of ABX / GEM reduced tumor volume by 90% compared to ABX / GEM alone, 50 days after the first drug administration (P=0.001). Some mice achieved complete remission. Even at day 120, 50% of the mice in the ABX / GEM+ACT group had a tumor volume of 50 mm compared to 10% in the ABX / GEM alone group. 3 All mice maintained a stable complete remission with tumor volume less than 50 mm3. Similar activity was observed with ONI treatment. At 50 days after the start of the study, the ONI+ACT group induced an 85% reduction in tumor volume compared to the ONI alone group (P=0.024). At 120 days, 60% of mice in the ONI+ACT® group had tumor volumes of 50 mm3 or less. 3 ACT administered immediately prior to chemotherapy also showed improved efficacy, although slightly less than when administered after chemotherapy. Overall, these findings demonstrate the potential utility of ACT to improve the therapeutic efficacy of chemotherapy agents such as ABX / GEM and ONI in PDAC and provide solid preclinical evidence to support evaluation in patients. It also provides insight into the design of clinical trials.

[0151] Example 3: Therapeutic effect in a subcutaneous mouse model of hepatocellular carcinoma (HCC) - cancer immunotherapy using reovirus ± acoustic cluster therapy This example reports the effect of ACT combined with oncolytic virus for the treatment of HCC. This disease model resembles PDAC in many important features (see J Gastrointest Oncol. 2018 Feb; 9(1): 180-195. doi: 10.21037 / jgo.2017.06.09), thus providing relevant data to support the present invention.

[0152] Oncolytic viruses are a form of cancer immunotherapy that uses viruses to infect and destroy cancer cells. Viruses are particles that infect or invade our cells and then use the cell's genetic machinery to make copies of themselves, which then spread to surrounding uninfected cells. Recently, viruses have been used to target and attack already formed tumors. Such viruses are known as oncolytic viruses and present a promising approach to cancer treatment. Cancer cells often have reduced antiviral defenses, making them susceptible to infection. After infection, these oncolytic viruses can cause the cancer cells to "burst", killing them and releasing cancer antigens. These antigens can then stimulate an immune response that can seek out and eliminate any remaining tumor cells nearby and potentially elsewhere in the body.

[0153] Viruses of the Reoviridae family (Reovirus) have been extensively investigated for the treatment of PDAC (NCT01280058, NCT02620423, NCT03723915) and clear evidence or therapeutic efficacy has been confirmed. However, a factor that may reduce the efficacy of Reovirus is its limited uptake by cancer cells. In this regard, it is expected that combining ACT with viral therapy may potently increase viral uptake, thus leading to an increase in therapeutic efficacy.

[0154] Study Design: The therapeutic effect level was examined when Acoustic Cluster Therapy (ACT) and a composition for use according to the present invention (referred to as ACT) was combined with an oncolytic reovirus.

[0155] The ACT procedure applied was as described in Example 2.

[0156] HCC tumor xenografts were implanted subcutaneously into balb / C mice. 7 Cancer cells were injected into the flank and allowed to grow freely for approximately 21 days before enrollment in the study. Mice (N=5 per group) were treated with reovirus alone or in combination with ACT. In addition, a phosphate buffered saline control group was examined. Virus was administered six times in total on days 0, 5, 7, 10, 14, and 16. 1×10 cells / day were administered. 7 A dose of plaque forming units (PFU) was administered intravenously immediately prior to ACT administration. Body weight and tumor size (volume) were monitored by caliper measurement twice weekly during the study period (25 days).

[0157] result: The results for mean tumor volume and standard error of the mean (SEM) are set forth in Table 6 below and illustrated in FIG.

[0158] [Table 6]

[0159] FIG. 7 provides the therapeutic effect of ACT combined with oncolytic reovirus for the treatment of hepatocellular carcinoma. The Y-axis is mm 3 Figure 1 shows tumor volumes at 10 min after ACT. The x-axis shows time in days since study initiation. Grey triangles below the x-axis indicate days of treatment. Treatment groups are as follows: saline control (inverted triangles), oncolytic virus alone (filled squares), and oncolytic virus with ACT (filled circles).

