Treating infection

Abstract

The present invention relates to ultrasound-mediated delivery of antimicrobial agents to sites of infection, and in particular for the treatment of infectious diseases. Accordingly, the present invention provides cluster compositions and pharmaceutical compositions for use in the delivery and preparation of antimicrobial agents for the administration and treatment of infectious diseases.

Description

[Technical Field]

[0001] The present invention relates to ultrasound-mediated delivery of antimicrobial agents to a site of infection, and in particular for the treatment of infection. Accordingly, the present invention provides cluster compositions and pharmaceutical compositions for use in the preparation and delivery of antimicrobial agents for the administration and treatment of infection. [Background technology]

[0002] Antibacterial agents are of paramount importance in modern medicine for treating or preventing the spread of an ever-increasing variety of infections caused by microorganisms such as bacteria, parasites, viruses, and fungi. Antibacterial, antifungal, antiviral, and anthelmintic agents all have numerous and widespread uses and are prescribed and used in large quantities against a variety of infections.

[0003] The problem with many common antimicrobials is their relatively low effectiveness and the high doses required. Unused drugs persist in the body and / or the environment, promoting increasing drug resistance. Antimicrobial resistance is a serious public health concern worldwide, threatening our ability to treat common infectious diseases and resulting in long-term illness, disability, and death. Without effective antimicrobials, major surgery, medical procedures such as cancer chemotherapy, and diabetes management become extremely risky. Unless action is taken, the mortality burden due to antimicrobial resistance will skyrocket to 10 million lives per year by 2050, with a cumulative cost of $100 trillion in global economic output. Therefore, reducing antibiotic use is a critical goal.

[0004] Another common problem with such antimicrobials is their side effects, which range from diarrhea and vomiting to headaches and fatigue, to secondary infections such as candidiasis after a course of antimicrobials. Antimicrobial toxicity can be dose-limiting and sometimes leads to longer treatment periods. Finding ways to prevent such side effects is important for each patient as well as for the healthcare system.

[0005] In view of the above, there is a need for new and alternative compositions and methods for the treatment of subjects suffering from infectious diseases. Summary of the Invention

[0006] The inventors have discovered that acoustic cluster therapy (ACT®) can be used to target and increase the uptake of antimicrobial agents at the site of infection, thereby enhancing efficacy and effectively reducing toxicity through increased exposure. ACT, as described in WO 2015 / 047103, is a concept for ultrasound-mediated targeted delivery in which a microbubble / microdroplet cluster composition is administered with a therapeutic agent, and ultrasound irradiation of the target pathology can result in increased therapeutic efficacy compared to using the therapeutic agent alone. While commonly used antimicrobial treatment methods and compositions typically require systemic treatment with very high doses, even when the infection is highly localized, ACT maximizes clinical efficacy by enabling targeted treatment and high exposure to the antimicrobial agent at the site of infection. This allows for the use of lower doses of antimicrobial agents, limiting off-target side effects. Dosage reduction is also economically beneficial, potentially resulting in significant savings, and further provides opportunities for the reuse of antibiotics, ensuring their longevity and addressing the issue of antimicrobial resistance. Furthermore, the use of ACT can also improve the delivery rate of antimicrobial agents, resulting in longer periods of time in the tissue. Various drugs, particularly compounds used to treat infections, are time and concentration dependent.

[0007] In one aspect, the present invention provides a pharmaceutical composition for use in a method for treating an infectious disease, wherein the pharmaceutical composition comprises: (a) A cluster composition comprising a suspension of clusters in an aqueous biocompatible medium, said clusters having an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and comprising: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, a cluster composition, wherein the microbubbles and microdroplets of the first component and the second component have opposite surface charges and form the clusters via attractive electrostatic interactions; and (b) an antimicrobial agent selected from the group of antibiotics, antifungals, antivirals, antiparasitics, or combinations thereof, provided as a separate composition from (a). Includes.

[0008] The present invention therefore provides a microbubble / microdroplet cluster composition for use in a method of treating an infectious disease, wherein (a) the cluster composition comprises a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters through attractive electrostatic interactions; and The method comprises: (a) a cluster composition, and (b) an antimicrobial agent selected from the group of antibiotics, antifungals, antivirals, antiparasitics, or combinations thereof, provided as a separate composition from (a). This includes administering

[0009] The present invention further provides the microbubble / microdroplet cluster composition described above for use in a method for delivering an antimicrobial agent or for treating a subject suffering from an infection, the method comprising: (i) administering to the subject at least one antimicrobial agent selected from the group consisting of an antibiotic, an antifungal, an antiviral, an antiparasitic, or a combination thereof; (ii) administering to said subject a microbubble / microdroplet cluster composition, wherein at least one antimicrobial agent is administered before, and / or simultaneously with, and / or after the cluster composition; (iii) activating a phase shift of the diffusible component of the second component of the cluster composition from step (ii) by ultrasonic irradiation of a region of interest in the subject; (iv) further insonifying the area of ​​interest to promote extravasation of the antimicrobial agent administered in step (i). Includes.

[0010] In another aspect, the present invention provides a system for localized delivery of an antimicrobial agent to a target site, the system comprising: (a) A cluster composition comprising a suspension of clusters in an aqueous biocompatible medium, said clusters having an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and comprising: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, a cluster composition, wherein the microbubbles and microdroplets of the first component and the second component have opposite surface charges and form the clusters via attractive electrostatic interactions; and (b) an antimicrobial agent selected from the group including antibiotics, antifungals, antivirals, antiparasitics, or combinations thereof, provided as a separate composition from (a) or together with (a); Includes.

[0011] In yet another aspect, the present invention provides a method for preparing a subject for subsequent treatment with an antimicrobial agent, the method comprising the step of administering to the subject a cluster composition comprising a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and are as follows: a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing said microdroplets, said oil comprising a diffusible component capable of diffusing into said gas microbubbles to at least transiently increase their size; Including, the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters through attractive electrostatic interactions; The method further comprises: activating a phase shift of a diffusible component of a second component of the cluster composition by ultrasonic irradiation of a region of interest in the subject; and Further ultrasound irradiation to promote extravasation of the administered antimicrobial agent. Includes. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1 provides a visualization of the two major classes of antibacterial agents defined by their PK / PD properties. In both panels of Figure 1, the curves show the evolution of the drug's plasma concentration (y-axis) over time (x-axis). The top panel shows that for drugs with %T > MIC, the drug's therapeutic effect is related to the proportion of time the drug's plasma concentration is above a certain minimum inhibitory concentration (MIC). The bottom panel shows that for drugs with AUC / MIC (area under the drug plasma concentration curve) or Cmax / MIC, the therapeutic effect is related to the ratio of AUC or Cmax to MIC. [Figure 2] Figure 2 visualizes the cluster size versus the effect of the in vivo product, with the Y-axis showing the calculated correlation coefficient of grayscale enhancement from US imaging (i.e., the amount of bubbles deposited after activation) and the X-axis showing the cluster diameter (μm). [Figure 3] Figure 3 provides a sketch of the equipment setup used in Study 2 of Example 2 for the application of the ACT sonoporation procedure, including ultrasound activation and enhancement. Numbers indicate: 1—amplifier, 2—signal generator, 3—0.5-2.7 MHz switch box, 4—dual-frequency transducer, 5—water-filled cone, 6—water-filled bag, 7—ultrasound gel, 8—mouse in prone position, 9—ear bars, 10—acoustic absorber pad. [Figure 4]Figure 4 provides box plots of results from Study 2 of Example 2: ACT-induced delivery of nanoparticles across the blood-brain barrier. Top: Representative photographs from near-infrared fluorescence (NIRF) imaging of CCPM nanoparticle uptake into brain tissue. Control and ACT-treated brains at 1 hour and 24 hours after ACT treatment. Bottom left panel: The Y-axis shows uptake measured by NIRF as a percent of the injected dose per gram of brain tissue for the control and ACT groups at 1 hour and 24 hours after ACT treatment. Filled circles indicate individual observations. Lines and asterisks indicate statistical significance between groups (***p<0.001) obtained from a t-test. Bottom right panel: The Y-axis shows uptake for the control and ACT groups at 1 hour after ACT treatment, measured by confocal microscopy as a percent of brain area containing nanoparticles. Filled circles indicate individual observations. Lines and asterisks indicate statistical significance between groups (*p<0.05) obtained from a Mann-Whitney Rank Sum Test. [Figure 5] Figure 5 shows the dose-response of levofloxacin treatment of mouse thigh infections. The Y-axis shows the logarithm of bacterial density in infected tissue (Log Colony Forming Units / g), and the X-axis shows the levofloxacin dose (mg / kg). The dotted line indicates the dose required for 50% killing. [Figure 6] Figure 6 shows the logarithm of bacterial density within infected tissue in an evaluation of levofloxacin (20 mg / kg) versus levofloxacin (20 mg / kg) + ACT (2 mL / kg) + US treatment of mouse thigh infections. Column A shows results from the control group (saline), column B shows results from the levofloxacin alone group, and column C shows results from the levofloxacin + ACT + US group. DETAILED DESCRIPTION OF THE INVENTION

[0013] definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0014] As used herein, acoustic cluster therapy (ACT), as further defined below, involves the administration of a cluster composition (see definition below) in combination with at least one therapeutic agent, followed by the application of ultrasound to a targeted area of ​​interest (e.g., infected tissue) in a subject. The term "ACT treatment" or "ACT procedure" is used to describe the administration of the cluster and the ultrasound application, and thus includes steps (iii) and (iv) in addition to the administration of the cluster.

[0015] The terms "treating," "treatment," and "therapy" (and grammatical variations thereof) are used interchangeably herein and refer to 1) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder, such as preventing a disease, in a subject suffering from or exhibiting a pathology or symptom of the disease, condition, or disorder (i.e., prophylactic treatment, preventing further development of the pathology and / or symptom); or 2) alleviating the symptoms of a disease; or 3) ameliorating a disease; e.g., improving a disease, condition, or disorder (i.e., reversing the pathology and / or symptom) in a subject suffering from or exhibiting a pathology or symptom of the disease, condition, or disorder. Treatment may relate to reducing the amount of parasites and / or arthropods. The term may relate to the use and / or administration of a pharmaceutical, active pharmaceutical ingredient (API), and / or pharmaceutical composition.

[0016] As used herein, the terms "administer," "administration," and "administering" mean (1) providing, giving, dispensing, and / or prescribing a formulation, preparation, or composition according to the present disclosure, either by or under the direction of a healthcare professional or authorized representative, and (2) injecting, drinking, or ingesting a formulation, preparation, or composition according to the present disclosure by a subject themselves.

[0017] As used herein, "subject" means any human or non-human animal selected for treatment or therapy, and includes, but may be limited to, "patient," particularly a human patient suffering from an infectious disease. Neither term should be construed as requiring the supervision (full-time or otherwise) of a medical professional (e.g., a physician, nurse, nurse practitioner, physician assistant, attendant, clinical researcher, etc.) or a scientific researcher.

[0018] As used herein, the term "therapeutically effective amount" means an amount of a therapeutic agent (antibacterial agent) effective to produce a desired therapeutic effect in a subject, at a reasonable benefit / risk ratio applicable to any treatment.

[0019] The terms "microbubbles" or "regular contrast microbubbles" are used herein to refer to microbubbles with diameters ranging from 0.2 to 10 μm, typically with an average diameter between 2 and 3 μm. Regular contrast microbubbles include commercially available agents such as Sonazoid (GE Healthcare), Optison (GE Healthcare), Sonovue (Bracco Spa), and Definity (Lantheus Medical Imagin), as well as preclinical agents such as Micromarker (VisualSonics Inc.), Polyson L (Miltenyi Biotec GmbH), and Imagent® (IMCOR Pharmaceuticals Inc., San Diego, CA, USA).

[0020] The term HEPS / PFB microbubbles is used herein to describe microbubbles formed by reconstituting the first component (as provided in Example 1) with 2 mL of water.

[0021] As used herein, the terms "phase shift bubbles," "large, phase shift bubbles," "large, activated bubbles," and "activated bubbles" are used to refer to large (>10 μm) bubbles formed after ultrasound (US)-induced activation of the cluster composition.

[0022] The term "microdroplet" is used herein to refer to emulsion microdroplets having diameters in the range of 0.2 to 10 microns.

[0023] "Insonation" or "US irradiation" is a term used to describe exposure to or treatment with ultrasound.

[0024] As used herein, the term "resonance frequency" or "microbubble resonance frequency" refers to the acoustic resonance frequency of a single bubble in an infinite matrix domain (ignoring the effects of surface tension and viscous damping). The resonance frequency is given by: TIFF2023529237000001.tif1542 (where a is the bubble radius, γ is the polytropic coefficient, pA is the ambient pressure, and ρ is the matrix density.)

[0025] The term "deposit tracer" is used herein in reference to activated phase-shift bubbles to imply that the transient mechanical trapping of large bubbles in the microcirculation will result in the local deposition of phase-shift bubbles in tissue reflecting the amount of blood flowing through the tissue microcirculation at the time of activated bubble deposition. Thus, the number of trapped "deposited" phase-shift bubbles will be linearly dependent on tissue perfusion at the time of deposition.

[0026] In this specification, the term "phase shift (process)" is used to describe the phase transition of a substance from a liquid state to a gas state. Specifically, it refers to the transition (process) of the state change of the oil component of the cluster composition microdroplets from a liquid to a gas state.

[0027] As used herein, the term "bi-phasic" refers to a system that contains two phases, specifically a liquid and a gas phase, such as the microbubble (gas) and microdroplet (liquid) components of a cluster composition.

[0028] As used herein, the terms "therapy delivery / therapeutic agent(s)" and "drug delivery / drug(s)" are both understood to include the delivery of at least one therapeutically active agent. The therapeutic agent is for the treatment of an infection and, i.e., an antibacterial agent or a combination of two or more antibacterial agents.

[0029] The term "first component" (or 1st component, or C1) is used herein to describe the dispersed gas (microbubble) component. The term "second component" (or 2nd component, or C2) is used herein to describe the dispersed oil phase (microdroplet) component that contains the diffusible component.

[0030] The term "cluster composition" is used herein to describe a composition resulting from the combination, e.g., mixing, of a first (microbubble) component with a second (microdroplet) component. Thus, a cluster composition having the properties as further described herein refers to a composition ready for administration to a subject and formulated for use in acoustic cluster therapy.

[0031] The term "diffusible component" is used herein to refer to a chemical component of the oil phase of a second component that is capable of diffusing in vivo into microbubbles in a first component and transiently increasing their size.

[0032] As used herein, the term "pharmaceutical composition" has its conventional meaning, particularly a form suitable for mammalian administration. The composition preferably comprises two separate compositions: a cluster composition (a) and a therapeutic agent (b), both of which are suitable for mammalian administration, such as parenteral, intraperitoneal, or intramuscular injection, administered by the same or different routes of administration. The term "in a form suitable for mammalian administration" refers to a composition that is sterile and pyrogen-free, does not contain compounds that produce excessive toxicity or adverse effects, and is formulated at a biocompatible pH (approximately pH 4.0 to 10.5). Such compositions are formulated to prevent precipitation upon contact with biological fluids (e.g., blood), contain only biologically compatible excipients, and are preferably isotonic.

[0033] As used herein, the term "sonometry (system)" refers to a measurement system that uses acoustic techniques to dynamically size and count activated phase-shifting bubbles.

[0034] The term "reactivity" as used herein describes the ability of microbubbles in a first component and microdroplets in a second component to form clusters of microbubbles / microdroplets upon mixing.

[0035] As used herein, the term "microbubble / microdroplet cluster" or "cluster" or "cluster composition" refers to a group of microbubbles and microdroplets that are held together in a single particle, cohesive entity, by electrostatic attraction. As used herein, the term "clustering" refers to the process of forming clusters of microbubbles of a first component and microdroplets of a second component.

[0036] In medical ultrasound systems, acoustic power is usually expressed as the "Mechanical Index" (MI), which is the peak negative pressure (PNP) of the ultrasound field divided by the square root of the center frequency of the ultrasound field, Fc (MHz) (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). TIFF2023529237000002.tif1324 Regulatory requirements for medical US imaging are an MI of less than 1.9. During US imaging with microbubble contrast agents, an MI of 0.7 or less is recommended to avoid adverse bioeffects such as microbleeds and irreversible vascular damage, and using an MI of 0.4 or less is considered "best practice." When MI is referred to in the text, it should be understood to reflect in-situ MI, i.e., MI applied to the targeted region of interest (see The British Medical Ultrasound Society (BMUS) Guidelines for the safe use of diagnostic ultrasound equipment 2009).

