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WO2025034892A2 - Agent de contraste ultrasonore pour une liaison à un mucus respiratoire ou une adhérence à celui-ci et ses procédés d'utilisation - Google Patents

Agent de contraste ultrasonore pour une liaison à un mucus respiratoire ou une adhérence à celui-ci et ses procédés d'utilisation Download PDF

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Publication number
WO2025034892A2
WO2025034892A2 PCT/US2024/041327 US2024041327W WO2025034892A2 WO 2025034892 A2 WO2025034892 A2 WO 2025034892A2 US 2024041327 W US2024041327 W US 2024041327W WO 2025034892 A2 WO2025034892 A2 WO 2025034892A2
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contrast agent
ultrasound
mucus
subject
agent particles
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WO2025034892A3 (fr
Inventor
Melissa Champlin CAUGHEY
Paul Alexander DAYTON
Kazuya James TSURUTA
Phillip Gregory DURHAM
Jacob Reed MCCALL
Phillip Wayne CLAPP
Andrew WEITZ
David Brooks HILL
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University of North Carolina at Chapel Hill
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University of North Carolina at Chapel Hill
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/48—Diagnostic techniques
    • A61B8/481—Diagnostic techniques involving the use of contrast agents, e.g. microbubbles introduced into the bloodstream
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B8/00—Diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/52—Devices using data or image processing specially adapted for diagnosis using ultrasonic, sonic or infrasonic waves
    • A61B8/5215—Devices using data or image processing specially adapted for diagnosis using ultrasonic, sonic or infrasonic waves involving processing of medical diagnostic data
    • A61B8/5223—Devices using data or image processing specially adapted for diagnosis using ultrasonic, sonic or infrasonic waves involving processing of medical diagnostic data for extracting a diagnostic or physiological parameter from medical diagnostic data
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/007—Pulmonary tract; Aromatherapy
    • A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0078—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a nebulizer such as a jet nebulizer, ultrasonic nebulizer, e.g. in the form of aqueous drug solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings

Definitions

  • the subject matter described herein relates to an ultrasound contrast agent with a mucus targeting ligand and/or a cationic surface charge optimized for mucus adhesion and methods for using the ultrasound contrast agent for respiratory evaluation of a subject, including evaluating mucociliary clearance of the subject and evaluation of pulmonary structures such as mucosal thickness, airway edema and mucus hypersecretion.
  • the subject matter described herein also relates to a method for measuring lung function and structure using ultrasound and contrasting agents targeted to the lung.
  • the subject matter described herein describes administering via inhalation an ultrasound contrast agent optimized for mucus adhesion and then applying ultrasound imaging techniques to evaluate lung structure, pulmonary structures such as mucosal thickness, airway edema and mucus hypersecretion, and quantifying mucociliary clearance of the subject.
  • the subject matter described herein also relates to a method and a system for delivering an acoustically activatable agent to the respiratory tract Attorney Docket No.421/538 PCT of a subject to disrupt mucus in the respiratory tract and/or to deliver a therapeutic agent to the respiratory tract of the subject.
  • BACKGROUND Evaluation of lung function is important in testing efficacy of treatments for respiratory diseases, such as cystic fibrosis.
  • Mucociliary clearance is the mechanism by which the lungs expel particles and foreign pathogens from the airways.
  • mucociliary clearance is evaluated using gamma scintigraphy, which requires the subject to inhale and clear radioactive particles from the lungs while the lungs are imaged to monitor decrease in radioactivity as the gamma particles are expelled. While gamma scintigraphy is effective, repeated exposure of the lungs to ionizing radiation is undesirable. Accordingly, there exists a need for an improved inhalable contrast agent that avoids the need to expose the subject to ionizing radiation.
  • Airway evaluation is also important for risk stratification of burn inhalation injury, the leading cause of all fire-related fatalities.
  • the current method for airway evaluation is bronchoscopy, which is an invasive procedure that is not easily performed at the scene of injury, particularly where first responder skill levels may vary. Accordingly, there exists a need for rapid, noninvasive, and accessible imaging technologies to aid clinical decision making and management of burn inhalation injury.
  • An inhalable ultrasound contrast agent for evaluation of respiratory function or structure of a subject includes a particle of material comprising a core.
  • the inhalable ultrasound contrast agent further includes a shell surrounding the core.
  • the inhalable ultrasound contrast agent further includes a mucus-targeting ligand located in or on the shell, wherein the mucus- targeting ligand binds with or adheres to respiratory mucus.
  • the core comprises a liquid phase at room temperature and atmospheric pressure.
  • Attorney Docket No.421/538 PCT According to another aspect of the subject matter described herein, the core comprises a perfluorocarbon.
  • the shell comprises a lipid.
  • the mucus-targeting ligand comprises chitosan.
  • the mucus-targeting ligand comprises hyaluronan.
  • the mucus-targeting ligand comprises a material with a cationic surface charge.
  • the particle comprises a nanodroplet.
  • the particle comprises a microbubble.
  • a method for evaluating respiratory function or structure of a subject includes providing a mixture including a liquid and ultrasound contrast agent particles, each of the ultrasound contrast agent particles comprising a core, a shell surrounding the core, and a mucus-targeting material located in or on the shell, wherein the mucus-targeting material comprises a material that binds with or adheres to respiratory mucus.
  • the method further includes nebulizing the mixture to make the ultrasound contrast agent particles inhalable.
  • the method further includes allowing a subject to inhale the nebulized mixture.
  • the method further includes applying ultrasound energy to image the ultrasound contrast agent particles within the subject.
  • the method further includes using results of the imaging to evaluate respiratory function or structure of the subject.
  • the material that binds with or adheres to respiratory mucus comprises a mucus- targeting ligand.
  • Attorney Docket No.421/538 PCT According to another aspect of the subject matter described herein, the material that binds with or adheres to respiratory mucus comprises a material with a cationic surface charge.
  • applying the ultrasound energy includes applying the ultrasound energy with a mechanical index that induces stable cavitation of the ultrasound contrast agent particles.
  • the mechanical index is about 0.19.
  • applying the ultrasound energy includes applying the ultrasound energy at a frequency corresponding to a resonant frequency of the ultrasound contrast agent particles.
  • using the results of the imaging to evaluate respiratory function or structure of the subject includes using results of the imaging to evaluate mucociliary clearance of the subject.
  • using the results of the imaging to evaluate respiratory function or structure of the subject includes using results of the imaging to evaluate lung damage caused by trauma or disease.
  • applying the ultrasound energy includes applying the ultrasound energy to the ultrasound contrast agent particles to disrupt respiratory mucus within the subject.
  • a method for disrupting mucus in a respiratory tract of a subject includes providing a mixture including a liquid and energy-activatable agent particles. The method further includes nebulizing the mixture to make the energy-activatable agent particles inhalable. The method further includes Attorney Docket No.421/538 PCT administering the energy-activatable agent particles into the respiratory tract of the subject.
  • the method further includes applying energy to the energy- activatable agent particles within the respiratory tract of the subject, causing the energy-activatable agent particles to cavitate and disrupt respiratory mucus.