[0160] As can be seen from the results displayed in Table 6 and illustrated in Figure 7, treatment with reovirus alone did not show significant inhibition of tumor growth at the doses investigated when compared to the PBS control group. However, when the same dose of virus was combined with ACT treatment, a significant and pronounced inhibition of tumors was observed, with a greater than 95% reduction in tumor volume at day 25 compared to virus alone. At day 25, the p-value was calculated using a two-sided ANOVE test (nonparametric Kruskal-Wallis test with Dunns multiple comparison correction) with a 95% confidence interval, p=0.037.

[0161] Thus, this study confirms a strong synergistic effect when combining reovirus immunotherapy treatment with ACT according to the present invention. As the disease model used in the examples resembles PDAC in many important characteristics, a similar synergistic effect would be expected for the treatment of PDAC.

[0162] Example 4 (Prospective). Pilot study of Acoustic Cluster Therapy (ACT) with PD-1 antibody in a syngeneic mouse model for pancreatic cancer. Introduction: Despite great success in many cancer types, cancer immunotherapy, such as immune checkpoint inhibitors, fails to show any meaningful efficacy in patients with pancreatic ductal adenocarcinoma (PDAC). Three main factors contribute to this inefficacy: 1) low tumor immunogenicity, 2) low cell recruitment, and 3) cytokine-induced T cell inactivation. Interstitial desmoplasia with high interstitial fluid pressure physically impedes T cell infiltration, and chemically induced intratumoral hypoxia releases immunosuppressive cytokines from stromal cells, suppressing T cell activation [Hilmi et al., World J Gastroenterol 24, 2137-2151]. Acoustic Cluster Therapy (ACT), a novel approach for ultrasound (US)-mediated drug delivery [Sontum et al., International Journal of Pharmaceutics 495 (2015) 1019-1027], uses a dispersion of clusters of negatively charged microbubbles and positively charged microdroplets to increase the localized permeability of blood vessels upon localized US exposure, allowing drug extravasation into tumor tissue. Using ACT techniques to increase vascular permeability in PDAC can increase the delivery of immunotherapeutics that increase T cell infiltration and improve T cell activation. This could therefore have a major impact on the outcome of immunotherapy in patients with PDAC.

[0163] Objective: The aim of this study was to evaluate the antitumor activity of ACT combined with a PD-1 antibody in a syngeneic mouse model of pancreatic cancer.

[0164] Experimental design A - Evaluation of the antitumor activity of ACT combined with PD-1 antibody using a syngeneic mouse model for pancreatic cancer.

[0165] The ACT procedure applied will be as described in Example 2.

[0166] In this study, a syngeneic mouse PDAC model will be used. This model uses a mouse PDAC cell line derived from the KPC genetically engineered mouse model [Hingorani et al., Cancer Cell 7, 469-483][Lee et al., Curr Protoc Pharmacol 73, 14 39 11-14 39 20] that has been shown to closely recapitulate human PDAC. Cells will be inoculated subcutaneously into the flank of C57BL / 6J (BLACK 6) mice. To evaluate the efficacy of ACT-based therapy, mice will be enrolled in four treatment groups: (1) isotype IgG control, (2) isotype IgG + ACT only, (3) PD-1 antibody, (4) PD-1 antibody + ACT, 14 mice / group.

[0167] PD-1 antibody (0.2 mg / mouse) will be administered ip on days 0, 3, 7, and 10. Tumors will be measured with calipers and tumor volumes will be calculated using the formula: Tumor volume = (a x b2 / 2), where "b" is the smallest diameter and "a" is the largest diameter. Established tumors will reach approximately 50-150 mm 3 After reaching 100 mg / kg, mice are pair-matched to various treatment groups to reduce variability in tumor size between groups. Antibody and ACT are administered according to schedule on the same day (4 doses total). Individual tumors in each mouse are allowed to grow to the approximate endpoint (tumor volume of 1,500 mm). 3 At the time when the tumor growth index (TGI) reaches a predefined endpoint, the mice are sacrificed in regulated CO2. The tumor growth inhibition index (TGI) is calculated by comparing the tumor measurements in the control group with those in the other test groups.

