Compositions and methods for detecting and treating alzheimer's disease

Abstract

To provide microbubbles and / or nanodroplets labeled with diagnostic and / or therapeutic ligands useful for the detection and treatment of Alzheimer's disease or related diseases and conditions, and to provide emulsions thereof, and methods of preparing and using them.SOLUTION: Provided is a microscale bubble and / or nanoscale droplet conjugated to one or more first ligands having a binding affinity to beta amyloid or tau protein, and one or more second ligands capable of degrading or otherwise metabolizing beta amyloid or tau protein, the first ligand being selected, for example, from the structures shown in the figures, and the second ligand being an enzyme or antibody, or a fragment thereof, selected from the group consisting of insulin-degrading enzyme (IDE), neprilysin (NEP), endothelin-converting enzyme (ECE) and the like, and antibodies against tau and amyloid beta.SELECTED DRAWING: Figure 1b

Description

[Technical field]

[0001] Priority claims and related patent applications This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 650,239, filed March 29, 2018, the entire contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] The present invention relates to pharmaceutical compositions and methods for their preparation and use in diagnosis and therapy. More particularly, the present invention relates to microbubbles and / or nanodroplets, and emulsions thereof, labeled with diagnostic and / or therapeutic ligands useful for the detection and treatment of Alzheimer's disease, or related diseases and conditions, and methods for their preparation and use.

[0003] 2. Background of the Invention Alzheimer's disease (AD) is an irreversible, progressive neurodegenerative disease that gradually destroys memory, thinking, and ultimately the ability to perform the simplest tasks. Over 30 million people worldwide suffer from AD. AD currently ranks as the sixth leading cause of death in the United States, accounting for 60% to 70% of dementia cases. Patients in the advanced stages of the disease suffer from symptoms that often include language problems, disorientation, withdrawal from family and society, and other behavioral issues, eventually leading to loss of physical function and ultimately death. Properly diagnosing AD requires a process that includes a thorough examination and a series of clinical evaluations and eliminations.

[0004] AD is characterized by the accumulation of amyloid plaques between nerve cells (neurons) in the brain. Beta amyloid (or amyloid beta, Aβ) is a peptide of 36-43 amino acids that is implicated in AD as the main component of the amyloid plaques found in the brains of AD patients. Beta amyloid is derived from the amyloid precursor protein (APP), which is cleaved by beta and gamma secretases to generate beta amyloid. Beta amyloid molecules can aggregate to form flexible soluble oligomers that can exist in several forms. Studies have shown that certain misfolded oligomers can induce other beta amyloid molecules to also adopt misfolded oligomeric forms, potentially setting off a chain reaction similar to prion infection. The oligomers are toxic to nerve cells. (Hamley 2012 Chemical Reviews 112(10):5147-92; Haass et al. 2007 Nature Reviews Molecular Cell Biology 8(2):101-12.)

[0005] Another protein implicated in AD is the tau protein (or tau protein), which also forms such prion-like misfolded oligomers. Studies have shown that misfolded beta-amyloid can cause tau to misfold. AD pathology is associated with tau proteins that have become defective and no longer properly stabilize microtubules. (Nussbaum et al. 2013 Prion.(1):14-9; Pulawski et al. 2012 Applied Biochemistry and Biotechnology 166(7):1626-43.)

[0006] No drugs have been clearly shown to slow or halt the progression of AD. Several drugs are currently used to treat the cognitive problems of AD, including acetylcholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists, but the benefit of their use is very limited.

[0007] Thus, there remains an urgent need and a significant challenge for novel, safe and reliable diagnostic tools and therapeutic agents for AD. Summary of the Invention

[0008] The present invention is based in part on the discovery of novel microbubbles and nanodroplets, and emulsions thereof, which are designed to target beta amyloid and / or tau proteins for improved detection of AD by ultrasound.The present invention is also based in part on the discovery of novel microbubbles and / or nanodroplets, and emulsions thereof, which are designed to target beta amyloid and / or tau proteins for improved treatment of AD by ultrasound.The present invention further relates to pharmaceutical compositions and methods of preparation and use thereof.

[0009] The targeted microbubbles and / or nanodroplets can be acoustically activated and they have at least one, preferably two (or more) ligands. The first ligand is a motif that binds to beta amyloid or tau protein for detection and localization purposes. The second ligand may include a second different ligand and / or an enzyme for degrading beta amyloid or tau protein. The present invention detects and increases the efflux of misfolded and / or aggregated beta amyloid and / or tau protein from the brain to treat AD.

[0010] In one aspect, the invention generally relates to microscale gas bubbles (microbubbles) or nanoscale liquid droplets (nanodroplets) (sometimes referred to as "microscale or nanoscale gas / droplets" or "microscale gas bubbles and / or nanoscale droplets") conjugated with one or more first ligands having binding affinity for beta amyloid and one or more second ligands capable of degrading or otherwise metabolizing beta amyloid.

