Compositions and methods of detecting and treating thrombosis and vascular plaques

Abstract

To provide an improved pharmaceutical composition for the detection and treatment of thrombosis and related diseases and conditions.SOLUTION: By using an aqueous emulsion or suspension of nanodroplets having one or more fibrin-binding ligands attached thereto, imaging is performed to detect the presence of vascular thromboses or vascular plaques. Further, by applying ultrasound to the target region of a subject after administration of the emulsion, thrombosis or vascular plaques can be treated.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claims and related patent applications This application claims the benefit of priority from U.S. Provisional Application Serial No. 62 / 857,766, filed June 5, 2019, the entire contents of which are incorporated herein by reference for all purposes. [Background technology]

[0002] Technical field of the invention The present invention relates to pharmaceutical compositions and methods for their preparation and diagnostic or therapeutic use. More particularly, the present invention relates to targeted microbubbles and / or nanodroplets, and emulsions thereof, labeled with diagnostic and / or therapeutic ligands useful for the detection and destruction of vascular thrombosis (e.g., fibrin clots) and vascular plaque, and methods for their preparation and use.

[0003] Background of the Invention Cardiovascular disease (CVD) is a leading cause of death and disability worldwide. Thrombosis is the underlying cause of many types of CVD, including venous thromboembolism (VTE), ischemic heart disease, and ischemic stroke. Efforts to remove obstructive thrombi by angioplasty / stenting, thromboembolization, mechanical disruption, and / or biochemical lysis have met with varying degrees of success. These techniques are typically time-consuming and expensive to perform and often carry a substantial risk of bleeding complications.

[0004] Microbubbles have been used to enhance coronary sonothrombolysis in the treatment of acute myocardial infarction (MI) and non-acute ischemic stroke. In both MI and ischemic stroke, thrombi cause arterial occlusion, depriving downstream tissue of blood flow and causing ischemia and potential cell death. Thrombi are variably composed of fibrin and platelets and may contain many red blood cells trapped within the reticulum.

[0005] Fibrin, also known as factor Ia, is a fibrous, non-globular protein involved in blood clotting. It is present in high concentrations in both venous and arterial thrombosis, demonstrating high sensitivity for fibrin-targeted therapies. At the same time, fibrin is not present in the circulating blood, potentially enabling high specificity for these therapies. In addition to protein-based approaches, we also describe small cyclic peptides that exhibit high affinity for fibrin and high selectivity over fibrinogen. Potential advantages of small peptides compared to antibodies include faster blood clearance and the ability to penetrate the fibrin network, both of which improve target-to-background ratios.

[0006] Inflammation and endothelial dysfunction are key critical events in the progression of atherosclerosis. Expression of endothelial cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), has been shown to play a key role in leukocyte recruitment and is often increased at sites of pathological inflammation. Persistent expression of VCAM-1 on dysfunctional endothelial cells mediates the adhesion, rolling, and tethering of mononuclear leukocytes, facilitating their migration into developing atherosclerotic plaques. Therefore, VCAM-1 is a target not only for early detection by imaging but also for therapeutic drug delivery.

[0007] Ultrasound can be used to disrupt blood clots, but there is a trade-off between time / efficiency and damage to healthy tissue. Agents that can locally amplify sound, such as microbubbles, can accelerate disruption while delivering lower energy. A caveat with the use of bubbles is their size (1-5 microns), which can impede access to the interior of the clot. While blood clots exhibit a porous matrix, the interstices within the mass typically preclude the entry of micron-sized structures.

[0008] Thus, there remains a continuing need for improved therapeutic agents and methods for the detection and treatment of thrombosis and related diseases and conditions. Efforts to improve the safety, effectiveness, and efficiency of thrombectomy have potentially great clinical impact. Summary of the Invention

[0009] The present invention is based, in part, on novel microbubbles and nanodroplets and emulsions thereof with targeting capabilities for select biomarkers useful in the diagnosis and treatment of certain diseases and conditions, particularly thrombosis. These carriers can target various protein targets, such as fibrin and VCAM-1, to improve the detection or disruption of thrombi, platelets, and vascular plaques that occur in cardiovascular disease. The present invention further relates to pharmaceutical compositions and methods for their preparation and use.

[0010] In one aspect, the present invention generally relates to aqueous emulsions or suspensions of microbubbles and / or nanodroplets bearing one or more fibrin-binding ligands.

[0011] In another aspect, the present invention generally relates to aqueous emulsions or suspensions of microbubbles and / or nanodroplets bearing one or more VCAM-1 binding ligands.

[0012] In yet another aspect, the present invention generally relates to aqueous emulsions or suspensions comprising microbubbles and / or nanodroplets bearing one or more fibrin-binding ligands as disclosed herein and microbubbles and / or nanodroplets bearing one or more VCAM-1-binding ligands as disclosed herein.

[0013] In yet another aspect, the present invention generally relates to a method for detecting vascular thrombus or vascular plaque, the method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and imaging a portion of the subject to detect the presence of vascular thrombus or vascular plaque.