[0168] Assessment of the increased presence of immune cells using the activity of B-ACT.

[0169] To evaluate changes in the tumor immune microenvironment due to treatment, four mice from each group are sacrificed 1 hour after the last drug administration on day 10. Blood and tumor tissues are collected. Half of the tumor tissue from each mouse is snap frozen and the other half is formalin-fixed paraffin embedded (FFPE processing). FFPE tissues are analyzed using hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) including immune markers: F4 / 80, CD3, CD4, CD8, Foxp3.

[0170] Results (prospective): The results will show that, compared to anti-PD1 treatment alone, the combination with ACT results in meeting one or more of the following therapeutic efficacy endpoints: a significant increase in TGI, a significant increase in median overall survival, a significant increase in tumor infiltration of immune cells.

[0171] This study will therefore confirm the strong synergistic effects of combining immunotherapy treatment with anti-PD1 with ACT according to the present invention.

[0172] Example 5 (Prospective) Preparation of Cluster Compositions with Various Microbubble and Microdroplet Components To demonstrate that the present invention is applicable to various chemical compositions of C1 and C2, several formulations have been manufactured or commercially procured and the in vitro attributes of the resulting cluster compositions have been examined.

[0173] C1 Example The commercially available microbubble US imaging agents Sonovue (Bracco Spa, Italy) and Micromarker (VisualSonics Inc, USA) are procured and used as C1 components. Sonovue is a sulfur hexafluoride microbubble stabilized with a membrane of distearoylphosphatidylcholine, sodium dipalmitoylphosphatidylglycerol, palmitic acid and PEG4000 and is provided in a lyophilized form to be reconstituted with 5 mL of aqueous matrix. Micromarker is a perfluorobutane / nitrogen microbubble stabilized with phospholipids, polyethylene glycol, and fatty acids and is provided in a lyophilized form to be reconstituted with 0.7 mL of aqueous matrix.

[0174] C2 Example Microdroplets (C2) containing the following diffusible components: Perfluorodimethylcyclobutane, 2-(trifluoromethyl)perfluoropentane, and perfluorohexane would be prepared as follows:

[0175] 790 mg of distearoylphosphatidylcholine (DSPC) and 8.1 mg of stearylamine (SA) are weighed into a 250 mL round-bottom flask and 50 mL of chloroform is added. The sample is heated in hot water until a clear solution is obtained. The chloroform is removed by rotary evaporation to dryness at 350 mmHg and 40 °C, followed by further drying in a desiccator at 50 mmHg overnight. Then 160 mL of water is added and the flask is rotary evaporated again and the lipids are rehydrated for 25 min at full rotation speed and a water bath temperature of 80 °C. The resulting lipid dispersion is transferred to an appropriate vial and stored in the refrigerator until use.

[0176] Emulsions are prepared by transferring 1 mL aliquots of the chilled lipid dispersion into 2 mL chromatography vials. To each of the six vials, 100 μm of fluorocarbon oil is added as detailed above. The chromatography vials are shaken for 75 seconds on a CapMix (Espe, GmbH). The resulting emulsions are washed three times by centrifugation and removal of the supernatant, after which an equal volume of 5 mM TRIS buffer is added. The vials are immediately chilled on ice, pooled, and stored cold until use.

[0177] Coulter counter analysis is performed to determine the volume concentration and diameter of the microdroplets, and the emulsion is then diluted with 5 mM TRIS buffer to give a dispersed phase concentration of 4 μL of macrodroplets / mL.