[0011] In another aspect, the invention generally relates to micro- or nano-scale bubbles / droplets conjugated with one or more first ligands having binding affinity to tau protein and one or more second ligands capable of degrading or otherwise metabolizing tau protein.

[0012] In yet another aspect, the invention generally relates to aqueous emulsions or suspensions containing the disclosed microscale or nanoscale gas / droplets.

[0013] In yet another aspect, the present invention generally relates to a method for detecting beta amyloid, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale or nanoscale gas / droplets disclosed herein, and imaging a portion of the subject to detect the presence of beta amyloid.

[0014] In yet another aspect, the present invention generally relates to a method for detecting tau protein, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale or nanoscale bubbles / droplets disclosed herein, and imaging a portion of the subject to detect the presence of tau protein.

[0015] In yet another aspect, the present invention generally relates to a method for diagnosing or assessing risk of Alzheimer's disease, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale or nanoscale bubbles / droplets disclosed herein, and imaging a portion of the subject to diagnose or assess Alzheimer's disease in the subject.

[0016] In yet another aspect, the present invention generally relates to a method for treating Alzheimer's disease, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale or nanoscale bubbles / droplets disclosed herein, and applying ultrasound to a target region of the subject's brain. [Brief description of the drawings]

[0017] [Figure 1a] FIG. 1a shows exemplary chemical structures of ligands that bind to aggregated tau aggregates or Aβ plaques. [Figure 1b] FIG. 1b shows exemplary chemical structures of ligands that bind to aggregated tau aggregates or Aβ plaques.

[0018] [Diagram 2] FIG. 2 shows exemplary chemical structures of PEGylated phospholipids with reactive functional groups, DSPE-PEGn-NHS ester (A) and DSPE-PEGn-DBCO.

[0019] [Diagram 3] Figure 3 shows an exemplary chemical reaction between ligands with binding affinity to tau aggregates or to Aβ plaques and phospholipids: an amine group in a small molecule reacts with an NHS-ester group to generate an amide linker (A), and an azide group in a small molecule reacts with an alkyne group in dibenzylcyclooctyne (DBCO) via copper-free click chemistry to generate a triazole linker (B).

[0020] [Figure 4] FIG. 4 shows that an exemplary DSPE-PEG-ligand conjugate is incorporated into a microbubble formulation to generate targeted microbubbles for the detection of tau aggregates or Aβ plaques in Alzheimer's disease.

[0021] [Diagram 5]FIG. 5 shows that exemplary proteins such as insulin degrading enzyme (IDE), neprilysin (NEP), endothelin converting enzyme (ECE), angiotensin converting enzyme (ACE), plasmin, matrix metalloproteinase (MMP), phosphatase, alkaline phosphatase (AP) as well as antibodies against tau and amyloid beta are conjugated to DSPE-PEG-NHS ester via lysine or to DSPE-PEG-maleimide and conjugated to cysteine ​​amino acids within their structures.

[0022] [Figure 6] Figure 6 shows that exemplary DSPE-PEG-ligands and DSPE-PEG-enzymes are incorporated into microbubble formulations to generate targeted microbubbles carrying the enzymes. Nanoscale droplets made from MBs localize the enzymes to areas where tau aggregates or Aβ plaques form, accelerating the degradation and clearance of these proteins.

[0023] [Figure 7] FIG. 7 shows an exemplary size analysis of targeted microbubbles.

[0024] [Figure 8] FIG. 8 shows an exemplary size analysis of the targeted nanodroplets.

[0025] [Figure 9] Figure 9 shows exemplary data on the effect of MB, targeted MB and targeted nanodroplets on tau aggregates. MB means the use of microbubbles only, MB+US means the use of MB with ultrasound, t2CMB+US means the use of targeted MB with compound 2C and ultrasound. Similarly for 4C and 4A. t2CND+US means the use of targeted ND with compound 2C (n=3 samples / condition, bars are averages, error bars are standard error). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention provides novel constructs of micro- and / or nano-bubbles / droplets and their emulsions targeting beta-amyloid and tau proteins for superior detection and treatment of Alzheimer's disease using ultrasound.

[0027] Ultrasound has been used to open the blood-brain barrier. (U.S. Patent No. 5,752,515.) Microbubbles lower the cavitation threshold, facilitating the opening of the blood-brain barrier. In Alzheimer's disease mode, microbubbles are used with ultrasound to open the blood-brain barrier and facilitate the entry of antibodies against beta-amyloid. (Jordao, et al. 2010 PloS one 5.5, el0549.)

[0028] In one aspect, the present invention generally relates to micro- or nano-scale bubbles / droplets that are conjugated to one or more first ligands having binding affinity for beta amyloid, and one or more second ligands capable of degrading or otherwise metabolizing beta amyloid.