[0014] In yet another aspect, the present invention generally relates to a method for diagnosing or assessing thrombosis, the method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and imaging a portion of the subject to diagnose or assess thrombosis in the subject.

[0015] In yet another aspect, the present invention generally relates to a method for disrupting or destroying vascular thrombosis or vascular plaque, said method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and applying ultrasound to a target area of ​​an organ of said subject having vascular thrombosis or vascular plaque, thereby disrupting or reducing said vascular thrombosis or vascular plaque.

[0016] In yet another aspect, the present invention generally relates to a method for treating thrombosis or arterial plaque, said method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and applying ultrasound to a target area of ​​said subject.

[0017] In yet another aspect, the present invention generally relates to a method for performing sonothrombolysis, the method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and applying ultrasound to a target area of ​​the subject. [Brief explanation of the drawings]

[0018] [Figure 1]FIG. 1 shows that a fibrin-binding peptide (FBP) with an azide functional group is conjugated with DSPE-PEG5000-DBCO to form a product with a dibenzocyclooctatriazole linker. [Figure 2] FIG. 2 shows that an FBP with an amine functionality is coupled with a DSPE-PEG5000-NHS ester to form a product with an amide linker. [Figure 3] Figure 3 shows that perfluorobiphenyl sulfide was oxidized to produce a more active sulfone derivative, which was then reacted with DSPE-PEG5000-amine to produce DSPE-PEG5000-PFPhSO2. Finally, DSPE-PEG5000-PFPhSO2 was reacted with an amine-containing FBP to produce the conjugated final product. [Figure 4] Figure 4 shows that the conjugation of FBP with DSPE-PEG5000-DBCO (A), DSPE-PEG5000-NHS ester (B), and DSPE-PEG5000-PFPhSO2 (C) was confirmed by MS data. [Figure 5] Figure 5 shows that FBP tagged with 5(6)-carboxytetramethylrhodamine N-succinimidyl ester produces FBP-Rh (MW = 2100.75 Da) (top), and DK-12 tagged with 5(6)-carboxytetramethylrhodamine N-succinimidyl ester produces DK-12-Rh (MW = 2182.49 Da) (bottom). [Figure 6] FIG. 6 shows the results of an in vitro affinity binding assay comparing the fluorescence of a control peptide (DK12) (rhodamine labeled) with the fluorescence of a fibrin-binding peptide (rhodamine labeled). [Figure 7] Figure 7 shows an overview of the targeted MB. In MBs, a spherical shell was formed by combining various phospholipids, and the interior was filled with a perfluorocarbon gas, preferentially octafluoropropane. Target-binding ligands, including VCAM-1 ligand or FBP (indicated by a green star), were attached to the surface shell of the bubble via a PEG linker. [Figure 8] Figure 8 shows the size distribution of various types of MBs with different FBP-conjugated phospholipids and MPEG control (A) and the number-weighted average of all samples (B). [Figure 9] Figure 9 shows the gas content of MB. The gas content of all four samples was measured by GC. [Figure 10] FIG. 10 shows TEM micrographs of (A) fibrin-binding peptide-targeted microbubbles and (B) fibrin-binding peptide-targeted nanodroplets. [Figure 11] FIG. 11 shows TEM micrographs of (A) fibrin-binding peptide-targeted microbubbles penetrating a fibrin clot, and (B) fibrin-binding peptide-targeted nanodroplets penetrating a fibrin clot. [Figure 12] Figure 12 shows that the VCAM-1 ligand was conjugated to the DSS linker via the N-terminal amine group, and DSPE-PEG2K-amine was conjugated to the other head of the DSS linker to form the VCAM-1-DSPE-PEG2K conjugate. [Figure 13] FIG. 13 shows exemplary fluorescence data regarding the breakdown of a fibrin clot. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides novel microbubbles and nanodroplets, and emulsions thereof, with targeting capabilities for select biomarkers, which are useful as diagnostic probes and therapeutic agents for certain diseases and conditions, particularly thrombosis and arterial plaque. These microbubbles and / or nanodroplets can target various protein targets, such as fibrin and VCAM-1, to improve the detection and / or disruption of blood clots (e.g., thrombi, platelets, and vascular plaque) that occur in many cardiovascular diseases. The targeted microbubbles and / or nanodroplets can be acoustically activated in situ to cause clot disruption. The present invention also provides pharmaceutical compositions and methods of preparation and use thereof.

[0020] A key feature of the present invention is that the acoustically active nanodroplets are nanoscale, typically smaller than the size of microbubbles, e.g., in the range of about 100 nm to about 300 nm, which allows the droplets to penetrate thrombus more easily, significantly improving sonothrombolysis efficiency and clinical efficacy.

[0021] Another key feature of the present invention is the use of low temperature and high pressure to condense fluorocarbon microbubbles (e.g., octafluoropropane microbubbles) into nanodroplets (e.g., octafluoropropane nanodroplets). Despite the boiling point of octafluoropropane (-34°C) being significantly lower than body temperature, the nanodroplets remain condensed after intravenous (IV) administration and subsequently reform into microbubbles after entering an acoustic field.

