Stable microbubble suspensions as enhancement agents for ultrasound echography

Abstract

Disclosed are injectable suspensions of gas filled microbubbles in an aqueous carrier liquid usable as contrast agents in ultrasonic echography. The suspensions comprise amphipathic compounds of which at least one may be a laminarize phospholipid as a stabiliser of the microbubbles against collapse with time and pressure. The concentration of phospholipids in the carrier liquid is below 0.01% wt but is at least equal to or above that at which phospholipid molecules are present solely at the gas microbubble-liquid interface. Also disclosed is a method of preparation of the stable suspensions of air or gas filled microbubbles.

1 . An injectable suspension of gas filled microbubbles in an aqueous carrier liquid, usable as contrast agent in ultrasonic echography, comprising at least 10 7  microbubbles per millilitre and amphipathic compounds at least one of which is a phospholipid stabilizer of the microbubbles against collapse, characterized in that the concentration of the phospholipids in the carrier liquid is below 0.01% by weight while being equal to or above that at which the phospholipid molecules are present solely at the gas microbubble-liquid interface.  
     
     
         2 . The injectable suspension of  claim 1 , in which the concentration of microbubbles per millilitre is between 10 8  and 10 10 .  
     
     
         3 . The injectable suspension of  claim 1 , in which the concentration of phospholipids is above 0.00013% wt.  
     
     
         4 . The injectable suspension of any preceding claims, in which the liquid carrier further comprises water soluble poly- and oligosaccharides, sugars and hydrophilic polymers such as polyethylene glycols as stabilizers.  
     
     
         5 . The injectable suspension of any preceding claim, in which the phospholipids are at least partially in lamellar or laminar form and are selected from lecithins such as phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol phosphatidylinositol, cardiolipin and sphingomyelin.  
     
     
         6 . The injectable suspension of  claim 4  or  5 , further containing substances affecting the properties of phospholipids selected from phosphatidylglycerol, phosphatidic acid, dicetylphosphate, cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propylgallate, ascorbyl palmitate and butylated hydroxytoluene.  
     
     
         7 . The injectable suspension of  claim 1 ,  2  or  3 , in which the phospholipids are in the form of powders obtained by freeze-drying or spray-drying.  
     
     
         8 . The injectable suspension of  claim 1 , containing about 10 8 -10 9  microbubbles per millilitre with the microbubble size between 0.5-10 μm showing little or no variation under storage.  
     
     
         9 . The injectable suspension of  claim 1 , in which the liquid carrier further comprises up to 50% by weight non-laminar surfactants selected from fatty acids, esters and ethers of fatty acids and alcohols with polyols such as polyalkylene glycols, polyalkylenated sugars and other carbohydrates, and polyalkylenated glycerol.  
     
     
         10 . The injectable suspension of any preceding claim, in which the microbubbles are filled with SF 6 , CF 4 , freons or air.  
     
     
         11 . A method of making suspensions of air or gas filled microbubbles comprising selecting at least one film forming surfactant, converting the surfactant into a powder, contacting the powder with air or another gas and admixing the powder surfactant with an aqueous liquid carrier to form said suspension, characterised by introducing the suspension into a container, forming a layer of the gas filled microbubbles in the upper part of the container, separating the layer of the microbubbles formed, and washing the microbubbles with an aqueous solution saturated with the microbubble gas.  
     
     
         12 . The method of  claim 11 , in which prior to converting into the powder, the film forming surfactant is at least partially lamellarized.  
     
     
         13 . The method of  claim 12 , in which prior to contacting withair or another gas the partially lamellarized surfactant is admixed with the aqueous liquid carrier.  
     
     
         14 . The method of claims  12  or  13 , in which the liquid carrier further contains stabiliser compounds selected from hydrosoluble proteins, polypeptides, sugars, poly- and oligo-saccharides and hydrophilic polymers.  
     
     
         15 . The method of  claim 12 , in which the conversion is effected by coating the surfactant onto particles of soluble or insoluble materials leaving the coated particles for a while under air or a gas, and admixing the coated particles with an aqueous liquid carrier.  
     
     
         16 . The method of  claim 12 , in which the conversion is effected by sonicating or homogenising under high pressure an aqueous solution of film forming lipids, this operation leading, at least partly, to the formation of liposomes.  
     
     
         17 . The method of  claim 16 , in which prior to contacting of at least partially lamellarized surfactant with air or another gas the liposome containing solution is freeze-dried.  
     
     
         18 . The method of claims  16  and  17 , in which the water solution of film forming lipids also contains viscosity enhancers or stabilisers selected from hydrophilic polymers and carbohydrates in weight ratio relative to the lipids comprised between 10:1 and 1000:1.  
     
     
         19 . A method of preparation of a suspension of air or gas filled microbubbles comprising a film forming surfactant, a hydrophilic stabiliser and an aqueous liquid carrier, characterised by dissolving the film forming surfactant and the hydrophilic stabiliser in an organic solvent, freeze drying the solution to form a dry powder, contacting the powder with air or another gas and admixing said powder with the aqueous carrier.  
     
     
         20 . The method of  claim 19 , in which the hydrophilic stabiliser is polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, glycolic acid, malic acid or maltol.  
     
     
         21 . The method of  claim 19  or  20 , in which the organic solvent is tertiary butanol, 2-methyl-2-butanol or C 2 Cl 4 F 2 .  
     
     
         22 . A method of making an injectable suspension of gas-filled microbubbles according to  claim 1 , which comprises suspending laminarized phospholipids, and optionally other additives, in an aqueous carrier liquid, said phospholipids having been in contact with said gas prior or after being suspended, under conditions such that a concentration of said microbubbles sufficient to provide an echographic respose is formed in the suspension, allowing a portion of said phospholipids to form a stabilization layer around said bubbles and thereafter depleting the carrier liquid of the excess of phospholipids not involved in microbubble stabilization.