[0037] "Activation" in this context refers to the induction of a phase shift in microbubble / microdroplet clusters by ultrasound (US) irradiation.

[0038] The term "time-dependent antimicrobial agent" or "%T>MIC" agent refers to an antimicrobial agent that has a therapeutic effect relative to the time that the plasma concentration of the agent is above some minimum level defined as the minimum inhibitory concentration (MIC), i.e., the minimum concentration of the agent that exerts an inhibitory effect on the microorganisms it is intended to kill.

[0039] The term "infectious disease" refers to an illness caused by an infectious disease.

[0040] The term "concentration-dependent antimicrobial agent" or "Cmax agent" refers to an antimicrobial agent whose therapeutic effect is related to the ratio of the antimicrobial agent's maximum plasma concentration (Cmax) to its MIC. This class is intended to include antimicrobial agents described by an alternative PK / PD parameter describing the area under the curve (AUC) of drug plasma concentration over the dosing interval ("AUC / MIC agents").

[0041] As used herein, the term "antimicrobial agents" refers to compounds (drugs, chemicals, or other substances) that kill or slow the growth of microorganisms. Antimicrobial agents include antibacterial agents (antibiotics), antiviral agents, antifungal agents, and antiparasitic agents.

[0042] As used herein, the term "infection" refers to the invasion of a subject's body tissues and / or organs by a disease-causing pathogen, and / or the proliferation of said pathogen, and / or the response of the host tissues / organs to said pathogen and / or any substances produced thereby. Infection can be caused by any one or more pathogens from the list including, but not limited to, viruses, bioids, prions, bacteria, fungi, parasites, arthropods, etc.

[0043] As used herein, the term "site of infection" refers to one or more sites, e.g., tissues, organs, body parts, where an infection is present. An infection may be systemic.

[0044] Reference to a specific drug is intended to include any drug containing the same active ingredient or ingredients and having a corresponding mechanism of action, for example generic drugs.

[0045] Details of the invention Thus, delivery of antimicrobial agents to infection sites and treatment of infectious diseases according to the present invention can be achieved through the use of a two-component microbubble / microdroplet formulation system (i.e., cluster composition) in which microbubbles in the first component are physically attached to micron-sized emulsion microdroplets in the second component by electrostatic attraction prior to administration. The present invention employs ACT technology to generate large, phase-shifted bubbles in vivo from compositions containing administered microbubble / microdroplet clusters, which facilitate the delivery and uptake of another antimicrobial agent administered before, simultaneously, and / or afterward. When clusters in the cluster composition are exposed to ultrasound, the volumetrically vibrating microbubbles initiate vaporization (phase shift) of the attached microdroplets. The resulting enlarged bubbles have been shown to deposit in capillary-sized blood vessels in vivo and can be excited with low-frequency ultrasound, inducing biomechanical effects that increase drug penetration in the exposed tissue.

[0046] The compositions for use in the treatment of infectious diseases according to the present invention provide improved uptake of antimicrobial agents specifically at the site of infection, resulting in beneficial treatment. Mixing the first component with the second component prior to administration is a prerequisite for the efficient formation of such microbubble / microdroplet clusters. The therapeutic effect of the antimicrobial agent is significantly increased compared to administration of the agent alone due to biomechanical mechanisms in microvessels, as further explained below. The present disclosure further demonstrates that the specific use of ACT technology, which involves two steps of ultrasound irradiation at different frequencies and mechanical indices, allows for increased delivery of separately administered antimicrobial agents (multiple antimicrobial agents). In one embodiment, ultrasound irradiation of the region of interest at a first frequency is performed in step (iii) to activate a phase shift in the diffusible component, followed by further ultrasound irradiation of the region of interest at a second, lower frequency to promote extravasation of the antimicrobial agent.

[0047] The clusters can be easily activated in vivo using low-power conventional medical imaging ultrasound, i.e., a first frequency in the range of 1-10 MHz, with a first MI of less than 1.9, e.g., less than 0.7, preferably less than 0.4, e.g., less than 0.3, to induce a liquid-gas transition (phase transition) of the diffusible components. A therapeutic agent, i.e., an antimicrobial agent or a combination of two or more antimicrobial agents, is administered in conjunction with the cluster composition, i.e., pre-administered, co-administered, and / or post-administered, as a conventional pharmaceutical formulation, according to the drug's approved route. The large activated bubbles are temporarily retained within the microvasculature of the tissue into which they are inserted, and further application of low-frequency ultrasound, e.g., a second frequency in the range of 0.2-1 MHz, most preferably at an MI of 0.4-0.6 MHz, can be utilized to promote drug uptake at the site of infection. Activated phase-shifting bubbles are approximately 10 times larger in diameter than regular contrast microbubbles (>10 μm diameter, typically 20 μm diameter), and larger than the diameter of a microcapillary, allowing for: trapping of activated bubbles in microvasculature; avoiding pauses in blood flow and rapid washout of the agent; intimate contact of activated bubbles with the endothelium; and bioeffects during post-activation US procedures that are orders of magnitude greater than regular contrast microbubbles, while still avoiding inertial cavitation mechanisms.

[0048] The present invention therefore provides a microbubble / microdroplet cluster composition for use in a method of treating an infectious disease, wherein (a) the cluster composition comprises a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters through attractive electrostatic interactions; and The method comprises: (a) a cluster composition, and (b) an antimicrobial agent selected from the group of antibiotics, antifungals, antivirals, antiparasitics, or combinations thereof, provided as a separate composition from (a). This includes administering

[0049] In such a method of use or treatment using the microbubble / microdroplet cluster composition described above, the method comprises: (i) administering to the subject at least one antimicrobial agent selected from the group consisting of an antibiotic, an antifungal, an antiviral, an antiparasitic, or a combination thereof; (ii) administering to said subject a microbubble / microdroplet cluster composition, wherein at least one antimicrobial agent is administered before, and / or simultaneously with, and / or after the cluster composition; (iii) activating a phase shift of the diffusible component of the second component of the cluster composition from step (ii) by ultrasonic irradiation of a region of interest in the subject; (iv) further insonifying the area of ​​interest to promote extravasation of the antimicrobial agent administered in step (i). Includes.

[0050] Hereinafter, step (iii) will also be referred to as the "activation step," and step (iv) will also be referred to as the "enhancement step."

[0051] Below, details and preferred embodiments of the present cluster composition, first component (microbubbles), second component (microdroplets), ultrasound treatment, infectious disease and antimicrobial agents are disclosed and further described in detail.

[0052] Cluster composition: The cluster composition, i.e., the combination of the first and second components, comprises clusters of gas microbubbles and oil microdroplets, i.e., a dispersion containing microbubbles and microdroplets, respectively, that combine to form stable microbubble / microdroplet clusters. The ACT treatment according to the present invention involves the use of a cluster preparation in which microbubbles, e.g., negatively charged microbubbles, are combined with microdroplets, e.g., positively charged microdroplets, where these clusters can be activated by ultrasound. The mixture of these microbubbles and microdroplets produces clusters of small microbubble-microdroplets held together by electrostatic forces. The microdroplets typically contain an oil component with a boiling point below 50°C and low solubility in blood. Analytical methods for quantitatively detecting and characterizing the clusters are described in Example 1. In this context, the term "cluster" refers to a group of microbubbles and microdroplets held together by electrostatic attraction into a single particle, a cohesive entity. The content and size of clusters in the cluster composition are essentially stable for a period of time (e.g., >1 h) after combining the first and second components in vitro. That is, the clusters do not spontaneously disintegrate, form larger aggregates, or spontaneously activate (phase shift), and are essentially stable for a period of time even after dilution and continuous stirring. Therefore, various analytical techniques requiring dilution and / or stirring can detect and characterize clusters in the cluster composition. Furthermore, the stability of the cluster composition allows for necessary clinical procedures (e.g., reconstitution, dose withdrawal, and administration) to be performed.

[0053] Each cluster in the cluster composition contains at least one microbubble and at least one microdroplet, typically 2 to 20 or 2 to 50 individual microbubbles / microdroplets. Clusters typically have an average diameter in the range of 1 to 10 μm and are therefore free-flowing in the vascular system. Clusters in the cluster composition are further characterized and separated from individual microbubbles and microdroplets by a circularity parameter. The circularity of a two-dimensional feature (e.g., a projection of a microbubble, microdroplet, or microbubble / microdroplet cluster) is the ratio of the perimeter of a circle having the same area as the feature divided by the actual perimeter of the feature. Thus, a perfect circle (i.e., a two-dimensional projection of a spherical microbubble or microdroplet) has a theoretical circularity value of 1, and any other geometric feature (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.

[0054] According to the present invention, compositions containing clusters having an average size in the range of 1 to 10 μm, particularly 3 to 10 μm, and defined by a circularity of <0.9 are considered particularly useful. In one embodiment, the average cluster size is in the range of 3 to 10 μm, e.g., 4 to 9 μm, e.g., 5 to 7 μm. Clusters in this size range flow freely in the vasculature before activation, and they are easily activated by US irradiation, generating activated bubbles large enough to temporarily deposit and reside in the microvasculature. The microbubbles within the clusters allow efficient energy transfer of ultrasound energy in the diagnostic frequency range (1 to 10 MHz), i.e., the activation step (step iii), and allow evaporation (phase shift) of the emulsion microdroplets and diffusion of the evaporated liquid into microbubbles and / or fusion between the vapor bubbles and the microbubbles at a low MI (less than 1.9, e.g., less than 0.7, preferably less than 0.4). The activated bubbles then expand further from inward diffusion of matrix gas (eg, blood gases) to reach a volume-weighted median diameter of greater than 10 μm, eg, greater than 15 μm but less than 40 μm.

[0055] 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 efficient phase-shifting events. The number and size characteristics of the clusters are strongly related to the composition's efficiency, i.e., its ability to form large, activated (i.e., phase-shifted) bubbles in vivo, and have been found to be a prerequisite for intended functionality in vivo. The number and size characteristics can be controlled through various formulation parameters, including, 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 between the microbubbles and the microdroplets, and the composition of the aqueous matrix (e.g., buffer concentration, ionic strength, etc.). The average equivalent circular diameter of the clusters formed is preferably greater than 3 μm, more preferably between 5 and 7 μm, but desirably less than 10 μm. 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. In one embodiment, based on the results shown in Tables 5 and 6 of WO 2015 / 047103, the composition should contain at least 0.6 million / mL of clusters with an average size of 5 to 10 μm. In another embodiment, the composition for administration should contain at least 3 million / mL of clusters with diameters between 5 and 10 μm. This minimum value, according to Figure 11 of Applicant's WO 2015 / 047103, would ensure an enhancement of greater than 150 GS units, as well as a certain minimum level of product efficacy and therapeutic benefit. In another embodiment, the concentration of clusters in the size range of 1 to 10 μm should be at least 10 million / mL, e.g., at least about 25 million / mL.

[0056] The size of the activated bubbles can be engineered by varying the size distribution of the microdroplets in the emulsion and the size characteristics of the clusters (see Example 1 of WO 2015 / 047103). After administration, the clusters are activated to generate larger bubbles by applying external ultrasound energy under image control, for example, from a clinical ultrasound imaging system. The large phase-shifted bubbles generated are typically 10 μm or larger in diameter. Low MI energy levels, 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-transfer techniques (e.g., acoustic microdroplet evaporation (ADV)). Due to their large size, the activated bubbles temporarily reside within microvasculature and can be spatially localized in target tissues or organs, such as infected tissues or organs, by spatially localized ultrasound irradiation, i.e., irradiation tailored to the pathological region of interest. Thus, after administration of the cluster composition, the clusters are activated within, at, or near the site of infection by the deposition of ultrasonic energy at the site of infection. The large activated bubbles (greater than 10 μm in diameter) that are generated undergo acoustic resonance at low ultrasonic frequencies (less than 1 MHz, typically 0.5 MHz).

[0057] Those skilled in the art will understand that the additional irradiation of large activated bubbles with low-frequency ultrasound for the compositions, methods, and systems of the present invention further enhances the uptake of antimicrobial agents. The ultrasound procedure is described in further detail below under Ultrasound Procedures. Thus, for example, application of low-frequency ultrasound during the enhancement step (step iv)) has been found to create mechanical (shear force and microstreaming) and / or thermodynamic bioeffect mechanisms to increase vascular permeability and sonoporation, thereby increasing the delivery and retention of antimicrobial agents in target tissues. This mechanism has been shown to increase vascular permeability and thus allow for the delivery of larger drug doses (see WO 2015 / 047103). Furthermore, this can improve the distribution of drugs in infected tissues and increase the uptake of drugs into diseased cells. Furthermore, the biomechanical effects on endothelial cells can result in the generation of biochemical signals and the development of immune responses, further enhancing therapeutic efficacy.

[0058] Comparing the compositions, methods, and systems of the present invention with methods using free-flowing, conventional contrast microbubbles, the large phase-shifted microbubbles of the present invention are entrapped in blood vessel segments, and the microbubble surface is in intimate contact with the endothelium. Furthermore, the volume of activated bubbles is typically 1,000 times larger than that of conventional microbubbles. When irradiated at equal MIs at frequencies close to the resonance for both bubble types (0.5 MHz for phase-shifted microbubbles and 5 MHz for conventional contrast microbubbles), the absolute volume displacement during oscillation in phase-shifted bubbles has been shown to be nearly three orders of magnitude larger than that of conventional contrast microbubbles. Therefore, insonification of phase-shifted bubbles will result in a completely different level of biomechanical effects, with significantly larger size and penetration depth effects than those observed with conventional contrast microbubbles. The bioeffects observed with free-flowing, conventional contrast microbubbles are believed to be dependent on a cavitation mechanism, raising safety concerns such as microbleeding and irreversible vascular damage. Larger phase-shifting bubbles can be vibrated in a softer manner (low MI, e.g., less than 0.3), avoiding cavitation mechanisms but inducing sufficient mechanical function to promote uptake of antimicrobial agents from the vasculature and into target tissues. The capture of large phase-shifting bubbles would also function as a deposition tracer. This would further enable quantification of the number of activated clusters and tissue perfusion, and contrast imaging of tissue vasculature would allow identification of the spatial extent of the pathology to be treated.

[0059] In the systems or methods of the present invention, or in pharmaceutical compositions for use, the cluster compositions may contain any of a wide range of gases and first stabilizers for the first microbubble component, and oils and second stabilizers for the second microdroplet component. Example 5-2 of WO 2015 / 047103 provides results from a study in which various first components were explored in terms of gas and first stabilizer. This example demonstrates that useful cluster compositions according to the present invention were formed in almost all combinations explored. Furthermore, in Example 5-3 of the same publication, various oils were explored for the second component. This example demonstrates that stable cluster compositions activated by US irradiation were formed with various oil components, preferably 1-10. -4 Less than M, e.g., 1 to 10 -5 The present disclosure also provides a cluster composition that can be formed with a water solubility of less than 1000 M. Both examples are incorporated herein by reference. Furthermore, in Example 6 of the present disclosure, a second component containing various oils is combined with a first component containing various gases and first stabilizers. All of these combinations demonstrate that the cluster compositions of the present invention can contain a wide range of gases and first stabilizers for the first microbubble component, as well as oils and second stabilizers for the second microdroplet component.

[0060] Thus, in a preferred embodiment, the cluster composition comprises a first component comprising perfluorinated gas microbubbles stabilized with a first stabilizer selected from the group consisting of polymers, proteins, phospholipids, and surfactants. In a more preferred embodiment, the cluster composition comprises a second component comprising halogenated oil microdroplets stabilized with a second stabilizer selected from the group consisting of polymers, proteins, phospholipids, and surfactants. Thus, in one embodiment, the cluster composition comprises the above combination of a first component and a second component.

[0061] In some embodiments, only the cluster composition without the antimicrobial agent is administered to a subject to prepare the subject for subsequent administration of the antimicrobial agent. In such embodiments, the administration of the cluster composition and application of ultrasound treatment is preparation for treatment, rather than the administration being treatment.