[0022] Yet another important feature of the present invention is that nanodroplets bearing one or more targeting ligands can be acoustically activated locally in situ. High specificity can be achieved due to the absence of fibrin in circulating blood. The small peptides used herein as targeting ligands exhibit high affinity for fibrin and high selectivity over fibrinogen. These small peptides offer the advantage of faster bloodstream clearance and the ability to penetrate the fibrin network, resulting in improved target-to-background ratios.

[0023] Yet another important feature of the present invention is the unique formulation disclosed herein, which provides the nanodroplets with sufficient enhanced stability for manipulation and handling during preparation, storage, and treatment procedures.

[0024] U.S. Patent No. 9,801,959 B2 and PCT / US19 / 24713, filed March 28, 2019, are incorporated herein by reference in their entirety for all purposes.

[0025] In one aspect, the present invention generally relates to aqueous emulsions or suspensions of microbubbles and / or nanodroplets bearing one or more fibrin-binding ligands.

[0026] In certain embodiments, each of said microbubbles and / or nanodroplets is bound to a plurality of said fibrin-binding ligands.

[0027] In certain embodiments, the one or more fibrin-binding ligands comprise a fibrin-binding peptide having from about 11 to about 16 amino acids.

[0028] In certain embodiments, the fibrin-binding peptide is selected from the Tn6, Tn7, or Tn10 family (Table 1). [Table 1]

[0029] In certain embodiments, the fibrin-binding ligand is attached to the microbubbles and / or nanodroplets via a bifunctional spacer, preferably a polyethylene glycol (PEG) group, and preferably has a number-average molecular weight (MW) in the range of about 1,000 to about 10,000 daltons (e.g., about 2,000 to about 10,000, about 3,000 to about 10,000 daltons, about 4,000 to about 10,000 daltons, about 1,000 to about 8,000 daltons, about 1,000 to about 6,000 daltons, about 3,000 to about 7,000 daltons, or about 4,000 to about 6,000 daltons), and more preferably about 5,000 daltons. The PEG group is covalently attached to a lipid anchor, preferably a phospholipid.

[0030] In certain embodiments, the phospholipid composition comprises dipalmitoylphosphatidylcholine ("DPPC"). DPPC is a zwitterionic compound and a substantially neutral phospholipid. In certain embodiments, the composition comprises a PEGylated lipid.

[0031] Examples of lipids include phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt),

[0033] (ammonium salt), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt). Dipalmitoylphosphatidylethanolamine ("DPPE") is a preferred lipid, preferably present in formulations with other lipids at a concentration of 5 to 20 mole percent, most preferably 10 mole percent.

[0032] In certain embodiments, the microbubbles and / or nanodroplets are filled with a gaseous material.

[0033] In certain embodiments, the gaseous material comprises a fluorinated gas. As used herein, the term "fluorinated gas" refers to a hydrofluorocarbon containing hydrogen, fluorine, and carbon, or a compound containing only carbon and fluorine atoms (also known as a perfluorocarbon), or a compound containing sulfur and fluorine. In the context of the present invention, the term may refer to a material whose molecular structure is composed of carbon and fluorine, or sulfur and fluorine, and which is a gas at room temperature and pressure.

[0034] In certain embodiments, the fluorinated gas is selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof.

[0035] In certain embodiments, the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, and mixtures of two or more thereof.

[0036] In a particular embodiment, the fluorinated gas comprises octafluoropropane.

[0037] In certain embodiments, the aqueous emulsion or suspension further comprises a stabilizer.

[0038] In certain embodiments, the stabilizer is selected from the group consisting of D(+) trehalose dihydrate, propylene glycol, glycerol, polyethylene glycol, glucose, and sucrose.

[0039] In certain embodiments, the gaseous material further comprises an appropriate percentage of a non-fluorinated gas or gas mixture, such as about 2% to about 20% air or nitrogen (e.g., about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 2% to about 15%, about 2% to about 10%, about 2% to about 5% air or nitrogen).

[0040] In certain embodiments, the fluorocarbon within the microbubbles and / or nanodroplets is present in a condensed, i.e., liquid, state.

[0041] In another aspect, the invention generally relates to aqueous emulsions or suspensions of microbubbles and / or nanodroplets bearing one or more VCAM-1 binding ligands.

[0042] In certain embodiments, each of said microbubbles and / or nanodroplets is bound to a plurality of said VCAM-1 binding ligands.

[0043] In certain embodiments, the one or more VCAM-1 binding ligands are VCAM-1 binding peptides having from about 8 to about 16 amino acids.

[0044] In certain embodiments, the VCAM-1 binding peptide is selected from the B2702p1~20 peptide (Table 2). [Table 2]

[0045] In certain embodiments, the VCAM-1 binding ligand is attached to the microbubbles and / or nanodroplets via a PEG linker disclosed herein.

[0046] In certain embodiments, the microbubbles and / or nanodroplets are filled with a gaseous material.

