Nitroxylated proteins and methods for the use thereof

Abstract

Succinimide-activated nitroxyl compounds and methods for the synthesis of such compounds are provided. The invention also relates to the use of succinimide-activated nitroxyl compounds to prepare nitroxylated proteins, for example nitroxylated heme proteins (e.g., nitroxylated hemoglobin and nitroxylated myoglobin). The nitroxylated proteins are optionally also conjugated to a polyalkylene oxide (PAO), for example to a polyethylene glycol (PEG). Polynitroxylated heme proteins are useful as oxygen therapeutic agents (OTAs). The invention further relates to pharmaceutical compositions of the nitroxylated proteins and methods for the use of nitroxylated proteins in the treatment of various conditions.

What is claimed is: 
     
         1 . A nitroxylated protein having the structure (VI): 
       
         
           
           
               
               
           
         
         wherein 
         Z represents the protein, and the protein comprises a hemoglobin tetramer;
 each of R 1 , R 2 , R 3 , and R 4  are independently C 1 -C 4  alkyl; 
 
         X is oxygen, sulfur, nitrogen, phosphorus, or silicon; 
         Y is CH 2 ; 
         m is 0 or 1; 
         p is the average number of nitroxyl groups conjugated to the protein; and 
         N is a nitrogen of the protein. 
       
     
     
         2 . The nitroxylated protein of  claim 1 , wherein each of R 1 , R 2 , R 3 , and R 4  is —CH3. 
     
     
         3 . The nitroxylated protein of  claim 1 , wherein m is 0. 
     
     
         4 . The nitroxylated protein of  claim 1 , wherein m is 1. 
     
     
         5 . The nitroxylated protein of  claim 1 , wherein the N-terminal amino group of the protein is nitroxylated. 
     
     
         6 . The nitroxylated protein of  claim 1 , wherein at least one epsilon (ε)-amino group of a lysine residue of the protein is nitroxylated. 
     
     
         7 . The nitroxylated protein of  claim 1 , wherein p is about 1 to about 25. 
     
     
         8 . The nitroxylated protein of  claim 1 , wherein p is at least about 2. 
     
     
         9 . The nitroxylated protein of  claim 8 , wherein p is at least about 10. 
     
     
         10 . The nitroxylated protein of  claim 7 , wherein p is about 15 to about 20. 
     
     
         11 . The nitroxylated protein of  claim 1 , wherein the hemoglobin tetramer comprises a cross-linked aa dimer or a cross-linked R R  dimer. 
     
     
         12 . The nitroxylated protein of  claim 1 , wherein the hemoglobin tetramer comprises about seventeen nitroxylated amino groups. 
     
     
         13 . The nitroxylated protein of  claim 1 , wherein the nitroxylated protein is conjugated to a polyethylene glycol (PEG). 
     
     
         14 . The nitroxylated protein of  claim 13 , wherein the PEG is a maleimide-PEG. 
     
     
         15 . The nitroxylated protein of  claim 14 , wherein the maleimide is linked to the PEG via an ethylene linker. 
     
     
         16 . The nitroxylated protein of  claim 14 , wherein the maleimide-PEG is conjugated to a thiol moiety of the protein selected from the group consisting of an intrinsic thiol moiety of a cysteine residue of the protein, a thiol moiety of a thiolated lysine residue of the protein, and a combination thereof, and the maleimide-PEG has the structure (VIII) 
       
         
           
           
               
               
           
         
         wherein 
         Z represents the protein, 
         S is a thiol of the protein, 
         R 3 ′ is an alkylene or phenylene group, 
         X is a terminal group, 
         m′ is the average number of activated-PEG polymers conjugated to the protein, and 
         n′ represents the average number of oxyethylene units of a PEG having an average molecular weight of about 2,000 to about 20,000 Daltons. 
       
     
     
         17 . The nitroxylated protein of  claim 16 , wherein R 3 ′ is ethylene, m′ is about 6 to about 10, and X′ is methoxy (—OCH 3 ) or carboxylate (—COOH). 
     
     
         18 . The nitroxylated protein of  claim 13 , wherein the PEG is a succinimidyl valerate PEG (SVA-PEG). 
     