[0178] Preparation of the cluster compositions will be performed by reconstituting Sonovue or Micromarker with 5 mL or 0.7 mL, respectively, of each of the above C2 components.

[0179] Results (prospective) Upon mixing of components C1 and C2, all six combinations are expected to contain more than 10 million clusters per mL with average diameters between 3 and 10 μm.

Claims

1. A pharmaceutical composition for use in a method for treating pancreatic cancer in a human diagnosed with pancreatic cancer, said pharmaceutical composition comprising: (a) a cluster composition comprising a suspension of clusters in an aqueous biocompatible solvent, said clusters having an average diameter in the range of 3-10 μm and a circularity of less than 0.9, and a cluster concentration of clusters in the size range of 3-10 μm of at least about 10 million / mL, said cluster composition comprising: (i) a first component comprising gas microbubbles and a first stabilizing agent for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizing agent for stabilizing said microdroplets, said oil phase comprising a diffusible component capable of diffusing into said gas microbubbles and thereby increasing their size at least temporarily; wherein the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via electrostatic interactions; (b) a therapeutic agent selected from the group consisting of chemotherapeutic agents and immunotherapeutic agents or a combination thereof, wherein said therapeutic agent comprises said chemotherapeutic agent, and when said chemotherapeutic agent comprises paclitaxel, said paclitaxel is an albumin-bound form of paclitaxel, and at least one of said therapeutic agents is provided as a separate composition from (a).

23. A pharmaceutical composition comprising:

2. The pharmaceutical composition according to claim 1 for use according to claim 1, wherein the gas of the gas microbubbles of the first component comprises sulfur hexafluoride, C3-C6 perfluorocarbons, nitrogen, air or mixtures thereof.

3. 3. The pharmaceutical composition of claim 1 or 2 for use as claimed in claim 1, wherein the first stabilizer and the second stabilizer each have a net electrostatic charge that is opposite to the other.

4. The pharmaceutical composition of claim 3 for the use of claim 1, wherein the first stabilizer and the second stabilizer each independently comprise a phospholipid, a protein, a polymer, a polyethylene glycol, a fatty acid, a positively charged surfactant, a negatively charged surfactant or a mixture thereof.

5. The pharmaceutical composition according to any one of claims 1 to 4 for use as claimed in claim 1, wherein the oil phase of the microdroplets of the second component comprises a partially or fully halogenated hydrocarbon or a mixture thereof.

6. A pharmaceutical composition according to any one of claims 1 to 5 for use according to claim 1, wherein said clusters have an average diameter in the range of 4 to 9 μm, preferably in the range of 5 to 7 μm.

7. The pharmaceutical composition according to any one of claims 1 to 6 for the use according to claim 1, wherein said therapeutic agent is selected from the group of alkylating agents, antimetabolites, microtubule inhibitors, topoisomerase inhibitors, cytotoxic antibiotics, immunotherapeutic agents and cytokine therapeutic agents.

8. The pharmaceutical composition according to any one of claims 1 to 7 for the use according to claim 1, wherein the therapeutic agent is formulated in a vehicle comprising the agent in the form of a liposome, a conjugate, a nanoparticle or a microsphere.

9. The pharmaceutical composition according to any one of claims 1 to 8 for the use according to claim 1, wherein said therapeutic agent is selected from the group of immunotherapeutic agents such as nab-paclitaxel, liposomal irinotecan, and checkpoint inhibitors.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the use is for the treatment of pancreatic ductal adenocarcinoma (PDAC).

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the use is for the treatment of pancreatic ductal adenocarcinoma at any of stages 0 to IV.

12. The use comprises the steps of: (i) administering said pharmaceutical composition to a mammalian subject suffering from pancreatic cancer, wherein at least one therapeutic agent is administered separately, prior to, and / or together with, and / or subsequent to said cluster composition; (ii) optionally imaging the clusters of the pharmaceutical composition using ultrasound imaging to identify target areas for treatment within the subject; (iii) activating a phase shift of the diffusible component of the second component of the cluster composition from step (i) by ultrasonic irradiation of a target area within the subject; (iv) promoting extravasation of the therapeutic agent administered in step (i) by further exposure to ultrasound.