  • a method for delivering a therapeutic agent to a respiratory tract of a subject includes providing a mixture including a liquid and acoustically-activatable agent particles, where at least some of the particles carry a therapeutic agent.
  • the method further includes nebulizing the mixture to make the acoustically-activatable agent particles inhalable.
  • the method further includes administering the acoustically-activatable agent particles into the respiratory tract of the subject.
  • the method further includes applying acoustic energy to the acoustically-activatable agent particles, causing the particles to cavitate, burst, and deliver the therapeutic agent into the respiratory tract of the subject.
  • a system for implementing the methods of either of the preceding two paragraphs is provided.
  • s system for evaluating respiratory function or structure of a subject is provided.
  • the system includes a mixture including a liquid and ultrasound contrast agent particles, each of the ultrasound contrast agent particles comprising a core, a shell surrounding the core, and a mucus-targeting material located in or on the shell, wherein the mucus-targeting material comprises a material that binds with or adheres to respiratory mucus.
  • the system further includes a nebulizer for nebulizing the mixture to make the ultrasound contrast agent particles by a subject.
  • the system further includes an ultrasound imaging system including at least one ultrasound transducer for applying ultrasound energy to the ultrasound contrast agent particles within the subject, detecting ultrasound energy scattered by the ultrasound contrast agent particles, and generating, from the detected ultrasound energy, an image of the ultrasound Attorney Docket No.421/538 PCT contrast agent particles usable to evaluate respiratory function or structure of the subject.
  • applying the ultrasound energy includes applying the ultrasound energy to the ultrasound contrast agent particles to disrupt respiratory mucus within the subject.
  • the subject matter described herein can be implemented in software in combination with hardware and/or firmware.
  • the subject matter described herein can be implemented in software executed by a processor.
  • the subject matter described herein can be implemented using a non-transitory computer readable medium having stored thereon computer executable instructions that when executed by the processor of a computer control the computer to perform steps.
  • Exemplary computer readable media suitable for implementing the subject matter described herein include non-transitory computer-readable media, such as disk memory devices, chip memory devices, programmable logic devices, and application specific integrated circuits.
  • a computer readable medium that implements the subject matter described herein may be located on a single device or computing platform or may be distributed across multiple devices or computing platforms.
  • Figure 1 is an image of gamma scintigraphy of the lungs
  • Figure 2A is a schematic diagram of a particle of an ultrasound contrast agent comprising a nanodroplet
  • Figure 2B is a schematic diagram a particle of an ultrasound contrast agent comprising a microbubble
  • Figure 3 is an image of a ligand conjugated microbubble or nanodroplet contrast agent suspended in liquid inside of an aerosolized particle
  • Figure 4 is a graph illustrating particle deposition fraction versus aerosolized particle size
  • Figures 5A-5C are images illustrating and ex vivo porcine trachea in agar ( Figure 5A), cross-sectional imaging of the trachea before exposure to a prototype nebulized contrast agent that did not include mucus targeting ligands ( Figure 5B), and cross-sectional imaging of the
  • Figures 12A and 12B illustrate time-lapse movement of fluorescently labeled TAP microbubbles on human bronchial epithelial cell culture.
  • Figure 12A illustrates a panel from a transmitted light video
  • Figure 12B illustrates a panel from epifluorescence video. The panels show the same field of view, with fluorescently labeled particles similar in size and motion to those seen in the transmitted light images.
  • Figure 13 illustrates mucociliary transport rate measured across 8 fields of view by tracking the motion of TAP microbubble fluorescence versus motion of visible debris on human mucociliary transport cultures.
  • Figure 14 illustrates cytotoxic (lactate dehydrogenase secretion) and proinflammatory response (interleukin-8 release) induced by exposing human bronchial epithelial cell cultures to TAP microbubble contrast agent, TAP microbubble vehicle, and saline.
  • Figure 16 illustrates randomly selected representations of mouse lung histology, 7-days post exposure to 50 ⁇ L of TAP microbubble contrast agent or saline control, via oropharyngeal aspiration to the lung.
  • Figure 17 is a block diagram of a system for evaluating respiratory function or structure of a subject;
  • Figure 18 is a flow chart illustrating an exemplary process for using ultrasound contrast agents with mucus-targeting material to evaluate respiratory structure and/or function of a subject;
  • Figure 19 is a flow chart illustrating exemplary steps of a method for disrupting mucus in the respiratory tract of a subject;
  • Figure 20 is a flow chart illustrating an exemplary process for delivering a therapeutic agent to a respiratory tract of a subject.
  • HEMT cystic fibrosis
  • CF cystic fibrosis
  • bronchoscopy is the current method for assessing inhalation injury, it is an invasive procedure requiring Attorney Docket No.421/538 PCT sedation and intubation. Tracheal intubation and bronchoscopy are not always advisable, particularly at the scene of injury, where first responder skill levels may vary. These invasive procedures carry the risk of tracheospasm, hemorrhage, and arrythmia. Thus, there is a clinical need for a noninvasive, accessible, and portable imaging technology for at-the-scene evaluation of lung and airway pathology.
  • gamma scintigraphy is the current method for assessing MCC, which is quantified visually by the percent clearance of technetium-99m labeled sulfur colloid, a radiolabeled particle.
  • the subject matter described herein includes an inhalable ultrasound contrast agent for MCC evaluation. Ultrasound is accessible, inexpensive, non-ionizing, and suitable for pediatric use, but is underutilized in pulmonology applications due to poor image quality.
  • the subject matter described herein includes a nebulized contrast agent for ultrasound imaging applications, which is inhaled to coat the airway linings for visualization of the pulmonary structures.
  • an inhalable ultrasound contrast agent without mucus targeting ligands or cationic surface charge was preliminarily evaluated the in vitro toxicity and in vivo tolerability, with no significant bioeffects noted.
  • the subject matter described herein includes optimized microbubble and nanodroplet ultrasound contrast agents for muco- adhesion, using targeting ligands informed by drug delivery research, 7 and / or cationic surface charges used in inhaled drug delivery research (reference: Fromen et al) 52 , and the use of such contrast agents for a non-ionizing, clinical diagnostic test for assessment of MCC and pulmonary pathology.
  • an in vitro assay will be used to evaluate mucociliary clearance of the ultrasound contrast agents.
  • the assessment of mucociliary clearance using the inhalable contrast agent will be validated using gamma scintilography.
  • Nasal MCC defined by the percent clearance of ultrasound contrast agent over 10 minutes, will be compared to percent clearance of technetium-99m labeled sulfur colloid on gamma scintigraphy, in healthy and affected mice with moderate or severely impaired ciliary function. It is believed that nasal MCC assessed by contrast-enhanced ultrasound will correlate well with gamma scintigraphy.
  • Ultrasound as a non-ionizing alternative for MCC assessment Ultrasound is accessible, inexpensive, non-ionizing, and suitable for pediatric use, but is underutilized in pulmonology applications due to poor image quality.