[0047] In certain embodiments, the gaseous material comprises a fluorinated gas.

[0048] In certain embodiments, the fluorinated gas is selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof.

[0049] In certain embodiments, the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, and mixtures of two or more thereof.

[0050] In a particular embodiment, the fluorinated gas comprises octafluoropropane.

[0051] In certain embodiments, the aqueous emulsion or suspension further comprises a stabilizer.

[0052] In certain embodiments, the stabilizer is selected from the group consisting of D(+) trehalose dihydrate, propylene glycol, glycerol, polyethylene glycol, glucose, and sucrose.

[0053] In yet another aspect, the present invention generally relates to aqueous emulsions or suspensions comprising microbubbles and / or nanodroplets bearing one or more fibrin-binding ligands as disclosed herein, and microbubbles and / or nanodroplets bearing one or more VCAM-1-binding ligands as disclosed herein.

[0054] In certain embodiments of the aqueous emulsions or suspensions disclosed herein, the microbubbles and / or nanodroplets are coated with a film-forming material.

[0055] In certain embodiments, the film-forming material comprises one or more lipids.

[0056] In certain embodiments, the lipid comprises a phospholipid or a mixture of phospholipids.

[0057] Any suitable lipid can be utilized. The lipid chain of the lipid can vary in length from about 10 to about 24 carbons (e.g., about 10 to about 20, about 10 to about 18, about 12 to about 20, about 14 to about 20, about 16 to about 20, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24). More preferably, the chain length is about 16 to about 18 carbons in length.

[0058] In some embodiments, the microscale or nanoscale bubbles have a diameter in the range of about 10 nm to about 10 μm (e.g., about 10 nm to about 5 μm, about 10 nm to about 1 μm, about 10 nm to about 500 nm, about 10 nm to about 100 nm, about 50 nm to about 10 μm, about 100 nm to about 10 μm, about 1 μm to about 10 μm). In some embodiments, the microscale or nanoscale particles or bubbles have a diameter of about 10 nm to about 100 nm. In some embodiments, the microscale or nanoscale particles or bubbles have a diameter of about 100 nm to about 1 μm. In some embodiments, the microscale or nanoscale particles or bubbles have a diameter of about 1 μm to about 10 μm.

[0059] In certain embodiments, the microbubbles and / or nanodroplets are microbubbles having a microscale size ranging from about 0.5 to about 10 microns (e.g., about 1 μm to about 10 μm, about 2 μm to about 10 μm, about 5 μm to about 10 μm, about 0.5 μm to about 5 μm, about 0.5 μm to about 2 μm, about 1 μm to about 5 μm).

[0060] In certain embodiments, the microbubbles and / or nanodroplets are nanodroplets having a nanoscale size ranging from about 100 nm to about 800 nm (e.g., from about 100 nm to about 500 nm, from about 100 nm to about 300 nm, from about 120 nm to about 280 nm). In certain embodiments, the microbubbles and / or nanodroplets are nanodroplets having a nanoscale size ranging from about 120 nm to about 280 nm.

[0061] In certain embodiments, the microbubbles and / or nanodroplets do not include microbubbles and / or nanodroplets having a size outside of about 120 nm to about 280 nm (i.e., substantially all microbubbles and / or nanodroplets are nanodroplets having a nanoscale size in the range of about 120 nm to about 280 nm).

[0062] In certain embodiments, the aqueous emulsion or suspension is in homogenized form.

[0063] In certain embodiments, the aqueous emulsion or suspension further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

[0064] In yet another aspect, the present invention generally relates to a method for detecting vascular thrombus or vascular plaque, the method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and imaging a portion of the subject to detect the presence of vascular thrombus or vascular plaque.

[0065] In yet another aspect, the present invention generally relates to a method for diagnosing or assessing thrombosis or atherosclerosis, the method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and imaging a portion of the subject to diagnose or assess thrombosis in the subject.

[0066] In yet another aspect, the present invention generally relates to a method for disrupting or destroying vascular thrombosis or vascular plaque, said method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and applying ultrasound to a target area of ​​an organ of said subject having vascular thrombosis or vascular plaque, thereby disrupting or reducing said vascular thrombosis or vascular plaque.

[0067] In yet another aspect, the present invention generally relates to a method for treating thrombosis, atherosclerosis, or arterial plaque, said method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and applying ultrasound to a target area of ​​said subject.

[0068] In yet another aspect, the present invention generally relates to a method for performing sonothrombolysis, the method comprising administering to a subject in need thereof an aqueous emulsion or suspension disclosed herein, and applying ultrasound to a target area of ​​the subject.

[0069] In certain embodiments of the method, the fluorinated gas comprises perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof.

[0070] In certain embodiments of the method, the fluorinated gas comprises octafluoropropane.

[0071] In certain embodiments of the method, the microbubbles and / or nanodroplets are microbubbles having a microscale size ranging from about 0.5 to about 10 microns.

[0072] In certain embodiments of the method, the microbubbles and / or nanodroplets are nanodroplets having a nanoscale size ranging from about 120 nm to about 280 nm.

