Long-lasting aqueous dispersions or suspensions of pressure-resistant gas-filled microvesicles and methods for the preparation thereof

Abstract

One can impart outstanding resistance against collapse under pressure to gas-filled microvesicle used as contrast agents in ultrasonic echography by using as fillers gases whose solubility in water, expressed in liter of gas by liter of water under standard conditions, divided by the square root of the molecular weight does not exceed 0.003.

1. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas-filled microvesicles suspended in an aqueous liquid carrier phase, the microvesicles being either  microbubbles bounded by an evanescent gas/liquid interfacial closed surface, or microballoons bounded by a material envelope  filled with a physiologically acceptable gas wherein the gas is bounded by a stabilizing layer of one or more film forming phospholipids in lamellar or laminar form at the gas/liquid interface, said method comprising the step of forming the microvesicles in the presence of a physiologically acceptable gas, or gas mixture comprising a physiologically acceptable gas, or filling preformed microvesicles with said gas, or said gas mixture, said  the physiologically acceptable gas being  selected from the group consisting of SF 6 , SeF 6 ,  CF 4 , CBrF 3 , C 4 F 8 , CClF 3 , CCl 2 F 2 ,  C 2 F 6 , C 2 ClF 5 , CBrClF 2 , C 2 Cl 2 F 4 , CBr 2 F 2   and C 4 F 10 , said microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas-filled microvesicles is in  injected into the bloodstream of a patient. 
     
     
       2. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas-filled microvesicles suspended in an aqueous liquid carder  carrier phase, the microvesicles being either  microbubbles bounded by an evanescent gas/liquid interfacial closed surface, or microballoons bounded by a material envelope  filled with a physiologically acceptable gas wherein the gas is bounded by a stabilizing layer of one or more film forming phospholipids in lamellar or laminar form at the gas/liquid interface, said method comprising the steps of:
 preforming the microvesicles or precursors thereof under an atmosphere of a first gas; and  
 substantially substituting at least a fraction of said first gas with a second gas which is a physiologically acceptable gas, or gas mixture comprising a physiologically acceptable gas, said  the physiologically acceptable gas being  selected from the group consisting of SF 6 , SeF 6 ,  CF 4 , CBrF 3 , C 4 F 8 , CClF 3 , CCl 2 F 2 ,  C 2 F 6 , C 2 ClF 5 , CBrClF 2 , C 2 Cl 2 F 4 , CBr 2 F 2   and C 4 F 10 , said microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas-filled microvesicles is injected into the bloodstream of a patient.  
 
     
     
       3. The method of  claim 2 , in which the gas used in the first step allows effective control of the average size and concentration of the microvesicles in the carrier liquid, and the physiologically acceptable gas added in the second step ensures prolonged useful echogenic life of the suspension for in-vivo ultrasonic imaging. 
     
     
       4. The method of  claim 1 , in which the aqueous phase carrying the microbubbles contains dissolved film-forming surfactants in lamellar or laminar fog said surfactants stabilizing the microbubbles boundary at the gas/liquid innerface. 
     
     
       5. The method of  claim 4 , in which said surfactants comprise one or more phospholipids. 
     
     
       6. The method of  claim 5 , in which at least pan of the phospholipids are in the form of liposomes. 
     
     
       7. The method of claim  5    1 , in which m  at least one of the phospholipids is a diacylphosphatidyl compound wherein the acyl group is a C 16  fatty acid residue or a higher homologue thereof. 
     
     
       8. The method of  claim 1 , in which the microballoon material envelope is made of an organic polymeric membrane. 
     
     
       9. The method of  claim 8 , in which the polymers of the membrane are selected from the group consisting of polylactic or polyglycolic acid and their copolymers, reticulated serum albumin, reticulated haemoglobin polystyrene, and esters of polyglutamic and polyaspanic acids. 
     
     
       10. The method of  claim 1 , in which the forming of vesicles with said physiologically acceptable gas is effected by alternately subjecting dry precursors thereof to reduced pressure and restoring the pressure with said gas, and dispersing the precursors in a liquid carrier. 
     