  • 4,5 Sonographic image quality can be improved by intravenously-administered microbubble contrast agents, which are currently FDA-approved for diagnostic cardiac imaging by ultrasound. Outside of the United States, microbubble contrast agents are also used to evaluate the pancreas, liver, kidney, and breast. 14
  • no ultrasound contrast agents Attorney Docket No.421/538 PCT have been developed to enhance pulmonary imaging, nor has an inhalable route of delivery been previously described. Our team has been working on technologies to enhance airway imaging by ultrasound, using an inhalable contrast agent.
  • the contrast agent described herein is believed to be a safe, inhalable, ultrasound contrast agent that will adhere to the respiratory mucus layer to facilitate non-invasive and non- ionizing monitoring of MCC in patients with recurrent need for thoracic imaging.
  • Microbubble and nanodroplet ultrasound contrast agents are composed of a lipid or albumin outer shell filled with an inert, high molecular weight gas such as sulfur hexafluoride or octafluoropropane (both of which are FDA approved), 14 or alternatively, decafluorobutane (pending FDA approval).
  • phase-change nanodroplets composed of liquid perfluorocarbon encapsulated in a polymer shell, have also been developed as for use as ultrasound contrast agents. 15 To date, none have been FDA approved, although at least one company is moving them towards regulatory approval.
  • the phase change nanodroplets can also be encapsulated in a lipid shell.
  • the phase change nanodroplets may be stable with the core primarily in liquid form at room temperature (25 Attorney Docket No.421/538 PCT degrees Celsius) and atmospheric pressure.
  • the inhalable ultrasound contrast agent described herein may be formed of microbubbles, phase change nanodroplets, or both.
  • Microbubble and nanodroplet stability One benefit of nanodroplets is that they are substantially more stable than microbubbles. Stability of the contrast agent is likely to be an important consideration for the development of an MCC imaging protocol, which will infer ciliary clearance of the contrast agent based on the elapsed time until the imaging signal disappears.
  • contrast bubbles to acoustic pressure induces stable cavitation (i.e., oscillating swelling and contraction), the mechanism by which nonlinear echoes are produced to generate a contrast signal.
  • cavitation i.e., oscillating swelling and contraction
  • transient cavitation is induced, causing bubbles to rupture.
  • the ultrasound system’s mechanical index is typically lowered from 0.4 to ⁇ 0.19.
  • a mechanical index of 0.4 is well within the FDA approved range for diagnostic imaging and does not cause bioeffects but is sufficient to destroy contrast bubbles.
  • Nondestructive subharmonic imaging techniques whereby the ultrasound system’s transmission frequency is in sync with the contrast bubbles’ oscillating resonant frequency, are also used to prevent transient cavitation.
  • Intravenously-administered microbubble contrast agents have been functionalized by conjugating adhesion ligands onto the outer shell. 18,19 With this technique, vascular endothelial growth factors, such as VEGFR2, P- and E-selectin, have been successfully targeted to image regions of angiogenesis or vascular endothelial dysfunction. 14,20
  • a inhalable contrast agent targeting the respiratory mucus layer has not been previously developed but may be particularly effective for sonographic pulmonary evaluation and assessment Attorney Docket No.421/538 PCT of MCC.
  • the epithelium of the large (but not small) airways is coated by gel- forming mucus at the air-epithelium interface.
  • a particle of an inhalable contrast agent may include a core, a shell surrounding the core, and one or more mucus-targeting ligands located in or on the shell.
  • FIG. 2A is a schematic diagram of a particle of an ultrasound contrast agent comprising a nanodroplet
  • Figure 2B is a schematic diagram a particle of an ultrasound contrast agent comprising a microbubble.
  • an inhalable ultrasound contrast agent nanodroplet particle 200 includes a core 202, a shell 204 surrounding the core, and one or more mucus targeting materials 206 located in or on shell 204.
  • Core 202 may be a perfluorocarbon that is stable at room temperature and atmospheric pressure.
  • Shell 204 may be a lipid, a polymer, or other suitable material.
  • Mucus targeting material 206 may be a material designed to adhere to respiratory mucus.
  • an inhalable contrast agent microbubble particle 208 includes a core 210, shell 204, and mucus targeting ligands 206.
  • Core 210 may be a gaseous material, such as a perfluorocarbon, that has changed phase and increased in diameter in response to application of ultrasound energy.
  • Shell 204 may be of the same composition described above for nanoparticle 200.
  • Mucus targeting material 206 may likewise be of the same material described above with regard to nanoparticle 200. It is understood that Attorney Docket No.421/538 PCT microbubble 208 may be formed from nanoparticle 200 after nanoparticle 200 is inhaled and exposed to ultrasound energy. In another example, microbubble 208 may be inhaled as a microbubble instead of a nanodroplet that changes phase inside of the body of the subject.
  • microbubble or nanodroplet contrast agents will be suspended in liquid droplets (illustrated in Figure 3) generated by a nebulizer.
  • the deposition of an inhaled particle is primarily determined by its MMAD (mass median aerodynamic diameter), which influences impaction, sedimentation, and diffusion properties.
  • 24 Particles with nanoscale MMADs are transported through the airways by Brownian motion. 24,25
  • the movement and deposition of particles with micron-scale MMADs is driven by laminar or turbulent airflow, inertial impaction, gravity.
  • aerosolized droplets are expected to impact more readily against the inner surfaces of the upper airways, due to inertial impaction. 24 Upon surface impact, the aerosolized carrier droplets will rupture, releasing the microbubble or nanodroplet payload onto the airway surface, as we previously demonstrated using ex vivo pig tracheas 6 (described in the next section).
  • the deposition of aerosols is also influenced by airflow dynamics, which can be modified by patient instruction. For example, tachypnea (i.e., fast and shallow Attorney Docket No.421/538 PCT breathing) increases the deposition and impaction of aerosolized particles in the extrathoracic airways (e.g., trachea), while limiting deposition in the more distal airways and lung.
  • microbubble or nanodroplet contrast agents with mucus targeting ligands can be provided to the subject using a nebulizer, which generates aerosolized liquid (e.g., water) droplets, each of which encapsulate and carry one or more particles of the contrast agent.
  • aerosolized liquid e.g., water
  • the subject can then inhale the aerosolized liquid droplets including the particles of the contrast agent.
  • Preliminary Results Prototype nebulized contrast agent enhances ultrasonic imaging of an ex vivo trachea To evaluate the feasibility of enhancing airway imaging with a prototype nebulized contrast agent, we constructed an imaging phantom by encasing a fresh ex vivo porcine trachea in agar ( Figure 5A).
  • Contrast microbubbles were formulated from distearoylphospatidylcholine (DSPC) and distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) and filled with inert, high molecular weight decafluorobutane gas.
  • the microbubbles were activated by mechanical agitation for 45 seconds and nebulized using a custom-modified Pari LL nebulizer. 29 Imaging was performed using a Siemens Sequoia ultrasound system, with contrast pulse sequencing (CPS) mode selected to optimize signal enhancement.