[0073] In certain embodiments of the method, the microbubbles and / or nanodroplets do not include microbubbles and / or nanodroplets having a size outside of about 120 nm to about 280 nm (i.e., substantially all of the microbubbles and / or nanodroplets are nanodroplets having a nanoscale size in the range of about 120 nm to about 280 nm). Hereinafter, embodiments of the present invention will be described. Article A-1. an aqueous emulsion or suspension of nanodroplets with one or more fibrin-binding ligands attached thereto via polyethylene glycol (PEG) linkers, the PEG linkers having a number average molecular weight (MW) ranging from about 1,000 to about 10,000 daltons; the nanodroplets are coated with a film-forming material comprising a mixture of phospholipids; the nanodroplets have a size in the range of 100 nm to 300 nm; the nanodroplets are filled with a gaseous material comprising a fluorinated gas selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof; The aqueous emulsion or suspension may further contain stabilizers. Article 2. The aqueous emulsion or suspension described in clause A-1, wherein each of the nanodroplets is bound to a plurality of the fibrin-binding ligands. Article 3. The aqueous emulsion or suspension described in clause A-1, wherein all of the nanodroplets are bound to a plurality of the fibrin-binding ligands. Article A-4. The aqueous emulsion or suspension described in clause A-1, wherein the one or more fibrin-binding ligands are fibrin-binding peptides having 11 to 16 amino acids. Article 5. The fibrin-binding peptides are listed in the following table: [Table 3] The aqueous emulsion or suspension according to clause A-4, selected from: Article A-6. The aqueous emulsion or suspension according to clause A-1, wherein the fluorinated gas comprises octafluoropropane. Article A-7. The aqueous emulsion or suspension according to any one of clauses A-1 to A-6, wherein the stabilizer comprises trehalose. Article A-8. The aqueous emulsion or suspension according to any one of clauses A-1 to A-6, wherein the stabilizer comprises D(+) trehalose dihydrate. Article A-9. An aqueous emulsion or suspension described in any one of clauses A-1 to A-6, wherein the aqueous emulsion or suspension comprises nanodroplets carrying one or more fibrin-binding ligands and nanodroplets carrying one or more VCAM-1-binding ligands. Article A-10. The aqueous emulsion or suspension of any one of clauses A-1 to A-6, wherein the nanodroplets have a nanoscale size in the range of 120 nm to 280 nm. Article A-11. The aqueous emulsion or suspension according to any one of clauses A-1 to A-6, wherein the aqueous emulsion or suspension is in a homogenized form. Article A-12. The aqueous emulsion or suspension according to any one of clauses A-1 to A-6, wherein the aqueous emulsion or suspension further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

[0074] As used herein, "emulsion" refers to a heterogeneous system consisting of at least one immiscible liquid dispersed within another in the form of droplets that can vary in size from nanometers to microns. Emulsion stability varies widely, with emulsions taking anywhere from seconds to years to separate. A suspension may consist of solid particles or droplets in a bulk liquid phase. As an example, an emulsion of dodecafluoropentane can be prepared using a phospholipid or fluorosurfactant, with the conjugate incorporated into the emulsion in a ratio of about 0.1 mole percent to about 1 mole percent, or even up to 5 mole percent, relative to the surfactant used to stabilize the emulsion.

[0075] In certain embodiments, the emulsion or suspension further comprises a pharmaceutically acceptable excipient, carrier, or diluent. Each excipient, carrier, or diluent must be "acceptable" in the sense of being compatible with the other components of the emulsion or suspension and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable excipients, carriers, or diluents include, but are not limited to, normal saline, phosphate buffered saline, propylene glycol, glycerol, and polyethylene glycols, such as PEG400 or PEG3350MW.

[0076] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to a living animal (human or non-human). The subject may be a mammal. The term "mammal" or "mammalian" refers to any animal within the taxonomic category Mammalia. A mammal may be a human or a non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat, and mouse. The term "subject" does not exclude an individual who is completely normal or normal in all respects with respect to a disease or condition.

[0077] As used herein, the terms "treatment" or "treating" a disease or disorder refer to a method of reducing, delaying, or ameliorating such condition, or one or more symptoms of such disease or condition, before or after its onset. Treatment may be directed at one or more effects or symptoms of the disease and / or underlying pathology. The treatment may be any reduction, and may be, but is not limited to, complete ablation of the disease or symptoms of the disease. The degree of such reduction or prevention, as measured by standard techniques, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, compared to an equivalent untreated control. [Example]