     
       11. The method of  claim 1 , in which the filling of the microballoons with said physiologically acceptable gas is effected by flushing the suspension with said gas under ambient pressure. 
     
     
       12. The method of  claim 1 , in which the microvesicles are made under an atmosphere composed at least in part of said gas. 
     
     
       13. A method of making a contrast agent for ultrasonic echography which consists of gas-filled microvesicles  microbubbles suspended in an aqueous liquid carrier phase, the microvesicles  microbubbles having resistance against collapse resulting from pressure increases effective when the said suspensions are injected into the bloodstream of a patient, and the microbubbles being filled with a physiologically acceptable gas wherein the gas is bounded by a stabilizing layer of one or more film forming phospholipids in lamellar or laminar form at the gas/liquid interface, said method comprising the step of forming the microvesicles  microbubbles in the presence of a physiologically acceptable gas or gas mixture comprising a physiologically acceptable gas, or filling preformed microvesicles with said gas or said gas mixture,  said physiologically acceptable gas being  selected from the group consisting of SF 6 , SeF 6 ,  CF 4 , CBrF 3 , C 4 F 8 , CClF 3 , CCl 2 F 2 ,  C 2 F 6 , C 2 ClF 5 , CBrClF 2 , C 2 Cl 2 F 4 , CBr 2 F 2   and C 4 F 10 , said gas or at least a gas in said gas mixture  being such that, under standard conditions, the pressure difference ΔP between pressures at which the bubble counts are about 75% and 25% of the original bubble count is at least 25Torr. 
     
     
       14. An aqueous suspension made according to the method of  claim 13 , wherein the physiologically acceptable gas is such that, under standard conditions, and at a rate of pressure increase to the suspension of about 100 Torr/min, the pressure difference ΔP between pressures at which the bubble counts are about 75% and 25% of the original bubble count is at least 25Torr. 
     
     
       15. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas- filled microvesicles suspended in an aqueous liquid carrier phase, the microvesicles being microbubbles filled with a gas mixture wherein the gas mixture is bounded by a stabilizing layer of one or more film forming phospholipids in lamellar or laminar form at the gas/liquid interface, said method comprising the step of forming the microvesicles in the presence of the gas mixture comprising a physiologically acceptable gas, selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , and microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas - filled microvesicles is injected into the bloodstream of a patient.    
     
     
       16. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas- filled microvesicles suspended in an aqueous liquid carrier phase, the microvesicles being microballoons consisting of a physiologically acceptable gas bounded by an organic polymer envelope at the gas/liquid interface, said polymer envelope formed from one or more polymers selected from the group consisting of polylactic or polyglycolic acid and their copolymers, denatured albumin, reticulated hemoglobin, and esters of polyglutamic and polyaspartic acids, said method comprising the step of forming the microvesicles in the presence of said physiologically acceptable gas selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas - filled microvesicles is injected into the bloodstream of a patient.    
     
     
       17. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas- filled microvesicles suspended in an aqueous liquid carrier phase, the microvesicles being microballoons consisting of a gas mixture bounded by an organic polymer envelope at the gas/liquid interface, said polymer envelope formed from one or more polymers selected from the group consisting of polylactic or polyglycolic acid and their copolymers, denatured albumin, reticulated hemoglobin, and esters of polyglutamic and polyaspartic acids, said method comprising the step of forming the microvesicles in the presence of the gas mixture comprising a physiologically acceptable gas, selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas - filled microvesicles is injected into the bloodstream of a patient.    
     
     
       18. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas- filled microvesicles suspended in an aqueous liquid carrier phase, the microvesicles being microbubbles filled with a gas mixture wherein the gas mixture is bounded by a stabilizing layer of one or more film forming phospholipids in lamellar or laminar form at the gas/liquid interface, said method comprising the steps of:      preforming the microvesicles or precursors thereof under an atmosphere of a first gas; and        substantially substituting at least a fraction of said first gas with a second gas which is the gas mixture comprising a physiologically acceptable gas selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas - filled microvesicles is injected into the bloodstream of a patient.      
     