  • the mechanical index was set to 0.19 to prevent destruction of the contrast microbubbles by acoustic energy.
  • the trachea is easily imaged by ultrasound without obstruction from bone, we anticipate that it will be the clinical target for future imaging protocols to evaluate MCC, which will monitor the percentage of cleared contrast bubbles over time. In this situation, a single imaging plane of the trachea is likely to be sufficient for MCC assessment.
  • existing technologies for 3D ultrasound could be used, by rendering imaging views from the frontal and 2 lateral imaging planes of the trachea.
  • Prototype contrast agent is not toxic to human bronchial epithelial cells
  • hBE primary human bronchial epithelial
  • LDH Lactate dehydrogenase
  • Prototype contrast agent enhances in vivo airway imaging in a mouse model
  • the mouse was anesthetized by isoflurane (indicated by depressed breathing and testing for righting reflex) and was suspended with the tongue extended from the mouth and hung to the side.
  • a controlled amount (50 ⁇ L) of prototype Attorney Docket No.421/538 PCT contrast agent was instilled onto the oropharynx (back of the throat), using a gel loading micropipette tip.
  • the mouse was imaged using a 3D volumetric SonoVol Vega ultrasound platform. Contrast-enhanced delineation of the trachea is shown in Figure 7.
  • Prototype contrast agent enhances in vivo lung parenchyma imaging in a mouse model At 2 minutes post-inhalation, the prototype contrast agent became apparent in the lung parenchyma Figure 8A. Because the mouse was positioned laterally during aspiration, sedimentation of the contrast agent and resulting image enhancement was observed in the left lung. The mouse was closely observed for acute signs of respiratory distress (i.e., gasping, suspended breathing) during the imaging procedure, and was also monitored for signs of physical distress up to 4 weeks following exposure. No acute or long-term adverse sequelae were noted. For comparison, we show our contrast-enhanced murine lung ultrasound image next to a conventional lung ultrasound from a human patient.
  • respiratory distress i.e., gasping, suspended breathing
  • chitosan as the muco-adhesive targeting ligand, or alternatively hyaluronan. 7
  • a chitosan-lipid bioconjugate will be formulated. 41,42 As described, the bioconjugate will be added to a vial which will be sealed, with the headspace of the vial purged with 50 mL of perfluorocarbon gas. 33 The colloidal dispersion will then be agitated and cooled, to create functionalized microbubbles or nanodroplets.
  • MCT mucociliary transport
  • We will quantify the MCT rate for each contrast bubble formulation using superharmonic ultrasound imaging techniques (tracking the motion of contrast agent bubbles along the racetrack assay), which will be compared with the MCT rate quantified by video microscopy (tracking the motion of fluorescent beads), as an established control.
  • the human airway cells used to create the racetrack assays will be procured from the UNC Marsico Lung Institute, an activity deemed to be “Not Human Subjects Research” by our IRB.
  • Superharmonic Ultrasound Imaging For each racetrack assay, a controlled amount (1 ⁇ L) of activated candidate contrast agent will be diluted with 1 ⁇ L of saline and added to the mucus layer. Once deposited, the motion of individual contrast bubbles will be tracked using high frame rate superharmonic ultrasound. This technology achieves resolution beyond the diffraction limit of conventional coherent imaging and can resolve 25-50 micron single bubble displacements over 1 second. Our team member (Dr. Dayton) has extensive experience in the localization and tracking of ultrasound contrast agents by this technique.
  • ciliary cross-links are observed with both types of mucus binding ligands, we will abandon mucus-targeting optimization of our contrast agents and our focus for Aim 2 will instead be the evaluation of non-conjugated formulations.
  • Analytical Plan For both imaging modalities (i.e., video microscopy and superharmonic ultrasound), the mucociliary transport rate will be calculated across a 10 second interval, with measurements made in triplicate. Consistency in the measured mucociliary transport rate will be analyzed by calculating the coefficient of variation,46 grouping the 3 video microscopy measurements with 3 measurements for each candidate contrast agent to assess assay-specific variability in the MCT rate measured by the 2 modalities.
  • contrast signal clearance is similar to the approach used by gamma scintigraphy (described later in Aim 3).
  • contrast signal clearance By validating our assessment of MCC by ultrasound contrast agents both in vitro (tracking individual contrast bubbles) and in vivo, we expect to develop a sensitive, safe, and physiologically meaningful diagnostic test for MCC. Verify in vivo safety and tolerability of candidate contrast agents optimized for muco-adhesion, using in healthy mice. Contrast agent allocation: After validating ciliary clearance of our contrast bubbles in vitro, we will randomly assign 23 adult wild type C57BL/6 mice 47 (aged 8-12 weeks, weighing 20-25 g) to 3 exposure groups: 1).
  • saline control [N 3].
  • Mice will be anesthetized by intravenous injection and nebulized contrast agents (or saline control) will be administered via nose cone.
  • a controlled amount of activated candidate contrast agent (50 ⁇ L) or an equivalent dose of saline control will be released as an aerosol into the nose cone, for a duration of 30 seconds.
  • the nose cone will be removed, and an alcohol wipe will be used to remove any contrast agent that may have deposited on the fur surrounding the nose and head.
  • Images of the upper/nasal airways, trachea, and lung will be acquired by whole-body ultrasound using a SonoVol Vega with nondestructive subharmonic imaging techniques.
  • Contrast-enhanced ultrasound The elapsed time from appearance until dissipation of contrast signal will be recorded, within 3 distinct regions of interest: 1). Nasal airways, 2). Trachea, and 3). Lung.
  • in vivo tolerability The in vivo tolerability of our mucus- targeting, inhalable ultrasound contrast agents will be assessed by observing acute symptoms of respiratory distress (e.g., gasping, suspended breathing) during the ultrasound imaging session, as well as noting any post-exposure symptoms of malaise (e.g., lack of grooming or food refusal) within 7 days. The development of symptoms will be compared to the control group exposed to nebulized saline. We have selected healthy mice for the in vivo tolerability assessment, because symptomatic disease states (e.g., CF phenotype) would confound the attribution of adverse effects.
  • symptomatic disease states e.g., CF phenotype
  • mucus- targeting contrast agents will be well tolerated, both with the microbubble and nanodroplet formulation.
  • the risk of adverse events following exposure to mucus-targeting contrast agents is expected to be low.
  • the candidate contrast agent will be eliminated Attorney Docket No.421/538 PCT from consideration.
  • alternative formulations such as mucus- targeting by conjugated hyaluronan, or non-conjugated formulations will be substituted.
  • Nasal MCC will be quantified using an established gamma scintigraphy protocol for mice.
  • the nasal airways are selected for evaluation, because clearance of mucus in the upper and nasal airways is completely dependent on MCC, without confounding by other mechanisms such as coughing.
  • Our team has extensive experience with gamma scintigraphy and includes investigators (Drs. Bennett and Zeman) who pioneered the original imaging protocols that are in use today.
  • Gamma scintigraphy images will be acquired using an Intel Medical planar gamma camera and pinhole collimator.
