Contrast agents

Abstract

Microbubble dispersions stabilized by phospholipids predominantly comprising molecules which individually have an overall net charge exhibit advantageous stability, rendering them useful as efficacious contrast agents. An improved process for preparing microbubble-containing contrast agents is also disclosed, this comprising lyophilising an aqueous dispersion of gas microbubbles stabilized by one or more membrane-forming lipids to yield a dried product which may be reconstituted in an injectable carrier liquid to generate a microbubble-containing contrast agent.

1 . An aqueous dispersion of gas microbubbles stabilised by amphiphilic material consisting essentially of phospholipid predominantly comprising molecules which individually have an overall net charge.  
     
     
         2 . A size fractionated microbubble dispersion according to    claim 1   .  
     
     
         3 . A microbubble dispersion according to    claim 1    or    claim 2    wherein at least 75% of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         4 . A microbubble dispersion according to    claim 3    wherein substantially all of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         5 . A microbubble dispersion according to any of the preceding claims wherein the phospholipid is selected from naturally occurring, semisynthetic and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, cardiolipins, lyso forms of any of the foregoing and mixtures of any of the foregoing.  
     
     
         6 . A microbubble dispersion according to    claim 5    wherein acyl groups present in the phospholipid each contain about 14-22 carbon atoms.  
     
     
         7 . A microbubble dispersion according to    claim 5    or    claim 6    wherein one or more phosphatidylserines constitute at least 70% of the phospholipid.  
     
     
         8 . A microbubble dispersion according to    claim 7    wherein said phosphatidylserine or phosphatidylserines are selected from synthetic phosphatidylserine, semisynthetic phosphatidylserine, hydrogenated natural phosphatidylserine, hydrogenated semisynthetic phosphatidylserine, synthetic distearoylphosphatidylserine, synthetic dipalmitoylphosphatidylserine and synthetic diarachidoylphosphatidylserine.  
     
     
         9 . A microbubble dispersion according to any of the preceding claims wherein the gas is selected from air, nitrogen, oxygen, carbon dioxide, hydrogen, nitrous oxide, inert gases, sulphur fluorides, selenium hexafluoride, silanes, halogenated silanes, low molecular weight hydrocarbons, halogenated low molecular weight hydrocarbons, ethers, ketones, esters and mixtures of any of the foregoing.  
     
     
         10 . A microbubble dispersion according to    claim 9    wherein the gas comprises sulphur hexafluoride or a fluorinated low molecular weight hydrocarbon.  
     
     
         11 . A microbubble dispersion according to    claim 10    wherein said hydrocarbon is perfluorinated.  
     
     
         12 . A microbubble dispersion according to    claim 11    wherein said perfluorinated hydrocarbon comprises perfluoropropane, perfluorobutane or perfluoropentane.  
     
     
         13 . A microbubble dispersion according to    claim 7    wherein the gas is perfluorobutane.  
     
     
         14 . A microbubble dispersion according to any of    claims 10    to    13    characterised in that the microbubbles exhibit at least 90% recovery of size distribution and echogenic properties following exposure to an overpressure of 300 mm Hg for 90 seconds.  
     
     
         15 . A contrast agent for use in diagnostic studies, comprising a microbubble dispersion as claimed in any of the preceding claims in an injectable aqueous carrier liquid.  
     
     
         16 . A method of diagnostic imaging which comprises administering to a subject a contrast-enhancing amount of a contrast agent according to    claim 15    and imaging at least a part of said subject.  
     
     
         17 . A method according to    claim 16    wherein an MR image of said subject is generated.  
     
     
         18 . A method according to    claim 16    wherein an X-ray image of said subject is generated.  
     
     
         19 . A method according to    claim 16    wherein a scintigraphic or light image of said subject is generated.  
     
     
         20 . A method according to    claim 16    wherein an ultrasound image of said subject is generated.  
     
     
         21 . A method according to    claim 20    wherein the contrast agent is administered at a dose such that the amount of phospholipid administered is in the range 0.1-10 μg/kg body weight.  
     
     
         22 . A method according to    claim 21    wherein the dose is such that the amount of phospholipid administered is in the range 1-5 μg/kg body weight.  
     
     
         23 . A process for the preparation of a contrast agent comprising the steps: 
 i) dispersing gas in an aqueous medium containing a membrane-forming lipid to form a lipid-stabilised gas microbubble dispersion;    ii) lyophilising said dispersion to yield a dried lipid-containing product; and    iii) reconstituting said dried product in an aqueous injectable carrier liquid.    
     
     
         24 . A process as claimed in    claim 23    wherein the gas employed in step (i) is a fluorinated low molecular weight hydrocarbon.  
     
     
         25 . A process as claimed in    claim 24    wherein said hydrocarbon is perfluorinated.  
     
