Compositions for oxygen transport comprising a high oxygen affinity modified hemoglobin

Abstract

The present invention relates to blood products, and more particularly to compositions comprising a modified oxygenated hemoglobin having a high affinity for oxygen and methods for making such compositions. Such compositions according to the present invention have better stability to autooxidation and superior oxygen carrying characteristics.

1 . A blood substitute product comprising surface-modified oxygenated hemoglobin, wherein the surface-modified oxygenated hemoglobin has a P50 less than native stroma-free hemoglobin from the same animal source when measured under the same conditions.  
     
     
         2 . A composition useful as a blood substitute, comprising the blood substitute product of  claim 1  in an aqueous diluent.  
     
     
         3 . The blood substitute product of  claim 1 , wherein the surface-modified oxygenated hemoglobin has a P50 less than 10 torr.  
     
     
         4 . The blood substitute product of claim a, wherein the surface-modified oxygenated hemoglobin has a P50 less than 7 torr.  
     
     
         5 . The blood substitute product of  claim 1  produced by covalently attaching one or more polyalkylene oxides to the oxygenated hemoglobin.  
     
     
         6 . The blood substitute product of  claim 5 , wherein the polyalkylene oxide is polyethylene glycol according to the formula H(OCH 2 CH 2 ) n OH, where n is greater than or equal to 4.  
     
     
         7 . The blood substitute product of  claim 5  having a methemoglobin/total. hemoglobin ratio less than 0.10.  
     
     
         8 . A blood substitute product stable to autooxidation at 24° C. comprising a PEG-hemoglobin conjugate, wherein the methemoglobin/total hemoglobin ratio is less than 0.10 and the PEG-hemoglobin conjugate has a P50 less than 10 torr.  
     
     
         9 . A method of making a blood substitute product comprising the steps of: 
 a) preparing hemoglobin by oxygenating the hemoglobin having a methemoglobin/total hemoglobin ratio less than 0.10;    b) covalently attaching at least one polyalkylene oxide to the hemoglobin to form surface-modified oxygenated hemoglobin having a P50 less than 10 torr; and    c) suspending the surface-modified oxygenated hemoglobin in an auqeous diluent.    
     
     
         10 . The method of  claim 9 , wherein step a) further comprises isolating hemoglobin from red blood cells, wherein said hemoglobin has a methemoglobin/total hemoglobin ratio of 0.10 or greater and exposing the hemoglobin to the atmosphere for a time sufficient to lower the methemoglobin/hemoglobin ratio to less than 0.10.  
     
     
         11 . The method of  claim 10 , wherein step a) is carried out in the absence of a thiol-containing reducing agent.  
     
     
         12 . A method of using a blood substitute product to deliver oxygen to a tissue, comprising administering the composition of  claim 2  to a mammal in need thereof.  
     
     
         13 . The blood substitute product of  claim 1 , wherein the hemoglobin is horse hemoglobin.