  • mice will be anesthetized by inhalational isoflurane, and 1 ⁇ L of technetium99m-labeled sulfur colloid suspension will be delivered into the nose by inserting a catheter into the nostril. Mice will then be positioned under the pinhole collimeter for continuous imaging by the planar gamma camera.
  • the nasal MCC rate is not constant over time, with a rapid phase observed during the first 10 minutes, followed by a slower clearance rate from 10 to 60 minutes.
  • RI radioactivity intensity
  • imageJ image analysis software
  • 48 we will calculate the cumulative nasal MCC and the average nasal MCC rate, using established algorithms.
  • Analytical Plan The relationship between nasal MCC measured by contrast- enhanced ultrasound versus gamma scintigraphy will first be visualized by scatterplots. We will then analyze the within-subject agreement in nasal MCC measurements using Bland-Altman plots, and the within-subject correlation using Spearman or Pearson correlation coefficients.
  • the betaENaC-Tg mouse is a model of CF and expresses moderately impaired MCC.50
  • a gel mucus layer coats the large airways at the air-epithelium interface, 22 and can be targeted by ligands (e.g., chitosan, hyaluronan) 61 or by imparting a surface charge onto aerosolized compounds.
  • ligands e.g., chitosan, hyaluronan
  • 62 we designed a polydisperse contrast agent with diameters ranging from 1-5 ⁇ m, which is consistent with intravenously administered contrast agents that are used clinically. 20 The micron-scale diameter was selected to ensure the contrast agent remained within the airway surface layer with minimal epithelial absorption.
  • Mucus-targeting contrast agent was synthesized in house, using 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) and polyoxyethylene (40) stearate (PEG 40-S) to create the microbubble lipid outer shell. Because muco-adhesion is amplified by positive charge, 53 the lipid mixture included 1,2-distearoyl-3-trimethylammonimum-propane (TAP) to yield a cationic Attorney Docket No.421/538 PCT surface.
  • TEP 1,2-distearoyl-3-trimethylammonimum-propane
  • the cationic lipid mixture (12 mol% TAP, 34 mol% PEG40-S, and 54 mol% DSPC; TAP-lipid) 54 was heated in propylene glycol (PG) at 60 C for 1 hour with sonication to create a dissolved suspension.
  • PG propylene glycol
  • the aqueous lipid stock solution (4.67 mM total lipid concentration) contained 15% (v/v) propylene glycol and 5% (v/v) glycerol in phosphate-buffered saline (PBS) as the diluent (PBS-PGG).
  • PBS phosphate-buffered saline
  • TAP-lipid solutions containing 1-, 2-, and 3-mM total lipid were created by diluting the stock cationic lipid solution with PBS-PGG.
  • Lipid solutions (1.5 mL) were packaged into 3 mL serum vials, sealed with silicone septa, and the air in the headspace was removed and exchanged with perfluorobutane (C 4 F 10 ), an inert, high molecular weight gas.
  • C 4 F 10 perfluorobutane
  • TAP-lipid solutions and vials of lipid solution were stored at 4oC.
  • sealed vials containing room-temperature TAP-lipid solutions and perfluorocarbon gas were shaken in a VialMix (Lantheus Medical Imaging, N. Billerica, MA; 4500 rpm, 45 seconds).
  • TAP-microbubbles were determined for microbubbles prepared from 1, 2, and 3 mM TAP-lipid solutions using an Accusizer FX Nano system (Entegris, Billerica, MA). Authentication of mucus-targeting To verify the association of our cationic contrast agent with the respiratory mucus layer, we observed the movement and distribution of TAP microbubbles versus our standard, neutrally charged microbubbles relative to a target mucus sample.
  • Our neutral microbubble contrast agent was formulated in-house, as previously described, 64 from a 1 mM lipid solution containing a (9:1) molar ratio of DSPC to 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE- PEG2000) to create a neutral lipid shell (DSPC/P2K) around a core of C 4 F 10 gas.
  • Cationic (TAP) or neutral contrast agents were enriched for larger diameter microbubbles to facilitate capturing photomicrographs of microbubble-mucus interactions at 400X magnification.
  • TAP or neutral microbubbles were centrifuged (1-2 minutes, 160x g) in sealed 5-mL syringes. The smaller diameter microbubbles in the infranatant fluid were separated and discarded from the floating ‘cake’ of larger microbubbles. Larger microbubbles Attorney Docket No.421/538 PCT were resuspended in PBS-PGG, with the centrifugal enrichment repeated 3 times before determining the final concentration and size distribution of ‘large’ microbubbles. Clean glass slides were plated with respiratory mucus from an anonymous, healthy donor and allowed to dry overnight. A 1% bovine serum albumin (BSA) solution in PBS was used to rehydrate the mucus and block the rectangular area surrounding the mucus sample.
  • BSA bovine serum albumin
  • Mucociliary transport assay 43 was used to quantify the movement of TAP versus neutrally charged microbubbles on human bronchial epithelial cell cultures, with a fluorescent lipophilic cationic indocarbocyanine dye (Invitrogen, Waltham, MA, VybrantTM DiO) used for labeling.
  • a fluorescent lipophilic cationic indocarbocyanine dye Invitrogen, Waltham, MA, VybrantTM DiO
  • 65 the in vitro transport assay was created in house by affixing a central ring to a Millicell culture insert, which was etched to form circular “racetracks” around the perimeter. First passage primary hBECs were cultured and plated on the Millicell insert and allowed to grow for 2-3 weeks until confluent.
  • hBEC cultures achieve continuous directional mucociliary transport when grown on the patterned substrate.
  • Mucociliary transport assays are coated by a mucus layer at the air surface interface, allowing direct, in vitro quantification of mucociliary transport.
  • a culture was first incubated for 5 minutes with 1 ml of PBS with calcium and magnesium at 37°C to rehydrate the mucus.
  • the surface fluid was then removed and 20 ⁇ L volume of microbubble solution (either TAP or neutral contrast agent, concentrated to Attorney Docket No.421/538 PCT 200,000 microbubbles/ ⁇ L), was pipetted onto one position in the center of the track.
  • the culture was then placed in an OkoLab microscope incubator.
  • the incubator was maintained at 37°C and humidified air with 5% CO 2 flowing through the culture chamber at 400 ml/min.
  • the culture was allowed to equilibrate for ten minutes. Videos were captured from eight equally spaced fields in the center of the track using a Nikon Eclipse TE2000 microscope (Nikon Instruments Inc, Melville, NY, USA) and a Basler acA1300-200 um camera controlled by SAVA software (version 2.0.8W, Ammons Engineering, Clio, MI, USA). 67 At each field of view, a transmitted light video was taken using phase optics (20x ELWD objective) and a 120 frame/second capture rate.
  • a fluorescence video was taken of the same field of view with a 15 frames per second (fps) capture rate.
  • the fluorescence excitation was provided by a Lumencor SOLA 5-LCR-VG LED source (Lumencor; Beaverton, OR) and the light path used a Nikon B-2E/C filter block.
  • a particle position was manually recorded at two locations in each video.