[0062] First component (microbubbles) The first component comprises gas microbubbles containing a gas ("dispersed gas") and a first stabilizer for stabilizing the gas. The microbubbles may be similar to conventional ultrasound contrast agents commercially available and approved for use in some clinical applications, such as Sonazoid, Optison, Definity, or Sonovue, or similar agents used in preclinical applications, such as Micromarker and Polyson L. The first component may be in the form of an injectable aqueous dispersion or a lyophilized powder for reconstitution. Any biocompatible gas may be present in the microbubbles, and 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. Therefore, the gas may be, for example, air; nitrogen; oxygen; carbon dioxide; hydrogen; inert gases such as helium, argon, xenon or krypton; sulfur fluorides such as sulfur hexafluoride, disulfur decafluoride or trifluoromethyl sulfur pentafluoride; selenium hexafluoride; optionally halogenated silanes such as methylsilane or dimethylsilane; low molecular weight hydrocarbons (for example, containing up to 7 carbon atoms), for example, alkanes such as methane, ethane, propane, butane or pentane, cycloalkanes such as cyclopropane, cyclobutane or cyclopentane, alkenes such as ethylene, propene, propadiene or butene, or alkynes such as acetylene or propylene; ethers such as dimethyl ether; ketones; esters; halogenated low molecular weight hydrocarbons (for example, containing up to 7 carbon atoms); or any mixture thereof. Advantageously, the gas is a halogenated gas, for example, a perfluorinated gas.Advantageously, at least some of the halogen atoms in the halogenated gas are fluorine atoms, and thus the biocompatible halogenated hydrocarbon gas may be selected from, for example, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, fluoroethane, 1,1-difluoroethane, and perfluorocarbons. Representative perfluorocarbons include perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (e.g., perfluoro-n-butane, optionally in admixture with other isomers such as perfluoroisobutane), perfluoropentane, perfluorohexane, or perfluoroheptane; 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 perfluorocycloalkanes such as perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylcyclobutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluorocyclohexane, perfluoromethylcyclohexane, or perfluorocycloheptane. Other halogenated gases include methyl chloride, fluorinated (eg, perfluorinated) ketones such as perfluoroacetone, and fluorinated (eg, perfluorinated) ethers such as perfluorodiethyl ether.

[0063] 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 observed in the bloodstream of microbubbles containing such gases. Other gases with physicochemical properties that allow for the formation of highly stable microbubbles in the bloodstream may be similarly useful. Preferably, the dispersion gas comprises sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane (i.e., a C3-6 perfluorocarbon), nitrogen, air, or any mixture thereof, preferably sulfur hexafluoride or a C3-6 perfluorocarbon. In certain embodiments, the first component gas is selected from the group consisting of sulfur fluorides and halogenated low-molecular-weight hydrocarbons (e.g., containing up to 7 carbon atoms). In some embodiments, the dispersion gas comprises sulfur hexafluoride, perfluoropropane, or perfluorobutane, or any mixture thereof. In certain embodiments, the dispersion gas is perfluorobutane.

[0064] The dispersed gas may be in any convenient form using any suitable gas-containing ultrasound contrast agent formulation, for example, as a gas-containing component such as Sonazoid, Optison, Sonovue, or Definity, or as a preclinical agent such as Micromarker or PolySon L. The first component may also include a material for stabilizing 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 a first stabilizer, such as an aggregation-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, microparticle biodegradable polyaldehydes, microparticulated N-dicarboxylic acid derivatives of polyamino acid-polycyclic imides), a non-polymeric and non-polymeric wall-forming material, or a surfactant (e.g., a polyoxyethylene-polyoxypropylene block copolymer surfactant, such as Pluronic, a polymeric surfactant, or a film-forming surfactant, such as a phospholipid). Preferably, the dispersed gas is in the form of phospholipid-, protein-, or polymer-stabilized gas microbubbles. Thus, in one embodiment, the first stabilizer is selected from the group consisting of phospholipids, proteins, and polymers. Particularly useful surfactants include phospholipids containing molecules with an overall net negative charge, such as naturally occurring (e.g., from soybeans 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 supplemented with a negative surfactant such as a fatty acid, e.g., phosphatidylcholine plus palmitic acid, or may be a mixture of phospholipids with different charges, e.g., phosphatidylethanolamine and / or phosphatidylcholine and / or phosphatidic acid and / or phosphatidylserine.For the first stabilizer, i.e., to stabilize the microbubbles of the first component, different examples are shown in WO 2015 / 047103, Example 5, and Tables 9 and 10, where various microbubble formulations with different excipients are tested. The results show that the ACT concept used in the present invention is applicable to various microbubble formulations, also in terms of the composition of the stabilizing membrane. This will also be further demonstrated as suggested in Example 6 of the present disclosure.

[0065] The microbubble size of dispersed gas components intended for intravenous injection should preferably be less than 7 μm, more preferably less than 5 μm, and most preferably less than 3 μm or less to allow smooth passage through the pulmonary system, even within microbubble / microdroplet clusters.

[0066] Second component (Microdroplet) The second component comprises microdroplets containing an oil phase and a second stabilizer for stabilizing the microdroplets, the oil comprising a diffusible component. The diffusible component can undergo a phase shift upon US irradiation of the microbubbles / microdroplet clusters and / or diffuse and / or combine into the gas microbubbles of the first component to at least transiently increase their size. In the second component, the "diffusible component" is preferably a gas / vapor, volatile liquid, volatile solid, or precursor thereof that can generate gas, e.g., upon administration. The main requirement is that the component can generate or has the ability to generate sufficient gas or vapor pressure in vivo (e.g., at least 50 torr, and preferably greater than 100 torr) to facilitate the internal diffusion of gas or vapor molecules into the diffused gas. The "diffusible component" is preferably formulated as an emulsion of fine droplets (i.e., a stabilized suspension) in a suitable aqueous medium, because in such a system the vapor pressure in the aqueous phase of the diffusible component will be substantially the same as the vapor pressure of the pure component material, even in a very dilute emulsion.

[0067] The diffusible component in such microdroplets is advantageously liquid at processing and storage temperatures, which may be as low as -10°C if the aqueous phase contains a suitable antifreeze material, while at body temperature it is gaseous or exhibits a substantial vapor pressure. Suitable compounds may be selected from various lists of emulsifiable low-boiling liquids, for example, as shown in WO 94 / 16379 or WO 2015 / 047103, the contents of which are incorporated herein by reference. Specific examples of emulsifiable diffusible components include, for example, aliphatic ethers such as diethyl ether; polycyclic oils or alcohols such as menthol, camphor, or eucalyptol; heterocyclic compounds such as furan or dioxane; saturated or unsaturated and linear or branched, for example, n-butane, n-pentane, 2-methylpropane, 2-methylbutane, 2,2-dimethylpropane, 2,2-dimethylbutane, 2,3-dimethylbutane, 1-butene, 2-butene, Aliphatic hydrocarbons such as 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-yne, 1-butene, 2-butene, or 1,3-butanedione; cycloaliphatic hydrocarbons such as cyclobutane, cyclobutene, methylcyclopropane, or cyclopentane; and halogenated low-molecular-weight hydrocarbons containing, for example, up to seven 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 (e.g. pentafluoropropane, such as 1H,1H,3H-pentafluoropropane), fluoropropane, hexafluorobutane, nonafluorobutanes such as 2H-nonafluoro t-butane, decafluoropentanes such as 2H,3H-decafluoropentane), partially fluorinated alkenes (e.g., heptafluoropentenes such as 1H,1H,2H-heptafluoropent-1-ene, nonafluorohexenes such as 1H,1H,2H-nonafluorohexene), fluorinated ethers (e.g., 2,2,3,3,3-pentafluoropropyl methyl ether or 2,2,3,3,3-pentafluoropropyl difluoromethyl ether), 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; perfluorodiakenes such as perfluorobutene (e.g., perfluorobut-2-ene or perfluorobut-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.

[0068] Particularly useful in the present invention are 1 to 10 -4 Less than M, more preferably 1 to 10 -5 The diffusible component is a component having 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 significant portion of the mixture may contain compounds with higher water solubility. Based on water solubility, examples of suitable oils (diffusible components) are the following: perfluorodimethylcyclobutane, perfluoromethylcyclopentane, 2-(trifluoromethyl)perfluoropentane, and perfluorohexane.

[0069] It will be appreciated that mixtures of two or more diffusible components may be used in accordance with the present invention, if desired, and references herein to "diffusible component" shall be interpreted as including such mixtures.

[0070] The second component also includes a material for stabilizing the microdroplet dispersion, referred to herein 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 such as a phospholipid, a polymer, or a protein. The nature of such a material can significantly 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, for example, in EP 0 727 225 A1, the contents of which are incorporated herein by reference. Representative examples of useful surfactants (stabilizers) include fatty acids (e.g., straight-chain saturated or unsaturated fatty acids, e.g., containing 10 to 20 carbon atoms) and their carbohydrate and triglyceride esters, phospholipids (e.g., lecithin), fluorinated phospholipids, proteins (e.g., albumins such as human serum albumin), polymers such as polyethylene glycol and block copolymer surfactants (e.g., polyoxyethylene-polyoxypropylene block copolymers such as Pluronics, extended polymers such as 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 trade 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 that contain molecules with a neutral overall charge, such as distearoyl-sn-glycerol-phosphocholine (DSPC).As the second component, various stabilizers can be used to stabilize microdroplets.In addition, a wide range of ionic, preferably cationic, substances can be used to promote appropriate surface charge.

[0071] It will be understood that to promote electrostatic attraction interactions between the microbubbles of the first component and the emulsion microdroplets of the second component to achieve clustering, 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 microdroplets, a cationic surfactant may be added to the stabilizing structure. A wide range of cationic substances can be used, including compounds that are at least somewhat hydrophobic and / or substantially water-insoluble and have 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 to which a cationic surfactant has been added, such as a DSPC membrane containing stearylamine.

[0072] In one embodiment, the first stabilizer and the second stabilizer each independently comprise a phospholipid, a protein, a polymer, polyethylene glycol, a fatty acid, a positively charged surfactant, a negatively charged surfactant, or a mixture thereof.

[0073] In one embodiment, the first component comprises a dispersing gas selected from the group consisting of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, nitrogen, and air, or a mixture thereof, stabilized by a first stabilizer selected from the group consisting of phospholipids, proteins, and polymers, and the second component comprises a diffusible component selected from the group consisting of perfluorocarbons, such as perfluorocycloalkanes, stabilized by a second stabilizer selected from the group consisting of surfactants, polymers, and proteins. More particularly, the first stabilizer comprises a phospholipid, a protein, or a polymer optionally containing a negatively charged surfactant, and the second stabilizer comprises a phospholipid, a protein, or a polymer optionally containing a positively charged surfactant.

[0074] In one embodiment, the first component comprises a dispersing gas selected from the group of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, or mixtures thereof, stabilized with a first stabilizer 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 stabilizer selected from the group of surfactants, such as phospholipids, polymers, and proteins. More particularly, one of the stabilizers is selected from phospholipids.

[0075] In some embodiments, the cluster composition comprises a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1-10 μm and a circularity of <0.9, and have the following properties: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via attractive electrostatic interactions; The first and second stabilizers are both selected from the list including, but not limited to, aggregation-resistant surface films, film-forming proteins, polymeric materials, non-polymeric and non-polymeric wall-forming materials, surfactants, and preferably selected from proteins, polymers and phospholipids, and may be the same or different.

[0076] In some embodiments, the cluster composition comprises a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1-10 μm and a circularity of <0.9, and have the following properties: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via attractive electrostatic interactions; The first and second stabilizers are both surfactants.

[0077] The cluster composition comprises a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and have the following properties: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via attractive electrostatic interactions; the first and second stabilizers are both selected from the list including, but not limited to, aggregation-resistant surface films, film-forming proteins, polymeric materials, non-polymeric and non-polymeric wall-forming materials, surfactants, and preferably selected from proteins, polymers and phospholipids, and may be the same or different; The diffusible component is an emulsifiable low boiling liquid having an aqueous solubility of less than 1-10 −4 M, such as less than 1-10 −5 M.

[0078] In some embodiments, the cluster composition comprises a suspension of clusters in an aqueous biocompatible medium, wherein the clusters have an average diameter in the range of 1-10 μm and a circularity of <0.9, and have the following properties: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via attractive electrostatic interactions; the first and second stabilizers are both surfactants; The diffusive component is 1 to 10 -4 Less than M, e.g., 1 to 10 -5 It is an emulsifiable low-boiling liquid with an aqueous solubility of less than M.

[0079] The ACT concept for the delivery of antimicrobial agents or the treatment of infection, i.e., the compositions and methods for use of the present invention, is envisioned as a concept applicable to a wide range of combinations of components (first and second) and to a wide range of antimicrobial agents. Thus, any of the components listed for the first component, including a gas and a first stabilizer, can be combined with the components listed for the second component, including a diffusible component and a second stabilizer. In summary, in one embodiment, a two-component formulation system for the preparation of microbubble / microdroplet cluster compositions for use in accordance with the methods of the present invention comprises the following: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles, wherein the gas of the gas microbubbles is selected from the group of halogenated gases, preferably perfluorinated gases, and most preferably perfluorobutane; and the first stabilizer is selected from the group of phospholipids, proteins, and polymers optionally containing a negatively charged surfactant, and more preferably phospholipids, and most preferably hydrogenated egg phosphatidylserine sodium (HEPS-Na); (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size, the oil being selected from the group of aliphatic ethers, heterocyclic compounds, aliphatic hydrocarbons, halogenated low molecular weight hydrocarbons and perfluorocarbons, preferably perfluorocarbons, and most preferably perfluoromethyl-cyclopentane (pFMCP); and the second stabilizer is selected from the group of phospholipids, proteins, and polymers optionally containing a negatively charged surfactant, more preferably a phospholipid containing a positively charged surfactant, and most preferably 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC)-containing stearylamine (SA), The microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters through attractive electrostatic interactions.

[0080] The first and second components of the two-component formulation system are combined immediately prior to intended use to prepare a microbubble / microdroplet cluster composition and for use within an appropriate timeframe according to the methods of the present invention. Thus, in one embodiment of the present invention, the method includes preparing the microbubble / microdroplet cluster composition prior to the administration step. In a preferred embodiment, the microbubble / microdroplet cluster composition is prepared by reconstituting the first component (microbubbles) in dry powder form with the second component (microdroplets) in fluid form. It will be appreciated that mixing the first and second components can be accomplished in a variety of ways known to those skilled in the art, 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 prior to reconstitution with a fluid (e.g., water for injection or buffer), etc. It will also be understood that other components can affect the ability of microbubbles and microdroplets to form clusters upon mixing, including, but not limited to, the level of surface charge on the microbubbles / microdroplets, the concentration of microbubbles / microdroplets in the two components, the size of the microbubbles / microdroplets, the composition and concentration of ions, the composition and concentration of excipients (e.g., buffers or tonicity components), etc. (see WO 2015 / 047103, Example 1). Such component and composition characteristics can also affect the size and stability (both in vitro and in vivo) of the resulting clusters and can be important factors influencing biological attributes (e.g., efficacy and safety profile). It should also be understood that not all of the microbubbles / microdroplets in a cluster composition will be present in a clustered form, and a significant portion of the microbubbles and / or microdroplets may exist in a free (unclustered) form along with the microbubble / microdroplet cluster populations. Additionally, the manner in which the two components are mixed can affect the following aspects, including, 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.

[0081] The microdroplet size of the dispersed diffusible component in emulsions intended for intravenous injection is preferably less than 7 μm, more preferably less than 5 μm, and most preferably less than 4 μm, to facilitate unimpeded passage through the pulmonary system, and greater than 0.5 μm, more preferably greater than 1 μm, and most preferably greater than 2 μm, while retaining sufficient volume for activated bubble retention in the microvasculature. In a preferred embodiment, the average diameter of the microdroplets in the second component is 2-4 μm. Growth of the dispersed gas phase in vivo can occur, for example, simultaneously with the expansion of any encapsulating material (if this material 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 stretching of the encapsulating material and / or its interaction with ultrasound can substantially increase its porosity. While such collapse of the encapsulating material has often been found to result in a rapid loss of echogenicity due to outward diffusion and dissolution of the exposed gas, the inventors have found that the exposed gas exhibits inherent stability when using the compositions of the present invention. While not wishing to be bound by theoretical calculations, the inventors surmise that exposed gas, e.g., in the form of released microbubbles, may be stabilized, e.g., against microbubble collapse, by a supersaturated environment created by diffusible components that provide an inward pressure gradient to counteract the outward diffusion tendency of the microbubble gas. The substantial absence of encapsulating material may cause the exposed gas surface to exhibit highly favorable acoustic properties, as manifested by high backscatter and low energy absorption at typical imaging frequencies (e.g., as represented by a high backscatter:attenuation ratio), and this reverberation effect may persist for significant periods, even during continuous ultrasound exposure.

[0082] Ultrasonic Procedures - Activation and Enhancement Processes It will be appreciated that the microbubbles of the first component and the larger bubbles formed after activation of the clusters are different in size and therefore respond differently to an applied US field. Thus, when using a cluster composition in a method of treatment or delivery, the US fields applied in steps iii) (the activation step) and iv) (the enhancement step) need to be different and carefully selected to promote increased extravasation of the co-administered antimicrobial agent.

