# US 9,750,821 B2 · Gas-filled microvesicle assembly for contrast imaging > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,750,821 B2 - Document ID: US9750821B2 - Status: Granted - Kind: B2 - Application number: 10/584,327 - Filed: 2004-12-21 - Published: 2007-03-29 - Granted: 2017-09-05 - Earliest claimed: 2003-12-22 - Assignees: Bracco Suisse SA - Applicants: Michel Schneider; Philippe Bussat; Feng Yan; Anne Senente - Inventors: Michel Schneider; Philippe Bussat; Feng Yan; Anne Senente - Web page: https://patentgrep.com/patent/US9750821B2 ## CPC - A61K 47/6925: {the form being a microcapsule, nanocapsule, microbubble or nanobubble} - A61K 41/0028: {Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds} - A61K 49/225: {Microparticles, microcapsules (gas-filled to be classified in A61K49/223)} - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Assembly comprising a gas-filled microvesicle and a structural entity which is capable to associate through an electrostatic interaction to the outer surface of said microvesicle (microvesicle associated component—MAC), thereby modifying the physico-chemical properties thereof. Said MAC may optionally comprise a targeting ligand, a bioactive agent, a diagnostic agent or any combination thereof. The assembly of the invention can be formed from gasfilled microbubbles or microballoons and a MAC having a diameter of less than 100 pm, in particular a micelle and is used as an active component in diagnostically and/or therapeutically active formulations, in particular for enhancing the imaging in the field of ultrasound contrast imaging, including targeted ultrasound imaging, ultrasound-mediated drug delivery and other imaging techniques such as molecular resonance imaging (MRI) or nuclear imaging. ## Claims ### Claim 1 An assembly comprising a gas-filled microvesicle bearing a first overall net charge and a component associated with said microvesicle wherein said component is a micelle, which bears a second overall net charge opposite in sign to said first net charge, consisting of one or more amphiphilic compounds; and a targeting ligand, bioactive agent, or a combination thereof, and wherein said associated component has a diameter of 100 nm or lower, and is substantially free of magnetic particles. ### Claim 2 (depends on 1) An assembly according to claim 1 wherein said associated component has a diameter of 80 nm or lower. ### Claim 3 (depends on 1) An assembly according to claim 1 wherein said associated component has a diameter of 50 nm or lower. ### Claim 4 (depends on 1) An assembly according to claim 1 wherein said one or more amphiphilic compounds is selected from the group consisting of (C2-C10) organic acids, organic fatty acids comprising a (C12-C24) aliphatic chain, pharmaceutically acceptable salts thereof, esters thereof with polyoxyethylene; polyionic (alkali) salts; organic amines; amides; quaternary amine salts; amino acids; phospholipids; esters of mono- or oligo-saccharides with (C12-C24)organic fatty acids; organic sulfonates; perfluoroorganic acids; polymeric surfactants; and mixtures thereof. ### Claim 5 (depends on 1) An assembly according to claim 1 wherein the ratio between the number of charges per mole of microvesicles and the number of charges per mole of the associated component is from about 10:1 to about 1:10. ### Claim 6 (depends on 5) An assembly according to claim 5 wherein said ratio is of about 3:1 or less. ### Claim 7 (depends on 5) An assembly according to claim 5 wherein said ratio is of about 2:1 or less. ### Claim 8 (depends on 1) An assembly according to claim 1 wherein said microvesicle is a microbubble stabilized by an envelope comprising an amphiphilic film-forming compound or a microballoon having a material envelope. ### Claim 9 (depends on 8) An assembly according to claim 8 wherein said amphiphilic film-forming compound is a phospholipid. ### Claim 10 (depends on 8) An assembly according to claim 8 wherein said envelope or material envelope comprises a phospholipid or a lipid bearing a positive or negative net charge. ### Claim 11 (depends on 10) An assembly according to claim 10 wherein said phospholipid or lipid is selected fioni the group consisting of phosphatidylserine derivatives, phosphatic ic acid derivatives, phosphatidylglycerol derivatives, polyethyleneglycol modified phosphatidylethanolamines, ethylphosphatidylcholine derivatives and the respective lyso-forms; cholic acid salts; deoxycholic acid salts; glycocholic acid salts; (C12-C24) fatty acid salts thereof; alkylainmonium salts comprising at least one (C10-C20) alkyl chain; tertiary or quaternary ammonium salts comprising at least one (C 10-C20) acyl chain linked to the nitrogen atom through a (C3-C6) alkylene bridge; and mixtures thereof. ### Claim 12 (depends on 8) An assembly according to claim 8 wherein the material envelope of said microballoon comprises a polymeric material, a proteinaceous material, a water insoluble lipid or any combination thereof. ### Claim 13 (depends on 8) An assembly according to claim 8 wherein the material envelope of said microballoon comprises an ionic biodegradable polymer. ### Claim 14 (depends on 1) An assembly accordine to claim 1 wherein said one or more amphiphilic compounds is selected from the group consisting of a polyethyleneglycol modified phospholipid; an alkylammonium salt comprising at least one (C10-C20) alkyl chain; a tertiary or quaternary ammonium salt comprising at least one (C10-C20) acyl