10. The pharmaceutical composition according to any one of claims 1 to 9, comprising an Acoustic Cluster Therapy (ACT) treatment comprising:

13. A pharmaceutical composition according to any one of claims 1 to 9 for use as claimed in claim 12, wherein the cluster composition can be administered within a time window of 3 hours after the first component and the second component are mixed.

14. The pharmaceutical composition according to any one of claims 1 to 9 for use according to claim 12 or 13, wherein step (iii) of activating the phase shift is carried out using ultrasonic irradiation at a frequency of 1 to 10 MHz and a mechanical index of 0.1 to 0.

7.

15. The pharmaceutical composition according to any one of claims 1 to 9 for use according to any one of claims 12 to 14, wherein step (iv) of promoting extravasation is carried out using ultrasonic irradiation at a frequency of 330 to 650 kHz and a mechanical index of 0.15 to 0.

4.

16. The pharmaceutical composition according to any one of claims 1 to 9 for use according to any one of claims 12 to 15, wherein steps (i) to (iv) of the ACT treatment are repeated 1 to 4 times.

17. A pharmaceutical composition according to any one of claims 1 to 9 for the use according to any one of claims 12 to 16 as part of a combination drug therapy.

18. A pharmaceutical composition according to any one of claims 1 to 9 for use according to any one of claims 12 to 17, wherein the ultrasonic irradiation of step (iii) that activates a phase shift is initiated immediately after step (i) and is followed immediately thereafter by the ultrasonic irradiation of step (iv) that promotes extravasation.

19. The pharmaceutical composition according to any one of claims 1 to 9 for use according to any one of claims 12 to 18, wherein the ultrasonic irradiation in step (iii) lasts for 30 to 120 seconds, followed by the ultrasonic irradiation in step (iv) lasting for 3 to 10 minutes.

20. A pharmaceutical composition according to any one of claims 1 to 9 for use according to any one of claims 12 to 19, wherein one to five therapeutic agents are administered simultaneously or sequentially over a time span, wherein at least one ACT treatment, e.g. one to five, is administered during the same period of time.

21. A pharmaceutical composition according to any one of claims 1 to 9 for use according to any one of claims 12 to 20, wherein a broadband or dual frequency US transducer is used for both the activating ultrasonic irradiation in step (iii) and the enhancing ultrasonic irradiation in step (iv).

22. 1. A method for delivering a therapeutic agent comprising the steps of: (i) administering to a subject suffering from PDAC the pharmaceutical composition of claim 1; wherein at least one therapeutic agent is administered separately prior to, and / or together with, and / or after the cluster composition, and prior to or after any of steps (ii)-(iii); (ii) optionally imaging the clusters of pharmaceutical composition using ultrasound imaging to identify the target areas for treatment within the subject; (iii) insonifying a target area within the subject to induce a phase shift of the diffusible component of the second component of the cluster composition from step (i); (a) the microbubbles of the cluster are expanded by the diffusive component of step (iii) to result in an expanded bubble that is localized in the target area due to a temporary blockage of microcirculation in the target area by the expanded bubble; and (b) promoting extravasation of the therapeutic agent administered in step (i). activating the compound; (iv) further promoting the extravasation of the therapeutic agent administered in step (i) by further irradiating with ultrasound. A pharmaceutical composition according to any one of claims 1 to 9 for use in a method comprising the steps of:

23. The use, compared to using the therapeutic agent alone, provides one of the following: Improved uptake of therapeutic agents, inhibition of tumor growth, induced tumor shrinkage, reduction in tumor volume, improved survival time, increased proportion of treated subjects experiencing a complete response A pharmaceutical composition according to any one of claims 1 to 9 for use according to any one of claims 1 to 21 to provide an enhancement in the therapeutic effect of one or more forms of

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