[0029] In another aspect, the invention generally relates to micro- or nano-scale bubbles / droplets that are conjugated to one or more first ligands having binding affinity to tau protein and one or more second ligands capable of degrading or otherwise metabolizing tau protein.

[0030] In certain embodiments, said first ligand is a compound listed in FIG. 1 (referring to FIGS. 1a and 1b) or a derivative thereof.

[0031] In certain embodiments, the second ligand is an enzyme or an antibody, or a fragment thereof.

[0032] In certain embodiments, each microscale or nanoscale bubble / droplet is conjugated to a plurality of said first ligands.

[0033] In certain embodiments, each microscale or nanoscale bubble / droplet is conjugated to a plurality of said second ligands.

[0034] In certain embodiments, the first ligand is conjugated to the microscale or nanoscale bubble / droplet via a PEG linker.

[0035] In certain embodiments, the second ligand is conjugated to the microscale or nanoscale bubble / droplet via a PEG linker.

[0036] In certain embodiments, the microscale or nanoscale bubbles / droplets are filled with gas and / or liquid material. In certain embodiments, the microscale or nanoscale bubbles / droplets are filled with gas material. In certain embodiments, the microscale or nanoscale bubbles / droplets are filled with liquid material.

[0037] In certain embodiments, the gaseous material comprises a fluorinated gas. The term "fluorinated gas" as used herein refers to hydrofluorocarbons, which contain hydrogen, fluorine and carbon, or compounds that contain only carbon and fluorine atoms (also known as perfluorocarbons), as well as compounds that contain sulfur and fluorine. In the context of the present invention, this term may refer to materials whose molecular structure consists of carbon and fluorine, or sulfur and fluorine, and that are gases at normal temperature and pressure.

[0038] 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.

[0039] In certain embodiments, the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, and mixtures of two or more thereof.

[0040] In certain embodiments, the gaseous material further comprises a suitable percentage of a non-fluorinated gas or gas mixture, for example, 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).

[0041] In certain embodiments, the fluorocarbon within the microscale or nanoscale bubbles / droplets is present in a condensed, i.e., liquid, state.

[0042] In certain embodiments, the micro- or nano-scale bubbles / droplets are coated with a film-forming material. In certain embodiments, the film-forming material comprises one or more lipids. In certain embodiments, the lipid comprises a phospholipid or a mixture of phospholipids.

[0043] Any suitable lipid may be utilized. The lipid chain of the lipid may vary in length from about 10 to about 24 (e.g., from about 10 to about 20, from about 10 to about 18, from about 12 to about 20, from about 14 to about 20, from about 16 to about 20, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) carbons. More preferably, the carbon chain length is from about 16 to about 18.

[0044] In some embodiments, the microscale or nanoscale bubbles / droplets of the present invention are capable of degrading and / or metabolizing both beta amyloid, tau protein.

[0045] In some embodiments, the microscale or nanoscale bubbles have a diameter ranging from 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 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.

[0046] As used herein, the terms "microscale" and "nanoscale" refer to microbubble sizes in the micrometer and nanometer ranges, respectively.

[0047] In certain embodiments of the methods disclosed herein, the microscale or nanoscale bubbles / droplets have a microscale size ranging from about 0.5 μm to about 10 μm (e.g., from about 1 μm to about 10 μm, from about 2 μm to about 10 μm, from about 5 μm to about 10 μm, from about 0.5 μm to about 5 μm, from about 0.5 μm to about 2 μm, from about 1 μm to about 5 μm).

[0048] In certain embodiments of the methods disclosed herein, the microscale or nanoscale bubbles / droplets have 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).

[0049] In another aspect, the invention generally relates to aqueous emulsions or suspensions containing the disclosed microscale or nanoscale gas / droplets.

[0050] As used herein, "emulsion" refers to a heterogeneous system consisting of at least one immiscible liquid dispersed in another in the form of droplets that can vary in size from nanometers to microns. Emulsion stability varies widely, and the time until the emulsion separates can be from a few seconds to years. The suspension can consist of solid particles or droplets in a bulk liquid phase. As an example, an emulsion of dodecafluoropentane can be prepared with a phospholipid or fluorosurfactant, and the complex can be incorporated into the emulsion in a ratio of about 0.1 mole percent to about 1 mole percent, or even 5 mole percent, relative to the surfactant used to stabilize the emulsion.

[0051] In certain embodiments, the emulsion or suspension further comprises a pharma- ceutically acceptable excipient, carrier, or diluent. Each excipient, carrier, or diluent must be "acceptable" in the sense that it is compatible with the other components of the emulsion or suspension and is not harmful to the patient. Some examples of materials that can serve as pharma-ceutically acceptable excipients, carriers, or diluents include, but are not limited to, normal saline, phosphate buffered saline, propylene glycol, glycerol, and polyethylene glycol, such as PEG400 or PEG3350MW.