chain linked to the nitrogen atom through a (C3-C6) alkylene bridge; a (C12-C24) fatty acid salt; a polymeric surfactant; and mixtures thereof. ### Claim 15 (depends on 1) An assembly according to claim 1 wherein said one or more amphiphilic compounds is selected from the group consisting of (C12-C24) fatty acid di-esters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphaddylethanolamine, phosphatidylserine, sphingomyelin, and mixtures thereof. ### Claim 16 (depends on 1) An assembly according to claim 1 wherein said one or more amphiphilic compounds is selected from the group consisting of a phospholipid, a lipid hearing a positive or negative net charge, a polymeric ionic surfactant, and mixtures thereof. ### Claim 17 (depends on 16) An assembly according to claim 16 wherein said one or more amphiphilic compounds is selected from the group consisting of phosphatidylserine derivatives, phosphatidic acid derivatives, phosphatidylglycerol derivatives, polyethyleneglycol modified phosphatidylethanolamines, ethylphosphatidylcholine derivatives and the respective lyso-forms; cholic acid salts; deoxycholic acid salts; glycocholic acid salts; (C12-C24) fatty acid salts thereof; alkylammonium salts comprising at least one (C10-C20) alkyl chain; tertiary or quaternary ammonium salts comprising at least one (C10-C20) acyl chain linked to the nitrogen atom through a (C3-C6) all bridge; and mixtures thereof. ### Claim 18 (depends on 1) An aqueous suspension of a physiologically acceptable liquid comprising an assembly according to claim 1. ### Claim 19 (depends on 1) An assembly according to claim 1, wherein an aqueous suspension of said assembly in a pharmaceutically acceptable carrier shows a ζ-potential which is decreased of at least 50% in absolute value with respect to the ζ-potential of an aqueous suspension in the same carrier of the gas-filled microvesicles forming said assembly. ### Claim 20 (depends on 19) An assembly according to claim 19 wherein said ζ-potential is decreased of at least 75% in absolute value. ### Claim 21 (depends on 19) An assembly according to claim 19 wherein said ζ-potential is decreased of about 100% or more in absolute value. ### Claim 22 (depends on 1) A method for preparing an assembly according to claim 1, which comprises admixing a preparation comprising gas-filled microvesicles or a precursor thereof with a preparation comprising a component or a precursor thereof to be associated to said microvesicles. ### Claim 23 (depends on 22) A method according to claim 22 which comprises: 1) preparing a first aqueous suspension comprising a gas-filled microvesicle; 2) preparing a second aqueous suspension comprising a component to be associated with said gas-filled microvesicle; 3) admixing said two suspensions, to obtain an aqueous suspension comprising said assembly. ### Claim 24 (depends on 22) A method according to claim 22 which comprises: 1) preparing a first aqueous suspension comprising a gas-filled microvesicle; 2) freeze-drying said first suspension, to obtain a first lyophilized product; 3) preparing a second suspension comprising a component to be associated with said gas-filled microvesicle; 4) freeze-drying said, second suspension, to obtain a second, lyophilized product; 5) reconstituting said first and said second lyophilized product with a physiologically acceptable aqueous carrier in the presence of a gas, to obtain an aqueous suspension comprising the assembly. ### Claim 25 (depends on 24) A method according io claim 24, wherein step 5) comprises the steps of: a) reconstituting the second lyophilized product with a physiologically acceptable aqueous carrier to obtain a suspension comprising the component to he associated to the gas-filled microvesicle; and b) reconstituting the first lyophilized product with said suspension in the presence of a gas. ### Claim 26 (depends on 22) A method according to claim 22 which comprises: 1) preparing an aqueous emulsion comprisina an organic solvent, a nhospholipid and a lyoprotecting agent; 2) preparing an aqueous suspension comprising a component to be associated vith a gas-filled microvesicle; 3) admixing said aqueous suspension with said aqueous emulsion; and 4freeze drying the mixture to remove the water and the organic solvent, to obtain a lyophilized product comprising said assembly. ### Claim 27 (depends on 1) A pharmaceutically active formulation comprising an assembly according to claim 1. ### Claim 28 (depends on 1) A method for ultrasound diagnostic imaging which comprises administering a contrast-enhancing amount of an aqueous suspension of an assembly according to claim 1. ### Claim 29 (depends on 1) A method of therapeutic treatment which comprises administering a therapeutically-effective amount of an aqueous suspension of an assembly comprising a bioactive agent as defined in claim 1. ### Claim 30 (depends on 1) The assembly according claim 1, wherein said component associated with said microvesicle has a mean dimension which is at least 10 times smaller than the mean dimension of said microvesicle. ### Claim 31 (depends on 1) The assembly according to claim 1, wherein said component associated with said microvesicle has a mean dimension which is from 10 to 1000 times smaller than the mean dimension of said microvesicle. ### Claim 32 (depends on 1) The assembly according to claim 1, wherein the mean diameter in number of said assembly is not higher than about 30% the mean diameter of said microvesicle.