  • transport assays were cultured from the same healthy, human donor. As a validation, the calculated transport of fluorescence was compared with the transport rate of visible debris within the same field of view, captured on transmitted light video at 15 fps.
  • Lactate dehydrogenase (LDH) and proinflammatory cytokine interleukin-8 (IL-8) were quantified according to manufacturer instructions (CyQUANT LDH Cytotoxicity Assy, Attorney Docket No.421/538 PCT Invitrogen and OptEIA Human IL-8 ELISA, BD Biosciences, respectively) in the basolateral medium following a 24-hour apical exposure to either 25 ⁇ L of undiluted TAP-contrast (with the microbubbles activated by mechanical agitation), 25 ⁇ L of undiluted vehicle control (TAP lipids), 25 ⁇ L saline control, or a no-treatment control.
  • TAP-contrast with the microbubbles activated by mechanical agitation
  • TAP lipids undiluted vehicle control
  • 25 ⁇ L saline control or a no-treatment control.
  • TAP alpha tumor necrosis factor alpha
  • mice In vivo safety, tolerability, and imaging feasibility
  • mice were imaged in contrast mode using a FUJIFILM Vevo F2 ultrasound machine interfaced with a 15.0 – 29.0 MHz transducer, capturing at a frame rate of 30 fps and dynamic range of 57.65 dB.
  • the trachea was imaged in longitudinal orientation, with the ultrasound transducer positioned by a stereotactic clamp. Mice were observed during the procedure for signs of distress, and for 7 days post-procedure by Attorney Docket No.421/538 PCT monitoring weight, grooming, and body condition score.
  • mice After monitoring for acute effects for 7 days, mice were sacrificed. The trachea and lungs were harvested and preserved in formalin, with tissues fixed with hematoxylin and eosin (H&E) stain. Histology was evaluated by an independent, veterinary pathologist who was blinded to the exposure condition.
  • H&E hematoxylin and eosin
  • large (4 ⁇ m) TAP microbubbles were suspended in saline and induced to flow across human respiratory mucus plated on a microscope slide, the microbubbles oriented in a spatial arrangement in proximity to the mucus ( Figure 11A).
  • our standard, neutrally charged microbubbles demonstrated no affinity for mucus (Figure 11B), and freely flowed as a bulk liquid on the microscope slide, in a uniform distribution with no clustering around the mucus sample.
  • TAP-microbubbles Unlike TAP-microbubbles, these neutrally charged microbubbles appeared to float above the mucus layer on a different plane, with no interaction or adhesion to the sample.
  • TAP-microbubbles When imaged by high frame rate video microscopy on mucociliary transport assays, TAP-microbubbles were easily visualized as bright, fluorescent clouds on epifluorescence video ( Figures 12A and 12B), with transport successfully tracked in 8 fields of view in the center of the track and evenly distributed along the full length of the track.
  • fluorescently tagged neutral microbubbles were faint and poorly visualized, and transport rates could not be quantified.
  • the mean ciliary beat frequency for the in vitro assay was 12.2 ⁇ 0.8 Hz, with a mean active ciliated area of 29.2 ⁇ 16 %, across the 8 fields of view. There was no observable evidence of cross-linking between TAP-microbubbles and the cilia, in any of the 8 fields of view. Epithelial absorption was observed to minimal and temporary, averaging at 0.8% across all fields of view during the 60 second observation period.
  • TAP-microbubble contrast agent associated with mucus and traveled with the mucus layer in vitro, by evaluating mucociliary transport assays composed of human bronchial epithelial cells.
  • the microbubbles mixed into the air-surface layer, without absorption into the epithelium, promoting the feasibility of TAP contrast-enhanced ultrasound imaging as a means to evaluate mucociliary transport.
  • Our TAP-microbubble contrast agent demonstrated no cytotoxic or proinflammatory response to human bronchial epithelial cells cultures with 24hr of exposure, nor were any ciliary cross-links observed, which suggests feasibility for clinical use, pending further evaluation.
  • TAP-microbubble contrast agent When administered to mice, TAP-microbubble contrast agent enhanced tracheal ultrasound imaging and was well tolerated in vivo, with no adverse effects noted on lung histology.
  • One clinical application for our optimized contrast agent may be evaluation of mucociliary clearance, by quantifying the overall percent clearance of visible microbubbles across a specified time interval (similar to the approach used by gamma scintigraphy). Assessment of mucociliary clearance time could potentially be performed using commercially available equipment. If deemed safe for clinical use, the contrast agent could be administered to the patient as an aerosol using a medical grade nebulizer.
  • Flash-replenishment time is accessible with clinical scanners and commercially available image analysis packages, 68 but its safe use in pulmonary applications, and particularly in patients with CF, has not been evaluated.
  • microbubbles are destroyed by increasing the ultrasound system insonation power, rupturing the microbubbles through induced cavitation. It is possible that the violent destruction of microbubbles lodged within the mucus layer may adversely affect ciliary function.
  • TAP contrast agent could potentially be used to measure mucociliary transport rate.
  • the in vitro transport rates that we calculated for TAP microbubbles on microscopy ranged from 90 to 150 ⁇ m/s, velocities that are too low to be captured by conventional Doppler, but within the range quantifiable by acoustic angiography, a contrast-enhanced superharmonic ultrasound imaging technique.
  • Mucociliary transport rate could also be measured by tracking the motion of individual microbubbles along a specified distance in the trachea (similar to invasive particle tracking by fiberoptic bronchoscopy 55-57 or intranasal optical coherence tomography 58 ).
  • Super-resolution ultrasound imaging is capable of tracking individual microbubbles in the circulation, and may also have applications in pulmonary imaging. Although not performed by routine imaging equipment, super-resolution ultrasound imaging achieves resolution beyond the diffraction limit of conventional coherent imaging and can resolve 25-50 micron single bubble displacements over 1 second. 33,34 In future preclinical work, we will evaluate the accuracy of super-resolution ultrasound imaging against video microscopy in mice with and without a CF phenotype.
  • Figure 17 is a block diagram of a system for evaluating respiratory function or structure of a subject.
  • the system includes a mixture 1700 including a liquid and ultrasound contrast agent particles, where each of the inhalable ultrasound contrast agent particles comprises a core, a shell surrounding the core, and a mucus-targeting material located in or on the shell.
  • the liquid may be water, and the mucus-targeting material comprises a material that binds with or adheres to respiratory mucus.
  • Mixture 1700 can include any of the ultrasound contrast agent particle formulations described herein.
  • the system further includes a nebulizer 1702 for nebulizing the mixture to make the particles of the ultrasound contrast agent inhalable by a subject 1704.
  • Nebulizer 1702 can be any suitable device for aerosolizing mixture 1700 to make mixture 1700 suitable for inhaling by subject 1704.
  • subject 1704 is a human subject.
  • subject 1704 can be a preclinical mammalian subject, such as a mouse.