     
         19 . The nitroxylated protein of  claim 18 , wherein the SVA-PEG is conjugated to an amino moiety of the protein selected from an ε-amino moiety of a lysine residue of the protein, an α-amino moiety of a terminal valine residue of the protein, or a combination thereof, and the SVA-PEG conjugated to an ε-amino moiety of a lysine residue of the protein or an α-amino moiety of a terminal valine residue of the protein has the structure (IX) 
       
         
           
           
               
               
           
         
         wherein: 
         Z is the protein, 
         N is an amino group of the protein, 
         X″ is a terminal group, 
         m″ is the number of activated-PEG polymers conjugated to the protein, and 
         n″ is the average number of oxyethylene units of a PEG having an average molecular weight of from about 2,000 to about 20,000 Daltons. 
       
     
     
         20 . The nitroxylated protein of  claim 19 , wherein X″ is methoxy (—OCH 3 ) or carboxylate (—COOH), and m″ is on average from about 6 to about 10 PAO molecules per tetramer. 
     
     
         21 . The nitroxylated protein of  claim 1 , wherein the hemoglobin tetramer is deoxygenated, liganded with CO, liganded with NO, or liganded with a mixture of CO and NO. 
     
     
         22 . A pharmaceutical composition comprising the nitroxylated protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         23 . The pharmaceutical composition of  claim 22 , wherein the pharmaceutically acceptable carrier comprises physiological saline, a saline-glucose mixture, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs-Ringer's solution, Hartmann's balanced saline, heparinized sodium citrate-citric acid-dextrose solution, an acetate solution, a multiple electrolyte solution, a lactiobionate solution, s polyethylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol, an ethylene oxide-propylene glycol condensate, a filler, a salt, a physiological buffer, a carbohydrate, an alcohol, a poly alcohol, an anti-oxidant, an anti-bacterial agent, an oncotic pressure agent, a reducing agent, or a combination thereof. 
     
     
         24 . A method of treatment comprising administering the nitroxylated protein of  claim 1  to a subject in need thereof, wherein the method is:
 for the treatment of acute liver failure, beta thalassemia, a burn, chronic critical limb ischemia, carbon dioxide or cyanide poisoning, chronic obstructive pulmonary disease (COPD), congestive heart failure, hypoxia, malaria, organ ischemia, peripheral vascular disease, porphyria, pre-eclampsia in pregnancy, sepsis, sickle cell disease, retinal disease, an intra-ocular condition, testicular torsion, trauma, shock, traumatic brain injury, ulcers, vasospasm, or a combination thereof; 
 for the treatment of non-traumatic hemorrhagic shock, pre-hospital setting trauma, traumatic hemorrhagic shock, acute lung injury, adult respiratory distress syndrome, traumatic brain injury, stroke, solid tumor cancer, organ degradation (ex-vivo), organ degradation (in recipient), severe sepsis, septic shock, myocardial infarction, cardiac ischemia, cardiogenic shock, acute heart failure, pulmonary embolism, or a combination thereof; 
 as an adjunct to angioplasty, as an adjunct for plastic surgery, or as an adjunct in implanting a ventricular assist device; as a blood substitute, a cardioprotectant, a cryopreservative, a hemodialysis adjunct, an oncology agent, an organ preservative, a performance enhancement agent, a surgery adjunct, or a wound healing agent; in imaging; to improve lung function; or a combination thereof; 
 as an adjunct to thoracic aortic repairs, as an adjunct to cardiopulmonary bypass, or as a priming solution for cardiopulmonary bypass; 
 for veterinary treatment of loss of blood due to injury, hemolytic anemia, infectious anemia, bacterial infection, Factor IV fragmentation, hypersplenation and splenomegaly, hemorrhagic syndrome in poultry, hypoplastic anemia, aplastic anemia, idiopathic immune hemolytic conditions, iron deficiency, isoimmune hemolytic anemia, microangiopathic hemolytic anemia, parasitism, or surgical-anesthesia induced brain damage; or 
 for delivering oxygen, nitric oxide, carbon monoxide or mixtures thereof to tissue and reducing nitrite to nitric oxide (NO) in microvasculature wherein following administration, the hemoglobin becomes unliganded and converts nitrite to nitric oxide in the microvasculature.