[0052] In certain embodiments, the emulsion or suspension is in homogenized form.

[0053] As used herein, "homogenized" refers to an emulsion or suspension prepared in the form of vigorous mixing. Homogenization can be accomplished by any of several processes used to make a mixture of two mutually insoluble liquids uniform throughout. This is generally accomplished by turning one liquid into a state consisting of very small particles uniformly distributed throughout the other liquid. Homogenization is usually accomplished using equipment such as ultra turrax type, ultrasonic probe mixer / homogenizer, or high pressure homogenizer, which emulsifies or suspends the components of the mixture by forcing them through small openings or valves whose internal size can be adjusted under high pressure.

[0054] In yet another aspect, the present invention generally relates to a method for detecting beta amyloid, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale or nanoscale bubbles / droplets disclosed herein, and imaging a portion of the subject to detect the presence of beta amyloid.

[0055] In yet another aspect, the present invention generally relates to a method for detecting tau protein, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale or nanoscale bubbles / droplets disclosed herein, and imaging a portion of the subject to detect the presence of tau protein.

[0056] In yet another aspect, the present invention generally relates to a method for diagnosing or assessing risk of Alzheimer's disease, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing microscale or nanoscale bubbles / droplets as disclosed herein, and imaging a portion of the subject to diagnose or assess Alzheimer's disease in the subject.

[0057] In yet another aspect, the present invention generally relates to a method for treating Alzheimer's disease, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale or nanoscale bubbles / droplets disclosed herein, and applying ultrasound to a target region of the subject's brain.

[0058] In yet another aspect, the present invention generally relates to a method for disrupting or reducing beta amyloid aggregates, comprising administering to a subject in need thereof an aqueous emulsion or suspension containing the microscale bubbles and / or nanoscale droplets disclosed herein, and applying ultrasound to a target area of ​​an organ of the subject having the beta amyloid aggregates, thereby disrupting or reducing the beta amyloid aggregates.

[0059] In yet another aspect, the present invention generally relates to a method for disrupting or reducing tau protein aggregates, comprising administering to a subject in need thereof an aqueous emulsion or suspension comprising the microscale bubbles and / or nanoscale droplets disclosed herein, and applying ultrasound to a target area of ​​an organ of the subject having tau protein aggregates thereby disrupting or reducing those tau protein aggregates.

[0060] In certain embodiments, the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, and mixtures of two or more thereof. In certain embodiments of the methods disclosed herein, the fluorinated gas comprises perfluoropropane, perfluorobutane, or perfluoropentane, or mixtures of two or more thereof.

[0061] In certain embodiments of the methods disclosed herein, the microscale or nanoscale bubbles / droplets have a microscale size ranging from about 0.5 μm to about 10 μm (e.g., from about 1 μm to about 10 μm, from about 2 μm to about 10 μm, from about 5 μm to about 10 μm, from about 0.5 μm to about 5 μm, from about 0.5 μm to about 2 μm, from about 1 μm to about 5 μm).

[0062] In certain embodiments of the methods disclosed herein, the microscale or nanoscale bubbles / droplets have 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).

[0063] 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 classification of mammals. 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 individuals who are completely normal or normal in all respects with respect to a disease or condition.

[0064] As used herein, the term "treatment" of a disease or disorder or "treating" a disease or disorder refers to a method of alleviating, delaying, or ameliorating such condition or one or more symptoms of such disease or condition before or after it occurs. Treatment may be directed at one or more effects or symptoms of the disease and / or underlying pathology. Said treatment may be any alleviation, and may be, but is not limited to, complete elimination of the disease or symptoms of the disease. The degree of such alleviation or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique, compared to an equivalent untreated control.

[0065] As shown in Figures 1-5, one or more ligands are selected that bind to beta amyloid and / or tau proteins. The ligands are attached to a bifunctional spacer, preferably a polyethylene glycol (PEG) group, and the PEG group preferably has a number average molecular weight (MW) in the range of about 1,000 to about 10,000 daltons (e.g., about 2000 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, about 4,000 to about 6,000 daltons), and more preferably about 5,000 daltons.

[0066] Enzymes or antibodies can be used as the second ligand. Preferred enzymes aid in the metabolism of beta amyloid and / or tau protein. The second ligand is also preferably linked via a bifunctional spacer, preferably PEG, and the PEG has a MW of about 1,000 to about 10,000 daltons (e.g., about 2000 to about 10,000, about 3000 to about 10,000 daltons, about 1,000 to 6,000 daltons, about 1,000 to about 5,000 daltons, about 1,000 to about 4,000 daltons), more preferably about 1,000 to about 2,000 daltons.