     
         26 . A process as claimed in    claim 23    wherein the gas employed in step (i) is sulphur hexafluoride.  
     
     
         27 . A process as claimed in any of    claims 23    to    26    wherein the lipid-containing aqueous medium employed in step (i) further contains one or more additives selected from viscosity enhancers and solubility aids for the lipid.  
     
     
         28 . A process as claimed in    claim 27    wherein said additive or additives are selected from alcohols and polyols.  
     
     
         29 . A process as claimed in any of    claims 23    to    28    wherein the membrane-forming lipid comprises at least one phospholipid.  
     
     
         30 . A process as claimed in any of    claims 23    to    29    wherein the membrane-forming lipid consists essentially of phospholipid and predominantly comprises molecules which individually have an overall net charge.  
     
     
         31 . A process as claimed in any of    claims 23    to    30    wherein the lipid-stabilised dispersion formed in step (i) is washed prior to being lyophilised.  
     
     
         32 . A process as claimed in any of    claims 23    to    31    wherein the lipid-stabilised dispersion is size fractionated prior to being lyophilised.  
     
     
         33 . A process as claimed in any of    claims 23    to    32    wherein a cryoprotectant and/or lyoprotectant is added to the lipid-stabilised dispersion formed in step (i) prior to it being lyophilised.  
     
     
         34 . A process as claimed in    claim 33    wherein said cryoprotectant and/or lyoprotectant is selected from alcohols, polyols, aminoacids, carbohydrates and polyglycols.  
     
     
         35 . A process as claimed in    claim 34    wherein said cryoprotectant and/or lyoprotectant is a physiologically tolerated sugar.  
     
     
         36 . A process as claimed in any of    claims 23    to    35    wherein said dried product is reconstituted by hand-shaking the product in the carrier liquid.  
     
     
         37 . A lyophilised residue of a suspension of gas microbubbles in an amphiphilic material-containing aqueous medium wherein the amphiphilic material consists essentially of phospholipid predominantly comprising molecules which individually have an overall net charge.  
     
     
         38 . A lyophilised residue according to    claim 37    wherein at least 75% of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         39 . A lyophilised residue according to    claim 38    wherein substantially all of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         40 . A lyophilised residue according to any of claims  37  to  39  wherein the phospholipid is selected from naturally occurring, semisynthetic and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, cardiolipins, lyso forms of any of the foregoing and mixtures of any of the foregoing.  
     
     
         41 . A lyophilised residue according to    claim 40    wherein acyl groups present in the phospholipid each contain about 14-22 carbon atoms.  
     
     
         42 . A lyophilised residue according to    claim 40    or    claim 41    wherein one or more phosphatidylserines constitute at least 70% of the phospholipid.  
     
     
         43 . A lyophilised residue according to    claim 42    wherein said phosphatidylserine or phosphatidylserines are selected from synthetic phosphatidylserine, semisynthetic phosphatidylserine, hydrogenated natural phosphatidylserine, hydrogenated semisynthetic phosphatidylserine, synthetic distearoylphosphatidylserine, synthetic dipalmitoylphosphatidylserine and synthetic diarachidoylphosphatidylserine.  
     
     
         44 . A lyophilised residue according to any of    claims 37    to    43    wherein the gas is selected from air, nitrogen, oxygen, carbon dioxide, hydrogen, nitrous oxide, inert gases, sulphur fluorides, selenium hexafluoride, silanes, halogenated silanes, low molecular weight hydrocarbons, halogenated low molecular weight hydrocarbons, ethers, ketones, esters and mixtures of any of the foregoing.  
     
     
         45 . A lyophilised residue according to    claim 44    wherein the gas comprises sulphur hexafluoride or a fluorinated low molecular weight hydrocarbon.  
     
     
         46 . A lyophilised residue according to    claim 45    wherein said hydrocarbon is perfluorinated.  
     
     
         47 . A lyophilised residue according to    claim 46    wherein said perfluorinated hydrocarbon comprises perfluoropropane, perfluorobutane or perfluoropentane.  
     
     
         48 . A lyophilised residue according to any of    claims 37    to    47    derived from a size fractionated microbubble suspension.  
     
     
         49 . A microbubble-releasing matrix containing gas-filled substantially spherical cavities or vacuoles surrounded by layers of membrane-forming lipid material.  
     
     
         50 . A matrix according to    claim 49    wherein the matrix structural material is a carbohydrate.  
     
     
         51 . A matrix according to    claim 49    or    claim 50    wherein the membrane-forming lipid material comprises at least one phospholipid.  
     
     
         52 . A matrix according to any of    claims 49    to    51    wherein the membrane-forming lipid material consists essentially of phospholipid predominantly comprising molecules which individually have an overall net charge.  
     