[0078] Example 1. Preparation of fibrin-targeted bioconjugates Three conjugation strategies were used to prepare peptide-phospholipid conjugates with different linkers. (1) A fibrin-binding peptide (FBP) with a mini-PEG linker and an azide functional group was directly conjugated with N-[dibenzocyclooctyl(polyethylene glycol-5000)]carbamyl-distearoylphosphatidylethanolamine (ammonium salt) (DSPE-PEG5000-DBCO) to produce a product with a dibenzocyclooctatriazole linker (Scheme 1). (2) A FBP with a mini-PEG linker and an amine functional group was conjugated with [(succinimidyloxyglutaryl)aminopropyl,polyethylene glycol-5000]carbamyl-distearoylphosphatidylethanolamine (sodium salt) (DSPE-PEG5000-NHS ester) to produce a product with an amide linker (Scheme 2). (3) The third strategy consisted of first reacting N-[aminopropyl(polyethylene glycol-5000)]-carbamyl-distearoylphosphatidyl-ethanolamine (sodium salt) (DSPE-PEG5000-amine) with 6,6′-sulfonylbis(1,2,3,4,5-pentafluorobenzene) (PFPhSO2) to produce DSPE-PEG5000-PFPhSO2. The FBP with a miniPEG linker and amine was then coupled with DSPE-PEG5000-PFPhSO2 to generate the product with a perfluorobenzene linker (Scheme 3).

[0079] FIG. 1 shows that an FBP with an azide functionality is coupled with DSPE-PEG5000-DBCO to generate a product with a dibenzocyclooctatriazole linker.

[0080] FIG. 2 shows that an FBP with an amine functionality is coupled with DSPE-PEG5000-NHS ester to produce a product with an amide linker.

[0081] Figure 3 shows that perfluorobiphenyl sulfide was oxidized to produce a more active sulfone derivative, which was then reacted with DSPE-PEG5000-amine to produce DSPE-PEG5000-PFPhSO2. Finally, DSPE-PEG5000-PFPhSO2 was reacted with an amine-containing FBP to yield the conjugated final product.

[0082] All products were purified by high-pressure liquid chromatography (HPLC) and characterized by mass spectrometry (MS) (Figure 4).

[0083] Figure 4 shows that the conjugation of FBP with DSPE-PEG5000-DBCO (A), DSPE-PEG5000-NHS ester (B), and DSPE-PEG5000-PFPhSO2 (C) was confirmed by MS data.

[0084] Example 2. Fibrin-targeted and non-targeted microbubble formulations A mixture of dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE), N-(carbonyl-methoxypolyethylene glycol 5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DPPE-MPEG5000), and DSPE-PEG5000-FBP conjugate was used to formulate targeted microbubbles (MBs) (Figure 2). For nontargeted microbubbles, DSPE-PEG5000-FBP was replaced with N-(carbonyl-methoxypolyethylene glycol 5000)-carbamyldistearoylphosphatidylethanolamine, sodium salt (DSPE-MPEG5000). Vials containing conjugated phospholipids with amide, dibenzocyclooctatriazole, and perfluorobenzene linkers were designated ester, DBCO, and PFPhSO2, respectively. A control sample containing DSPE-MPEG5000 was designated MPEG for the purposes of the experiment.

[0085] Figure 5 shows a schematic diagram of targeted MBs, where various phospholipid combinations form a spherical shell, the interior of which is filled with a perfluorocarbon gas, preferentially octafluoropropane. FBPs (represented by green stars) are attached to the surface shell of the bubbles via a PEG linker.

[0086] All vials containing a mixture of phospholipids in solution were filled with octafluoropropane gas (OFP). Two to four samples from each series of vials were tested for size measurement using a NiComp Acusazie 780 instrument (Figure 8). Our results showed that the ester, DBCO, PFPhSO2, and MPEG samples all formed MBs. However, the size distribution varied for MBs composed of various FBP-conjugated products. Vials containing DBCO and PFPhSO2 samples exhibited a smaller population of bubbles (-10%) with diameters of 0.56–1.06 μm compared to the ester and MPEG vials. In contrast, the DBCO and PFPhSO2 samples exhibited larger populations (7% and >2%) of bubbles with diameters of 1.06–2.03 and 2.03–5.99 μm, respectively, compared to the ester and MPEG vials (Figure 8A). No significant differences were observed in the number-weighted means of the different samples (Figure 8B).

[0087] Figure 8 shows the size distribution of various types of MBs containing various FBP-conjugated phospholipids and MPEG controls (A) and the number-weighted average of all samples (B). The gas content of each series of vials was analyzed by GC using two to four samples from each group (Figure 9).

[0088] Figure 9 shows that the gas content of all four samples was measured by GC. In this experiment, the ester sample showed the greatest percentage of gas content, while the PFPhSO2 and MPEG vials showed the least amount of OFP gas. However, the GC results confirmed that the gas filling process resulted in a gas content of over 80%, which is highly efficient for the formation of MB.

[0089] Example 3. Preparation of VCAM-1-targeted bioconjugates The bioconjugate was prepared by activating the VCAM-1 ligand in the presence of diisopropylamine and dimethylformamide, and then reacting the activated peptide with DSPE-PEG5000-NH2 to form the final product, which was purified by HPLC.

[0090] FIG. 12 shows the preparation of a DSPE-PEG2000-VCAM ligand bioconjugate.