  • the system further includes an ultrasound imaging system 1706 including at least one ultrasound transducer 1708 for applying ultrasound energy to the particles of the ultrasound contrast agent within the subject, detecting ultrasound energy scattered by the ultrasound contrast agent particles, and generating, from the detected ultrasound energy, an image of the ultrasound contrast agent particles usable to evaluate respiratory function or structure of the subject.
  • ultrasound imaging system 1706 may be a super-resolution ultrasound imaging system of the type described in commonly-assigned, co-pending U.S. patent application publication no. 2022/0211350, filed October 27, 2021, the disclosure of which is incorporated herein by reference in its entirety.
  • the super-resolution ultrasound imaging system includes a dual-frequency ultrasound transducer with non- overlapping -6 dB bandwidths for transmit and receive.
  • the system further includes a super-resolution processor capable of generating images including Attorney Docket No.421/538 PCT the spatial locations of individual contrast agent particles.
  • the super- resolution processor may be implemented using computer-executable instructions stored in a memory of ultrasound imaging system 1706 and executed by a processor of ultrasound imaging system 1706.
  • Figure 18 is a flow chart illustrating an exemplary method for using ultrasound contrast agents with mucus-targeting ligands and/or cationic surface charges to evaluate respiratory function or structure of a subject.
  • the method includes providing a mixture including a liquid and ultrasound contrast agent particles, each of the ultrasound contrast agent particles comprising a core, a shell surrounding the core, and a mucus-targeting material in or on the shell, wherein the mucus- targeting material comprises a material that binds with or adheres to respiratory mucus.
  • the mucus-targeting material comprises a mucus-targeting ligand that binds with respiratory mucus.
  • the mucus-targeting material may include a material with a cationic surface charge.
  • the method further includes nebulizing the mixture to make the particles of the ultrasound contrast agent inhalable.
  • the method further includes allowing a subject to inhale the nebulized mixture.
  • the method further includes applying ultrasound energy to image the particles of the ultrasound contrast agent within the subject.
  • ultrasound energy may be applied using an ultrasound transducer external to the subject at a frequency and power level that causes the ultrasound contrast agent to induce stable cavitation of the ultrasound contrast agent.
  • the ultrasound energy may be applied to produce a therapeutic response in the respiratory tract of the subject.
  • ultrasound energy may be applied to the mucus- targeting ultrasound contrast agent in the subject after the ultrasound contrast agent has been inhaled and adhered to respiratory mucus to cause the ultrasound contrast agent particles to cavitate and disrupt a mucus plug.
  • Attorney Docket No.421/538 PCT the method further includes using results of the imaging to evaluate respiratory function or structure of the subject.
  • the imaging results may be used to evaluate mucociliary clearance in subjects with cystic fibrosis or lung damage in burn victims.
  • a process for disrupting mucus in the respiratory tract of a subject is provided.
  • Figure 19 is a flow chart illustrating exemplary steps of a process for disrupting mucus in the respiratory tract of a subject.
  • the process includes providing a mixture including a liquid and energy- activatable agent particles.
  • the energy-activatable agent particles can be microbubbles, nanodroplets, or a combination of nanodroplets and microbubbles.
  • the energy-activatable agent particles can include the contrast agent particles described above which include a mucus- targeting material located in or on the shells of the particles. In another example, the particles may not include a mucus-targeting material.
  • the process further includes nebulizing the mixture to make the energy-activatable agent particles inhalable.
  • the method further includes administering the particles of the energy-activatable agent into a respiratory tract of the subject.
  • the process further includes applying energy to the energy-activatable agent particles within the respiratory tract of the subject, causing the particles to cavitate and disrupt respiratory mucus.
  • energy may be applied using an ultrasound transducer external to the subject at a frequency and power level that causes the ultrasound contrast agent to cavitate and decrease the viscosity of respiratory mucus, including mucus plugs.
  • mechanical or thermal energy may be applied to the energy-activatable agent particles to cause the particles to cavitate and disrupt the respiratory mucus. The application of energy may cause shear or shock waves in the particles of the energy-activatable agent.
  • the energy delivered to the particles of the energy-activatable agent may be Attorney Docket No.421/538 PCT delivered at a frequency in a range of 100 kHz to 3 MHz and/or with a mechanical index within a range from 0.2 to 3.0.
  • a process for delivering a therapeutic agent to a respiratory tract of a subject is provided.
  • Figure 20 is a flow chart illustrating an exemplary process for delivering a therapeutic agent to a respiratory tract of a subject. Referring to Figure 20, in step 2000, the process includes providing a mixture including a liquid and acoustically-activatable agent particles, where at least some of the particles carry a therapeutic agent.
  • the acoustically-activatable agent particles can be microbubbles, nanodroplets, or a combination of nanodroplets and microbubbles.
  • the acoustically-activatable agent particles can include the contrast agent particles described above which include a mucus-targeting material located in or on the shells of the particles. In another example, the particles may not include a mucus-targeting material.
  • therapeutic agents that may be carried by the acoustically- activatable agent particles include mRNA or DNA for gene therapy, for example, to treat cystic fibrosis.
  • mRNA or DNA for gene therapy for cystic fibrosis includes mRNA or DNA that encodes a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, such as a corrector, a potentiator, a stabilizer, or an amplifier.
  • CFTR modulators must be repeatedly delivered to the airway of a CF subject to be effective, and acoustically-activatable agent particles are believed to be candidates for such repeated delivery.
  • a therapeutic agent that may be carried by the acoustically-activatable contrast agent particles is a disulfide disrupter, such as tris(2-carboxyethyl)phosphine (TCEP).
  • the process further includes nebulizing the mixture to make the acoustically-activatable agent particles inhalable.
  • the process further includes administering the particles of the acoustically- activatable agent into a respiratory tract of the subject. For example, a physician or a technician may allow a subject to inhale the nebulized mixture.
  • Attorney Docket No.421/538 PCT the process includes applying acoustic energy to the acoustically-activatable particles within the respiratory tract of the subject to release the therapeutic agent within the respiratory tract of the subject.
  • an ultrasound transducer may apply ultrasound energy to the acoustically-activatable particles causing the particles to inertially cavitate, burst or rupture, and deliver the therapeutic agent to the respiratory tract of the subject.
  • the subject matter described herein may also include a system for implementing the subject matter of Figure 19 or Figure 20.
  • the system may include the same or similar components to the system illustrated in Figure 17 except that the ultrasound contrast agent particles may be replaced with the energy-activatable particles to implement the process of Figure 18 or acoustically-activatable particles carrying the therapeutic agent to implement the process of Figure 19.
  • the ultrasound imaging system may be replaced with an energy source of the appropriate type (ultrasound, thermal, or mechanical to implement the process of Figure 19 or an acoustic energy source (including ultrasound) to implement the process of Figure 20.
  • a method for evaluation of lung function or structure of a subject includes first administering a contrast agent through inhalation so that the contrast agent is deposited within the lung of the subject.
  • the method further includes acquiring ultrasound images of the distribution and/or movement of the contrast agent within the subject’s lungs.
  • the method further includes evaluating respiratory function or structure of the subject based on the distribution or movement of the contrast agent.