[0083] Activation process The microbubbles of the first component are typically 2-3 μm in diameter, and their acoustic resonance is in the diagnostic frequency range (1-10 MHz). When the cluster composition is administered to a subject, activation of the clusters is easily achieved using standard diagnostic ultrasound imaging pulses, such as those used in conventional medical ultrasound abdominal and cardiac applications, with a mid- to low-range mechanical index (MI) of less than 1.9, e.g., less than 0.7, and preferably less than 0.4, e.g., less than 0.3, but greater than 0.1. Such irradiation (activation irradiation) vaporizes the oil in the microdroplets, forming large (10 μm or larger diameter) oil droplets. Because the median diameter of microcapillaries in the human body is approximately 7 μm, the activated bubbles are temporarily trapped within the microvessels of the area of ​​interest (i.e., the site of infection) due to their size. After activation, the gas within the large bubbles begins to diffuse into the bloodstream, and the bubbles slowly shrink until they are small enough to move and become free-flowing after approximately 5-10 minutes. Activation under medical ultrasound imaging control allows for spatially targeted activation of the clusters in the tissue region illuminated by the ultrasound field, i.e., the region of interest, i.e., the site of infection. This method therefore allows for spatially targeted extravasation of the co-administered antimicrobial agent specifically at the site of infection.

[0084] In a preferred embodiment, the US field applied during the activation step has a first frequency in the range of 1-10 MHz and an MI less than 1.9, preferably less than 0.7, most preferably less than 0.4.

[0085] In one embodiment, activation, i.e., US irradiation at the first frequency, begins immediately after each administration of the cluster composition, e.g., within 20 seconds, and continues for, e.g., 60-120 seconds. In one embodiment, imaging, if performed, is performed before and during injection of the cluster composition, and thereafter, an activation clock is started when influx of contrast agent is observed.

[0086] Optional Imaging Step The clusters are not activated at low MI (below a cluster activation threshold of about 0.1), which allows standard medical ultrasound contrast imaging to be performed, for example, to identify associated pathologies without cluster activation. Therefore, in one embodiment, the method includes imaging using a low MI contrast imaging mode (MI<0.15, e.g., MI<0.1) to image the microbubble components, i.e., dispersed gas, without activating the clusters to identify pathological areas (sites of infection) for treatment. Thus, because the clusters are not activated at low MI (below the activation threshold), standard medical ultrasound contrast imaging can be performed before the activation step, for example, to identify microvascular pathologies, e.g., target tissues.

[0087] enhancement process After activation, the generated large phase-shifted bubbles, due to their larger size than microcapillaries, are temporarily trapped within the microvasculature of the irradiated region of interest, i.e., the site of infection. The resulting large phase-shifted bubbles are approximately 1,000 times the volume of the evaporated emulsion microdroplets (a 20 μm bubble diameter formed from a 2 μm diameter oil microdroplet). The scattering cross-section of these large phase-shifted bubbles is several orders of magnitude larger than that of the micron-sized microbubbles contained 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 the basic imaging modes of diagnostic imaging systems. The mechanical resonant frequency of the large phase-shifted bubbles is also an order of magnitude lower than that of the microbubbles contained in the clusters before activation: approximately 3-5 MHz versus 0.5 MHz. Application of an acoustic field commensurate with the resonant frequency of the large phase-shifted bubbles generates a relatively large range of vibrations in MIs within the scope of medical diagnosis. Applicant investigated the attributes of the US field applied during the second irradiation step (enhancement step) and its effect on the functionality of the applied procedure. Surprisingly, contrary to the teachings of WO 2015 / 047103, which discloses a preferred frequency range between 0.2 and 1 MHz and suggests an MI of less than 0.4, the applicant has found that the functionality of the concept is highly sensitive to these parameters. Based on these studies, the applicant has found that the preferred frequency range is 0.4 to 0.6 MHz, and that the applied MI should be kept greater than 0.1 but less than 0.3. When using a lower frequency and a higher MI during the enhancement step (iv), the applicant has surprisingly found that the induced activated bubble vibrations are too strong, leading to a significant loss of efficacy and vascular damage. On the other hand, when the frequency is high and the MI is low, the induced bubble vibrations are too weak, leading to a lack of sufficient biomechanical effect and, therefore, a significant loss of therapeutic efficacy.Thus, while low-frequency ultrasound in the range of 0.05-2 MHz, e.g., 0.1-1.5 MHz, or e.g., 0.2-1 MHz, can be used, a preferred embodiment employs frequencies of 0.4-0.6 MHz combined with an MI between 0.1-0.3 to generate a bioeffect mechanism that enhances the uptake of the administered drug and thus facilitates extravasation. In one embodiment, the frequency of irradiation in the enhancement step is lower than that of irradiation in the activation step. Exploiting the resonance effect of activated bubbles allows for better control of the initiation of these bioeffects at lower acoustic intensities and frequencies than possible with other techniques. The fact that large phase-shifted bubbles are activated under image control (enabling spatial targeting of large activated bubbles within tissue) and deposited within the microvasculature within tissue, coupled with their extended residence time, allows for more efficient and controlled implementation of the drug delivery mechanism.

[0088] Further application of ultrasound after activation therefore effectively promotes extravasation through an opened biological barrier and enhances the therapeutic effect of the antimicrobial agent delivered to the site of infection.

[0089] Irradiation with low-frequency ultrasound follows the activation step and typically lasts for 3 to 10 minutes, e.g., about 5 minutes. Preferably, step (iii) is followed immediately by step (iv). Dual-frequency transducers can be beneficially used in treatment for both the activation and enhancement steps. By using such transducers, switching from activation irradiation in step (iii) to enhancement in step (iv) can be performed without any delay. Applying an enhancement field immediately after activation can be important for the resulting therapeutic effect. In this regard, it would be beneficial to apply irradiation for both activation and enhancement using a broadband or dual-frequency US transducer. That is, a transducer capable of providing sufficient US pressure (i.e., MI) across all frequencies required by the preferred ranges mentioned. For example, a transducer capable of providing MI of up to 0.4 at both 1-10 MHz and 0.1-1 MHz, more preferably at 0.4-0.6 MHz.

[0090] The dual action concept for drug delivery to the site of infection and treatment of infection, i.e., compositions for use in the present invention, is envisioned to be applicable to a wide range of combinations of first and second components, and to a variety of infections and antibacterial agents.

[0091] In the systems or methods of the present invention, or in pharmaceutical compositions for use, an antimicrobial agent can be loaded into the microdroplets of the second component for release at a target site in vivo upon activation. Example 6 of WO 2015 / 047103 provides the results of a fluorescence microscopy study on activated bubbles loaded with Nile Red fluorescent dye. It is demonstrated that after activation, the loaded substance is homogeneously expressed on the surface of the activated bubbles and thus in close contact with the endothelial wall, making it accessible for extravasation. Examples 8 and 5-4 of the same publication elucidate the concept for achieving such loading. Both examples are incorporated herein by reference.

[0092] Therapeutic agents for infectious diseases and treatment The infectious disease treated using the compositions or methods of the present invention may be subclinical or silent, without clinically evident infection, or may be clinically evident. The infection may be latent. The infection may be caused by a primary pathogen or an opportunistic pathogen. In some embodiments, the infection is a primary infection. In other embodiments, the infection is a secondary infection. The infection may be a mixed infection, an ectopic infection, a nosocomial infection, and / or a community-acquired infection.

[0093] In some embodiments, the infection results from an invasive medical procedure, such as, for example, a surgical incision, a catheter, an IV drip, a subcutaneous injection, a blood sample and / or a biopsy site.

[0094] In some embodiments, the infection is selected from the group including, but not limited to, common cellulitis, ear infection, eye infection, sinusitis, food poisoning, skin infection, furuncles, folliculitis, scalded skin syndrome, common wound infection, necrotizing fasciitis, lung infection, pneumonia, toxic shock syndrome, actinomycosis, nocardiosis, meningitis, and sepsis.

[0095] Some vaccines require multiple doses to generate a strong immune response. In some embodiments, ACT treatments according to the present invention can enhance this immune response and reduce the number of vaccinations required. In some embodiments, the infectious disease treated using a composition or method according to the present invention is a bacterial infection. The infection can be caused by one or more Gram-positive and / or Gram-negative bacteria. In certain embodiments, the infection is caused by one or more bacteria from the list including, but not limited to, Staphylococcus, Staphylococcus aureus, Hemophilus, Hemophilus influenzae, Pseudomonas, Pseudomonas aeruginosa, Streptococcus, Streptococcus pneumoniae, Streptococcus Group A, Group B, Group C, Group D, Group G, Mycobacterium, Mycobacterium tuberculosis, Clostridium, and Enterobacterium eriaceae. In certain embodiments, the infection is caused by an antibiotic-resistant strain of bacteria.

[0096] The pharmacology of antibacterial drug therapy can be divided into two distinct components. The first of these components is pharmacokinetics (PK), which examines how a drug is handled in the body, including absorption, distribution, metabolism, and excretion. The second component is pharmacodynamics (PD), which examines the interrelationship between drug PK, in vitro potency (usually minimum inhibitory concentration [MIC]), and therapeutic outcome (usually efficacy, or sometimes drug toxicity). The time course of antibacterial activity reflects the interrelationship between PK and PD. The PK / PD relationship allows for the translation of microbiological activity into clinical practice and is essential for ensuring that antibacterial agents achieve successful outcomes. Many studies have shown that antibacterial agents can be broadly divided into two classes: those that exhibit concentration-dependent killing and long-lasting effects (e.g., aminoglycosides, fluoroquinolones, etc.), for which the peak plasma concentration relative to the minimum inhibitory concentration (MIC) of the infectious organism (Cmax / MIC) is the primary PK / PD parameter most correlated with efficacy; and those that exhibit time-dependent killing and minimal to moderately persistent effects (e.g., β-lactam and macrolide classes), for which the time that the drug concentration exceeds the MIC (expressed as a percentage of the dosing interval), i.e., %T>MIC, is the primary parameter determining efficacy. The differences between these two classes of antibacterial agents are illustrated in Figure 1.

[0097] In Example 2, it is clearly shown that application of the ACT procedure according to the present invention significantly increases the concentration of co-administered drugs in target tissue compartments. Study 1 of Example 2 demonstrates a 100% increase in the uptake of drug-mimetic molecules, particularly in tissues targeted by US irradiation. Furthermore, Study 2 of Example 2 demonstrates that ACT significantly increased the blood-brain barrier (BBB) ​​permeability of large nanoparticulate drug constructs when the two-step irradiation approach of the present invention was applied. The blood-brain barrier (BBB) ​​represents the body's most robust vascular barrier. Unless compromised, it is completely closed to therapeutic agents larger than approximately 4-500 daltons, prevailing in drug therapy for most diseases and disorders of the central nervous system (CNS). ACT resulted in a greater than 290-470% increase in extravasation into the brain parenchyma localized in the irradiation region of interest. Thus, ACT enables the delivery of large and small drug constructs across even the body's most robust vascular barrier (BBB). These two studies therefore demonstrate the ability of ACT to locally increase the concentration of co-administered therapeutic agents in target tissue compartments and indirectly demonstrate beyond reasonable doubt that this concept enhances the therapeutic efficacy of antibacterial agents. Based on PK / PD characteristics, combining ACT with Cmax-dependent drugs may be particularly beneficial, since a key feature of the ACT procedure is increasing the tissue concentration of the co-administered drug. However, drugs in the %T > MIC class may also benefit from ACT, as ACT can also improve drug distribution in target tissues, i.e., increasing drug penetration and increasing the chance of reaching pathogenic microorganisms. Furthermore, for drugs with %T > MIC, achieving higher concentrations may be important when resistance increases the MIC, such as in multidrug-resistant (MDR) Gram-negative bacterial infections, such as prostate cancer.In this case, even though prolonged infusion is usually indicated, the treatment regimen typically begins with a "loading dose" to rapidly achieve plasma concentrations higher than the MIC in infected tissues [N.J. Rhodes et al., "ImpactACT of Loading Doses on the Time to Adequate Predicted Beta-LACTam Concentrations in Prolonged and Continuous Infusion Dosing Schemes," Clinical Infectious Diseases, Volume 59, Issue 6, September 15, 2014, Pages 905-907]. In this case, i.e., in combination with a loading dose, ACT can be a highly clinically valuable intervention. For example, with a loading dose of meropenem, infusion can last up to 30 minutes, making it ideal for simultaneous ACT procedures. Finally, after the ACT procedure, increased permeability of the vascular barrier can persist for some time, i.e., more than 1 hour or even 2-3 hours. In this case, the time during which tissue concentrations exceed the MIC can be significantly extended, and the therapeutic efficacy of T%>MIC drugs can be significantly improved.

[0098] Therefore, because the therapeutic efficacy of all antibacterial agents is, to some extent, dose / concentration dependent relative to the agent alone, combination with ACT is likely to lead to improved outcomes with both classes of agents in the treatment of any localized / localized infection. The therapeutic efficacy of levofloxacin treatment in combination with ACT for staphylococcal infections in mice is clearly demonstrated in Example 3 and will be demonstrated in prospective Examples 4 and 5.

[0099] Antibacterial agents and drug classes useful under the present invention include, but are not limited to, anti-infectives; amebicides; aminoglycosides; anthelmintics; anthelmintics such as antiprotozoal agents, ectoparasitic agents; antifungals such as azole antifungals, echinocandins, polyenes; antimalarials such as antimalarial combinations, antimalarial quinolines; antituberculous agents such as aminosalicylates, antituberculous combinations, diarylquinolines, hydrazide derivatives, nicotinic acid derivatives, rifamycin derivatives, streptomyces derivatives; adamantane antivirals, antiviral boosters, antiviral combinations, antiviral interferons, chemokine receptor antagonists, integrase strand transfer inhibitors, neuraminidase inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nonstructural protein 5A (NS5A) inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors, purine These include antivirals such as cephalosporins; carbapenem antibiotics; carbapenem / β-lactamase inhibitors; cephalosporins such as cephalosporins / β-lactamase inhibitors, first-generation cephalosporins, fourth-generation cephalosporins, next-generation cephalosporins, second-generation cephalosporins, and third-generation cephalosporins; glycopeptide antibiotics; glycylcyclines; leprostatin antibiotics; lincomycin derivatives; macrolide derivatives such as ketolides and macrolides; various antibiotics; oxazolidinone antibiotics; penicillins such as aminopenicillins, antibacterial penicillins, β-lactamase inhibitors, natural penicillins, and penicinase-resistant penicillins; quinolones; streptogramins; sulfonamides; tetracyclines; urinary tract anti-infectives; and new classes of agents under development, such as immunotherapeutics, e.g., checkpoint inhibitors.

[0100] In a preferred embodiment, the antibacterial agent used in combination with the ACT, i.e., in the methods of the present invention, is selected from a Cmax-dependent class of drugs.

[0101] In another preferred embodiment, the antibacterial agent used in combination with the ACT is selected from the class of %T>MIC dependent.

[0102] In one embodiment, the antimicrobial agent is an antibiotic, in another embodiment, the antimicrobial agent is an antifungal agent, in yet another embodiment, the antimicrobial agent is an antiviral agent, and in yet another embodiment, the microbial agent is an antiparasitic agent.

[0103] Antibacterial agents used in the treatment of bacterial infections are antibiotics. Antibiotics that may be used in the compositions or methods according to the invention may have any mechanism of action known to those skilled in the art. Non-limiting examples include agents that act on bacterial cell walls, such as bacitracin, cephalosporins, cycloserine, fosfomycin, penicillin, ristocetin, and vancomycin; agents that affect or have an ablative effect on cell membranes, such as colistin, novobiocin, and polymyxins; agents that affect the cellular machinery of replication, signaling, and protein synthesis by acting on the ribosome, e.g., aminoglycosides, tetracyclines, chloramphenicol, clindamycin, cycloheximide, fucidin, lincomycin, puromycin, rifampicin, other streptomycins, and macrolide antibiotics such as erythromycin and oleandomycin; agents that affect nucleic acid metabolism, e.g., fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, griseofulvin, rifamycin; and agents that affect intermediary metabolism, such as sulfonamides, trimethoprim, and antituberculous drugs such as isoniazid and paraaminosalicylic acid. Antibiotics can be broad-spectrum or "narrow-spectrum." These antibiotics can have one or more primary mechanisms of action. They can be used separately or in combination with other agents, such as in combination with other antibacterial agents.

[0104] In some embodiments, the antibacterial agent is an antibiotic selected from the list including amoxicillin, ceftriaxone, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, levofloxacin, sulfamethoxazole and trimethoprim, amoxicillin and clavulanic acid, levofloxacin.