     
         53 . A matrix according to    claim 52    wherein at least 75% of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         54 . A matrix according to    claim 53    wherein substantially all of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         55 . A matrix according to any of    claims 52    to    54    wherein the phospholipid is selected from naturally occurring, semisynthetic and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, cardiolipins, lyso forms of any of the foregoing and mixtures of any of the foregoing.  
     
     
         56 . A matrix according to    claim 55    wherein acyl groups present in the phospholipid each contain about 14-22 carbon atoms.  
     
     
         57 . A matrix according to    claim 55    or    claim 56    wherein one or more phosphatidylserines constitute at least 70% of the phospholipid.  
     
     
         58 . A matrix according to    claim 57    wherein said phosphatidylserine or phosphatidylserines are selected from synthetic phosphatidylserine, semisynthetic phosphatidylserine, hydrogenated natural phosphatidylserine, hydrogenated semisynthetic phosphatidylserine, synthetic distearoylphosphatidylserine, synthetic dipalmitoylphosphatidylserine and synthetic diarachidoylphosphatidylserine.  
     
     
         59 . A matrix according to any of    claims 49    to    58    wherein the gas is selected from air, nitrogen, oxygen, carbon dioxide, hydrogen, nitrous oxide, inert gases, sulphur fluorides, selenium hexafluoride, silanes, halogenated silanes, low molecular weight hydrocarbons, halogenated low molecular weight hydrocarbons, ethers, ketones, esters and mixtures of any of the foregoing.  
     
     
         60 . A matrix according to    claim 59    wherein the gas comprises sulphur hexafluoride or a fluorinated low molecular weight hydrocarbon.  
     
     
         61 . A matrix according to    claim 60    wherein said hydrocarbon is perfluorinated.  
     
     
         62 . A matrix according to    claim 61    wherein said perfluorinated hydrocarbon comprises perfluoropropane, perfluorobutane or perfluoropentane.  
     
     
         63 . A frozen microbubble-releasing aqueous dispersion comprising gas microbubbles stabilised by amphiphilic material comprising at least one phospholipid.  
     
     
         64 . A frozen microbubble dispersion according to    claim 63    wherein said amphiphilic material consists essentially of phospholipid predominantly comprising molecules which individually have an overall net charge.  
     
     
         65 . A size fractionated frozen microbubble dispersion according to    claim 63    or    claim 64   .  
     
     
         66 . A frozen microbubble dispersion according to    claim 64    or    claim 65    wherein at least 75% of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         67 . A frozen microbubble dispersion according to    claim 66    wherein substantially all of the phospholipid consists of molecules which individually have an overall net charge.  
     
     
         68 . A frozen microbubble dispersion according to any of    claims 64    to    67    wherein the phospholipid is selected from naturally occurring, semisynthetic and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, cardiolipins, lyso forms of any of the foregoing and mixtures of any of the foregoing.  
     
     
         69 . A frozen microbubble dispersion according to claim  68  wherein acyl groups present in the phospholipid each contain about 14-22 carbon atoms.  
     
     
         70 . A frozen microbubble dispersion according to    claim 68    or    claim 69    wherein one or more phosphatidylserines constitute at least 70% of the phospholipid.  
     
     
         71 . A frozen microbubble dispersion according to    claim 70    wherein said phosphatidylserine or phosphatidylserines are selected from synthetic phosphatidylserine, semisynthetic phosphatidylserine, hydrogenated natural phosphatidylserine, hydrogenated semisynthetic phosphatidylserine, synthetic distearoylphosphatidylserine, synthetic dipalmitoylphosphatidylserine and synthetic diarachidoylphosphatidylserine.  
     
     
         72 . A frozen microbubble dispersion according to any of    claims 63    to    71    wherein the gas is selected from air, nitrogen, oxygen, carbon dioxide, hydrogen, nitrous oxide, inert gases, sulphur fluorides, selenium hexafluoride, silanes, halogenated silanes, low molecular weight hydrocarbons, halogenated low molecular weight hydrocarbons, ethers, ketones, esters and mixtures of any of the foregoing.  
     
     
         73 . A frozen microbubble dispersion according to    claim 72    wherein the gas comprises sulphur hexafluoride or a fluorinated low molecular weight hydrocarbon.  
     
     
         74 . A frozen microbubble dispersion according to    claim 73    wherein said hydrocarbon is perfluorinated.  
     
     
         75 . A frozen microbubble dispersion according to    claim 74    wherein said perfluorinated hydrocarbon comprises perfluoropropane, perfluorobutane or perfluoropentane.  
     
     
         76 . A microbubble-containing contrast agent prepared by a process as claimed in any of    claims 24    to    26    wherein the membrane-forming lipid consists essentially of phospholipid and predominantly comprises molecules which individually have an overall net charge, characterised in that the microbubbles exhibit at least 90% recovery of size distribution and echogenic properties following exposure to an overpressure of 300 mm Hg for 90 seconds.  
     