[0091] Example 4. VCAM-1 targeted microbubble formulations The targeted microbubble formulations included dipalmitoylphosphatidylcholine (DPPC), dipalmitoyl-sn-glycerophosphatidylethanolamine-polyethylene glycol-2000-OMe (DPPE-MPEG-2000), and a lipid-ligand bioconjugate composed of either DPPE-PEG2000-NH- or DPPE-PEG2000-C(=O)-ligands linked to the ligand via a suberoyl linker (Sub). The conjugates were used at approximately 1 mol% of the total phospholipid. The microbubbles were prepared by adding DPPC (90 mol%), DPPE-PEG2000 (9 mol%), and the targeted phospholipid-PEG2000-linker-peptide conjugate (1%) to propylene glycol stirred at 50–65°C until the solids were completely dissolved. Next, the warm solution of phospholipids in propylene glycol was added in several portions to a solution of 5% glycerol (volume %) in phosphate-buffered saline (PBS) with stirring at 50–65°C. The solution was stirred for 5–10 minutes. The solution was then transferred to serum vials, immediately stoppered, and crimp-capped. The solution was allowed to warm to ambient temperature and then stored at 4°C. A portion of 25–50 2 mL nominal serum vials was filled with 1.5 mL aliquots of the cooled phospholipid solution, followed by a slight vacuum and purging with perfluorobutane gas. The vials were then quickly stoppered and crimp-capped. The vials were stored at 4°C until use. Once warmed to ambient temperature, they were agitated for 45 seconds at 75 Hz (4500 rpm) on a Bristol Myers Squibb vial mixer to form the microbubbles.

[0092] Example 5. Preparation of nanodroplets A lipid suspension was prepared from a mixture of DPPC (82%), DPPE (10%), DPPE-MPEG5000 (7%), and DSPE-MPEG5000-FBP bioconjugate (1%) at a total lipid concentration of 0.75 mg / mL in propylene glycol (10.35 mg / mL) by heating at 75°C for 1 hour. The lipid suspension was mixed with an aqueous solution of sodium chloride (4.78 mg / mL), monosodium hydrogen phosphate (2.34 mg / mL), disodium hydrogen phosphate (2.16 mg / mL), and glycerol (12.62 mg / mL) to form the final solution. The final solution was used to fill vials (1.5 mL / vial), which were then gassed with perfluoropropane gas before being sealed and crimped. The vials were incubated in an ice bath at -15 to -18°C for 3 minutes. In addition to the aforementioned excipients, 3 w / v% glucose, 0.25 w / v%, 0.5 w / v%, and 1.0 w / v% D(+) trehalose dihydrate were also added as excipients. The vials were agitated for 45 seconds using an amalgam shaker (Vialmix, BMS Medical Imaging Inc., 4500 rpm) until a milky appearance was formed, indicating the formation of microbubbles (MBs). The vials were then incubated in an ice bath at -15 to -18 °C for 3 minutes. The vials were then pressurized with N2 at 40 to 80 psi until a more transparent appearance was formed, indicating the formation of nanodroplets (NDs). The vials were then incubated in an ice bath at -15 to -18 °C for 10 minutes. The vials were then kept at room temperature for 1 hour and then stored under various conditions.

[0093] Microbubbles designated MVT-100 were used as a comparison standard. All samples were subjected to particle sizing using an AccuSizer 780 (PSS. NiComp Particle Sizing Systems) and a Nanobrook 90 Plus (Brookhaven) size analyzer to measure MB and ND sizes, respectively. The average size of MVT-100 MB and fibrin-targeted MB was 1-3 microns. The results are shown in the table below. The average size of nanodroplets derived from MVT-100 rapidly increased and then decreased as the perfluoropropane gas was lost from the nanodroplets. 3% glucose provided a protective effect, but not as much as D(+) trehalose dihydrate. 1% D(+) trehalose dihydrate was preferred because it resulted in nanodroplets that were stable for 24 hours.

[0094] Example 6. Disintegration of fibrin clots by FTMB All wells of a 24-well plate were coated with fibrin by adding fibrinogen and thrombin and leaving the plate overnight. Briefly, 160 μL of fibrinogen (1.75 μM in PBS) was added to each well in the presence of 30 μM thioflavin. Subsequently, 40 μL of thrombin (7.5 units / mL in PBS) was added to each well. The plate was incubated overnight at room temperature in the dark. Fibrin clots were visualized under a contrast phase microscope. [Table 4] [Table 5]

[0095] MB was activated (Vial Mix vortex, 45 seconds). The final stock solution of each MB formulation was made at 500 μL in 5.2 mL PBS. The fibrin-coated wells were washed with PBS (1.0 mL x 1) before adding MB to the wells. MB was incubated in the fibrin-coated wells for 3 minutes.

[0096] Ultrasound was delivered to each well for 30 seconds (parameters: 2000 mW, PRF 10, 10 ms burst length, frequency 590 Hz).

[0097] The supernatant was collected and spun down at 10,000 rpm for 15 minutes at room temperature. The emitted fluorescence was measured in a dark 96-well plate. Thioflavin fluorescence was measured at 485 nm (λ excitation = 450 nm; λ emission = 485 nm).