[0105] In some embodiments, the antibiotic is amoxicillin / clavulanate, amoxil, Augmentin, azithromycin, azithromycin Dose Pack, Bactrim, Bactrim DS, ceftriaxone, cefuroxime, Cipro, Cleocin, Flagyl, Keflex, Levaquin, levofloxacin, penicillin VK, sulfamethoxazole / trimethoprim, vancomycin, Zithromax, Rocephin, Avelox, Ceftin, minocycline, Vibramycin, Doxi 100, moxifloxacin, penicillin V potassium, Septra, Zyvox, Apoamoxi, cilastatin / imipenem, cefazolin, Dorix, Dorix MPC, gentamicin, Monodox, Molgidox, Oraxyl, Septra DS, Cipro IV, Cipro XR, Cleocin HCl, Flagyl. 375, Flagyl IV, Linezolid, Tobramycin, Amocran, Ampicillin, Augmentin XR, Chloramphenicol, Cleocinpedia, Cleocin phosphate, Cotrimoxazole, Minocin, Tetracycline, Vancocin, Vancocin HCl, Dinacef, Achromycin V, Acticite, Aratet, Azactam, Bicillin LA, Brodospec, Chloromycetin, Dynacin, Garamycin, Lincocin, Minocin for injection, Sulfatrimpediacal, Tobi, Unastin, Vancocin HCl Pervil, Ximino, Ampicillin / Sulbactam, Avelux IV, Aztreonam, Bactocil, Cefotetan, Cefoxitin, Chloromycetin sodium succinate, Declomycin, Demeclocycline, Lincomycin, Nafcillin, Oxacillin, Penicillin g Select from a list including benzathine, penicillin g potassium, penicillin G procaine, penicillin g sodium, pfizerpen, primaxin iv, procaine penicillin, sivextro, tedizolid, tobi podhar.

[0106] In some embodiments, the antibiotic is amoxicillin / clavulanate, amoxil, Augmentin, azithromycin, bactrim, flagyl, keflex, levaquin, levofloxacin, penicillin VK, sulfamethoxazole / trimethoprim, vancomycin, zithromax, rocephin, avelox, ceftin, minocycline, vibramycin, moxifloxacin, penicillin V potassium, septra, zyvox, apoamoxi, cilastatin / imipenem, cefazolin, doliquis, gentamicin, monodox, molgidox, oraxyl, septra DS, cipro IV, cipro XR, cleocin HCl, flagyl. 375, linezolid, tobramycin, amocran, ampicillin, chloramphenicol, cleocin phosphate, cotrimoxazole, minocin, tetracycline. Vancocin, dinacef, achromycin V, acticyte, dynasyn, garamycin, lincocin, unasyn, vancocin hydrochloride, palbrez, ximino, ampicillin-sulbactam, avelox IV, aztreonam, bactocil, cefoxitin, cefotetan, cefoxitin, lincomycin, nafcillin, oxacillin, penicillin g benzathine, penicillin g potassium, penicillin g procaine, penicillin g sodium, procaine penicillin, sibextro, tedizolid.

[0107] In some embodiments, the antibiotic is selected from the list including Augmentin, Azithromycin, Cefuroxime, Flagyl, Flagyl ER, Amoxil, Cipro, Keflex, Bactrim, Bactrim DS, Levaquin, Zithromax, Avelox, Cleocin, Vancomycin, Rocephin.

[0108] In some embodiments, the infection treated using a composition or method according to the invention is a fungal infection. The infection may be from one or more of the Ascomycota, which includes yeasts such as Candida, filamentous fungi such as Aspergillus, Pneumocystis species, and dermatophytes, a group of organisms that cause infections of the skin and other superficial structures in humans, and the Basidiomycota, which includes the human pathogenic genus Cryptococcus.

[0109] Although bacteria and fungi are different phylogenetically distant groups, a list of similarities should also be taken into consideration. Both fungi and bacteria have cell walls. Most bacteria and all fungi obtain their energy through aerobic respiration. Both groups have cell membranes composed of phospholipids. Some yeasts and most bacteria reproduce by binary fission. Certain bacteria and fungi have the ability to produce antibiotics. Many species in both groups are pathogens of humans, animals, and plants. Both groups have the ability to survive harsh environmental conditions by producing specialized thick-walled spores, although they differ in their formation and structure. The antibacterial agent used to treat fungal infections is an antifungal agent. The antifungal agent that can be used in the composition or method according to the present invention can have any mechanism of action known to those skilled in the art. They can be used separately or in combination with other antibacterial agents.

[0110] In some embodiments, the antifungal agent is selected from the list including, but not limited to, polyenes, imidazoles, azoles such as triazoles and thiazoles, arylamines, echinocandins.

[0111] In some embodiments, the antifungal is selected from the list including clotrimazole, econazole, miconazole, terbinafine, fluconazole, ketoconazole, amphotericin, gallium nitrate.

[0112] In one embodiment, the antifungal agent is the drug Ganite, gallium nitrate. Although gallium has no natural function in biology, gallium ions interact with cellular processes in a manner similar to iron(III). When gallium ions are mistakenly taken up by bacteria such as Pseudomonas instead of iron(III), the ions interfere with respiration and the bacteria die. This occurs because iron is redox active, allowing the transfer of electrons during respiration, whereas gallium is not redox active.

[0113] In some embodiments, the infectious disease treated using the compositions or methods according to the present invention is a viral infection. The antibacterial agent used to treat a viral infection is antiviral. The antiviral agent that can be used in the compositions or methods according to the present invention can have any mechanism of action known to those skilled in the art. They can be used individually or in combination with other antibacterial agents.

[0114] In some embodiments, the antiviral agent is idoxuridine, trifluridine, brivudine, vidarabine, entecavir, telbivudine, foscarnet, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, lamivudine + zidovudine, abacavir, abacavir + lamivudine + zidovudine, emcitabine, nevirapine, delavirdine, efavirenz, etravirine, rilpivirine , saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir-ritonavir, atazanavir, fosamprenavir, tipranavir, darunavir, darunavir + cobicistat, atazanavir + cobicistat, traprevir, boceprevir, simeprevir, asunaprevir, vaniprevir + ribavirin + PegIFNα-2b, paritaprevir, grazoprevir, raltegravir, Elvitegravir, dolutegravir, dolutegravir + abacavir + lamivudine, dolutegravir + lamivudine, RSV-IGIV, palivizumab docosanol, enfuvirtide, maraviroc, VZIG, valizig, acyclovir, ganciclovir, famciclovir, valacyclovir, penciclovir, valganciclovir, cidofovir, tenofovir disoproxil fumarate, adefovir dipivoxil tenofovir disoproxil fumarate + emtricitapine, tenofovir disoproxil fumarate + efavirenz + emtricitabine, tenofovir disoproxil fumarate + rilpivirine + emtricitabine, tenofovir disoproxil fumarate + cobicistat + emtricitabine + elvitegravir, tenofovir alafenamide + cobicistat + emtricitabine + elvitegravir. Tenofovir alafenamide + rilpivirine + emtricitabine, tenofovir alafenamide + emtricitabine, sofosbuvir + ribavirin, sofosbuvir + ribavirin + PegIFNα, daclatasvir + asunaprevir, ledipasvir + sofosbuvir, sofosbuvir + simeprevir, ombitasvir + dasabuvir + paritaprevir + ritonavir, ombitasvir + paritaprevir + ritonavir, daclatasvir + sofosbuvir, elbasvir + grazoprevir, amantadine, ribavirin, rimantadine, zanamivir, oseltamivir.The drug is selected from a list including laninamivir octanoate, peramivir, favipiravir, peginterferon alfa-2b, interferon alfacon-1, peginterferon alfa-2b plus ribavirin, peginterferon alfa-2a, fomivirsen, podofilox, imiquimod, and sinecatequinz.

[0115] In some embodiments, the antiviral agent is selected from the group consisting of idoxuridine, trifluridine, brivudine, vidarabine, entecavir, telbivudine, foscarnet, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emcitabine, nevirapine, delavirdine, efavirenz, etravirine, rilpivirine, saquinavir-ritonavir, indinavir, nelfinavir, amprenavir, lopinavir-ritonavir, atazanavir, fosamprenavir, and tipuravir. The drug may be selected from the list including: navir, darunavir, telaprevir, boceprevir, simeprevir, asunaprevir, paritaprevir, grazoprevir, raltegravir, elvitegravir, dolutegravir, RSV-IGIV, palivizumab, docosanol, enfuvirtide, maraviroc, VZIG, valizig, acyclovir, ganciclovir, famciclovir, valacyclovir, penciclovir, valganciclovir, cidofovir, tenofovir disoproxil fumarate, adefovir dipivoxil, amantadine, ribavirin, rimantadine, zanamivir, oseltamivir, laninamivir octanoate, peramivir, favipiravir, peginterferon alfa-2b, interferon alfacon-1, peginterferon alfa-2a, fomivirsen, podofilox, imiquimod, and sinecatechins.

[0116] In some embodiments, the antiviral agent is selected from the list including idoxuridine, trifluridine, brivudine, didanosine, zalcitabine, emtricitabine, nevirapine, delavirdine, efavirenz, etravirine, rilpivirine, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir-ritonavir, atazanavir fosamprenavir, tipranavir, darunavir, telaprevir, boceprevir, simeprevir, asunaprevir, paritaprevir, grazoprevir, RSV-IGIV, palivizumab, rimantadine, zanamivir, oseltamivir, laninamivir octanoate, peramivir, and favipiravir.

[0117] In some embodiments, the infectious disease treated using the compositions or methods according to the present invention is a parasitic infection. The parasite causing the infection may be a single-celled microorganism (protozoa) and / or a multicellular organism with an organ system (helminth). The antimicrobial agent used to treat the parasitic infection is an anthelmintic. The anthelmintic that can be used in the compositions or methods according to the present invention may have any mechanism of action known to those skilled in the art. They can be used separately or in combination with other antimicrobial agents.

[0118] In some embodiments, the infection treated using a composition or method according to the invention is a parasitic infection.

[0119] There is a need for treatments against parasites that can cross the blood-brain barrier (BBB) ​​and invade the central nervous system (CNS). Common anthelmintics do not cross the BBB and are therefore ineffective when parasites cross the BBB. Therefore, there is a great need for anthelmintics that can facilitate crossing the BBB. As demonstrated by Example 5, ACT can deliver even large drug molecules and constructs across the BBB and into the brain parenchyma. Therefore, in a preferred embodiment, the compositions and methods of the present invention are for the treatment of infectious diseases of the CNS.

[0120] Taenia solium, also known as the pig tapeworm, is known to cause epileptic seizures and other neurological disorders in humans through the ingestion of eggs containing infectious larvae. Decomposition of the eggshell occurs in the intestine, allowing the larvae to escape and enter the bloodstream. Once in the bloodstream, the larvae can colonize various body tissues. The larvae can cross the BBB and invade the central nervous system, where the embryos develop into fluid-filled cysts, causing a condition known as neurocysticercosis, one of the most dangerous parasitic central nervous system infections in the world. Diagnosis of neurocysticercosis is difficult due to the lack of specific clinical symptoms. Niclosamide is the drug of choice for the treatment of T. saginata and Taenia solium (pig tapeworm) infections, with a cure rate of approximately 90%. It is non-toxic because it is not absorbed. Alternative treatments for cestodeworm infection have varying degrees of safety.

[0121] Naegleria fowleri (commonly known as the brain-eating amoeba) is a single-celled, free-living parasite that thrives in warm waters. This parasite can cause meningoencephalitis, a rare brain infection that can lead to severe encephalitis. This amoeba can also cause many other neurological symptoms in the host and has a near-100% mortality rate. When amoeba-containing water enters the nose, the parasite can travel via the olfactory nerve, which detects odor molecules and transmits signals to the brain. This parasite has been detected in South America and Asia, but cases have also been reported in Australia, the United States, and the United Kingdom. Naegleria fowleri infection is diagnosed based on microscopic examination of central nervous system fluid, where active amoebas can be detected. Perhaps the most widely accepted drug for the treatment of N. fowleri infection is amphotericin B, which has been studied in vitro and has been used in several case reports. Other anti-infectives used in case reports include fluconazole, miconazole, miltefosine, azithromycin, rifampin, etc. In some embodiments, the anthelmintic drug is selected from the list including chloroquine, quinine, mefloquine, primaquine, Fancidar (sulfadoxine and / or pyrimethamine), doxycycline, atovaquone-proguanil, artemether-lumefantrine.

[0122] In some embodiments, the anthelmintic agent is albendazole, amphotericin B, artemether-lumefantrine, artesunate, atovaquone, atovaquone / proguanil, azithromycin, benznidazole, bithionol, chloroquine phosphate, ciprofloxacin, clarithromycin, clarithromycin, crotamiton, dapsone, dapsone plus trimethoprim, dapsone plus atovaquone, dapsone plus clindamycin, dapsone plus pentamidine, dapsone plus primaquine, dapsone plus pyrimethamine, diethylcarbamazine, diloxane furoate, doxycycline, eflortine, eflortine plus nifurtimox, fluconazole, flucytosine, fumagrin, fumagrin plus albendazole, furazolidone, iodoquinol, ibex The drug may be selected from the list including lumectin, liposomal amphotericin B, malathion, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metronidazole, miltefosine, niclosamide, nifurtimox, nifurtimox / eflornithine, nitazoxanide, oxamniquine, paromomycin, pentamidine, permethrin, praziquantel, prednisone, primaquine, pyrantel, piperonyl butoxide-containing pyrethrins, pyrimethamine, quinacrine, quinidine gluconate, quinine, quinine dihydrochloride, quinine sulfate, rifampin, sodium stibogluconate, spinosad, sulfadiazine, suramin, tetracycline, tinidazole, triclabendazole, trimethoprim / sulfamethoxazole.

[0123] In some embodiments, the anthelmintic drug is selected from the list consisting of amphotericin B, azithromycin, benznidazole, bithionol, chloroquine phosphate, ciprofloxacin, clarithromycin, clindamycin, diethylcarbamazine, diloxanide furoate, doxycycline, and eflortine, or from the list including fluconazole, flucytosine, fumagillin, furazolidone, iodoquinol, ivermectin, liposomal amphotericin B, malathion, mebendazole, mefloquine, meglumine antimoniate, niclosamide, nifurtimox, nifurtimox / eflonithine, prednisone, primaquine, pyrantel, pyrimethamine, and quinine.

[0124] The infections treated using the compositions or methods according to the invention may be in tissues and / or organs in any part of the subject's body.

[0125] In one embodiment, the infection is bacterial meningitis, such as an infection caused by any one or more of Neisseria meningitidis, Haemophilus influenzae, Escherichia coli, Streptococcus agalactiae, Streptococcus pneumoniae, or Listeria monocytogenes.

[0126] In one embodiment, the infection is otitis media, such as acute otitis media, such as otitis media with effusion, such as pneumococcal infection.

[0127] In one embodiment, the infection is an eye infection, for example an infection caused by one or more of Staphylococcus spp., Streptococcus spp.

[0128] In one embodiment, the infection is a sinusitis, such as acute sinusitis, chronic sinusitis, etc., for example an infection by one or more of Haemophilus influenzae, Moroxella catarrhalis, Streptococcus pneumoniae.

[0129] In one embodiment, the infection is an upper respiratory tract infection, for example, an infection caused by one or more of Haemophilus influenzae, Streptococcus pyogenes.

[0130] In one embodiment, the infection is pneumonia, for example, an infection caused by one or more of Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter eriaceae, Haemophilus influenzae, Streptococcus pneumoniae.

[0131] In one embodiment, the infection is a skin infection, for example an infection caused by one or more of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes.

[0132] In one embodiment, the infection is gastritis, such as infection with Helicobacter pylori.

[0133] In one embodiment, the infection is food poisoning, for example an infection caused by one or more of Campylobacter, Escherichia coli, Salmonella enterica, Shigella, Staphylococcus aureus, or Listeria.

[0134] In one embodiment, the infection is a urinary tract infection, such as an infection with one or more of Escherichia coli, Klebsiella, Pseudomonas aeruginosa, Proteus, or Enterococcus.

[0135] In one embodiment, the infection is a sexually transmitted disease, for example, an infection caused by one or more of Chlamydia trachomatis, Neisseria gonorrhoeae, and Haemophilus ducreyi.

[0136] In certain embodiments, the infection is one or more of an infection in the central nervous system; aspergilloma; acute bacterial cholangitis; catheter-associated and / or complicated UTI.