[0067] As shown in Figures 2-5, the PEG is covalently attached to a lipid anchor, preferably a phospholipid.

[0068] In certain embodiments, the phospholipid composition comprises dipalmitoylphosphatidylcholine ("DPPC"), phospholipid 1. DPPC is a zwitterionic compound and is a substantially neutral phospholipid. In certain embodiments, the phospholipid composition comprises a polyhydroxy head group, and / or a Na + , K + , Li + , and NH4 +and a second phospholipid 2 comprising a head group of greater than 350 daltons with a counterion. In certain embodiments, the phospholipid 2 comprises a phospholipid 3 comprising a sodium cation and a glycerol head group attached to a phosphoryl moiety. Phospholipid 4 comprises an ammonium counterion and a polyethylene glycol ("PEG") head group attached to a phosphoryl moiety. In certain embodiments, the composition comprises a PEGylated lipid. In certain embodiments, the MW of the PEG group is about 1,000 to about 10,000 daltons. In certain embodiments, the PEG group MW is about 2,000 to about 5,000 daltons. In certain embodiments, the PEG group MW is about 5,000 daltons.

[0069] 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), Examples of phospholipids include 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). Phospholipid 5 stands for dipalmitoyl phosphatidylethanolamine, or DPPE. PE, especially DPPE, is the preferred lipid in the present invention, preferably in formulations containing other lipids at a concentration between 5 and 20 mole percent, most preferably at a concentration of 10 mole percent.

[0070] Fluorocarbons for use as gaseous precursors in the compositions of the present invention include partially or fully fluorinated carbons, preferably saturated, unsaturated or cyclic perfluorocarbons.Preferred perfluorocarbons include, for example, perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures thereof.More preferably, the perfluorocarbon is perfluorohexane, perfluoropentane, perfluoropropane or perfluorobutane. EXAMPLES

[0071] Example 1 Preparation of complexes [ka] Scheme 1: Preparation of the conjugate: Example of compound 2C: DSPE-PEG-5K-DBCO (A) and AD-2C (B) were dissolved in acetonitrile (ACN) and reacted at room temperature overnight in a molar ratio of 1 (A):3 (B). The products were purified and separated using a Sep-Pack column. The same procedure was used to synthesize compounds 4A (D) and 4C (E). Preparation of microbubbles:

[0072] The bioconjugates (1% molar ratio) were mixed separately with DPPC (82% molar ratio), DPPE (10% molar ratio), and DPPE-MPEG-5K (7% molar ratio) to generate microbubbles. The phospholipids were dissolved in propylene glycol while heating to 75 °C for 30 min. This solution was added to the salts contained in the MVT-100 formulation. The final solution was dispensed into vials (1.5 mL each) and gassed with octafluoropropane (OFP). Microbubble Size Analysis:

[0073] The vials were activated and analyzed for microbubbles concentration and size distribution. [Table 1]

[0074] An exemplary size analysis of targeted microbubbles is shown in FIG. Preparation of nanodroplets

[0075] The vial containing the microbubble formulation was cooled in a cold bath (-15 to -18°C) for 3 min. The microbubbles were then activated and cooled in a cold bath (-15 to -18°C) for 3 min. Nitrogen (40 to 80 psi) was pumped into the vial until the milky white color of the solution turned cloudy. The vial was kept in the cold bath (-15 to -18°C) for 10 min and then at RT for 1 h. Size analysis of nanodroplets

[0076] Size analysis of the nanodroplets showed the samples to have effective diameters between 170 and 250 nm.

[0077] An exemplary size analysis of the targeted nanodroplets is shown in FIG. Effect of microbubbles / nanodroplets on tau protein aggregates in vitro

[0078] Tau protein forms aggregates in the presence of heparin. Thioflavin T ( k Excit =450nmA. Cmis Fluorescent probes such as tau (Tau(K18)P301L mutant preformed fibrils and protein monomers; 2 mg / mL) bind to tau. 24-well plates were incubated with tau protein (Tau(K18)P301L mutant preformed fibrils and protein monomers; 2 mg / mL) and heparin 0.03 M in aggregation buffer pH 7.4, Tris 20 mM, NaCl 100 mM, EDTA 1 mM, and incubated at 37 °C for 3-4 days in the presence of DTT 1 μM.

[0079] Figure 9 shows exemplary results for the effect of microbubbles (MB), targeted MB, and targeted nanodroplets on tau aggregates, where MB means the use of microbubbles only, MB+ultrasound (US) means the use of MB with ultrasound, t2CMB+US means the use of targeted MB with compounds 2C and ultrasound, and similarly for 4C and 4A. t2CND+US means the use of targeted ND with compound 2C (n=3 samples / condition, bars are the mean, error bars are standard error).

