Hemoglobin compositions
The invention provides compositions containing hemoglobin, particularly PEGylated hemoglobin. The PEGylated hemoglobin molecule is capable of transferring oxygen or carbon monoxide bound thereto to a tissue with which it is in proximity. Exemplary PEGylated hemoglobin formulations of the invention are virally inactivated. Various compositions of the invention include deoxygenated hemoglobin, which may be conjugated with one or more water-soluble polymer. PEGylated hemoglobin includes those species in which the iron atom of the hemoglobin molecule is not bound to oxygen or any other species, and hemoglobin molecules in which a species other than oxygen, e.g., carbon monoxide, is bound to the iron atom. The compositions of the invention are formulated as hypo-, iso- or hypertonic solutions of the PEGylated hemoglobin. The compositions are of use to treat and/or ameliorate disease, injury and insult by providing for the oxygenation of tissues and/organs.
1. An aqueous pharmaceutical formulation capable of delivering oxygen or carbon monoxide (CO) to tissue of a subject to whom said formulation is administered, said formulation comprising a covalent conjugate between a functional, native hemoglobin molecule and at least one molecule of poly(ethylene glycol), said formulation comprising:
a. a water-soluble hemoglobin fraction comprising a group of bovine hemoglobin molecules wherein each member of said group of bovine hemoglobin molecules
i. is bound to CO;
ii. is covalently conjugated to at least one molecule of said poly(ethylene glycol) through an amine moiety of an amino acid residue;
iii. is substantially free of chemical cross-linking agents;
iv. includes less than 5% cross-linked hemoglobin; and
iv. has a P 50 of from about 9 mm Hg to about 14 mm Hg; and
b. a water-soluble stabilizer fraction rendering said group of bovine hemoglobin molecules oxidation resistant, said water-soluble stabilizer fraction comprising, a stabilizing agent comprising a structural element more reactive with oxygen than said group of bovine hemoglobin molecules; and
c. an aqueous diluent fraction comprising, a pharmaceutically acceptable aqueous diluent in which said hemoglobin fraction is soluble,
said formulation being essentially free of viral activity, and stably comprising less than about 10% methemoglobin after storage at 45° C. for at least about 4 days.
2. The formulation according to claim 1 wherein each member of said group of bovine hemoglobin molecules is covalently conjugated to five or more molecules of said poly(ethylene glycol) through said amine moiety of five or more of said amino acid residues.
3. The formulation according to claim 1 wherein said amine moiety of said amino acid residue is an ε-amine moiety of a lysine residue.
4. The formulation according to claim 1 wherein said stabilizing agent is an amino acid.
5. The formulation according to claim 4 wherein said stabilizing agent is cysteine.
6. The formulation of claim 1 , wherein said aqueous diluent fraction comprises a phosphate buffered saline diluent.
7. The formulation of claim 1 , wherein said aqueous diluent fraction comprises a hypertonic saline diluent.
8. The pharmaceutical formulation according to claim 7 wherein said hypertonic saline diluent includes NaCl at a concentration of from about 2.5% to about 7.5%.
9. The formulation according to claim 1 , said formulation stably comprising less than about 10% methemoglobin after storage at 45° C. for at least about 15 days.