[0137] The penetration and distribution of antimicrobial agents into tissues depends on various factors, including drug properties such as molecular weight, protein binding, lipid solubility, and degree of ionization; target tissue properties such as tissue membrane function and angiogenesis; and the presence or absence of inflammation. Those skilled in the art will appreciate the need to select an infection that is insufficiently exposed to the antimicrobial agent and is sufficiently localized to benefit from ultrasound targeting.

[0138] Furthermore, providing localized treatment may reduce the stress on the immune system that can be caused by immune responses resulting from the treatment of systemic disease.

[0139] In some embodiments, the pharmaceutical compositions or methods or systems of the present invention provide an increased therapeutic effect compared to the use of an antimicrobial agent alone, hi certain embodiments, the increased therapeutic effect is in the form of one or more of the following: improved uptake of the antimicrobial agent, reduced microbial density / number, reduced volume / area of ​​the infected area, improved quality of life, complete eradication of the infection, improved overall survival, improved median survival, or improved progression-free survival.

[0140] In some embodiments, the infection treated using the compositions or methods of the present invention is associated with organ transplantation. Solid organ transplantation is an effective life-saving measure for thousands of patients worldwide with organ dysfunction syndrome. In 2008, more than 29,000 solid organ transplant procedures were performed in the United States alone. Despite significant advances in surgical techniques and immunosuppressive regimens, significant risks for post-transplant infection remain, with invasive fungal infections (IFI) being one of the most significant. The most commonly reported IFIs in organ transplant patients are invasive candidiasis, cryptococcosis, and invasive mold infections such as aspergillosis and zygomycosis. The incidence of IFI varies in frequency and specific etiology depending on the type of organ transplant procedure and transplant center. A deep understanding of the overall burden of IFI in this population is generally lacking. Transplanted organs include the heart, pancreas, kidney, liver, lung, bone, and cornea.

[0141] Pseudomonas infections are the most common post-transplant infection. Pseudomonas is a genus of Gram-negative gammaproteobacteria belonging to the family Pseudomonadaceae and includes 191 validated species. Members of this genus exhibit great metabolic diversity and can inhabit a variety of niches. Their ease of in vitro culture and the increasing number of genome sequences of Pseudomonas species have made this genus a center of great interest for scientific research. The most well-studied species are P. aeruginosa in its role as an opportunistic human pathogen, P. syringae as a plant pathogen, P. putida as a soil bacterium, and P. fluorescens, P. lini, P. migulae, and P. graminis as plant growth-promoting bacteria. Most Pseudomonas species are naturally resistant to penicillin and most related beta-lactam antibiotics, but a number are sensitive to piperacillin, imipenem, ticarcillin, and ciprofloxacin. Aminoglycosides such as tobramycin, gentamicin, and amikacin are other options for treatment.

[0142] In one embodiment, the infection treated using the composition or method according to the present invention is a prosthetic joint implant infection. Infections caused by Gram-positive and Gram-negative pathogens associated with foreign implants or intravascular catheters are very difficult to control with antimicrobial therapy. Removal of the implanted device is often unavoidable, and standard clinical treatment has been adopted. The ability of pathogens to adhere to materials and promote biofilm formation is the most important characteristic of their virulence.

[0143] In one embodiment, the infection treated using the composition or method according to the present invention is osteomyelitis, such as infection caused by one or more of S. aureus, Pseudomonas, and Enterobacteriaceae. Although bone is normally resistant to bacterial colonization, events such as trauma, surgery, the presence of a foreign body, or the placement of a prosthetic joint can disrupt bone integrity and lead to the development of bone infection. Osteomyelitis can also occur as a result of hematogenous spread after bacteremia. When an artificial joint becomes infected, microorganisms usually grow in a biofilm. Conventional treatment for osteomyelitis involves antimicrobial treatment combined with a surgical approach. The treatment process is prolonged due to poor penetration of the agent into the lesion and the frequent association with biofilm formation on implanted metal instruments.

[0144] The ACT treatment of the present invention has clear potential benefits in overcoming underexposure / pure penetration and addressing the biofilm problem. Advantageously, the infection is localized. ACT is: Reduced need for artificial implant removal; Shorter treatment duration; causing an early switch to oral therapy; and Shorter hospital stay It has potential advantages in that it is cost-effective.

[0145] In one embodiment, the infection treated using the compositions or methods of the present invention is bacterial endocarditis, such as infection with one or more of Staphylococci and Streptococci. Endocarditis is an infection of the endocardium, the inner lining of the ventricles and heart valves. Endocarditis is a new manifestation of an old problem. While previously associated with rheumatic heart disease in the pre-antibiotic and early eras, prosthetic valve replacement, hemodialysis, intravenous catheters, and immunosuppression are now major risk factors. The average patient is older and frail, with increased comorbidities and a high in-hospital mortality rate (10-20%). Long-term intravenous (IV) antibiotic therapy is recommended. Bacterial endocarditis represents a significant burden to patients and hospitals, with a median length of hospital stay of 43 days (France) and average hospital costs per patient exceeding $120,000 (US). Despite trends toward earlier diagnosis and surgical intervention, 1-year mortality rates have not improved for over 20 years.

[0146] Poor penetration into the clot may be improved by ACT. Vascularization exists regardless of inflammation, and anatomic-pathological studies have dispelled the myth of avascularity of the heart valves. ACT is reduction in prolonged hospital stays; Shortening long treatment periods; causing an early switch to oral therapy; and Ultimately, improved survival may be affected by

[0147] In one embodiment, the infection treated using the compositions or methods of the present invention is acute bacterial prostatitis. Prostatitis is a common urological disease in adult men, usually caused by the same bacteria that cause urinary tract infections. As many as 50% of men will experience an episode of prostatitis in their lifetime, and 2% to 3% of men will suffer from bacterial prostatitis. Prostate biopsies (more than 1 million per year in Europe) are a common cause. While prevention is the standard of care, a high percentage of patients still develop infections after biopsy. The prostate's acidic environment and lipid epithelium lead to low antibiotic exposure and a long treatment period, typically six weeks.

[0148] In a preferred embodiment, the infectious diseases that can be treated using the compositions or methods according to the present invention are selected from the group consisting of endocarditis, prosthetic joint infection (PJI) (or other foreign body infection), osteomyelitis, prostatitis, ventriculitis, brain abscess, aspergilloma, or acute bacterial cholangitis. In each disease, depending on the type of infection (i.e., the type of microorganism and its level of resistance to antibacterial agents), the present invention should be combined with the current standard of care antibacterial agents.

[0149] In a preferred embodiment, for the treatment of endocarditis, the one or more antibacterial agents are selected from the list of penicillin, penicillin G, ceftriaxone, vancomycin, gentamicin, nafcillin, oxacillin, ampicillin, streptomycin, sulbactam, and rifampin.

[0150] In a preferred embodiment, for the treatment of PJI, the one or more antibacterial agents are selected from the list of rifampin, nafcillin, cefazolin, geftriaxone, vancomycin, penicillin G, ampicillin, cefepime, meropenem, ertapenem, beta-lactams, ciprofloxacin, and ceftriaxone.

[0151] In a preferred embodiment, for the treatment of osteomyelitis, the one or more antibacterial agents are selected from the list of: dalbavantin, clindamycin, rifampin, clindamycin, bactrim, linezolid, quinolone, fluconazole, doxycycline, cephalosporin, trimethoprim-sulfamethoxazole, levofloxacin, moxifloxacin, linezolid, minocycline, metronidazole-clindamycin, penicillin, penicillin G, nafcillin, ampicillin, ampicillin-sulbactam, and cefazolin.

[0152] subject The subject to be treated may be a human or non-human mammalian subject. The subject may be male or female. In some embodiments, the subject is an adult (i.e., 18 years of age or older). In some embodiments, the subject is geriatric. In some embodiments, the subject is not geriatric. In still other embodiments, the subject is a child (i.e., under 18 years of age). In some embodiments, the subject also has another condition, such as a tumor. In other embodiments, the subject does not have a tumor. In some embodiments, the subject is an immunosuppressed individual. In some embodiments, the subject has undergone an organ transplant. In some embodiments, the subject is a trauma patient, a burn patient, a patient taking organ transplant medication, a diabetic patient, or an AIDS patient.

[0153] Administration route The cluster composition is preferably administered to the mammalian subject parenterally, preferably intravenously. The administration route can be selected from intraarterial, intramuscular, intraperitoneal, intratumoral, or subcutaneous administration. The antibacterial agent can be administered before, co-administered with, and / or after the cluster composition, or as a separate composition. In one embodiment, it can be loaded into the microdroplets of the cluster composition, although the practical implementation of this has yet to be resolved. The antibacterial agent is administered by a route appropriate for the type of agent and the formulation in which it is provided. Typically, the route is selected from the group including, but not limited to, oral, intravenous (IV), intramuscular (IM), intrathecal, subcutaneous (SC), sublingual, buccal, rectal, vaginal, ocular, auricular, nasal, inhalation, nebulization, dermal, and transdermal administration. Thus, the two compositions, ie, cluster composition (a) and antimicrobial composition (b), are administered via the same route of administration or via different routes of administration.

[0154] procedure The present invention can be used as a first line treatment, or a second line treatment, or any type of treatment.

[0155] It will be understood that the compositions for use, methods for treatment, and / or systems or methods for drug delivery of the present invention may be employed, for example, as part of a multi-drug treatment regime. In one embodiment, the pharmaceutical composition for use according to the present invention includes the use of one or more antibacterial agents. Furthermore, in one embodiment, several ACT treatments may be performed during the administration period of the antibacterial agent.

[0156] Thus, in one embodiment, two or more antimicrobial agents, such as 1 to 5 antimicrobial 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 to 5, is performed during the same period. In one embodiment, the following ACT procedure is provided: intravenous administration of a cluster composition followed by three consecutive local ultrasound (US) irradiations (activations) of the infected site either immediately before or after administration of the antimicrobial agents.

[0157] Several therapeutic agents may be used, and several ACT procedures may be applied during the treatment regimen. In one embodiment, the ACT treatment is performed when the active therapeutic molecule exhibits a maximum or near-maximum concentration in the blood after administration. Therefore, the timing of the ACT treatment(s) may vary depending on the pharmacokinetics of the antibacterial agent.

[0158] It will also be appreciated that the cluster composition may be used to prepare a subject for subsequent treatment with an antimicrobial agent.

[0159] The dosage of an antimicrobial agent is crucial in the treatment of serious infections, where rapid efficacy is essential. The higher the dose of antimicrobial agent that can be delivered to the site of infection, the greater the effect. In some embodiments, ACT is given close to the time when the antimicrobial agent reaches its maximum serum concentration in a particular compartment of the body (i.e., the site of infection) after administration of the agent.

[0160] The antimicrobial agent(s) are administered before, with, and / or after the cluster composition. In a preferred embodiment, the antimicrobial agent is administered after the administration of at least one of the cluster compositions. Performing ACT treatment before the administration of the antimicrobial agent, i.e., administering the cluster composition followed by a two-step US procedure, can achieve similar effect sizes as if the ACT treatment were initiated after the antimicrobial agent is administered (i.e., when the antimicrobial agent is in the bloodstream). This can be clinically beneficial because the ACT treatment can be performed before the administration of the therapeutic agent and treatment begins. Thus, in one embodiment, the cluster composition is administered and activated in vivo, and then the antimicrobial agent is administered. In another embodiment, the cluster composition is administered either immediately before or immediately after the administration of the antimicrobial agent(s).

[0161] Thus, in one embodiment, the present invention provides a pharmaceutical composition for use in a method of delivering an antibacterial agent, wherein the method comprises: (i) administering to a mammalian subject suffering from an infection a pharmaceutical composition as defined in the first aspect, wherein at least one antibacterial agent is administered before and / or together with and / or after the cluster composition and before steps ii)-iii) or after any of steps ii)-iii); (ii) optionally imaging the clusters of pharmaceutical compositions using ultrasound imaging to identify areas of interest for treatment within the subject; (iii) activating a phase shift of a diffusible component of the second component of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject, the step comprising: (a) the microbubbles of the cluster are expanded by the diffusive component of step (iii) to provide expanded bubbles that are localized in the region of interest due to the expanded bubbles temporarily blocking microcirculation in the region of interest; and (b) promoting extravasation of the antimicrobial agent administered in step (i); and (iv) promoting further extravasation of the antimicrobial agent administered in step (i) by further ultrasonic irradiation. Includes.

[0162] Accordingly, the present invention provides a microbubble / microdroplet cluster composition as described above for use in a method of delivering an antimicrobial agent to a subject suffering from an infection, the method comprising: (i) administering to the subject at least one antimicrobial agent selected from the group consisting of an antibiotic, an antifungal, an antiviral, an antiparasitic, or a combination thereof; (ii) administering to said subject a microbubble / microdroplet cluster composition, wherein at least one antimicrobial agent is administered before, and / or simultaneously with, and / or after the cluster composition; (iii) activating a phase shift of the diffusible component of the second component of the cluster composition from step (ii) by ultrasonic irradiation of a region of interest in the subject; (iv) promoting further extravasation of the antimicrobial agent administered in step (i) by further ultrasonic irradiation. Includes.

[0163] The duration of treatment can be guided by the severity and location of the infection and the clinical and microbiological course of the subject. Treatment can be performed as frequently and as many times as necessary depending on the treatment regimen. The ultrasound procedure, steps (iii) and (iv), is carried out as disclosed above.

[0164] The present invention further provides a method for delivering at least one antimicrobial agent to a mammalian subject, comprising: (i) administering to a mammalian subject a pharmaceutical composition defined in the first aspect; (ii) optionally imaging the microbubbles of the pharmaceutical composition using ultrasound imaging to identify areas of interest for treatment within the subject; (iii) activating a phase shift of a diffusible component of the second component of the cluster composition from step (i) by ultrasonic irradiation of a region of interest within the subject, the step comprising: (a) the microbubbles of the cluster are expanded by the diffusive component of step (iii) to provide expanded bubbles that are localized in the region of interest due to the expanded bubbles temporarily blocking microcirculation in the region of interest; and (b) said activating step (iii) promotes extravasation of the antimicrobial agent administered in step (i); and (iv) promoting further extravasation of the antimicrobial agent administered in step (i) by further ultrasonic irradiation. Includes.

[0165] As provided for the first aspect, the mammalian subject may be, for example, a subject suffering from an infectious disease.

[0166] In a further aspect, the present invention provides a method of treating an infection in a mammalian subject, the method comprising administering to the subject a pharmaceutical composition comprising: (a) A cluster composition comprising a suspension of clusters in an aqueous biocompatible medium, said clusters having an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and comprising: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, a cluster composition, wherein the microbubbles and microdroplets of the first component and the second component have opposite surface charges and form the clusters via attractive electrostatic interactions; and (b) an antimicrobial agent selected from the group of antibiotics, antifungals, antivirals, antiparasitics, or combinations thereof, provided as a separate composition from (a).

[0167] In yet another aspect, the present invention relates to the use of a cluster composition for preparing a subject for subsequent treatment with an antimicrobial agent, said cluster composition comprising a suspension of clusters in an aqueous biocompatible medium, said clusters having an average diameter in the range of 1 to 10 μm and a circularity of <0.9, and having the following: (i) a first component comprising gas microbubbles and a first stabilizer for stabilizing the microbubbles; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil comprises a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size; Including, A cluster composition, wherein the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via electrostatic attraction interactions. Such uses include the ultrasound procedures, steps (iii) and (iv) disclosed above.

[0168] Embodiments and features described in the context of one aspect, for example, an aspect relating to a composition for use, also apply to other aspects of the invention.

[0169] The present invention is not limited to the embodiments and examples shown. While various embodiments of the present disclosure have been described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications and changes, as well as variations and substitutions to the embodiments described herein, will be apparent to those skilled in the art without departing from the scope of the present invention. It will be understood that various alternatives to the embodiments described herein can be employed in implementing the present disclosure. Furthermore, it is intended that the appended claims cover such modifications and variations that fall within the true scope of the present invention.

[0170] It should be understood that any embodiment of the present disclosure may be optionally combined with any one or more of the other embodiments described herein.

[0171] It will be understood that each component, compound, or parameter disclosed herein is intended to be disclosed for use alone or in combination with one or more of the other components, compounds, or parameters disclosed herein. Furthermore, each amount / value or amount / value range of each component, compound, or parameter disclosed herein is also disclosed in combination with each amount / value or amount / value range disclosed for any other component(s), compound(s), or parameter(s) described herein, and any combination of amounts / values ​​or amount / value ranges for two or more components, compounds, or parameters disclosed herein is therefore also intended to be disclosed in combination with each other for purposes of this specification. Any feature described herein and combinations of such features are within the scope of the present invention, provided that the features are not mutually inconsistent.