[0098] In one example, the amplifier power level reading was 2,000 mW, while the power meter on the line with the transducer read approximately 100 mW. The estimated mechanical index of the ultrasound was approximately 0.28 MPa (Figure 9).

[0099] In another example, an MI of ultrasound greater than 0.40 megapascals is used for sonothrombolysis of the ND.

[0100] Example 8 Patients with acute STEMI were treated with nanodroplet-enhanced sonothrombolysis. The nanodroplet formulation contained MVT-100 + 1% D(+) trehalose dihydrate, which was formed via a proprietary cooling / pressurization process. The patient received intravenous administration of the nanodroplets (4 mL administered over a 30-minute infusion period during concurrent ultrasound treatment). The ultrasound protocol used was as described by Mathias (Mathias, Wilson, et al. 2016 J. Am. Coll. Cardiol. 67.21:2506-2515). Image-guided diagnostic high-mechanical index ultrasound (1.8 MHz; 1.1-1.3 mechanical index; 3 ms pulse duration) was applied, impinging on the apical four-, two-, and three-chamber views, including the at-risk area of ​​the myocardium. Following sonothrombolysis, the patient was treated with conventional angioplasty and stent placement. Thirty days after treatment, myocardial flow is improved and left ventricular ejection fraction is improved.

[0101] Example 9. Another patient with acute STEM is treated with fibrin-targeted nanodroplets using ultrasound parameters similar to those described in Example 1. Clearly, coronary revascularization is achieved more rapidly with the targeted nanodroplets than with non-targeted nanodroplets.

[0102] Example 10. Patients with acute ischemic stroke receive an IV infusion of three vials (6 mL total) of fibrin-targeted nanodroplets over a 60-minute period, concurrent with an IV infusion of t-PA. Ultrasound is applied throughout the time window using a 1 MHz probe at MU=1.0 for the same duration as the co-infusion of t-PA and nanodroplets. Blood flow is rapidly restored to the middle cerebral artery.

[0103] Example 11 The patient has extensive plaque in the left anterior descending coronary artery, causing a 90% blockage of the LAD. The patient receives an IV infusion of 6 mL of VCAM-1-targeted nanodroplets while undergoing ultrasound as in Example 1. This reduces the plaque and improves coronary blood flow.

[0104] Example 12 A patient has an acute peripheral arterial occlusion in the lower extremity. A clot is localized in the femoral artery, causing loss of blood flow to the leg. An IV infusion of fibrin-targeting nanodroplets is initiated. Ultrasound is applied percutaneously to the area of ​​the arterial occlusion using a 3D ultrasound transducer with a center frequency of 2 MHz, pulsed at 1.6 megapascals and an applied power of 2 seconds on, 2 seconds off. Simultaneously, the nanodroplets are infused IV for 2 hours at a rate of 2.0 cc / hour. The arterial occlusion is relieved and blood flow is restored to the leg.

[0105] Applicant's disclosure is described herein in preferred embodiments with reference to the figures, in which like numbers represent the same or similar elements. References throughout this specification to "one embodiment," "an embodiment," or similar language mean that a particular feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0106] The features, structures, or characteristics described in the applicant's disclosure may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present invention. However, one skilled in the relevant art will recognize that the applicant's compositions and / or methods may be practiced without one or more specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring aspects of the disclosure.

[0107] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0108] As used herein, unless specifically stated or clear from the context, the term "about" is understood to be within normal tolerances in the art, for example, within two standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless clear from the context, all numerical values ​​provided herein can be modified with the term about.

[0109] As used herein, unless specifically stated otherwise or clear from context, the term "or" is understood to be inclusive.

[0110] When used to define compositions and methods, the term "comprising" is intended to mean that the composition and method include the recited elements but do not exclude other elements. When used to define compositions and methods, the term "consisting essentially of" is intended to mean that the composition and method include the recited elements and exclude any other elements of essential importance to the composition and method. For example, "consisting essentially of" refers to the administration of pharmacologically active agents that are expressly recited and excludes pharmacologically active agents that are not expressly recited. The term "consisting essentially of" does not exclude pharmacologically inactive or non-pharmacologically active agents, such as pharmaceutically acceptable excipients, carriers, or diluents. When used to define compositions and methods, the term "consisting of" is intended to mean excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Although any method and material similar or equivalent to those described herein can also be used to implement or test this disclosure, preferred methods and materials are described herein.The methods described herein can be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0112] Incorporation by Reference This disclosure references and cites other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated herein by reference but that conflicts with existing definitions, descriptions, or other disclosed material expressly set forth herein is incorporated to the extent that no inconsistency arises between the incorporated material and the disclosed material. In the event of a inconsistency, the inconsistency should be resolved in favor of the present disclosure as the priority disclosure.

[0113] equivalent The representative examples are intended to aid in illustrating the invention and are not intended to, and should not be construed as, limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the examples and references to scientific and patent literature contained herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

[Claim 1] An aqueous emulsion or suspension of microbubbles and / or nanodroplets with one or more fibrin-binding ligands attached.

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