     
       19. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas- filled microvesicles suspended in an aqueous liquid carrier phase, the microvesicles being microballoons consisting of a physiologically acceptable gas bounded by an organic polymer envelope at the gas/liquid interface, said polymer envelope formed from one or more polymers selected from the group consisting of polylactic or polyglycolic acid and their copolymers, denatured albumin, reticulated hemoglobin, and esters of polyglutamic and polyaspartic acids, said method comprising the steps of:      preforming the microvesicles or precursors thereof under an atmosphere of a first gas; and        substantially substituting at least a fraction of said first gas with a second gas which is the physiologically acceptable gas selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas - filled microvesicles is injected into the bloodstream of a patient.      
     
     
       20. A method of making a contrast agent having resistance against collapse from pressure increases when used in ultrasonic echography, said contrast agent consisting of gas- filled microvesicles suspended in an aqueous liquid carrier phase, the microvesicles being microballoons consisting of a gas mixture bounded by an organic polymer envelope at the gas/liquid interface, said polymer envelope formed from one or more polymers selected from the group consisting of polylactic or polyglycolic acid and their copolymers, denatured albumin, reticulated hemoglobin, and esters of polyglutamic and polyaspartic acids, said method comprising the steps of      preforming the microvesicles or precursors thereof under an atmosphere of a first gas; and        substantially substituting at least a fraction of said first gas with a second gas which is the gas mixture comprising a physiologically acceptable gas selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said microvesicles having resistance against collapse resulting, at least in part, from pressure increases effective when a suspension of said gas - filled microvesicles is injected into the bloodstream of a patient.      
     
     
       21. The method of  claim 18 , in which the gas used in the first step allows effective control of the average size and concentration of the microvesicles in the carrier liquid, and the physiologically acceptable gas added in the second step ensures prolonged useful echogenic life of the suspension for in- vivo ultrasonic imaging.    
     
     
       22. The method of claims  19  or  20 , in which the gas used in the first step allows effective control of the average size and concentration of the microvesicles in the carrier liquid, and the physiologically acceptable gas added in the second step ensures prolonged useful echogenic life of the suspension for in- vivo ultrasonic imaging.    
     
     
       23. The method of  claim 15 , in which at least one of the phospholipids is a diacylphosphatidyl compound wherein the acyl group is a C 16    fatty acid residue or a higher homologue thereof.    
     
     
       24. The method of claims  16  or  17 , in which the forming of vesicles with said physiologically acceptable gas is effected by alternately subjecting dry precursors thereof to reduced pressure and restoring the pressure with said gas, and dispersing the precursors in a liquid carrier.  
     
     
       25. The method of claims  16  or  17 , in which the filling of the microballoons with said physiologically acceptable gas is effected by flushing the suspension with said gas under ambient pressure.  
     
     
       26. A method of making a contrast agent for ultrasonic echography which consists of gas- filled microbubbles suspended in an aqueous liquid carrier phase, the microbubbles having resistance against collapse resulting from pressure increases effective when the said suspension are injected into the bloodstream of a patient and the microbubbles being filled with a gas mixture wherein the gas mixture is bounded by a stabilizing layer of one or more film forming phospholipids in lamellar or laminar form at the gas/liquid interface, said method comprising the step of forming the microbubbles in the presence of the gas mixture comprising a physiologically acceptable gas selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said gas or at least a gas in said gas mixture being such that, under standard conditions, the pressure difference ΔP between pressure at which the bubble counts are about  75   %  and  25   %  of the original bubbles count is at least  25  Torr.    
     