[0172] It is understood that the lower limit of each range disclosed herein is interpreted as being disclosed in combination with the upper limit of each range disclosed herein for the same component, compound, or parameter. Thus, a disclosure of two ranges is interpreted as a disclosure of four ranges obtained by combining the lower limit with the upper limit of each range. For example, a disclosure of three ranges is interpreted as a disclosure of nine ranges by combining the lower and upper limits of each range. Furthermore, a specific amount / value of a component, compound, or parameter disclosed in the detailed description or examples is interpreted as a disclosure of either the lower or upper limit of a range, and therefore can be combined with other lower or upper limits, ranges, or specific amounts / values ​​of the same component, compound, or parameter disclosed elsewhere in this application to form a range of that component, compound, or parameter. [Example]

[0173] Example 1 Cluster Preparation, Analysis Tools, and Basic Properties WO 2015 / 047103, particularly the Examples therein (the contents of which reference is made are incorporated herein by reference), provides a description of analytical methodologies for characterizing cluster compositions, results from the use of clusters, and the like.

[0174] In the following examples, the first component is designated C1, the second component is designated C2, and the cluster composition, i.e., the composition resulting from the combination of the first and second components, is designated DP (drug product).

[0175] Example 1 provides a description of the analytical methodology for characterizing and quantifying microbubble / microdroplet clusters in DPs, illustrating relevant responses and attributes, including concentration, size, and circularity. It also provides details on analytical techniques for characterizing and quantifying activated bubble size and concentration. Furthermore, data on the stability of clusters after preparation is presented, along with a comparison of the properties of premixes versus co-injected DPs. Furthermore, details are provided on the operational steps for controlling cluster content and size in DPs.

[0176] Example 1 further provides the results of an in vivo study elucidating the influence of cluster characteristics on the product's effectiveness as a function of its ability to deposit large activated bubbles in the microcirculation. Further analysis of these data concludes that clusters with an average size of 3-10 μm, defined by a circularity of less than 0.9, contribute to the effectiveness of the cluster composition.

[0177] The microbubble / microdroplet clusters, designated DP, formed by combining C1 and C2 are critical to a key quality attribute of the composition, namely, its functionality for drug delivery. Therefore, analytical techniques for characterizing and controlling the formed clusters in terms of concentration and size are essential tools for evaluating the present invention and for pharmaceutical quality control (QC). Applicants have identified three different analytical tools that can be applied for this purpose: Coulter counting, flow particle image analysis (FPIA), and microscopy / image analysis.

[0178] In addition to these techniques applied to characterizing clusters in cluster compositions, analytical methodologies have been developed to study in vitro cluster activation, i.e., the generation of large activated bubbles by ultrasonic irradiation. This methodology, "sonometry," is described in detail in WO 2015 / 047103, E1-6. The primary reported responses from sonometry analysis are the number and volume of activated bubbles and their size distribution, both relative to time after activation. The activation reaction can also be examined by microscopy / image analysis, as described in detail in WO 2015 / 047103, E1-5.

[0179] Ingredients and Composition The first component (C1) in the compositions examined in the included 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, thereby ensuring a negative charge on the surface of the microbubbles. Each vial of C1 contains approximately 16 μL or 2-10 microbubbles, with an average diameter of approximately 2.0 μm.

[0180] The second component (C2) in the composition investigated in this example consisted of perfluoromethylcyclopentane (pFMCP) microdroplets stabilized by a 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) membrane with 3% mol / mol 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 has an average diameter of approximately 1.8 μm and contains approximately 4 μL or 0.8-10 microdroplets per mL.

[0181] 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 in the text.

[0182] Cluster Composition (DP) was prepared aseptically by reconstituting a vial of C1 with 2 mL of C2, followed by 30 seconds of manual homogenization. 2 mL was removed from the vial of C2 using a sterile, single-use syringe and needle. The contents of the syringe were added through the stopper of the vial of C1, and the resulting DP was homogenized.

[0183] As shown in WO 2015 / 047103, the first and second components, i.e., the microbubble and microdroplet formulations, can be varied. For example, as shown in Tables 9 and 10 of WO 2015 / 047103, both the gas and stabilizing film of the first component can be varied to prepare clusters with suitable properties expected to be useful in processing according to the present invention.

[0184] Cluster stability in cluster composition during analysis Clusters in DPs are formed and held together by electrostatic attractive forces between microbubbles and microdroplets. These forces are limiting, and clusters can disintegrate after formation through various pathways / influences, such as mechanical stress or thermodynamic (Brownian) motion. For accurate characterization, it is important that the clusters remain stable throughout the analysis period. This stability was investigated using all of the methodologies described above. To assess stability, three to five analyses were performed on a single DP sample over a period longer than 5 minutes. No significant changes in either concentration or size were observed in these replicate samples, indicating that microbubbles, microdroplets, and clusters are stable for periods longer than 5 minutes under the analysis conditions described above, i.e., after dilution with PBS or water and under continuous homogenization (stirring).

[0185] Formulation Many different formulation aspects can be explored to control cluster content and size in the 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 the microbubbles and microdroplets (e.g., SA%), the microdroplet size of C2, pH, the TRIS concentration in C2, and the concentration of the microbubbles and microdroplets. Additionally, chemical degradation of components, such as prolonged storage at elevated temperatures, can affect the ability of C1 and C2 to form clusters during DP preparation.

[0186] As reported in WO 2015 / 047103, in vitro characterization of 15 compositions allowed for the extraction of several important correlations that shed light on the properties and characteristics of the systems. We found that the size of the clusters formed also strongly correlated with the reactivity of the system. At relatively low levels of reactivity (e.g., less than 20%), only small (1-5 μm) and medium-sized (5-10 μm) clusters formed. As reactivity increased, larger clusters began to form: at R > approximately 20%, 10-20 μm clusters began to form, and at R > approximately 50%, 20-40 μm clusters began to form. The formation of larger clusters came at the expense of small and medium-sized clusters. We found clear optima between cluster concentrations of 1-5 μm and 5-10 μm. We found that the formation of larger clusters was detrimental to the efficacy of the composition, and that the likelihood of cluster formation needed to be appropriately balanced.

[0187] Based on the applicant's experiments and the results shown in Tables 5 and 6 of WO 2015 / 047103, the effect of the cluster composition (linear enhancement (GS)) is based at least on the cluster mean size and cluster concentration (million clusters / mL). Results are reported from a multivariate, principal component analysis (PCA) of the contribution of clusters of various size classes to the linear enhancement in ultrasound signals from canine myocardium (grayscale units) upon intravenous administration of the cluster composition and activation within the left ventricle. See Example 2 of WO 2015 / 047103. PCA was performed on data from 30 samples detailed in Tables 5 and 6 therein. The results indicate that small and medium-sized clusters (<10 μm) contribute significantly to the effect of the cluster composition, while larger clusters (>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, preferably 4-9 μm, more preferably 5-7 μm.

[0188] The cluster concentration and average diameter of the cluster composition prepared according to Example 1 was analyzed and found to have a cluster concentration of approximately 40-44 million / mL and an average cluster diameter of approximately 5.8-6.2 μm over several hours. 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 an optimal concentration of cluster diameter can be achieved.

[0189] [Table 1]

[0190] Cluster size influences efficacy. Figure 2 visualizes the relationship between cluster size and product efficacy, demonstrating that clusters with an average diameter in the 3-10 μm range are optimal for cluster composition efficacy. The Y-axis in Figure 2 shows the calculated correlation coefficient, i.e., contribution to myocardial enhancement, for cluster size classes (X variables) 1-5 μm, 5-10 μm, 10-20 μm, and 20-40 μm. Figure 2 is an alternative visualization of Figure 12 (left) in WO 2015 / 047103 and is based on the data provided in Table 2 below.

[0191] [Table 2]

[0192] In contrast to the co-injection of the two components taught in WO / 9953963, applying the concept of the present invention, i.e., preparing a cluster composition from C1 and C2 prior to administration, thus forming microbubble / microdroplet clusters, allows for an increase in efficacy of more than a factor of 10. The formation of microbubble / microdroplet clusters by the combination of the first and second components, and the administration of these pre-prepared clusters, is a prerequisite for the intended functionality in vivo.

[0193] Example 2 ACT-induced, localized delivery of drug-mimetic molecules and nanoparticles Two in-vivo studies were conducted to demonstrate that the ACT concept can enhance the localized delivery of antimicrobial agents such as antibiotics. In the first study (Study 1), tissue-specific delivery of a drug-mimetic chromophore (Evans Blue) was investigated in a mouse model. In the second study (Study 2), localized delivery of drug-mimetic nanoparticles across the blood-brain barrier was investigated in a mouse model.

[0194] Study 1: Tissue-specific delivery of Evans Blue The tissue-specific uptake of Evans Blue (EB, a fluorescent dye) was investigated in a mouse model. Under physiological conditions, the endothelium is impermeable to albumin, and Evans Blue-bound albumin remains confined within the blood vessels. Therefore, Evans Blue is often used as a model compound in drug delivery studies [Bohmer et al., J Controlled Release, 148, Issue 1, 2010, pp.18-24].

[0195] material and method Female Balb / c nude mice were used. Before the procedure, the mice were surgically anesthetized with a subcutaneous injection of a mixture of fentanyl (0.05 mg / kg), midazolam (5 mg / kg), and medetomidine (0.5 mg / kg). An intravenous cannula (BD Neoflon™ 24 GA) was placed in the tail vein. Patency was confirmed by injecting a small amount of 0.9% sodium chloride for injection, followed by a small amount of heparin (10 U / mL) to prevent blood clotting. The cannula hub was filled with 0.9% sodium chloride for injection to eliminate dead space and closed with a cap. The cannula was secured to the tail with surgical tape.

[0196] Evans Blue solution (50 mg EB / kg) was administered iv, immediately followed by 2 mL / kg of the cluster dispersion detailed in Example 1. Two groups (N=3 for each group) were compared: 1) EB + cluster dispersion, and 2) EB + cluster dispersion + ultrasound irradiation for activation and enhancement.

[0197] The left hind limb of the mouse was placed in a water bath equipped with two US transducers for balanced ultrasound exposure of the left thigh. Ultrasound-activated exposure was delivered for 45 seconds (MI = 0.8) from the time of injection using a VScan clinical ultrasound scanner (GE Healthcare) equipped with a 2 MHz probe. This was immediately followed by an enhancement step involving 5 minutes of 500 kHz ultrasound exposure (MI = 0.2) using a single-element transducer (Imasonic SAS). Thirty minutes after treatment, the animals were sacrificed, and tissue samples were collected from the thigh muscle of the treated (left) leg and the thigh muscle of the contralateral untreated leg. Evans Blue content was extracted and quantified. A dimensionless ratio of enhanced uptake in the treated muscle was obtained by dividing the concentration in the treated thigh muscle by the concentration in the untreated thigh muscle of each animal (matched pair). One-way analysis of variance was applied to the data.

[0198] result In animals that did not receive US irradiation, the mean ± SD EB ratio between left (treated) and right (untreated) was 1.1 ± 0.2, which was not significantly different, whereas in animals that received US irradiation, the mean ± SD EB ratio between left (treated) and right (untreated) was 2.0 ± 0.3, indicating that ACT treatment significantly (p < 0.05) increased EB uptake by approximately 100%.

[0199] Study 2 - Localized delivery of drug-mimetic nanoparticles to brain tissue The delivery of drug-mimetic nanoparticle agents to specific sites within the brain parenchyma was investigated in a mouse model. Models that examine the delivery of therapeutic agents across the BBB and into brain tissue are therefore often used as the "ultimate test" when evaluating various drug delivery concepts.

[0200] material and method The ACT cluster compositions investigated were as detailed in Example 1.

[0201] The nanoparticles investigated were core-crosslinked polymeric micelles (CCPMs) from Cristal Therapeutics (Maastricht, The Netherlands). These CCPMs were 70 nm in diameter and labeled with rhodamine B (Cy7) for imaging, and the formulation contained 44 mg / mL of polymer and 40 nmol / mL of Cy7. CCPM extravasation in healthy mouse brains was measured using near-infrared fluorescence (NIRF) imaging, and CCPM microdistribution in brain sections was imaged by confocal laser scanning microscopy (CLSM). Thirteen female albino BL6 mice (Janvier labs, France) purchased at 6–8 weeks of age were housed in groups of five in individually ventilated cages under specific pathogen-free conditions. The cages were enriched with housing, nesting material, and chew sticks and maintained in a controlled environment (20–23°C, 50–60% humidity) with a 12-h day / night cycle. Animals had free access to food and sterile water. All experimental procedures were approved by the Norwegian Food Safety Authority. A schematic diagram of the ultrasound setup is shown in Figure 3. A custom-made dual-frequency transducer (center frequencies 0.5 MHz and 2.7 MHz) [Andersen et al., A Harmonic Dual-Frequency Transducer for Acoustic Cluster Therapy, Ultrasound Med Biol 2019 Sep; 45(9):2381-2390] was mounted on a custom-made cone filled with degassed water. The transducer had a diameter of 42 mm, and the -3 dB widths of the 0.5 MHz and 2.7 MHz beam profiles were 16 mm and 6 mm, respectively, at a distance of 220 mm from the transducer surface. Signals were generated with a signal generator (33500B, Agilent Technologies, USA) and amplified using a 50 dB RF amplifier (2100L, E&I, USA). The amplifier is connected to a switch box, which allows switching from an activating US field to an enhancing US field.The bottom of the cone was covered with an optically and acoustically transparent plastic foil (Jula Norge AS, Norway) to form a pouch, and the animal was positioned prone on an acoustic absorber (Aptflex F28, Precision Acoustics, UK) with ultrasound gel for coupling between the acoustic absorber, the animal's head, and the acoustically transparent foil.

[0202] The ACT procedure used included activation and enhancement steps. Attenuation through the mouse skull was measured to be approximately 21±17% and 42±21% at frequencies of 0.5 MHz and 2.7 MHz, respectively. These values ​​were used to calculate the in situ sound pressure / MI. The following ultrasound parameters were used in each step: Activation: central frequency of 2.7 MHz, average acoustic pressure in situ corresponding to a mechanical index (MI) of 0.18, pulse length of 8 cycles, pulse repetition frequency of 1 kHz, and exposure time of 60 seconds. Enhancement: center frequency of 0.5 MHz, average sound pressure equivalent to 0.15 MI, pulse length of 4 cycles, pulse repetition frequency of 1 kHz, and exposure time of 300 seconds. One round of ACT consisted of a 2 mL / kg intravenous bolus of the cluster composition, followed by 360 seconds of ultrasound. Each animal received three rounds of ACT, totaling 75 μL of ACT formulation and 18 minutes of ultrasound. CCPM was injected intravenously immediately before the first ACT treatment.

[0203] Animals were anesthetized with 2% isoflurane (Baxter, USA) in medical air (78%) and oxygen (20%), followed by cannulation of the lateral tail vein. Hair was removed with a hair trimmer and depilatory cream (Veet, Canada). During ACT, animals were anesthetized with 1.5-2% isoflurane in medical air. Respiratory rate was monitored using a pressure probe (SA instruments, USA), and body temperature was maintained with external heating. Each animal underwent three rounds of ACT immediately after CCPM injection. Control animals were treated identically to the animals receiving ACT, but received three 50 μL saline injections at 6-minute intervals instead of the cluster composition.

[0204] Two time points were examined: 1 hour and 24 hours after the end of ACT treatment. At these time points, animals were euthanized by intraperitoneal injection of pentobarbital (200 μL) and kept under anesthesia until breathing ceased. Animals were then transcardially perfused with 30 mL of PBS, after which the brains were removed and imaged with a NIRF imager. Groups were: control / 1 hour N=3, control / 24 hour N=3, ACT / 1 hour N=5, and ACT / 24 hour N=2.

[0205] The excised brains were placed in a NIRF imager (Pearl Impulse Imager, LI-COR Biosciences Ltd., USA) to assess the amount of CCPM® accumulated in the brain. The brain was excited at 785 nm, and fluorescence emission was detected at 820 nm. Images were analyzed using ImageJ (ImageJ 1.51j, USA). Regions of interest (ROIs) were drawn around the brain, and the total brain fluorescence intensity was acquired and normalized to the brain wet weight. A standard curve was used to convert the total fluorescence intensity to the percentage of injected dose per gram of brain tissue (%ID / g). Results were plotted by time point and treatment group.