[0080] Each well was filled with 1.5 mL of saline and incubated with 200 μL of microbubbles or nanodroplets for 1 min. The ultrasonic conditions applied to each well were as follows: 10% duty cycle, 5000 mW, frequency 590 Hz, 30 s cycle (Sonic Concepts, TPO-200-02). Following application of ultrasonic waves (or mock application) to the wells, each content was transferred to an Eppendorf tube and centrifuged at 10,000 rpm for 25 min at room temperature. The middle liquid phase was dispensed (200 pL / well) into a black ELISA plate for fluorescence measurement at 480 nm. Fluorescence from protein aggregates was measured as it was released upon disruption of the aggregates.

[0081] Results show that microbubbles and ultrasound disrupt tau aggregates, but tau-targeted microbubbles and nanodroplets induce a much greater effect. In vitro experiments support the concept that ultrasound can be used in conjunction with tau-targeted microbubbles and nanodroplets to treat AD. Example 2

[0082] For a detailed description of the production of microbubbles, see, for example, US Pat. No. 9,801,959 B2.

[0083] The lipid blend was prepared by suspending a mixture of lipids, including DPPC and DPPE-MPEG-5000, DPPE, and DSPE-PEG5K-conjugates, in propylene glycol. The lipids suspended in propylene glycol were heated to 70±5°C until they dissolved. The resulting lipid solution was then added to an aqueous solution containing sodium chloride, phosphate buffer, and glycerol and mixed thoroughly by stirring. Each ml of the resulting lipid blend contained 0.75 mg of total lipids (consisting of 0.39 mg DPPC, 0.046 mg DPPE, 0.26 mg MPEG-5000-DPPE, and 0.05 mg DSPE-PEG5K-conjugates). Additionally, each mL of the lipid blend contained 103.5 mg propylene glycol, 126.2 mg glycerin, 2.34 mg sodium phosphate monobasic monohydrate, 2.16 mg sodium phosphate dibasic heptahydrate, and 4.87 mg sodium chloride in water for injection. The pH was 6.2-6.8. One mole percent of the complex shown in Figure 3a was added to the lipid suspension. The material was provided in a sealed vial with a headspace containing octafluoropropane (OFP) gas (>80%) with the remainder air. The vial was activated using a Vialmix modified dental amalgamator to generate microbubbles targeting beta amyloid / tau proteins. Example 3

[0084] The above was essentially repeated except that separate formulations were prepared using the conjugates shown in Figures 4 and 5. Example 4

[0085] A lipid suspension containing the complex was prepared as in Example 2. Microbubbles were formed by agitation by shaking for 45 seconds. A 2 mL vial containing the formed microbubbles was then immersed in a cold bath controlled at a temperature of about -15°C. Nitrogen gas (40-120 psi) was injected into the vial septum with a needle. Freezing of lipids was avoided by periodically observing the temperature of the vial contents and the cold bath solution. After pressurization with nitrogen gas, the needle was removed from the vial, leaving a pressure head on the solution. The vial was kept in the cold bath for 10-20 minutes and at room temperature for 10-120 minutes. Particle sizing was performed on the microbubbles prepared in Example 2 and the nanodroplets from Example 4. The average diameter of the microbubbles was about 1-2 microns, and the particle size of the nanodroplets was about 200 nanometers. Example 5

[0086] Brain imaging is performed with PET showing that tau is deposited into beta amyloid aggregates. The nanodroplets from Example 4 were 10×10 9 A dose of nanodroplets is administered intravenously to a patient with AD and focused ultrasound energy is applied to the brain at 1 Mhz and MI=1.6. The energy is pulsed at a frequency of 60 Hz. After treatment, PET imaging is repeated to show a reduction in tau protein deposits.

[0087] 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 an 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.

[0088] The described features, structures, or characteristics of the applicant's disclosure may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are recited 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 can 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 present disclosure.

[0089] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0090] As used herein, unless specifically stated or clear from the context, the term "about" is understood to be within normal tolerances in the art, e.g., within 2 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 otherwise clear from the context, all numerical values ​​provided herein can be modified with the term about.

[0091] As used herein, unless specifically stated otherwise or clear from the context, the term "or" is understood to be inclusive.

[0092] The term "comprising," when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. The term "consisting essentially of," when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements and exclude other elements of any essential importance to the compositions and methods. 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 inert agents, such as pharma- ceutical acceptable excipients, carriers, or diluents. The term "consisting of," when used to define compositions and methods, is intended to mean that it excludes 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.

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Any method and material similar or equivalent to those described herein can also be used to carry out or test this disclosure, but preferred method and material are described here.The methods listed herein can be carried out in any order that is logically possible, in addition to the specific order disclosed. Incorporation by Reference

[0094] In this disclosure, references and citations are made to other documents, such as patents, patent applications, patent publications, journals, books, papers, 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, references, or other disclosure material explicitly set forth herein is incorporated only to the extent that no conflict occurs between the incorporated material and the disclosure material of this application. In the event of a conflict, the conflict should be resolved in favor of the present disclosure as the preferred disclosure. sword etc.

