Pegylated Hemoglobin and Albumin and Uses Thereof

Abstract

The present invention provides PEGylated hemoglobins and PEGylated albumins comprising polyethylene glycol (PEG) conjugated to hemoglobin or to albumin, wherein the PEG is a maleimide PEG, an alkylamide PEG, an iodoacetamide PEG, a p-nitro thio-phenyl PEG, a vinyl sulfone PEG, or a mixed disulfide PEG; and PEGylated albumins and PEGylated hemoglobins comprising polyethylene glycol (PEG) attached to a thiolated amino group of albumin or hemoglobin, wherein the amino group is thiolated using dithio sulfo succinimidyl propionate (DTSSP) or dithiosuccinimidyl propionate (DTSP) or dithiobispropionimidate. The invention also provides methods of preparing PEGylated hemoglobins and PEGylated albumins comprising a) reacting hemoglobin or albumin with a thiolating agent and with a PEGylating agent, and b) capping unPEGylated reactive thiols of hemoglobin or albumin with N-ethyl maleimide.

1 . A PEGylated hemoglobin or a PEGylated albumin comprising a polyethylene glycol (PEG) conjugated to hemoglobin or to albumin, wherein the PEG is a maleimide PEG, an alkylamide PEG, an iodoacetamide PEG, a p-nitro thio-phenyl PEG, a vinyl sulfone PEG, or a mixed disulfide PEG. 
     
     
         2 . The PEGylated hemoglobin of  claim 1 , wherein the PEGylated hemoglobin is a carboxamidomethyl (CAM) PEGylated hemoglobin. 
     
     
         3 . The PEGylated hemoglobin or PEGylated albumin of  claim 1 , wherein the maleimide PEG is a maleimide phenyl PEG or a maleimide PEG comprising an alkyl linker. 
     
     
         4 . The PEGylated hemoglobin or PEGylated albumin of  claim 1 , wherein the PEG is attached to the albumin or to the hemoglobin via a linker and/or an extension arm. 
     
     
         5 . The PEGylated hemoglobin or PEGylated albumin of  claim 4 , wherein the linker comprises an alkyl aryl and/or heteroaryl group. 
     
     
         6 - 8 . (canceled) 
     
     
         9 . The PEGylated hemoglobin or PEGylated albumin of  claim 1 , wherein each PEG chain has a molecular weight of 3,000 to 5,000 daltons. 
     
     
         10 - 14 . (canceled) 
     
     
         15 . The PEGylated albumin of  claim 1 , wherein 6-18 PEG chains are conjugated to albumin. 
     
     
         16 - 18 . (canceled) 
     
     
         19 . The PEGylated hemoglobin of  claim 1 , wherein 2-8 PEG chains are conjugated to hemoglobin. 
     
     
         20 . (canceled) 
     
     
         21 . The PEGylated hemoglobin or PEGylated albumin of  claim 1  or claim  7 , wherein PEGylation does not alter the surface charge of albumin or hemoglobin. 
     
     
         22 . The PEGylated hemoglobin or PEGylated albumin of  claim 1  or claim  7 , wherein PEGylation does alter the surface charge of albumin or hemoglobin. 
     
     
         23 . (canceled) 
     
     
         24 . The PEGylated hemoglobin of  claim 1 , wherein the hemoglobin contains an intramolecular crosslink. 
     
     
         25 . The PEGylated crosslinked hemoglobin of  claim 24 , wherein crosslinking the hemoglobin increases molecular volume of the PEGylated crosslinked hemoglobin. 
     
     
         26 . (canceled) 
     
     
         27 . A method of preparing a PEGylated hemoglobin or a PEGylated albumin comprising;
 a) reacting hemoglobin or albumin with a thiolating agent to produce thiolated hemoglobin or thiolated albumin;   b) reacting the thiolated hemoglobin or the thiolated albumin with a PEGylating agent; and   c) capping unPEGylated reactive thiols of hemoglobin or albumin with N-ethyl maleimide.   
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 27 , wherein the PEGylating agent is an iodoacetamide PEG or a maleimide PEG. 
     
     
         30 . A method of preparing a PEGylated hemoglobin or a PEGylated albumin comprising;
 d) reacting hemoglobin or albumin with dithio sulfo succinimidyl propionate (DTSSP) or with dithiosuccinimidyl propionate (DTSP) or with or dithiobispropionimidate to thiolate the hemoglobin or albumin, and   e) reacting the thiolated hemoglobin or the thiolated albumin with a PEGylating agent.   
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 27 , wherein the hemoglobin contains an intramolecular crosslink. 
     
     
         33 . A PEGylated hemoglobin or a PEGylated albumin prepared by the method of  claim 27 . 
     
     
         34 . A composition comprising the PEGylated hemoglobin or the PEGylated albumin of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         35 . A blood substitute comprising the PEGylated hemoglobin or PEGylated albumin of  claim 1 . 
     
     
         36 . A method of treating a subject which comprises administering to the subject a PEGylated hemoglobin or a PEGylated albumin of  claim 1 .