Process for forming microbubbles with high oxygen content and uses thereof

Abstract

Formulations containing a carrier and microbubbles encapsulating oxygen gas, and methods for making and using the formulations are described herein. The formulations are manufactured by a process which includes high shear homogenization. The resulting microbubble suspension may be centrifuged to further concentrate the microbubbles. The resulting concentrated LOM suspension preferably has an oxygen content ranging from 50 to 99% (vol). Prior to administration to a patient, the viscosity of the LOM suspension may be reduced to the desired viscosity, preferably similar to the viscosity of the patient's blood. The resulting LOM formulation typically has an oxygen concentration ranging from 65 to 80% (vol). The microbubbles are formed from one or more lipids, preferably one or more phospholipids, most preferably DSPC, and preferably also contain one or more stabilizing agents/excipients, preferably cholesterol.

1 . A method for forming a concentrated oxygen-containing microbubble suspension comprising:
 (a) forming a precursor mixture comprising a fluid carrier and one or more lipids, and optionally comprising one or more stabilizing agents; and   (b) subjecting the precursor mixture to high shear homogenization using an in-line, closed system homogenizer, wherein oxygen gas is fed into the homogenizer, for a sufficient period of time and at a sufficient flow rate to produce a microbubble suspension.   
     
     
         2 . The method of  claim 1 , wherein the suspension is homogenized at a shear rate ranging from approximately 2,000 RPM to approximately 25,000 RPM, preferably from about 7,000 RPM to about 8,000 revolutions per minute (RPM). 
     
     
         3 . The method of  claim 1  wherein oxygen is fed into the homogenizer at a sufficient flow rate to encapsulate the oxygen in the microbubble suspension, with no free oxygen gas remaining, preferably the flow rate is sufficient to encapsulate the oxygen during one pass through the homogenizer. 
     
     
         4 . The method of  claim 3 , wherein the oxygen is fed into the homogenizer at a flow rate ranging from 25 milliliters per minute (mL/min) 2 liters per minute (L/min). 
     
     
         5 . The method of  claim 1 , wherein the one or more stabilizing agents are selected from the group consisting of cholesterol, polyoxyethylene-polyoxypropylene (Poloxamer 188), and Pluronic F108. 
     
     
         6 . The method of  claim 5 , wherein the stabilizing agent is cholesterol and the lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 
     
     
         7 . The method of  claim 1 , wherein the microbubble suspension further comprises one or more excipients selected from the group consisting of thinning agents, viscosity lowering agents, and combinations thereof. 
     
     
         8 . The method of  claim 1 , wherein the microbubble suspension comprises at least 50% oxygen by volume, preferably at least 70%, more preferably, at least 80%, more preferably at least 90%, more preferably at least 95%. 
     
     
         9 . The method of  claim 8 , wherein step (b) of  claim 1  is repeated via one or more additional passes through the homogenizer until the microbubble suspension contains the desired concentration of oxygen, wherein once this concentration of oxygen is attained no further processing steps are needed. 
     
     
         10 . The method of  claim 1 , further comprising centrifuging the microbubble suspension at a sufficient force and for a sufficient period of time to separate a microbubble phase from a liquid phase, wherein the microbubble phase comprises microbubbles having a size of predominantly 10 microns or lower. 
     
     
         11 . The method of  claim 1 , further comprising freezing the microbubble suspension, preferably at a temperature of approximately 0° C., wherein when the suspension is subsequently thawed by exposing to warmed water, convective heat, or a microwave for a period of less than 5 minutes, preferably less than 30 seconds, the size distribution and oxygen concentration of the microbubble suspension is the same as it was prior to the freezing step. 
     
     
         12 . A composition for rapidly delivering oxygen to a patient in need thereof comprising microbubbles in the form of a microbubble suspension made according to the method of  claim 1 , wherein the composition comprises at least 50% oxygen by volume. 
     
     
         13 . The composition of  claim 12 , wherein the composition comprises between about 50% to about 99% oxygen by volume, preferably from about 65% to about 80% oxygen by volume. 
     
     
         14 . The composition of  claim 12 , wherein viscosity of the composition is similar to the viscosity of human blood when measured under the same conditions. 
     
     
         15 . The composition of  claim 12 , wherein the composition is frozen, and wherein following thawing, the microbubbles have the same size distribution and oxygen concentration as the microbubbles had prior to freezing. 
     
     
         16 . A method for delivering oxygen to a patient, tissue or organ, comprising administering to the patient, tissue, or organ a composition comprising a suspension comprising microbubbles and a carrier,
 wherein the microbubbles comprise at least 50% oxygen by volume,   wherein the composition is administered in an effective amount to increase the concentration of oxygen in the patient's blood, tissue, or organ in need of oxygen within 0.5 seconds to 1 minute, preferably less than 10 seconds following administration.   
     
     
         17 . The method of  claim 16 , wherein the suspension is administered intravenously or intraosseously, as an infusion or bolus, or topically to a hypoxic tissue in the patient. 
     
     
         18 .- 19 . (canceled) 
     
     
         20 . The method of  claim 16 , wherein the suspension is topically applied to an infected wound in an effective amount to create hyperoxic conditions in order to reduce bacterial growth. 
     
     
         21 . The method of  claim 16 , wherein the patient is in cardiac arrest, and wherein the suspension is administered intravenously, intraosseously or intraarterially in an effective amount and for a sufficient period of time as an adjunct to the standard of care, wherein the standard of care comprises the steps of performing CPR, and/or administering epinephrine and/or antiarrhythmic agents. 
     
     
         22 . The method of  claim 21 , wherein the suspension is administered in an effective amount and for a sufficient period of time to enhance arterial oxygen content, cerebral oxygen delivery, myocardial oxygen delivery, and/or hemodynamic responses to resuscitative measures compared to the standard of care in the absence of the administration of the suspension.