[0095] The representative examples are intended to help illustrate 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 the 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. The embodiments of the present invention are listed below. (1) Microscale bubbles and / or nanoscale droplets that are conjugated to one or more first ligands having binding affinity for beta amyloid, and one or more second ligands capable of degrading or otherwise metabolizing beta amyloid. (2) Microscale bubbles and / or nanoscale droplets that are conjugated to one or more first ligands having binding affinity for tau protein and one or more second ligands capable of degrading or otherwise metabolizing tau protein. (3) The microscale bubbles and / or nanoscale droplets of (1) or (2), wherein the second ligand is an enzyme or an antibody, or a fragment thereof. (4) The microscale bubbles and / or nanoscale droplets of any one of (1) to (3), wherein the first ligand is a compound listed in FIG. 1 or a derivative thereof. (5) The microscale bubbles and / or nanoscale droplets of any one of (1) to (4), wherein each of the microscale bubbles and / or nanoscale droplets is conjugated to a plurality of the first ligands. (6) The microscale bubbles and / or nanoscale droplets of any one of (1) to (5), wherein each microscale bubble and / or nanoscale droplet is conjugated to a plurality of the second ligands. (7) The microscale bubbles and / or nanoscale droplets of any one of (1) to (6), wherein the first ligand is conjugated to the microscale bubbles and / or nanoscale droplets via a PEG linker. (8) The microscale bubbles and / or nanoscale droplets of any one of (1) to (7), wherein the second ligand is conjugated to the microscale bubbles and / or nanoscale droplets via a PEG linker. (9) The microscale bubbles and / or nanoscale droplets of any one of (1) to (8), wherein the microscale bubbles and / or nanoscale droplets are filled with a gaseous material. (10) The microscale bubbles and / or nanoscale droplets of (9) above, wherein the gaseous material comprises a fluorinated gas selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof. (11) The microscale bubbles and / or nanoscale droplets according to (10), wherein the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, and mixtures of two or more thereof. (12) The microscale bubbles and / or nanoscale droplets according to any one of (1) to (11), wherein the microscale bubbles and / or nanoscale droplets are coated with a film-forming material. (13) The microscale bubbles and / or nanoscale droplets of (12) above, wherein the film-forming material comprises one or more lipids. (14) The micro-scale bubbles and / or nano-scale droplets of (13), wherein the lipid comprises a phospholipid or a mixture of phospholipids. (15) The microscale bubbles and / or nanoscale droplets of any one of (1) to (14), wherein the microscale bubbles and / or nanoscale droplets have a microscale size in the range of 0.5 to 10 microns. (16) The microscale bubbles and / or nanoscale droplets of any one of (1) to (14), wherein the microscale bubbles and / or nanoscale droplets have a nanoscale size ranging from 120 nm to 280 nm. (17) The microscale bubbles and / or nanoscale droplets described in any one of (1) to (16), wherein the microscale bubbles and / or nanoscale droplets are capable of degrading or metabolizing both beta amyloid and tau protein through other means. (18) An aqueous emulsion or suspension containing microscale bubbles and / or nanoscale droplets according to any one of (1) to (17). (19) 19. The emulsion or suspension according to claim 18, wherein the emulsion or suspension is in a homogenized form. (20) The emulsion or suspension according to (18) or (19), further comprising a pharma- ceutically acceptable excipient, carrier, or diluent. (twenty one) A drug for treating Alzheimer's disease, comprising the microscale bubbles and / or nanoscale droplets according to any one of (1) to (17). (twenty two) A drug for disrupting or reducing beta amyloid aggregates or tau protein aggregates, comprising an aqueous emulsion or suspension containing microscale bubbles and / or nanoscale droplets according to any one of (1) to (17), A drug which, after administration to a subject in need thereof, destroys or reduces beta amyloid aggregates or tau protein aggregates by applying ultrasound to a target area of ​​an organ of the subject having the beta amyloid aggregates or tau protein aggregates. Further embodiments of the present invention are listed below. (A-1) 1. A method for treating a vascular endothelial cell comprising the steps of: (a) administering to a subject a subject, comprising administering to said subject a subject a subject, said administering to said subject a ... The first ligand is: [ka] [ka] is selected from The second ligand is an enzyme or antibody selected from the group consisting of insulin degrading enzyme (IDE), neprilysin (NEP), endothelin converting enzyme (ECE), angiotensin converting enzyme (ACE), plasmin, matrix metalloproteinase (MMP), phosphatase, alkaline phosphatase (AP) and antibodies against tau and amyloid beta, or a fragment