     
         77 . An aqueous dispersion of gas microbubbles stabilised by amphiphilic material consisting essentially of phospholipid predominantly comprising molecules which individually have an overall net charge, said dispersion having been prepared by: 
 i) dispersing gas in an aqueous medium containing said phospholipid to form a phospholipid-stabilised gas microbubble dispersion;    ii) lyophilising said dispersion to yield a dried phospholipid-containing product; and    iii) reconstituting said dried product in an aqueous medium.    
     
     
         78 . A lyophilised residue as claimed in    claim 48    wherein the gas is perfluorobutane and one or more phosphatidylserines constitute at least 70% of the phospholipid.  
     
     
         79 . A contrast agent composition comprising as a first component a lyophilised residue as claimed in    claim 78    and as a second component an injectable aqueous carrier liquid, said first and second components being contained respectively within first and second chambers of dual chamber storage means.  
     
     
         80 . An aqueous dispersion of gas microbubbles stabilised by amphiphilic material consisting essentially of phospholipid predominantly comprising molecules which individually have an overall net charge.  
     
     
         81 . A microbubble dispersion according to    claim 80    wherein substantially all of the phospholipid consists of molecules which individually have an overall net negative charge.  
     
     
         82 . A microbubble dispersion according to    claim 81    wherein one or more phosphatidylserines constitute at least 70% of the phospholipid.  
     
     
         83 . A microbubble dispersion according to    claim 80    wherein the gas comprises sulphur hexafluoride or a perfluorinated low molecular weight hydrocarbon.  
     
     
         84 . A microbubble dispersion according to    claim 83    wherein the gas is perfluorobutane.  
     
     
         85 . An ultrasound contrast agent for use in diagnostic studies, comprising a microbubble dispersion as claimed in    claim 80    in an injectable aqueous carrier liquid.  
     
     
         86 . A process for the preparation of a contrast agent comprising the steps: 
 i) dispersing gas in an aqueous medium containing a membrane-forming lipid to form a lipid-stabilised gas microbubble dispersion;    ii) lyophilising said dispersion in the presence of a cryoprotectant and/or lyoprotectant to yield a dried product comprising a cryoprotectant and/or lyoprotectant matrix containing gas-filled substantially spherical cavities or vacuoles surrounded by one or more layers of the membrane-forming lipid; and    iii) reconstituting said dried product in an aqueous injectable carrier liquid.    
     
     
         87 . A process as claimed in    claim 86    wherein the gas employed in step (i) is sulphur hexafluoride or a perfluorinated low molecular weight hydrocarbon.  
     
     
         88 . A process as claimed in    claim 86    wherein the lipid-containing aqueous medium employed in step (i) further contains one or more additives selected from alcohols and polyols.  
     
     
         89 . A process as claimed in    claim 86    wherein the membrane-forming lipid consists essentially of phospholipid and predominantly comprises molecules which individually have an overall net charge.  
     
     
         90 . A process as claimed in    claim 86    wherein, prior to step (ii), the lipid-stabilised dispersion formed in step (i) is washed and the microbubbles are size fractionated.  
     
     
         91 . A process as claimed in    claim 86    wherein the cryoprotectant and/or lyoprotectant is a physiologically tolerated sugar.  
     
     
         92 . A lyophilised residue of a microbubble dispersion as claimed in    claim 80   .  
     
     
         93 . A microbubble-releasing matrix comprising a matrix structural material containing a plurality of gas-filled substantially spherical cavities or vacuoles surrounded by layers of membrane-forming lipid material.  
     
     
         94 . A matrix according to    claim 93    wherein the matrix structural material is a carbohydrate.  
     
     
         95 . A matrix according to    claim 93    wherein the membrane-forming lipid material consists essentially of phospholipid predominantly comprising molecules which individually have an overall net charge.  
     
     
         96 . A matrix according to    claim 95    wherein substantially all of the phospholipid consists of molecules which individually have an overall net negative charge.  
     
     
         97 . A matrix according to    claim 96    wherein one or more phosphatidylserines constitute at least 70% of the phospholipid.  
     
     
         98 . A matrix according to    claim 93    wherein the gas comprises sulphur hexafluoride or a perfluorinated low molecular weight hydrocarbon.  
     
     
         99 . An ultrasound contrast agent composition comprising as a first component a matrix as claimed in    claim 97    wherein the matrix material is sucrose and the gas is perfluorobutane, and as a second component an injectable aqueous carrier liquid, said first and second components being contained respectively within first and second chambers of dual chamber storage means.  
     
     
         100 . A method of diagnostic ultrasound imaging which comprises administering to a subject a contrast-enhancing amount of an ultrasound contrast agent as claimed in    claim 85    and generating an ultrasound image of at least a part of said subject.