Contrast agents

Abstract

Microbubble dispersions stabilised 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 stabilised 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.