Nitric oxide-blocked cross-linked tetrameric hemoglobin

Abstract

The present invention includes compositions containing carboxamidomethylated cross-linked hemoglobin where the cysteine moiety of the hemoglobin includes a thiol protecting group and where the hemoglobin has a reduced ability to bind with nitric oxide. Preferably, the hemoglobin is deoxygenated, endotoxin free, and stroma free. The present invention also includes method of preparation, process of preparation and the method of use including supplementing blood volume in mammals and treating disorders in mammals where oxygen delivery agents are of benefit.

1 . A blood substitute suitable for administration to a human patient, the blood substitute comprising:
 a tetrameric hemoglobin; and   a pharmaceutically acceptable carrier;   wherein said tetrameric hemoglobin comprises four bovine polypeptides covalently cross-linked together; and   wherein said tetrameric hemoglobin includes at least one cysteine moiety chemically modified with a thiol-protecting group such that said at least one cysteine moiety is incapable of binding nitric oxide; and   wherein said tetrameric hemoglobin has a p50 for oxygen greater than that of whole human blood.   
     
     
         2 . The blood substitute of  claim 1 , wherein said tetrameric hemoglobin has a molecular weight greater than 60,000 daltons. 
     
     
         3 . The blood substitute of  claim 1 , wherein said blood substitute is endotoxin and stroma free. 
     
     
         4 . The blood substitute of  claim 1 , wherein said blood substitute is non-pyrogenic. 
     
     
         5 . The blood substitute of  claim 1 , wherein said blood substitute is deoxygenated. 
     
     
         6 . The blood substitute of  claim 1 , wherein said tetrameric hemoglobin comprises four bovine polypeptides covalently cross-linked together with bis 3′,5′ dibromo salicyl fumarate. 
     
     
         7 . The blood substitute of  claim 1 , wherein said tetrameric hemoglobin comprises four bovine polypeptides covalently cross-linked together with a poly-functional agent selected from the group consisting of: glutaraldehyde; succindialdehyde; activated polyoxyethylene; activated dextran; α-hydroxy aldehyde; glycolaldehyde; N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonic acid)-phenyl ester; m-maleimidobenzoic acid-N-hydroxysuccinimide ester; succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; m-maleimidobenzoyl-N-hydroxysuccinimide ester; m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; N-succinimidyl(4-iodoacetyl)aminobenzoate; sulfosuccinimidyl (4-iodoacetyl)aminobenzoate; succinimidyl 4-(p-maleimidophenyl)butyrate; sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; N,N′-phenylene dimaleimide; bis-imidate; acyl diazide; and aryl dihalide. 
     
     
         8 . The blood substitute of  claim 1 , wherein the thiol-protecting group is a carboxymethyl such that the tetrameric hemoglobin is a carboxymethylated tetrameric hemoglobin. 
     
     
         9 . The blood substitute of  claim 1 , wherein the thiol-protecting is one of: 4-pyridylmethyl; acetylaminomethyl; alkoxyalkyl; triphenylmethyl; carboxymethyl; acetyl; benzyl; benzoyl; tert-butoxycarbonyl; p-hydroxyphenacyl; p-acetoxybenzyl; p-methoxybenzyl; 2,4-dinitrophenyl; isobutoxymethyl; tetrahydropyranyl; acetamidomethyl; bezamidomethyl; bis-carboethoxyethyl; 2,2,2-trichloroethoxycarbonyl; tert-butoxycarbonyl; N-alkyl carbamate; and N-alkoxyalkyl carbamate. 
     
     
         10 . The blood substitute of  claim 1 , in combination with an oxygen impermeable polymer bag in which the blood substitute is enclosed. 
     
     
         11 . A blood substitute suitable for administration to a human patient, the blood substitute comprising:
 tetrameric hemoglobin; and   a pharmaceutically acceptable carrier;   wherein said tetrameric hemoglobin comprises four polypeptide chains derived from a non-human source covalently cross-linked together; and   wherein said tetrameric hemoglobin includes at least one cysteine moiety chemically modified with a thiol-protecting group such that said at least one cysteine moiety is incapable of binding nitric oxide; and   wherein said covalently cross-linked tetrameric hemoglobin has a p50 for oxygen greater than that of human whole blood.   
     
     
         12 . The blood substitute of  claim 11 , wherein said four polypeptide chains are bovine polypeptide chains. 
     
     
         13 . The blood substitute of  claim 11 , wherein said four polypeptide chains are porcine polypeptide chains. 
     
     
         14 . The blood substitute of  claim 11 , wherein said blood substitute is deoxygenated and non-pyrogenic. 
     
     
         15 . The blood substitute of  claim 11 , in combination with an oxygen impermeable polymer bag in which the blood substitute is enclosed. 
     
     
         16 . The blood substitute of  claim 11 , wherein said cross-linked tetrameric hemoglobin comprises hemoglobin proteins derived from a non-human source and cross-linked with bis 3′,5′ dibromo salicyl fumarate. 
     
     
         17 . The blood substitute of  claim 11 , wherein said cross-linked tetrameric hemoglobin comprises hemoglobin proteins derived from a non-human source and cross-linked with a poly-functional agent selected from the group consisting of: glutaraldehyde; succindialdehyde; activated polyoxyethylene; activated dextran; α-hydroxy aldehyde; glycolaldehyde; N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonic acid)-phenyl ester; m-maleimidobenzoic acid-N-hydroxysuccinimide ester; succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; m-maleimidobenzoyl-N-hydroxysuccinimide ester; m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester; N-succinimidyl(4-iodoacetyl)aminobenzoate; sulfosuccinimidyl (4-iodoacetyl)aminobenzoate; succinimidyl 4-(p-maleimidophenyl)butyrate; sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate; 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; N,N′-phenylene dimaleimide; bis-imidate; acyl diazide; and aryl dihalide. 
     
     
         18 . The blood substitute of  claim 11 , wherein the thiol-protecting group is a carboxymethyl such that the covalently cross-linked tetrameric hemoglobin is a covalently cross-linked carboxymethylated tetrameric hemoglobin. 
     
     
         19 . The blood substitute of  claim 11 , wherein the thiol-protecting is one of: 4-pyridylmethyl; acetylaminomethyl; alkoxyalkyl; triphenylmethyl; carboxymethyl; acetyl; benzyl; benzoyl; tert-butoxycarbonyl; p-hydroxyphenacyl; p-acetoxybenzyl; p-methoxybenzyl; 2,4-dinitrophenyl; isobutoxymethyl; tetrahydropyranyl; acetamidomethyl; bezamidomethyl; bis-carboethoxyethyl; 2,2,2-trichloroethoxycarbonyl; tert-butoxycarbonyl; N-alkyl carbamate; and N-alkoxyalkyl carbamate. 
     
     
         20 . A blood substitute suitable for administration to a human patient, the blood substitute comprising:
 tetrameric hemoglobin in a pharmaceutically acceptable carrier;   wherein said tetrameric hemoglobin comprises four polypeptide chains derived from a non-human source covalently cross-linked together with bis 3′,5′ dibromo salicyl fumarate; and   wherein said tetrameric hemoglobin includes at least one cysteine moiety chemically modified with a thiol-protecting group such that said at least one cysteine moiety is incapable of binding nitric oxide; and   wherein said tetrameric hemoglobin has a p50 for oxygen greater than 27 mm Hg.