  • the contrast agent comprises shelled particles, each of the particles comprising a core, a shell surrounding the core, and a mucus-targeting material located in or on the shell, where the mucus-targeting material comprises a material that binds with or adheres to respiratory mucus.
  • the shells of the contrast agent particles comprise a lipid, a protein, or a polymer.
  • the shells of the contrast agent particles comprise a phospholipid
  • the shells of the contrast agent particles comprise polyethylene glycol
  • the shells of the contrast agent particles comprise albumin.
  • the shells of the contrast agent particles comprise a phospholipid with a cationic charge
  • the material imparting the cationic charge comprises 1,2-distearoyl-3- trimethylammonimum-propane.
  • the cores of the contrast agent particles comprise a gas.
  • the gas in the cores in the contrast agent particles comprises a perfluorocarbon.
  • the gas in the cores of the contrast agent particles comprises a fluorinated compound.
  • the cores of the contrast agent particles comprise a liquid.
  • the liquid in the cores of the contrast agent particles comprises a perfluorocarbon.
  • the liquid in the cores of the contrast agent particles comprises a perfluorocarbon, which is a gas normally at room temperature and pressure, yet is metastable in a liquid state encapsulated within the shell of the contrast agent particles.
  • the cores of the contrast agent particles comprise perfluorobutane or perfluoropentane.
  • Attorney Docket No.421/538 PCT According to another aspect of the subject matter described herein, the cores of the contrast agent particles are a liquid when external to the lung, but convert into gas phase after administered into the lung. According to another aspect of the subject matter described herein, the cores of the contrast agent particles convert into gas phase after being administered into the lung and exposed to ultrasound energy.
  • administering the contrast agent comprises nebulizing the mixture to make the particles of the ultrasound contrast agent inhalable; allowing a subject to inhale the nebulized mixture; and allowing the inhaled contrast agent to distribute within the lung.
  • the ultrasound contrast agent particles prior to the nebulization, are suspended in an aqueous solution.
  • the aqueous solution comprises a salt.
  • the aqueous solution comprises glycerin and/or propylene glycol.
  • applying ultrasound energy to image the particles of the ultrasound contrast agent within the subject comprises application of an ultrasound imaging sequence specific for detection of the contrast agent and suppression of background signal from lung tissue.
  • the ultrasound energy is applied to the ultrasound contrast agent according to an imaging sequence after the ultrasound contrast agent has been inhaled by the subject.
  • the imaging sequence includes transmitting a pulse of ultrasound energy at a first frequency to induce the production of harmonic content from the ultrasound contrast agent microbubbles, where the harmonic content is at Attorney Docket No.421/538 PCT a second frequency greater than the first frequency.
  • the imaging sequence further includes receiving ultrasound scattered by the contrast agent microbubbles.
  • the imaging sequence further includes filtering the frequency content of the received ultrasound energy to select the frequency energy at the second frequency produced by the microbubbles, which results in reducing the background tissue signal and enhancing the microbubble signal.
  • the ultrasound transducer used to image the contrast agent particles in the subject’s airway includes at least two different ultrasound transducer elements, one for transmitting at the first frequency and another for receiving at the second frequency.
  • the ultrasound imaging sequence comprises transmitting two pulses of inverted phase, i.e., a first pulse with a first phase and a second pulse with a phase that is offset by 180 degrees from the phase of the first pulse.
  • the imaging sequence further includes receiving the ultrasound data from the subject and summing the received data from the two pulses, which cancels part of the signal from tissue and which results in reducing the background tissue signal and enhancing the microbubble signal.
  • the ultrasound imaging sequence comprises transmitting two pulses, a first pulse of an amplitude A and a second pulse of an amplitude 0.5A, receiving the ultrasound data from the subject, and subtracting the received data from the pulse with the amplitude A from data from twice the amplitude of the data from the pulse with the amplitude 0.5A, or equivalently the data from the pulse with the amplitude A from twice the amplitude of data from the pulse with the amplitude 0.5A, which results in reducing the background tissue signal and enhancing the microbubble signal.
  • the inhalable contrast agent includes a core which comprises a gas phase at room temperature and atmospheric pressure stabilized by a shell, and a mucus-targeting ligand is located in or on the shell, wherein the mucus- Attorney Docket No.421/538 PCT targeting ligand binds with or adheres to respiratory mucus.
  • a mucus-targeting ligand is located in or on the shell, wherein the mucus- Attorney Docket No.421/538 PCT targeting ligand binds with or adheres to respiratory mucus.
  • Livraghi-Butrico A Wilkinson KJ, Volmer AS, Gilmore RC, Rogers TD, Caldwell RA, Burns KA, Esther CR, Mall MA, Boucher RC, O’Neal WK, Grubb BR.
  • Lung disease phenotypes caused by over-expression of combinations of alpha, beta, and gamma subunits of the epithelial sodium channel in mouse Attorney Docket No.421/538 PCT airways. American Journal of Physiology-Lung Cellular and Molecular Physiology.2017;ajplung.00382.2. 51. Ostrowski LE, Yin W, Rogers TD, Busalacchi KB, Chua M, O’Neal WK, Grubb BR.
  • Mucoadhesive drug delivery system An overview. J Adv Pharm Technol Res. 2010;1:381. http://www.japtr.org/text.asp?2010/1/4/381/76436 54.
  • Christiansen JP French BA, Klibanov AL, Kaul S, Lindner JR. Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles. Ultrasound Med Biol. 2003;29:1759–1767. https://linkinghub.elsevier.com/retrieve/pii/S0301562903009761 55. Olivieri, D., Del Donno, M., Casalini, A., D’Ippolito, R. & Fregnan, G. B.

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Abstract

L'invention concerne un procédé permettant d'évaluer la fonction ou la structure respiratoire d'un sujet qui consiste à fournir un mélange comprenant un liquide et des particules d'agent de contraste ultrasonore, chacune des particules d'agent de contraste ultrasonore inhalables comprenant un noyau, une coque entourant le noyau, et un matériau de ciblage de mucus dans, ou sur, la coque, le ciblage de mucus comprenant un matériau qui se lie ou adhère au mucus respiratoire. Le procédé consiste en outre à nébuliser le mélange pour rendre inhalables les particules d'agent de contraste ultrasonore. Le procédé consiste en outre à permettre à un sujet d'inhaler le mélange nébulisé. Le procédé consiste en outre à appliquer de l'énergie ultrasonore pour imager les particules d'agent de contraste ultrasonore à l'intérieur du sujet. Le procédé consiste en outre à utiliser les résultats de l'imagerie pour évaluer la fonction ou la structure respiratoire du sujet.
PCT/US2024/041327 2023-08-07 2024-08-07 Agent de contraste ultrasonore pour une liaison à un mucus respiratoire ou une adhérence à celui-ci et ses procédés d'utilisation Pending WO2025034892A2 (fr)

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CN111729092B (zh) * 2020-06-28 2021-09-24 南京超维景生物科技有限公司 磁性超声造影剂组合物、磁性超声造影剂、磁性微泡超声造影剂及其制备方法

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