[0206] For confocal microscopy, the excised brains were mounted transversely on cork strips using Optimum Cutting Temperature Tissue Tek (Sakura, The Netherlands) and then gently immersed in liquid nitrogen. The first 500 μm of the frozen brain was removed from the top, and then 5 × 10 μm and 5 × 25 μm sections were cut transversely. This process was repeated every 800 μm throughout the brain.

[0207] result To investigate whether the increased permeability facilitated CCPM extravasation, the isolated brains were imaged with a NIRF imager. Representative NIRF images of control and nanoparticle-injected animals are shown in Figure 4 (top). Thus, clear accumulation could be observed in the brains that received ACT, in contrast to the control brains.

[0208] Quantitative analysis of NIRF images showed a statistically significant increase in accumulation (%ID / g) between ACT and control animals at both time points (Figure 4, bottom left). Compared to controls, ACT showed an increase in median %ID / g from 0.9%ID / g to 2.6%ID / g after 1 hour and from 0.8%ID / g to 2.2%ID / g after 24 hours. A 290% and 280% increase in %ID / g, respectively, was observed.

[0209] To confirm the increased accumulation of CCPM in brain tissue after ACT treatment and to examine the location of CCPM relative to blood vessels, brain sections were imaged by CLSM. Tile scans of ACT-treated brains showed several fluorescent "clouds" that were not observed in the brains of control animals. At 24 hours after ACT, tile scans of ACT-treated brains showed a cloud pattern similar to that of the 1-hour treatment group. The number of pixels representing CCPM was extracted from thresholded tiling scans of control and ACT-treated animals and normalized by the size of the ROI used to delineate the hemisphere. As can be seen in Figure 4 (bottom right), a clear and statistically significant 470% increase in CCPM was observed in sections 1 hour after ACT treatment compared to control brains.

[0210] To examine the location of CCPM relative to blood vessels, high-magnification CLSM images were acquired at different locations in both control and ACT-treated brains. In ACT-treated brains, CCPM were clearly extravasated, whereas in control brains, CCPM were mainly observed intravascularly or slightly offset from vascular staining.

[0211] Conclusion of Example 2 ACT induced a 100% increase in the uptake of the drug-mimetic molecule Evans Blue in US-irradiated tissue. Furthermore, ACT significantly increased the BBB permeability of large nanoparticle constructs, such as the 70 nm CCPM compound tested, when applied with our two-step ultrasound irradiation method. Furthermore, ACT increased extravasation into the brain parenchyma by more than 200% localized in the irradiated region of interest. These two studies therefore demonstrate the ability of ACT to locally increase the concentration of co-administered therapeutic agents in target tissue compartments and indirectly suggest that this concept may enhance the therapeutic efficacy of antibacterial agents, particularly Cmax-dependent antibiotics.

[0212] Example 3 Evaluation of ACT in Levofloxacin Treatment of Staphylococcal Thigh Infection in Mice The following study is being conducted to investigate the utility of ACT to enhance the antibacterial effect of levofloxacin (LEV) for the treatment of Staphylococcus aureus thigh infections in mice. LEV is a broad-spectrum, Cmax-dependent antibiotic used to treat a variety of infections, including but not limited to acute bacterial sinusitis, pneumonia, H. pylori, urinary tract infections, chronic prostatitis, and gastroenteritis.

[0213] Experimental Approach An established experimental model of neutropenic thigh infection is used, in which neutropenia is induced using cyclophosphamide. The thigh infection model provides a sensitive experimental system for early studies of antimicrobial efficacy in mammalian systems. This model is the most standardized for the evaluation of antimicrobial-microorganism interactions combined with antimicrobial pharmacokinetics, assessed in serum and tissue samples.

[0214] The challenge strain was a methicillin-susceptible strain of Staphylococcus aureus, inoculated into both thighs. Treatment was initiated 2 hours after inoculation, and the thighs were excised 26 hours later, and bacterial density (colony-forming units, CFU) in the excised tissue was measured. An initial dose-finding study was performed to determine the LEV ED 50 This study was carried out to determine the dose that induces a 50% reduction in bacterial density. LEV was administered intraperitoneally at 20 mg / kg.

[0215] Experimental group To demonstrate that ACT with LEV was significantly superior to LEV alone, three groups were evaluated: 1) vehicle control (saline), 2) LEV alone, and 3) LEV + ACT (see ACT procedure below).

[0216] ACT procedure The cluster composition investigated was as detailed in Example 1. The ultrasound device for application of the ACT sonoporation procedure was as detailed in Example 2 / Study 2, with US focused on the site of infection. The cluster composition was administered intravenously at 2 mL / kg, followed by localized ultrasound (US) irradiation of the infected site using a 45-second US activation field (2.7 MHz, MI 0.3) followed by a 5-minute US enhancement field (500 kHz, MI 0.2). The procedure was performed three times consecutively, starting immediately 10–15 minutes after LEV administration.

[0217] Bacterial density in excised thigh tissues from the various groups was determined by homogenizing excised tissue in sterile PBS, culturing diluted samples on growth medium plates, and counting the number of CFU.

[0218] To build a number with sufficient statistical power, experiments of the same design are conducted multiple times.

[0219] Results (interim report) Figure 5 shows the results from the dose-response of LEV in the model studied. Because the ED50 was estimated to be approximately 24 mg / kg, a dose of 20 mg / kg was considered for the ACT treatment study. Figure 6 shows the interim results for the three groups. Compared to the saline control, a dose of 20 mg / kg of LEV inhibited bacterial growth by 0.8 log units (approximately 6-fold). However, combining ACT sonoporation with the same dose of LEV inhibited bacterial growth by an additional 1.4 log units (approximately 25-fold).

[0220] Although the data numbers are small and therefore the observed differences are not statistically significant, the preliminary findings from this study clearly demonstrate that the ACT procedure of the present invention enhances the therapeutic efficacy of antimicrobial agents in treating infections.

[0221] Example 4 (Prospective) Evaluation of ACT in Daptomycin Treatment of Aphylococcal Thigh Infections in Mice The following study is planned to investigate the utility of ACT in enhancing the antibacterial effect of daptomycin (DPT), a standard Cmax-dependent antibiotic for Gram-positive infections, against staphylococcal thigh infections in mice.

[0222] method Animals (CD-1 mice) were made neutropenic by two intraperitoneal injections of cyclophosphamide hydrate (CPA) on day -4 (150 mg / kg, i.p.) and day -1 (100 mg / kg, i.p.) (day 0 is the day of infection). On the day of infection, animals were infected intramuscularly in the right thigh with 100 μL of a bacterial suspension of Staphylococcus aureus Xen29 (challenge: ~10 6 CFU / thigh). Treatment begins 2 hours post-infection (0 hours). Vehicle and DPT are administered subcutaneously. The dose of DPT is selected as the "first ineffective dose" determined in a dose-ranging study prior to the ACT therapy trial.

[0223] Three treatment groups will be examined (N=6 per group): 1) vehicle control (saline), 2) DPT alone, 3) DPT+ACT.

[0224] Animals are sacrificed 24 hours later and thighs are harvested and processed to obtain bacterial loads.

[0225] ACT procedure The cluster composition investigated is as detailed in Example 1. The ultrasound device for applying the ACT sonoporation procedure is as detailed in Example 2 / Study 2, with US focused on the site of infection. The cluster composition is administered intravenously at 2 mL / kg, followed by localized ultrasound (US) irradiation of the infected site using a 45-second US activation field (2.7 MHz, MI 0.3) followed by a 5-minute US enhancement field (500 kHz, MI 0.2). The procedure is performed three times consecutively, starting immediately 10-15 minutes after LEV administration.

[0226] Results (prospective) The results will show that treatment with DPT alone does not show a significant therapeutic effect over the vehicle control (i.e., no significant difference in cfu), but ACT in combination with DPT shows a marketable and significant therapeutic effect, with a greater than 10-fold reduction in cfu over DPT administration alone.

[0227] Example 5 (Prospective) Evaluation of ACT in Rifampicin Treatment of a Staphylococcal Human Foreign Body Infection Model in Mice The following study is designed to investigate the utility of ACTs in enhancing the antibacterial effect of rifampicin (RIF) for the treatment of Staphylococcus aureus human foreign body infections in mice. RIF is a standard Cmax-dependent antibiotic used to treat several types of bacterial infections, including tuberculosis, Mycobacterium avium complex, leprosy, and Legionnaires' disease.

[0228] method On the day of infection, animals (CD-1 mice) were anesthetized and surgically prepared. A subcutaneous pocket was created in the right flank and a polyethylene catheter (0.5 cm long) was inserted. Immediately after suturing the pocket, 100 μL / implant of Staphylococcus aureus Xen29 bacterial suspension (challenge: ~10 CFU / implant) was injected into the catheter. Treatment began 2 hours (0 h) postinfection. Vehicle and RIF were administered subcutaneously. The RIF dose was selected as the "first ineffective dose" determined in a dose-ranging study prior to the ACT therapy trial. Treatment was administered daily for 4 days.

[0229] Three treatment groups will be examined (N=6 per group): 1) vehicle control (saline), 2) RIF alone, 3) RIF+ACT.

[0230] Animals are sacrificed 5 days post-infection (24 hours after the last treatment) and implants are collected and processed to determine bacterial load (CFU / implant).

[0231] ACT procedure The cluster composition investigated is as detailed in Example 1. The ultrasound device for applying the ACT sonoporation procedure is as detailed in Example 2 / Study 2, with US focused on the site of infection. The cluster composition is administered intravenously at 2 mL / kg, followed by localized ultrasound (US) irradiation of the infected site using a 45-second US activation field (2.7 MHz, MI 0.3) followed by a 5-minute US enhancement field (500 kHz, MI 0.2). The procedure is performed three times consecutively, starting immediately 10-15 minutes after RIF administration.

[0232] Results (prospective) The results will show that treatment with RIF alone does not show a significant therapeutic effect over the vehicle control (i.e., no significant difference in CFU), but ACT in combination with RIF shows a marketable and significant therapeutic effect, reducing CFU by more than 10-fold over administration of RIF alone.

[0233] Example 6 (Prospective) Preparation of Cluster Compositions with Various Microbubble and Microdroplet Components To demonstrate that the present invention is applicable to a variety of chemical compositions of the first and second components (C1 and C2), several formulations are commercially manufactured or procured, and the in vitro attributes of the resulting cluster compositions are examined.

[0234] C1 Example Commercially available microbubble US imaging agents, Sonovue (Bracco Spa, Italy) and Micromarker (VisualSonics Inc, USA), were procured and used as components C1. Sonovue is a sulfur hexafluoride microbubble stabilized with a membrane of distearoylphosphatidylcholine, sodium dipalmitoylphosphatidylglycerol, palmitic acid, and PEG 4000, presented in a lyophilized form for reconstitution with 5 mL of aqueous matrix. Micromarker is a perfluorobutane / nitrogen microbubble stabilized with phospholipids, polyethylene glycol, and fatty acids, presented in a lyophilized form for reconstitution with 0.7 mL of aqueous matrix.

[0235] C2 Examples Diffusible Component: Microdroplet (C2) component containing perfluorodimethyl-cyclobutane, 2-(trifluoromethyl)perfluoropentane, and perfluorohexane can be prepared as follows.

[0236] Weigh 790 mg distearoylphosphatidylcholine (DSPC) and 8.1 mg stearylamine (SA) into a 250 mL round-bottom flask and add 50 mL chloroform. Heat the sample in warm water until a clear solution is obtained. Remove the chloroform by evaporating to dryness on a rotary evaporator at 350 mmHg and 40 °C, then further dry in a desiccator at 50 mmHg overnight. Then, add 160 mL of water, place the flask back on the rotary evaporator, and rehydrate the lipids for 25 minutes at full rotation speed and an 80 °C water bath temperature. Transfer the resulting lipid dispersion to an appropriate vial and store in the refrigerator until use.

[0237] Emulsions were prepared by transferring 1 mL aliquots of the cold lipid dispersion into 2 mL chromatography vials. Six vials were each supplemented with 100 μL of fluorocarbon oil, as detailed above. The chromatography vials were shaken for 75 seconds on a CapMix (Espe, GmbH). The resulting emulsions were washed three times by centrifugation to remove agranulocytes, followed by the addition of an equal volume of aqueous 5 mM TRIS buffer. The vials were immediately chilled on ice, pooled, and stored in the refrigerator until use.

[0238] Coulter counter analysis is performed to measure the volume concentration and diameter of the microdroplets, which are then diluted with 5 mM TRIS buffer to a dispersed phase concentration of 4 μL microdroplets / mL.

[0239] Cluster compositions are prepared by reconstituting Sonovue or Micromarker with 5 or 0.7 mL, respectively, of each of the C2 components listed above.

[0240] Results (prospective) Upon mixing of components C1 and C2, all six combinations are expected to produce greater than 10 million clusters per mL with an average diameter of 3-10 μm. This cluster composition is expected to be useful for delivery of antimicrobial agents and for use in the methods of treating infections according to the present invention.

Claims

1. A pharmaceutical composition for use in a method for the treatment of an infectious disease, comprising: (a) A cluster composition comprising a suspension of clusters in an aqueous biocompatible medium, said clusters having an average diameter in the range of 1-10 μm and a circularity of <0.9, and having the following: (i) a first component comprising gas microbubbles and a first stabilizing agent for stabilizing said microbubbles, the first stabilizing agent being selected from the group of phospholipids, proteins and polymers; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil phase comprises a halogenated low molecular weight hydrocarbon and the second stabilizer is selected from the group of surfactants, polymers and proteins, and the oil phase is a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size. Including, a cluster composition, wherein the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via attractive electrostatic interactions; and (b) an antimicrobial agent selected from the group including antibiotics, antifungals, antivirals, antiparasitics, or combinations thereof, provided as a separate composition from (a) or together with (a); 13. A pharmaceutical composition comprising:

2. 2. The pharmaceutical composition according to claim 1, for the use according to claim 1, wherein the antibacterial agent is an antibiotic or an antifungal agent.

3. 3. The pharmaceutical composition according to claim 1 or 2, for the use according to claim 1, wherein the infection is a bacterial infection or a fungal infection.

4. The pharmaceutical composition according to claim 1 or 2, for the use according to any one of claims 1 to 3, wherein the infection is a localized infection.

5. The pharmaceutical composition according to claim 1 or 2 for the use according to any one of claims 1 to 4, wherein the infection is an infection selected from the group comprising bacterial meningitis, otitis media, eye infection, sinusitis, upper respiratory tract infection, pneumonia, skin infection, gastritis, food poisoning, urinary tract infection, sexually transmitted disease.

6. The pharmaceutical composition according to claim 1 or 2, for the use according to any one of claims 1 to 5, wherein the infection is associated with organ transplantation.

7. The pharmaceutical composition of claim 1 or 2 for use in a method for delivering an antibacterial agent, the method comprising: (i) administering the pharmaceutical composition of claim 1 to a subject suffering from an infectious disease, wherein the at least one antibacterial agent is administered before and / or simultaneously with and / or after the cluster composition and before or after any of the following steps (ii) to (iii): (ii) optionally imaging the clusters of the pharmaceutical composition using ultrasound imaging to identify areas of interest for treatment in 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 region of interest in the subject, (a) the microbubbles of the cluster are expanded by the diffusive component of step (iii) to provide an expanded bubble that is localized in the region of interest due to a temporary blockage of microcirculation in the region of interest by the expanded bubble; and (b) a step performed in such a way as to promote extravasation of the antibacterial agent administered in step (i); (iv) optionally promoting further extravasation of the antimicrobial agent administered in step (i) by further ultrasound irradiation.

13. A pharmaceutical composition comprising:

8. A system for localized delivery of an antimicrobial agent to a target site, comprising: (a) A cluster composition comprising a suspension of clusters in an aqueous biocompatible medium, said clusters having an average diameter in the range of 1-10 μm and a circularity of <0.9, and having the following: (i) a first component comprising gas microbubbles and a first stabilizing agent for stabilizing said microbubbles, the first stabilizing agent being selected from the group of phospholipids, proteins and polymers; and (ii) a second component comprising microdroplets comprising an oil phase and a second stabilizer for stabilizing the microdroplets, wherein the oil phase comprises a halogenated low molecular weight hydrocarbon and the second stabilizer is selected from the group of surfactants, polymers and proteins, and the oil phase is a diffusible component capable of diffusing into the gas microbubbles to at least transiently increase their size. Including, a cluster composition, wherein the microbubbles and microdroplets of the first and second components have opposite surface charges and form the clusters via attractive electrostatic interactions; and (b) an antimicrobial agent selected from the group including antibiotics, antifungals, antivirals, antiparasitics, or combinations thereof, provided as a separate composition from (a) or together with (a); A system including:

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