     
       27. A method of making a contrast agent for ultrasonic echography which consists of gas- filled microballoons suspended in an aqueous liquid carrier phase, the microballoons having resistance against collapse resulting from pressure increases effective when the said suspensions are injected into the bloodstream of a patient and the microballoons consisting of a physiologically acceptable gas bounded by an organic polymer envelope at the gas/liquid interface, said polymer envelope formed from one or more polymers selected from the group consisting of polylactic or polyglycolic acid and their copolymers, denatured albumin, reticulated hemoglobin, and esters of polyglutamic and polyaspartic acids, said method comprising the step of forming the microballoons in the presence of the physiologically acceptable gas selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said gas or at least a gas in said gas mixture being such that, under standard conditions, the pressure difference ΔP between pressures at which the bubble counts are about  75   %  and  25   %  of the original bubble count is at least  25  Torr.    
     
     
       28. A method of making a contrast agent for ultrasonic echography which consists of gas- filled microballoons suspended in an aqueous liquid carrier phase, the microballoons having resistance against collapse resulting from pressure increases effective when the said suspensions are injected into the bloodstream of a patient and the microballoons consisting of a gas mixture bounded by an organic polymer envelope at the gas/liquid interface, said polymer envelope formed from one or more polymers selected from the group consisting of polylactic or polyglycolic acid and their copolymers, denatured albumin, reticulated hemoglobin, and esters of polyglutamic and polyaspartic acids, said method comprising the steps of forming the microballoons in the presence of the gas mixture comprising a physiologically acceptable gas selected from the group consisting of SF   6   , CF   4   , CBrF   3   , C   4   F   8   , CClF   3   , C   2   F   6   , C   2   ClF   5   , CBrClF   2   , C   2   Cl   2   F   4    and C   4   F   10   , said gas or at least a gas in said gas mixture being such that, under standard conditions, the pressure difference ΔP between pressures at which the bubble counts are about  75   %  and  25   %  of the original bubble count is at least  25  Torr.    
     
     
       29. An aqueous suspension made according to the method of  claim 26 , wherein the physiologically acceptable gas is such that, under standard conditions, and at a rate of pressure increase to the suspension of about  100  Torr/min, the pressure difference ΔP between pressures at which the bubble counts are about  75 %  and  25   %  of the original bubble count is at least  25  Torr.    
     
     
       30. An aqueous suspension made according to the method of claims  27  or  28 , wherein the physiologically acceptable gas is such that, under standard conditions, and at a rate of pressure increase to the suspension of about  100  Torr/min, the pressure difference ΔP between pressures at which the bubble counts are about  75 %  and  25   %  of the original bubble count is at least  25  Torr.    
     
     
       31. The method of claims  1  or  15 , wherein the physiologically acceptable gas is selected from the group consisting of CF 4   , C   2   F   6   , C   4   F   8   , or C   4   F   10 .  
     
     
       32. The method of  claim 1 , wherein the physiologically acceptable gas is CF 4 .  
     
     
       33. The method of  claim 1 , wherein the physiologically acceptable gas is C 2   F   6 .  
     
     
       34. The method of  claim 1 , wherein the physiologically acceptable gas is C 4   F   8 .  
     
     
       35. The method of  claim 1 , wherein the physiologically acceptable gas is C 4   F   10 .  
     
     
       36. The method of  claim 1 , wherein the physiologically acceptable gas is SF 6 .  
     
     
       37. The method of claims  16  or  17 , wherein the physiologically acceptable gas is selected from the group consisting of CF 4   , C   2   F   6   , C   4   F   8   , or C   4   F   10 .  
     
     
       38. The method of  claim 16 , wherein the physiologically acceptable gas is CF 4 .  
     
     
       39. The method of  claim 16 , wherein the physiologically acceptable gas is C 2   F   6 .  
     
     
       40. The method of  claim 16 , wherein the physiologically acceptable gas is C 4   F   8 .  
     
     
       41. The method of  claim 16 , wherein the physiologically acceptable gas is C 4   F   10 .  
     
     
       42. The method of  claim 16 , wherein the physiologically acceptable gas is SF 6 .  
     
     
       43. The method of  claim 1 , in which at least part of the phospholipids are in the form of liposomes.  
     
     
       44. The method of  claim 15 , in which at least part of the phospholipids are in the form of liposomes.