thereof; Microscale bubbles and / or nanoscale droplets. (A-2) The microscale bubbles and / or nanoscale droplets described in (A-1), wherein the first ligand has a binding affinity for beta amyloid, and the second ligand is capable of degrading or metabolizing beta amyloid other than by degradation. (A-3) The microscale bubbles and / or nanoscale droplets described in (A-1), wherein the first ligand has a binding affinity to tau protein, and the second ligand is capable of degrading or metabolizing tau protein other than by degradation. (A-4) The microscale bubbles and / or nanoscale droplets of (A-1), wherein each of the microscale bubbles and / or nanoscale droplets is conjugated to a plurality of the first ligands. (A-5) The microscale bubbles and / or nanoscale droplets of (A-1), wherein each microscale bubble and / or nanoscale droplet is conjugated to a plurality of said second ligands. (A-6) The microscale bubbles and / or nanoscale droplets of (A-1), wherein the first ligand is conjugated to the microscale bubbles and / or nanoscale droplets via a PEG linker. (A-7) The microscale bubbles and / or nanoscale droplets of (A-1), wherein the second ligand is conjugated to the microscale bubbles and / or nanoscale droplets via a PEG linker. (A-8) The micro-scale bubbles and / or nano-scale droplets of (A-1), wherein the micro-scale bubbles and / or nano-scale droplets are filled with a gaseous material. (A-9) The microscale bubbles and / or nanoscale droplets according to (A-1), wherein the gaseous material comprises a fluorinated gas selected from perfluoromethane, perfluoroethane, perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, perfluorohexane, perfluorocyclohexane, and mixtures of two or more thereof. (A-10) The microscale bubbles and / or nanoscale droplets according to (A-9), wherein the fluorinated gas is selected from perfluoropropane, perfluorocyclopropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorocyclopentane, and mixtures of two or more thereof. (A-11) The microscale bubbles and / or nanoscale droplets according to any one of (A-1) to (A-10), wherein the microscale bubbles and / or nanoscale droplets are coated with a film-forming material. (A-12) The microscale bubbles and / or nanoscale droplets according to (A-11), wherein the film-forming material comprises one or more lipids. (A-13) The microscale bubbles and / or nanoscale droplets according to (A-12), wherein the lipid comprises a phospholipid or a mixture of phospholipids. (A-14) The microscale bubbles and / or nanoscale droplets of any one of (A-1) to (A-10) above, wherein the microscale bubbles and / or nanoscale droplets have a microscale size in the range of 0.5 to 10 microns. (A-15) The microscale bubbles and / or nanoscale droplets according to any one of (A-1) to (A-10), wherein the microscale bubbles and / or nanoscale droplets have a nanoscale size ranging from 120 nm to 280 nm. (A-16) The microscale bubbles and / or nanoscale droplets described in any one of (A-1) to (A-10) are capable of decomposing or metabolizing both beta amyloid and tau protein through a process other than degradation. (A-17) An aqueous emulsion or suspension containing microscale bubbles and / or nanoscale droplets according to any one of (A-1) to (A-10). (A-18) The emulsion or suspension according to (A-17), wherein the emulsion or suspension is in a homogenized form. (A-19) The emulsion or suspension according to (A-17) or (A-18), further comprising a pharma- ceutically acceptable excipient, carrier, or diluent. (A-20) A drug for treating Alzheimer's disease, comprising the microscale bubbles and / or nanoscale droplets according to any one of (A-1) to (A-10). (A-21) A pharmaceutical agent for disrupting or reducing beta amyloid aggregates or tau protein aggregates, comprising an aqueous emulsion or suspension containing microscale bubbles and / or nanoscale droplets according to any one of (A-1) to (A-10), A drug which, after administration to a subject in need thereof, destroys or reduces beta amyloid aggregates or tau protein aggregates by applying ultrasound to a target area of ​​an organ of the subject having the beta amyloid aggregates or tau protein aggregates.

Claims

[Claim 1] 1. A method for treating a vascular endothelial cell comprising the steps of: (a) administering to a subject a subject, comprising administering to said subject a subject a subject a subject of administration a microscale gas bubble and / or nanoscale liquid droplet conjugated to one or more first ligands having a binding affinity for beta amyloid or tau protein, and one or more second ligands capable of degrading or otherwise metabolizing the beta amyloid or tau protein; The first ligand is: 【Chemistry 1】 【Chemistry 2】 is selected from The second ligand is an enzyme or antibody selected from the group consisting of insulin degrading enzyme (IDE), neprilysin (NEP), endothelin converting enzyme (ECE), angiotensin converting enzyme (ACE), plasmin, matrix metalloproteinase (MMP), phosphatase, alkaline phosphatase (AP) and antibodies against tau and amyloid beta, or a fragment thereof; Microscale bubbles and / or nanoscale droplets.

Images